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Index
Resumen ......................................................................................................................................................... 5
Abstract ........................................................................................................................................................... 7
1. Introduction ..................................................................................................................... 8
1.1 Cancer ................................................................................................................................................... 8
1.2 Pancreatic ductal adenocarcinoma (PDAC) ............................................................................. 9
1.3 The lipid membrane ....................................................................................................................... 10
1.3 Autophagy ......................................................................................................................................... 14
1.4 Lipid membrane alterations in cancer ..................................................................................... 18
1.5 Membrane-Lipid Therapy ............................................................................................................ 19
1.6 Objects of study: Mimetic triglycerides (TGMs) .................................................................. 20
2. Objectives ................................................................................................................................................ 22
3. Materials and methods..................................................................................................................... 23
3.1 Cell lines ............................................................................................................................................. 23
3.2 Cell culture ......................................................................................................................................... 23
3.2.1 Cell lines and growth conditions....................................................................................... 23
3.2.2 Thawing ...................................................................................................................................... 23
3.2.3 Cell passaging / maintaining .............................................................................................. 23
3.2.4. Freezing ..................................................................................................................................... 24
3.2.5 Cellular treatments ................................................................................................................. 24
3.3 Cell proliferation / cytotoxicity studies ................................................................................... 24
3.4 Cell cycle studies ............................................................................................................................. 25
3.5 Western Blot studies ...................................................................................................................... 26
3.5.1 Cell lysate ................................................................................................................................... 26
3.5.2 Protein quantification ............................................................................................................ 27
3.5.3 Western Blot ............................................................................................................................. 27
3.6 Thin Layer Chromatography ....................................................................................................... 29
3.6.1 Lipid extraction ........................................................................................................................ 29
3.6.2 Thin layer chromatography ................................................................................................. 30
3.7 In vivo studies ................................................................................................................................... 30
3.7.1 Experimental animals............................................................................................................. 30
3.7.2 Determination of the toxicity of the compounds TGM4 and TGM5. .................. 31
3.7.3 Antitumor studies ................................................................................................................... 31
3.8 Data analysis ..................................................................................................................................... 32
4. Results ....................................................................................................................................................... 33
4.1 Mimetic triglycerides (TGMS) inhibit cell viability and proliferation. .......................... 33
4.2 Determination of a safe dose to perform in vivo studies with TGM4 and TGM5... 36
4.3 TGM4 effect on the progression of Mia-PaCa-2 cell line xenograft in nude
immunodepressed mice ...................................................................................................................... 38
4.4 Effect of the TGM4 on the cell cycle. ....................................................................................... 40
4.5 Finding the type of cell death and related proteins behind the TGM4 effect. ........ 43
4.6 Lipid alterations produced by the treatment with TGM4 ................................................ 45
5. Discussion ............................................................................................................................................... 47
5.1 The TGMs, a novel library of compounds, are effective antiproliferative drugs. .... 47
5.2 The TGM4 is an effective antitumor drug, tested on Mia-PaCa-2 xenograft in vivo
models of pancreatic cancer. ............................................................................................................. 48
5.3 TGM4 produces an antiproliferative effect and death by autophagy in cancer cells
PANC-1. ..................................................................................................................................................... 50
6. Conclusions ............................................................................................................................................ 54
7. References ...................................................................................................................... 55
5
Resumen
El cáncer de páncreas de células ductales (PDAC en inglés) es la cuarta causa de muerte
por cáncer en EEUU y la sexta en Europa. Su difícil diagnóstico precoz, acompañado de
su alto índice de metástasis, hace de este cáncer una enfermedad fatal: la media de
supervivencia desde el diagnóstico es de solo 4-6 meses. La única terapia curativa es la
resección quirúrgica, y fuera de ella no hay tratamientos eficaces disponibles a día de
hoy. El cáncer de páncreas ofrece un escenario donde el desarrollo de nuevas terapias es
necesario, y este trabajo de fin de master estudia el uso de nuevos fármacos de diseño
que funcionan a través la Terapia Lipídica de Membrana como tratamiento contra el
cáncer pancreático.
En este trabajo se realizó un screening del efecto antitumoral in vitro de una nueva
librería de compuestos, los triglicéridos miméticos o TGMs. Estos compuestos
presentaron, en mayor o menor medida, efectos antiproliferativos destacables en una
línea celular de cáncer pancreático.
Se seleccionaron los compuestos TGM4 y TGM5 para realizar estudios in vivo utilizando
un modelo de inducción de tumores xenográficos en ratones inmunodeprimidos NUDE,
mediante la inyección subcutánea de células de cáncer de páncreas Mia-PaCa-2. Ambos
demostraron no ser tóxicos a las dosis estudiadas y ambos mostraron actividad
antitumoral provocando una reducción de la progresión y crecimiento de los tumores
inducidos respecto a los controles. Sin embargo, la eficacia del TGM4 fue mayor a la del
TGM5 y muy similar a la de la Gemcitabina, el fármaco de referencia para el tratamiento
del cáncer de páncreas en humanos. Por los resultados obtenidos, se seleccionó el TGM4
como compuesto del que determinar el mecanismo de acción.
En la investigación del mecanismo de acción del TGM4 se utilizaron células tumorales
ductales PANC-1. Se pudo determinar por citometría de flujo que el TGM4 producía
muerte celular y una disminución del porcentaje de células en fase G0/G1, datos
apoyados por la disminución de la proteína reguladora del ciclo celular Ciclina D3.
Estudios a nivel de proteína revelaron que el TGM4 produce la muerte celular a través de
un estrés reticular (indicado por la sobreexpresión de BIP) que desencadena a través de
CHOP la muerte por autofagia. La formación de autofagosomas fue corroborada por la
aparición de LC3B-II. También se observaron cambios en vías de señalización implicadas
en oncogénesis como son la vía de las MAPK y PI3K/AKT y en el proto-oncogen c-jun.
Finalmente, mediante cromatografía de capa fina se estudió como afectaba el TGM4 a la
composición de la membrana lipídica, revelando cambios en los fosfolípidos cardiolipina
y fosfatidilcolina.
En resumen, los resultados indican que los compuestos incluidos en la librería de TGMs
estudiada tienen efectos antiproliferativos en células tumorales. El TGM4 y TGM5 han
demostrado mantener el efecto en modelos in vivo, con resultados significativos en el
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caso del TGM4. Tras los estudios realizados, se puede decir que el TGM4 es una molécula
que por su efecto antiproliferativo a través de la inducción de la autofagia, y por su
aparente inocuidad y efectividad antitumoral en modelos in vivo, podría ser un fármaco
alternativo en el tratamiento del cáncer de páncreas.
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Abstract
Pancreatic cancer ductal cells (PDAC) is the fourth leading cause of cancer death in the
US and sixth in Europe. Its difficult early diagnosis accompanied by its high rate of
metastasis makes this a fatal disease: the median survival time from diagnosis is only 4-
6 months. The only curative therapy is surgical resection, and outside there are no
effective treatments available today. Pancreatic cancer requires the development of new
therapies, and this master thesis studies the use of novel synthetic lipidic drugs included
in the Membrane-Lipid Therapy as a treatment for pancreatic cancer.
In this work an in vitro screening to determine the antitumor effect of novel mimetic
triglycerides or TGMs library was performed. These compounds showed a greater or
lesser remarkable antiproliferative effects on a pancreatic cancer cell line.
The compounds TGM4 and TGM5 were selected for in vivo studies in xenograft tumor
models induced by pancreatic cancer cells, Mia-PaCa-2, injected subcutaneously in
immunodepressed nude mice. Both demonstrated to be non-toxic at the tested doses
and showed antitumor activity impairing the tumor growth and progression in
comparison to control. However, TGM4 showed greater efficacy than TGM5, being very
similar to gemcitabine, the standard of care drug for treating pancreatic cancer in
humans. From these results, TGM4 was selected to determine its mechanism of action.
Investigating the mechanism of action of TGM4, pancreatic ductal tumor cells PANC-1
were used. TGM4 produced cell death and decreased the percentage of cells in G0/G1
phase, as determined by flow cytometry. This result was supported by the decrease in
cell cycle regulatory protein cyclin D3. Furthermore, TGM4 provokes cell death through
a reticular stress (indicated by BIP overexpression) that triggers autophagy via CHOP. The
autophagosomes formation was corroborated by the LC3B-II protein modification.
Changes were also observed in signaling pathways involved in oncogenesis, such as the
MAPK and PI3K / AKT pathways and the proto-oncogene c-jun. Finally, Thin-Layer
Chromatography was use to study how TGM4 affected to the composition of the lipid
membrane, showing changes in the phospholipids cardiolipin and phosphatidylcholine.
In summary, the results indicate that the compounds included in the TGMs library have
antiproliferative effect on tumor cells. In fact, TGM4 and TGM5 have shown to be
antitumor drugs on in vivo models, with outstanding results in the case of TGM4. As
conclusion, TGM4 is a molecule which due to its antiproliferative effects through the
induction of autophagy, apparent safety and antitumor effectiveness in vivo models
could be an alternative drug in the treatment of pancreatic cancer.
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1. Introduction
1.1 Cancer
Cancer is the name given to a collection of diseases which share a common feature: a
transformation of cells leading to an abnormal and uncontrolled proliferation. Usually,
human cells grow and divide to form new cells as the body needs them, but if the new
cells are mutated or damaged, they die and healthy cells take their place, in cancer this
basic process is altered.
The uncontrolled proliferation leads to the formation of new cells when they are not
needed. Cancer cells differ from normal cells in many ways that allow them to grow out
of control and become invasive. These cells dividing without stopping may form a cell
mass commonly known as tumor, finally able to spread and invade subjacent tissues and
form new tumors in the process known as metastasis.
Figure 1. This illustration encompasses the six hallmark capabilities of tumor cells. From: Hanahan
& Weinberg, 2011.
The transformation of the cells from normal cells to tumor cells is mainly caused by the
sequential acquisition of mutations, which give to the cells the characteristics to grow
and evade the immune and homeostatic barriers (Hanahan and Weinberg, 2000). These
specific properties (Figure 1) are: immune evasion, redirection of metabolic energy,
invasiveness and metastasis, sustained angiogenesis, limitless replicative potential,
evasion of apoptosis, insensitivity to antiproliferative signals and self-sufficiency in
growth signals (Hanahan and Weinberg, 2011). The origin of these changes are extremely
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varied, ranging from endogenous factors such as DNA replication errors, loss of
heterozygosity, free radicals generated by metabolism, spontaneous mutations or
congenital genetic predisposition, but they can be also exogenous epigenetic factors
such as ionizing radiation, ultraviolet radiation or abundantly present carcinogens in the
current lifestyle.
Added to the internal nature of this kind of disease, the cells in a tumor are not exactly
equal, being possible to find, in a unique tumor, cells with highly different biochemical,
morphological and immunological features. In addition, cancer cells are able to ignore
signals that normally tell cells to stop dividing, as programmed cell death, or apoptosis,
which the organism uses to get rid of unneeded cells. Cancer cells may be able to
influence the normal cells, molecules, and blood vessels that surround and feed a
tumor—an area known as the microenvironment.
The genetic changes that contribute to cancer affect three main types of genes—proto-
oncogenes, tumor suppressor genes, and DNA repair genes. These changes are
sometimes called “drivers” of cancer.
In that scenario, proto-oncogenes are involved in normal cell growth and division.
However, when these genes are altered, usually being more active than normal, they may
become cancer-causing genes (or oncogenes), allowing cells to grow and survive when
they should not. On the other hand, tumor suppressor genes are also involved in
controlling and stopping cell growth and division. Cells with certain alterations in tumor
suppressor genes may grow in an uncontrolled manner. Finally, DNA repair genes are
involved in fixing damaged DNA, for example the mutations in proto-oncogenes or
tumor suppressor genes. Cells with mutations in DNA repair genes tend to develop
accumulate mutations in several genes. Together, these mutations may cause the cells to
become cancerous
As this introduction has tried to manifest, cancer is a highly complex disease, moreover,
it figure among the leading causes of morbidity and mortality worldwide, with
approximately 14 million new cases and 8.2 million cancer related deaths in 2012 (World
Cancer Report, 2014). The number of new cases is expected to rise by about 70% over
the next 2 decades.
1.2 Pancreatic ductal adenocarcinoma (PDAC)
The pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death
in the United States and sixth in Europe, despite of the relative short incidence of 10-
12:100.000 (Jemal et al. 2009). Every year more than 40.000 people are diagnosed with
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PDAC only in the United States, and more than 36.000 die every year due to this fatal
disease.
The clearest risk factor is the advanced age, but now it is known that smoking, diabetes,
obesity, or chronic inflammation of the pancreas, known as pancreatitis, are risk factors
to develop this cancer (Everhart & Wright 1995; Gapstur et al. 2000; Michaud et al. 2001;
de Gonzalez et al. 2003; Stolzenberg-Solomon et al. 2005)
The prognosis for those diagnosed with PDAC is fatal in the vast majority of cases. Fewer
than 5% of all PDAC patients are still alive 5 years after initial diagnosis (Yang et al. 2013).
The median survival time for PDAC patients is only 4-6 months from initial diagnosis.
Unfortunately, surgical resection is still the only potentially curative treatment (Riall et al.
2005) and chemotherapy or radiotherapy are used generally as palliative treatments.
The vast majority of pancreatic cancer patients are today primarily treated with a
palliative intent to reduce symptoms as well as to prolong life for some patients.
Gemcitabine is generally advised as the standard first-line treatment for pancreatic
cancer patients (Burris Ha et al. 1997). Gemcitabine is adeoxycytidine analogue that must
be phosphorylated to become active (gemcitabine diphosphate and gemcitabine
triphosphate). When activated, gemcitabine diphosphate inhibits ribonucleotide
reductase and reduces the intracellular pool of deoxynucleotide triphosphate required
for DNA synthesis.
Despite the bad prognosis, several hallmarks of the biology of this cancer are now
understood, and with unmet clinical needs new treatments are necessary to improve the
prognosis of this fatal disease.
1.3 The lipid membrane
It took nearly two hundred years after the development of the cell theory before a
complete cell membrane theory was developed to explain what separates the cells from
the outside world, but by the 19th century it was accepted that some form of semi-
permeable barrier must exist around a cell.
The composition of that membrane was correctly intuited by Quincke, who in a series of
elegant experiments noted that a cell generally forms a spherical shape in water and,
when broken in half, forms two smaller spheres, as the oil do. The idea of a semi-
permeable membrane means that the membrane is permeable to solvent but
impermeable to solute molecules.
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The fluid mosaic model, developed by S. J. Singer and G. L. Nicolson in 1972, consider
the membrane as a lipid bilayer, formed by several different lipids able to move through
the membrane, and where a large quantity of proteins are embedded. The membrane is
not a simple structure with the only function of separate the cell content from the outside,
it is a highly complex structure that allows the cell to communicate with his surround,
and that is the base for the multicellular life. Notwithstanding, the fluid mosaic model is
simplistic and incomplete.
An adequate view of the membrane based on the knowledge achieved in the last years
is that one proposed by Engelman (Engelman, 2005) (Figure 2), a membrane where
protein density is remarkable, where the lipids are heterogeneous and where the relation
between different types of lipids (1400 in average) and thousands of proteins is intensive.
Figure 2. A good perspective of the complex fluid mosaic model, where different proteins and
lipids are components of the lipid membrane. Taken from Pietzsch, J, 2004. The membranes of
eukaryotic cells contain, among others, three classes of lipids: glycerophospholipids,
sphingolipids, and cholesterol (CHO) or a closely-related sterol. Although the relative proportions
of these three lipid classes vary according to species or cell type, in vertebrates cholesterol is
typically present at levels of 30–40%, sphingolipids at levels of 10–20%, and glycerophospholipids
at levels of 40–60% of the total plasma membrane lipids (McMullen et al. 2004).
12
The glycerophospholipids consist of a glycerol backbone, two ester-linked fatty acyl
chains, and a phosphorylated alcohol, typically phosphorylcholine (PC),
phosphorylethanolamine (PE) or phosphorylserine (PS). The fatty acyl chains usually
contain typically between 16 and 20 carbon atoms in total, being the number of
unsaturations the main difference between the different fatty acyl chains.
The sphingolipids are based on a more complex alcohol, the sphingosine. The
sphingolipids contains a single amide-linked fatty acyl chain, which is usually saturated
and may contain up to 24 carbon atoms, and either a phosphorylated alcohol (usually
phosphorylcholine), or one or more sugar molecules linked to the hydroxyl terminus of
the sphingosine backbone.
Finally, cholesterol consists of a fused cyclic four-ring structure containing a single polar
hydroxyl group and an isooctyl side chain, the cyclic ring system being essentially planar
and rigid.
In reference to the heterogeneity of the membrane, the asymmetry in the lipid
composition of the membrane is well demonstrated: the inner mono-layer contains a
higher concentration of PS and PE, whereas the extracellular mono-layer is enriched in
PC and sphingomyelin (SM) (Meer et al. 2011). More complex structures as lipid rafts,
caveolaes or clusters offer an additional level of complexity.
Depending on the lipid composition and distribution, the lipids are organized in different
phases or structures that present characteristic biophysical properties, such as fluidity,
electric charge, cross sectional area, lateral pressure profile, surface packing and non-
lamellar-phase propensities.
13
Figure 3 . Membrane lipid structures. Examples of the relationship between lipid shapes, intrinsic
curvatures and lipid phases. A. Lipids with rectangular shapes (e.g. PC, SM) do not confer a
curvature strain forming lamellar phases. B. Lipids with a bulky polar head and only one acyl chain
(e. g. lisophospholipids) have an inverted cone shape inducing a positive curvature strain in
membranes. C. Lipids with a small polar head (e.g. PE, CHO, DAG) have a molecular shape that
resembles a truncated cone. They induce a negative curvature strain. D. Examples of phospholipid-
induced curvature strains in the membrane bilayer. From Lladó et al. 2014.
The most common lipid organization in the membrane is the lamellar phase which can
be subdivided in several types of lamellar sub-structures. These sub-structures can
change from one to another depending of lipid composition, pH, ionic strength, water
concentration or lateral pressure and the temperature that modulates the fluidity (Cullis
et al. 1979)
The lamellar α sub-structure (Lα), also known as fluid lamellar phase, liquid crystalline or
liquid disordered (Ld), is the structure found in most domains and regions of the cell
membrane, it is characterized by high quantities of PE and similar lipids, resulting in a
weaker surface pressure because the small polar heads of these lipids produce a lower
surface packing density and greater lipid and protein mobility. This lamellar structure can
evolve to a variety of more organized and less mobile structures, such as the gel phase
14
(Lβ), pseudo- crystalline (Lc), ripped (Pβ) membranes and the solid ordered (so or Lo)
(Yeagle, 2005).
In the LB state, the phospholipid hydrocarbon chains are in the fully extended all-trans
conformation, the thickness of the phospholipid bilayer is maximal, and both intra- and
intermolecular motion are more restricted than in La phase.
The Lo phase is only possible in the presence of CHO. CHO acts increasing the mechanical
rigidity and cohesiveness and reducing the permeability of phospholipid bilayers, also
reducing the rotational and lateral diffusion rates (Lladó et al. 2014). CHO is also a key
component of the lipid rafts. Lipid rafts are enriched in CHO and SM with the acyl chains
of the lipids extended and highly packed. The lipid structure can operate as regulator for
protein interactions, and for these reason, essential cellular regulation processes are
compartmentalized in these CHO and SM raft domains and not out of them (Lingwood
et al. 2010; Pike et al. 2006).
In addition to the lipids that form fluid and organized lamellar phases, the cell membrane
may form non-lamellar phases, induced by non-lamellar-prone lipids such as PE, CHO,
diacyl-glycerol (DAG) and acidic PS. These lipids are structurally characterized by a non-
cylinder shape, usually exhibing truncated or inverted cone shapes, which induces a
curvature stress into one of the layers of the cell membrane (Figure 3). If non-lamellar-
prone lipids are abundant, the membrane can adopt a conformation in which some lipids
adopt an extended shape with one of the acyl chain out of the bilayer allowing a better
access to the inner part of the cell membrane (Ibarguren et al. 2013). The curvature stress
induced by the non-lamellar-prone lipids is usually organized into hexagonal and cubic
phases among others (Luzzati et al. 2009).
As this long introduction has tried to reflex, the membrane is a complex structure, key as
an element for the cell concept, but also allowing the communication between the
surrounding and the cell, including the communication between cells.
1.3 Autophagy
As happens with proliferation, every process in the cells is wonderfully under control. The
controlled degradation of cytoplasmatic material, as macromolecules or even organelles,
is known as autophagy. There are three main types of autophagy, depending on the way
in which cytoplasmatic material are delivered to lysosomes: chaperone-mediated
autophagy, microautophagy and macroautophagy.
15
Autophagy plays an essential role in normal and pathological conditions such as
starvation, clearance of intracellular proteins and organelles, development, anti-aging,
elimination of microbes, cell death and tumor suppression. For this reason, autophagy is
now a marked target in cancer studies. Moreover, autophagy defects have been
associated with a wide range of disease, from microbial infection to chronic liver disease,
obesity, inflammatory bowel disease (IBD), aging, metabolic syndromes, Crohn’s disease,
Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and cancer (Rabinowitz &
White, 2010 ; Chen & Karantza 2011 ; Chen & White, 2011; Cheung & Ip, 2011).
The autophagy process (Figure 4) starts with a portion of the cytoplasm containing
material being engulfed by an isolation membrane called phagophore, which complete
elongation results in the formation of double membrane structure known as
autophagosome. These autophagosomes, containing the cytoplasmic material to be
degraded, are fused with lysosomes forming autolysosomes, which enzymes will degrade
the cytoplasmic material of the autophagosomes. Finally, the breakdown products are
released into the cytosol by permeases to recycle to use them in the cellular metabolism
(Rubinsztein et al. 2011)
Figure 4. Schematic diagram of the steps of autophagy. Autophagy begins with the formation of the
phagophore or isolation membrane. Phagophore elongation forms an autophagosome. The autophagosome
can engulf bulk cytoplasm. When the outer membrane of the autophagosome fuses a lysosome, it forms an
autophagolysosome. Finally, the sequestered material is degraded inside the autophagolyosome and
recycled. Meléndez & Levine, B, 2009
There are two major regulating pathways which control autophagy: The main regulating
mTOR dependent and mTOR independent.
The mTOR is the mammalian ortholog of the yeast protein kinase target of rapamycin
(TOR), which negatively regulates autophagy. In normal or non-pathological conditions,
when nutrients are present, the organism produce insulin and growth factors as signal of
nutrient abundance. The mTOR receive signals from insulin or growth factors from the
16
class 1 phosphatidylinositol-3-OH kinase (PI3K-I). PI3K-I, using the plasma membrane
lipid phosphatidylinositol-4,5-bisphosphate (PIP2) produce phosphatidylinositol-3,4,5-
trisphosphate (PIP3). The PIP3 recruits phosphoinositide-dependent kinase1 (PDK1),
phosphoinositide-dependent kinase 2 (PDK2) and protein kinase B (AKT/PKB) from the
cytosol to the plasma membrane (Mizushima & Levine, 2010). PDK1 and PDK2 function
is the phosphorylation of the third serine/threonine kinase, AKT. The phosphorylation in
specific amino acids (Thr 308 phosphorylated by PDK1 and Ser 473 phosphorylated by
PDK2) results in the activation of AKT. Activated AKT finally inactivates by
phosphorylation the tuberous sclerosis complex (TSC) 1/2. The inactivation of TSC 1/2
leads to activation of Rheb protein which subsequently activates mTORC1. The activation
of mTORC1 leads to inhibition of autophagy (Mizushima & Levine, 2010)
Nutrient starvation or presence of rapamycin, results in mTORC1 inactivation (complex
of mTOR). The inhibition of mTOR results in the translocation from the cytosol to the
endoplasmic reticulum (ER) of the ATG1 complex, comprising Unc-51-like kinase 1/2
(ULK1/2), ATG13, focal adhesion kinase family interacting protein of 200 kD (FIP200) and
ATG101. This translocation leads to the recruitment of class III PI3K complex, consisting
of VPS34 (Vascular protein sorting 34), VPS15, Beclin -1 and ATG14 to the ER site (Easton
& Houghton, 2006). The formation of phagophores is initiated when FIP200 and ATG13
are phosporylated by ULK1 (Rabinowitz & White, 2010; Levine et al. 2011). For nucleation
phase, VPS34 is activated by Beclin-1 (Funderburk et al. 2010) to generate
phosphatidylinositol 3-phosphate (PI3P), activating two ubiquitin-like pathways for the
third phase, the elongation.
Figure 5. Autophagy is regulated by a set of autophagy-related proteins (ATG proteins). In the
absence of amino acids or in response to other stimuli, ATG1 and a complex of the class III PI3K
(phosphoinositide 3-kinase) VPS34 and beclin 1 lead to the activation of downstream ATG factors
that are involved in the initiation (a), elongation (b) and maturation (c) of autophagy. a In amino-
acid-rich conditions, VPS34 contributes to mTOR activation and inhibition of ATG1 and
17
autophagy. b | The elongation and shape of the autophagosome are controlled by two protein
(and lipid) conjugation systems, similar to the ubiquitylation systems: the ATG12 and LC3
conjugation pathways, which include E1-activating and E2-conjugating enzymes. c | LC3
associated with the lumenal membrane remains trapped in the autophagosome and is degraded
during maturation into the autolysosome, which involves fusion of autophagosomes with late
endosomes, including endosomal multivesicular bodies and lysosomal organelles, and dissolution
of the internal membrane. VPS34 has a role in the formation of late endosomal multivesicular
bodies and lysosomal organelles contributing to the maturation stages of autophagy. Adapted
from: Levine & Deretic, 2007.
Atg 7, an ubiquitin-activating (E1-like) enzyme, activates the ubiquitin-like protein Atg12
through an ubiquitination-like process. Atg12 is subsequently transferred to Atg10, an
E2-like enzyme, which in turn conjugates with the lysine on Atg5 to create the conjugate
Atg5/Atg12. The conjugate complex binds to Atg16. The complex Atg12/5/16L is bound
to the outer layer of the isolated membrane of the phagophore (Levine et al. 2011),
enabling the second ubiquitin-like pathway to occur. The cysteine C-terminal residue of
the Atg8 (LC3) is cleaved by Atg4, a cysteine protease, to produce LC3I with a C-terminal
glycine residue. The cleaved LC3 I is conjugated to phosphatidylethanolamine (PE) by
Atg7 and Atg3 enzymes (Rabinowitz & White, 2010). This lipidated form of LC3 II is
attached to both outer and inner faces of phagophore. The cytoplasmic components are
recognized by p62/SQSTM1 and neighbor of BRCA1 gene 1 (NBR1) cargo receptor
proteins through the ubiquitin-interacting domains (UBA), and engulfed into the
phagophore by the interacting regions of LC3 (Gottlieb & Carreira, 2010). The
phagophore elongates to form an enclosed structure with double membrane known as
autophagosome, where target cargoes are engulfed in. At this moment, Atg12/5/16L is
released and Atg4 cleaves LC3 II from the outer surface of autophagosomes. The
autophagosome fuses with lysosome to form an acidified compartment by the help of
vacuolar proton ATPase (VPATPase). Finally, the cytoplasmatic materials of the
autophagosomes and the p62 and NBR1 proteins are degraded along with the other
components sequestered inside the autolysosomes of normal cells.
Notwithstanding the mTOR dependent is the main regulating pathway of autophagy,
there are also mTOR independent regulating pathways, being the phosphoinositol (PI)
signaling pathway the most important. In the PI pathway, the autophagy is negatively
regulated by intracellular level of free inositol and inositol 1,4,5-triphosphate (IP3) (Isakoff
et al. 2005) . The PI pathway is activated by G- protein coupled receptor after the
activation of the enzyme phospholipase C (PLC). PLC hydrolyzes PIP2 to form IP3 and
DAG. These IP3 are degraded by two enzymes, 5’-phosphatase and inositol
polyphosphate 1-phosphatase (IPPase), to form inositol monophosphate (IP1), finally
hydrolyzed by inositol monophosphatase (IMPase) into free inositol.
18
1.4 Lipid membrane alterations in cancer
Similar to regulation of gene expression, changes in the presence and levels of
membrane lipids species have been described in several human pathologies, associated
either with adaptive responses or with the etiology of the disease. In this regard,
numerous studies have shown that the lipid composition of tumor cell membranes is
altered with respect to non-tumor cells. This area of study has received little attention in
cancer research, mainly because structural and functional concepts of lipid alterations in
cancer are more difficult to understand than the functional role of certain proteins and
their genes in defining cancer cell phenotypes. Although it has not been shown a
common pattern of alterations characteristic for different kinds of tumors yet, certain
cancer induced lipid prolife changes have been described and should possess some
diagnostic values (Michalak, 2003).
In this context, a hallmark of cancer cells is the constitutive activation of the fatty acid
biosynthetic pathway, which produces saturated fatty acids (SFA) and monounsaturated
fatty acids (MUFA) to sustain the increasing demand of new membrane phospholipids
with appropriate acyl composition (Kuhajda 2006, Rashid et al. 1997, Swinnen et al. 2000).
In this regard, the increased levels of oleic acid, detected in several tumors, are related
to the activation of the fatty acids synthesis (Igal, 2010). The enzyme responsible for the
oleic acid synthesis is the steaoryl-COA desaturase (SCD).
Gangliosides, which are membrane-bound glycosphingolipid molecules, are frequently
aberrantly expressed in tumors (Hettmer et al. 2005). Ganglioside antigens on the cell
surface act as immunosupressors, and certains gangliosides, such as GD3 or GM2,
promote tumor associated angiogenesis (Birkle et al. 2003). The reduced levels of
Ceramide that were found in some types of cancer could be related to its pro-apoptotic
role (Riboni et al. 2002). One lipid alteration highly connected to the genetic alterations
found in cancer, is the elevated levels of PI(3,4,5)P3, formed by the activation of PI3K that
was observed in several tumors and contribute to oncogenesis through the PI3K/AKT
pathway (Vivanco & Sawyers, 2002)
It has been demonstrated in several studies that different types of lipids and their relative
abundance in the cell membrane can control numerous functions and regulate the
activity and localization of membrane proteins (Escribà et al. 1996;, Escribà et al. 1997;
Vögler et al. 2004). In the case of cancer, it has been shown that the proportion of
membrane lipids is altered in cases of breast, lung, pancreas, liver, prostate, brain and
colon cancer (Mikirova et al. 2004; Michalak et al. 2003).
19
1.5 Membrane-Lipid Therapy
As indicated, several studies have related a high number of important diseases with
structural or molecular disarranges at the lipid membrane, from where the most of the
pathways that regulate cellular functions began.
The role of proteins in the development of diseases is well known. Perhaps the most
direct relationship is in monogenic diseases where a single altered gene produces an
altered protein, with non-function or over-function which produces the disease. In the
case of multifactorial diseases, although there are more factors, it is known that part of
disease is due to the performance or non-performance of different proteins.
From the point of view of classical molecular medicine, the focus has always been to
develop drugs that target a protein that has been determined to be key to the disease
process in particular. This strategy has proven to be effective in many diseases and a very
high percentage of treatments are included in it. However, it has also shown that in
complex diseases, such as cancer, at the molecular level the mismatches are so abundant
that affecting one or two proteins with a drug in a try to reverse the disease,
unfortunately, is no sufficient.
Figure 6. Main difference of the two biological approaches mentioned in this master thesis for
the treatment of human pathologies. The molecular entities regulated by the treatment are
colored, whereas the molecular entities that are not affected by the therapy are shown as open
symbols. (a) Conventional chemotherapy is characterized by the interaction of a drug with a target
protein (gray). Upon drug binding, the activity of such a protein, the downstream elements and
gene expression are modulated (b) In membrane-lipid therapy, the clinical drug binds to
membrane lipids, regulating the structure of the membrane, with subsequent modulation of the
activity of a membrane protein and downstream events. Adapted from: Escribá, 2006).
If we consider that most of the cancer-related pathways are upstream activated in the
membrane and the lipid modifications occurring in cancer cells are associated with the
activation of proliferation and tumorigenesis, it is conceivable that lipid modifications
can regulate these pathological cell signaling pathways. The lipids control the interaction
20
and activity of many proteins, and not only that, the proteins bonded to the membrane
may alter the structure of this, being reciprocal the regulatory effect between proteins
and lipids.
Given the importance of the membrane, the strategy to develop specific therapies to
regulate the lipid membrane structure for the treatment of several diseases arising. The
Membrane-Lipid Therapy (MLT) seeks to regulate the participation of membrane lipids
in cellular functions by using lipid product being intercalated in the membrane and
regulate its structure, and therefore modifying the location and activity of membrane-
interacting proteins (Figure 6) (Reviewed in Escribá, 2015).
The relation between lipid structure and function, clearly known and accepted in the
protein world but not in the lipidic one, is the starting point for the rational development
of synthetic lipid compounds as effective therapeutic drugs. In addition, the lipid drugs
developed and tested on this group show a low toxicity profile, being the collateral
effects or toxicity of several cancer treatments a main trouble to overcome.
To demonstrate the efficacy of this novel approach, we can take as an example the
Minerval, a fatty acid analog to the oleic acid designed by Dr. Escribá, which works by
activating the sphingomyelin synthase (SMS). Currently Minerval is in Phase I / II clinical
trials (clinicaltrial.gov identifier NCT01792310) for the treatment of glioma and other
solid tumors. Preliminary data in those human studies are promising; several patients in
which the standard treatment had not worked showed a partial response or stable
disease after Minerval administration. As also mentioned, the toxicity profile of Minerval
is really positive, not having seen any SAE (Serious Adverse Event) at even high dosed
(up to 12 grams per day).
This master thesis can be included in the Membrane-Lipid Therapy, because the object
of study have been several synthetic triglycerides.
1.6 Objects of study: Mimetic triglycerides (TGMs)
Triglycerides are structures formed by a glycerol backbone and three fatty acid chains
attached via an ester bond (Figure 7). Physiologically involved in glucose metabolism and
fat, their presence at high levels is commonly used in clinical practice as marker of
atherosclerosis or heart disease risk.
When triglycerides are ingested with the diet, due to their large size, they cannot be
absorbed in the duodenum or enter cells until pancreatic lipase breaks the ester bond,
releasing the fatty acid chains. Thus, the absorbed product can be either free fatty acids,
monoglycerides (a glycerol molecule attached to a fatty acid) or diglycerides (one
21
molecule of glycerol with two fatty acids) which enter the cell through the FAT receptor.
Once in enterocytes, the triglycerides are re-formed from their fragments, and bind to
CHO and other proteins to form part of chylomicrons.
Figure 7. Simple representation of the structure of a triglyceride. A triglyceride is formed by a
glycerol backbone where three fatty acids chains are bonded. For this master thesis rationally
designed triglycerides has been used. The modifications where performed on the fatty acids
chains, even developing molecules with three different modified fatty acids chains.
The antitumor role of triglycerides is understood through the known antitumor effect of
some fatty acids that may be part of triglycerides. More than 10 years ago the effect of
fatty acids lauric, stearic, palmitic, oleic, linoleic, alpha-linolenic, gamma-linolenic,
arachidonic, docosahexaenoic and eicosapentaenoic was studied in different lines of
pancreatic cancer (Falconer et al. 1994). All the polyunsaturated fatty acids (PUFA) tested
had an inhibitory effect, with EPA being the most potent. SFA and MUFA fatty acids were
not inhibitory. Another clear example that shows the relation between structure and
function.
Triglycerides become very interesting objects of study when their potential use as
modified fatty acids carriers, such as Minerval, is exploited. The fact that a triglyceride
can carry up to three identical modified fatty acids with remarkable antitumor effect is
interesting, like happens in simple TGMs. The complexity and interest of their study
increases when we add two or three species of different fatty acids to the same molecule
of glycerol, all with a particularly physiological and therapeutic effect, and that is the case
of mixed TGMs.
Thus, this group has developed a battery of modified fatty acids that enhance their
antitumor activity. Furthermore, we have developed these triglycerides containing fatty
acids either separately or in mixed form, each possible combination results in a different
molecule with specific therapeutic effect against several pathologies, like cancer. The
antitumor efficacy against pancreatic cancer of these mimetic triglycerides or TGMs has
been the object of this master thesis study.
22
2. Objectives
The general aim of this master thesis was the investigation of the possible antitumor
activity of different lipid compounds developed by the Molecular and Cellular
Biomedicine group. Most of these compounds were TGMs or mimetic triglycerides,
triglycerides the fatty acids chains of which were modify under rational design.
Several compounds were screened for their antitumor potential in pancreatic
adenocarcinoma cell line Mia-PaCa-2. Of the analyzed compounds, the most interesting,
TGM4 and TGM5, were investigated in vivo with Mia-PaCa-2 xenograft tumors on
immunosuppressed mice. Finally, the mechanism of action of the TGM4 was studied.
The particular objectives of this master thesis were:
1. Determine the potential antitumor activity of the TGMs compounds developed by the
research group where this work was conducted.
2. Study the toxicity after chronic treatment with the compounds TGM4 and TGM5.
3. Determine the antitumor capacity of the compounds TGM5 and TGM4 in vivo, using a
human pancreatic tumor xenograft model.
4. Explore how the TGM4 exerts its antiproliferative effect; for this, three approaches were
planned:
4.1 Determine of the cell cycle alterations produced by TGM4 in pancreatic tumor
cells.
4.2 Investigate the TGM4 effects in pancreatic tumor cells through different key
proteins for the survival or cell proliferation, determining the mechanism through
TGM4 causes cell death.
4.3 Study of the lipid alterations produced by TGM4 in pancreatic cancer cells.
23
3. Materials and methods
3.1 Cell lines
Mia-PaCa-2 (Human pancreatic carcinoma) and PANC-1 (human pancreatic carcinoma
of ductal cells) were bought from ATCC (American Type Culture Collection).
3.2 Cell culture
3.2.1 Cell lines and growth conditions
Mia-PaCa-2 and PANC-1 monolayer cell lines were maintained and grown in 75 cm²
flasks with DMEM (Dulbecco's Modified Eagle Medium) with phenol red including 10%
of fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin. The
medium was also supplemented with D-glucose (4.5 g/L), L-Glutamine (4 mM) and
Sodium pyruvate (1 mM).
They were incubated in HEPA filtered cell incubator (Memmert GmbH Co, UK) at 37°C
with 95% humidified air and 5% CO2. Cell culture experiments were carried on laminar
vertical flow cabinet (Telstar S. A., Terrasa, Spain).
3.2.2 Thawing
Frozen cells in cryovials taken from nitrogen tank in cold room were defrosted at room
temperature. Then, they (2 ml) were transferred into T25/T75 tissue culture flask. The
volume was completed to 5/15 ml with DMEM complete medium and flasks were
incubated in CO2 incubator.
3.2.3 Cell passaging / maintaining
When the cells in flask reached 80% of confluence, the medium was discarded. The T75
flask was washed with 5 ml of PBS (137 mM NaCl, 2.7 mM KCl, 12 mM Na2HPO4 and 1.38
mM KH2PO4) in order to remove waste materials and serum which includes trypsin
inhibitors. Then 2 ml EDTA-trypsin was added and the flask was incubated in CO2
incubator for 2-5 min. When detachment was observed under inverted light microscopy,
flask was taken from incubator and growth medium was put in immediately to stop
trypsin activity, which large time exposure can cause damage in cells. Then the cells were
divided in the desired number of flasks, normally in 1:3-1:6 dilutions.
24
3.2.4. Freezing
If freezing of the cells was required, after trypsinization of cells, they were centrifuged at
600 x g for 5 minutes. The supernatant, which includes medium, dead cells and waste
products, was discarded. Pellet includes cells was re-suspended with freezing medium.
Freezing medium was prepared by mixing 10% of dimethylsulfoxide (DMSO), a
cryoprotectant that lowers the freezing point, and 90% FBS.
Cell suspension was put in cryovials, 2 ml of suspension for each. The cryovials were
stored at – 80°C to achieve gradual freezing and after three days they were placed in
liquid nitrogen tank (-190°C) for long term storage.
3.2.5 Cellular treatments
To dissolve the TGMs (lipid compounds), a stock solution of 100 mM of TGM was
prepared in full DMSO (Polar aprotic solvent). Then the stock solution was dissolved in
full DMEM medium to get the desired concentration, never surpassing the 0.5% of final
DMSO in the medium.
3.3 Cell proliferation / cytotoxicity studies
For cytotoxicity experiments, Mia-PaCa-2 and PANC-1 cell lines were seeded at a density
of 3x103 cells/well into 96 well plates and incubated at 37°C for 24 hours in CO2 incubator.
After 24h, when cells were attached, the medium was changed and replaced with new
medium containing one of the different TGMs studied. Different molecules were studied
at different times, the following table (Table 1) resume them.
TGM0 TGM5 TGM25
TGM1 TGM6 TGM46
TGM2 TGM12 TGM146
TGM4 TGM16
Table 1. List of the different molecules used for the studies of this master thesis, all forming part
of the TGMs library.
The cytotoxic effects were studied with Cell Proliferation XTT Kit (Roche Diagnostics, S.L.
Applied Science, Barcelona, Spain). The basis of this technique is the reduction of
tetrazolium salt XTT by living metabolically active cells to an orange colored formazan, a
reaction produced by succinate dehydrogenase enzymes of mitochondria respiratory
chain (Figure 8). Only the viable cells with intact mitochondrial and cellular membrane
25
have active dehydrogenases, thus the concentration of formazan formed is proportional
to the number of living cells. These bio-reduction of the tetrazolium salt is related with
the production of NAD(P)H through glycolysis.
Figure 8 Cell Proliferation Kit XTT employs 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide salt (XTT). Only in living cells mitochondria are capable to reduce XTT
to form an orange colored water soluble dye. Therefore, the concentration of the dye is
proportional to the number of metabolically active cells. Source: AppliChem
After incubate the cells the desired time with the different TGMs, the medium was
replaced with DMED without phenol red (it interferes on the absorbance of the formazan
product) mixed with the reagents XTT and electron-coupling, following manufacturer‘s
instructions. Then the cells were incubated at 37ºC until the color compound was formed.
Finally, the absorbance was read at a wave length of 495 nm in a plate reader (FLUOStar
OMEGA, BMG LABTECH, Germany)
For statistical confidence, 2 or 3 independent experiments were performed for each TGM
and each one was quadruplicate wells. According to dose-response curve (log (inhibitor)
vs. normalized response – Variable slope model) drawn according to percent viability, the
dose necessary to kill the 50% of the cells (IC50, inhibitory concentration 50) was
calculated with Graphpad Prism Version 5.
3.4 Cell cycle studies
The analysis of the effect of the TGM4 was performed through flow cytometry. PANC-1
cell lines were seeded at a density of 250.000 cells into 6 cm of diameter plates and
incubated at 37°C for 24 hours in CO2 incubator. After 24h the medium was changed and
replaced with new medium containing TGM4 at the desired concentration (20, 30, or 40
µM). The cells were incubated during 6, 12, 24, or 48 hours.
26
After the desired time of incubation, the cells contained
in the medium, both alive and dead were recovered by
centrifugation at 1000 x g for 5 minutes in 5 ml cytometry
tubes and resuspended in complete media. Then, the live
and attached cells were detached with EDTA-trypsin,
combined with the floating ones and centrifuged 1000 x
g for 5 minutes. The supernatant was totally discarded
and all the cells were stored at the tube.
To fix the cells, cold 70% ethanol was slowly added to the
tube while vortexing. To avoid the clumping of the cells
the solution was thoroughly pipetting. The tube was let
O/N at 4ºC. 24h later, the ethanol was discarded after a centrifugation at 2500 x g and
4ºC for 5 minutes. At this point PBS can be added to store the cells at 4ºC for at least a
week. To perform the cytometry the cells were washed with 1 ml of sodium citrate 38
mM pH 7.4. Then the sodium citrate was discarded after centrifuging at 2500 x g and 4ºC
for 5 minutes.
Finally, the cells were re-suspended in 500 µl of “Buffer A” solution formed by sodium
citrate 38 mM pH 7.4, 50 µg/ml of propidium iodide and 5 µg/ml of RNase A (Sigma-
Aldrich Co, St Louis, MO) and incubated at 37ºC for 20 minutes. After the addition of
propidium iodide the tubes were maintained permanently in the darkness until the
cytometry lecture. The flow cytometry was performed in a flow cytometer Beckman
Coulter Epics XL (Beckman Coulter S.A, Madrid, Spain). The different cell populations
corresponding to the different phases of the cell cycle (Sub-G1, G0/G1, S and G2/M) were
defined through their DNA quantity (Figure 9). The results were analyzed with the
software FlowJo (FlowJo, USA).
3.5 Western Blot studies
3.5.1 Cell lysate
For the immunoblot studies, 325.000 PANC-1 cells were seeded into 6 cm diameter plates
and incubated at 37°C for 24 hours in CO2 incubator. After 24h the medium was changed
and replaced with new medium containing TGM4 at a concentration of 30 µM.
At the desired time of treatment, the medium was discarded and the plates were washed
twice with cold PBS 1X and frozen at -80ºC until all the plates were collected. Then, 300
µl of lysis buffer (20 mM HEPES, 2 mM EDTA, 0.5 mM EGTA, 1.5 mM MgCl2, 1 mM
Figure 9. Relationship
between the cell cycle and the
DNA histogram. Source:
Ormerod & Novo, 2008.
27
cantaridine, 1 mM ortovanadate and a protein inhibitor cocktail from Roche) were added
to the plate. After 5 minutes from the addition of the lysis buffer, the cells were scrapped
and the lysate containing the protein was transferred to a new tube. To fully homogenize,
the samples were sonicated on ice twice for 5 seconds.
3.5.2 Protein quantification
The protein concentration determination of each sample was performed with the protein
quantification DCTM kit (Bio-Rad, Barcelona, Spain), a colorimetric assay used for the
quantification of proteins in presence of reducer agents and detergents. The DCTM
method is a modification of the classical Lowry method, based on the reduction of the
reagent Folin phenol, that modifies the color of the reagent, and changes are quantified
measuring the absorbance at 750 nm (Lowry et al. 1951)
The DCTM kit contains 3 reagents: A, S and B. The quantification was performed in a 96-
well plate. For each sample, triplicates were done. 5 µl of the sample were added to the
well, then 25 µl of A' reagent (mixing reagents A and S in a proportion 50:1) were added
to each well. Finally, 200 µl of the reagent B were added to the wells and then the plate
was maintained at room temperature for 15 minutes. Finally the plate absorbance was
read in a plate reader at 750 nm.
Protein concentrations were obtained by interpolation of the absorbance values on the
standard curve made with known concentrations of bovine serum albumin (BSA), from
0.2 to 6 mg/ml. Once the protein concentration of the samples was known, samples of
equal protein concentration were prepared to perform the experiments, diluting the
necessary samples with protein lysis buffer until achieve the desired concentration.
Protein samples were stored in ice or frozen at -20ºC to avoid the degradation during
the whole process.
3.5.3 Western Blot
Samples were mixed with loading buffer (Tris-HCl 12 mM pH 6.8, β-mercaptoethanol [-
ME], 1%, SDS 0.2%, bromophenol blue 0.01%, and glycerol 50%) in a 1:10 proportion and
boiled at 95ºC for 5 minutes. Then, 30 µg of whole cell lysate of each sample were loaded
on a 10 % SDS polyacrylamide gel. As a protein ladder standard, 2.5-5 µl of Precision Plus
Protein all blue standard (Bio-Rad) was loaded.
The acrylamide concentration in the gel determines the range of separation of proteins
during electrophoresis. These gels have two distinct areas, an area for concentration gel,
where the samples are loaded, and a zone for separation gel at the bottom, where the
28
samples are separated. The gel concentration ("stacking") is composed of acrylamide-
bisacrylamide 4%, Tris-HCl pH 6.8 166mM, SDS 0.1%, ammonium persulfate 1% and N,
N, N ', N'-tetramethylethylenediamine 0.1%. Gel separation ("resolving or running") is
composed of acrylamide-bisacrylamide 9.5%, Tris Base pH 8.8 1M, SDS 0.1%, ammonium
persulfate 0.4% and N, N, N ', N'- tetramethylethylenediamine 0.04%.
After loading all the samples in the SDS-polyacrilamide gels, SDS-PAGE (sodium dodecyl
sulfate-polyacrilamide gel, electrophoresis) was performed. (Laemmli, 1970).
The SDS, together with the -ME, has the ability to denature the proteins. The SDS gives
net negative charge to the protein allowing them to migrate through the gel
proportionally to its mass, since there is a constant charge/mass ratio (approximately 1
molecule of SDS per 2 amino acids, with a ratio SDS/protein of 1.4 g/g). . On the other
hand, the -ME breaks the disulfide bonds in the protein separating its subunits.
The electrophoresis was set at 90 V for the stacking phase and changed to 120-140 V for
the running phase. The electrophoresis buffer is composed of 19.2 mM Tris-base, 0.19 M
glycine pH 8.6, and 0.1% SDS. When the electrophoresis was finished, the proteins into
the gel were transferred to a nitrocellulose membrane (GE Healthcare, Kent, UK). The
transfer process was performed in cold conditions and applying a constant amperage of
350 -400 mA for 2 hours with buffer consisting of 19.4 mM Tris-base and 0.19 M glycine
and 20% ethanol.
After finishing the transfer, the membrane was blocked in 5% skim milk in TBS (50 mM
Tris-Cl, pH 7.6; 150 mM NaCl) for 30 minutes in order to prevent non-specific antibody
binding. Once blocked, the membrane was incubated in a primary antibody dilution
(1:1000) against the protein of interest and allowed to stir overnight at 4ºC. Primary
antibody solution was prepared containing 5% BSA and 0.1% Tween 20 (Sigma-Aldrich
Co., St. Louis, MO). The next day, the solution was removed of the membrane and 3
washes were performed for 5 minutes with TBS and Tween 20 0.1%. The membrane was
incubated for 1 hour in the dark with the secondary antibody (dilution 1: 5000)
conjugated with a fluorochrome (IRDye 800CW Donkey Anti-Mouse IgG (H + L) or IRDye
800CW Donkey anti Rabbit IgG (H + L), LI-COR Biosciences, USA). The secondary
antibody was prepared in 2.5% skim milk in TBS with 0.1% Tween 20. Then, the secondary
antibody was removed and the membrane was washed twice for 5 minutes TBS and 0.1%
Tween 20 and once with TBS for 5 minutes. Since the incubation with secondary antibody
the process was carried out maintaining the membrane in darkness.
The membranes were scanned in near infrared spectroscopy (Odyssey Infrared Imaging
System, LI-COR, Inc., Lincoln, NE, USA) with a resolution of 84 microns and analyzed with
Image StudioTM software (LI-COR, Inc., Lincoln, NE, USA) obtaining the values of
29
integrated optical density (DOI) of each band. The α-tubulin content in each sample was
used as a loading control.
3.6 Thin Layer Chromatography
The analysis of the TGM4 effect on cellular lipid composition was studied through Thin-
Layer Chromatography (TLC). TLC is based on the separation of a mixture of compounds
as they migrate with the help of a suitable solvent through a thin layer adsorbent material
which has been applied to an appropriate support. Different stationary phases and
several combination of polar/organic solvents are available to separate the mixture by
different characteristics. However, adsorption is the most common mechanism of
separation, where the sample is continually fractionated as migrates through the
adsorbent layer.
Competition for active adsorbent sites
between materials to be separated
and the developing solvent produces
continuous fractionation. A portion of
the material to be separated will be
found in the mobile phase and a
portion will be adsorbed to the solid
adsorbent particles. As the process
continues the several components move
different distances, depending on their
relative affinities for the adsorbent as compared with the migrating solvent (Figure 10).
The affinity of the components is mainly related to their polarity. The more polar
compounds are held back by the adsorbent while the less polar compounds advance
further. In these experiments silica (as adsorbent) pre-coated plates were used.
To the procedure, 300.000 PANC-1 cells were seeded into 6 cm diameter plates and
incubated at 37°C for 24 hours in CO2 incubator. After 24h the medium was changed and
replaced with new medium containing TGM4 at concentrations of 20 µM and 30 µM. For
the control, new medium without TGM4 was added. The cells were incubated during 12
and 48 hours, then the medium of the plated was discarded and the plates were washed
and frozen until their use.
3.6.1 Lipid extraction
For each plate 600 µl of hypotonic buffer (20 mM Tris pH 7.5 and 1 mM EDTA) was added
before cells were thawed. Then the plates were scrapped and the cells in the hypotonic
Figure 10. Scheme of a Thin Layer
Chromatography
30
buffer cell extracts were collected in 1.5 ml tubes. To fully homogenize the samples, they
were sonicated on ice 3 times 10 seconds on/10 seconds off at 20% of amplitude.
Neutral lipids or generally storage lipids are extracted with relatively non-polar solvents
as chloroform and/or petroleum ether, but membrane-associated lipids (the most
interesting for the MLT) requires polar solvents such as methanol to disrupt hydrogen
bindings or electrostatic forces. A mixture of Chloroform: Methanol (2:1) was used to the
lipid extraction for these experiments. To avoid the peroxidation of the extracted lipids,
all solvents were peroxide-free HPLC grade.
Then, 550 µl of each sample were transferred to a new glass tube, and 2.75 ml of a
chloroform: methanol mixture (2:1) was added. The samples were mixed by vortexing
and centrifuged for 10 minutes at 500 x g and 4ºC. After the centrifugation, three parts
were clearly observed, from the top to the bottom, the aqueous phase, a thin film formed
by the proteins, and the organic phase where the lipids were. The organic phase was
collected carefully, to avoid drag other components that will affect the separation of the
lipids in 2 ml tubes. The tubes were placed in an argon evaporator to evaporate the
organic solvents and, after that, the tubes containing only the lipids were stored at -20ºC.
3.6.2 Thin layer chromatography
Whatman silica gel-60 plates (20x20 cm, 250 µM, GE Healthcare, England) were heat-
activated at 110ºC for 1 hour, then the lipid samples where re-suspended in 40 µl of
chloroform and streaked onto the plates, this process was repeated twice. Phospholipids
were separated using chloroform/methanol/acetic acid/water (60:50:1:4 by volume)
which separated all major glycerophospholipids. Lipids were identified using
commercially available standards (Larodan, Sweden). To develop, plates were air-dried,
submerged in a solution containing 5% (W/v) H3PO4 and 4% (W/v) CuSO4, and charred
at 180ºC for 10 minutes (Gellerman et al. 2005). Lipids were finally quantified by photo-
densitometry using to the Quantity one software (Bio-Rad).
3.7 In vivo studies
3.7.1 Experimental animals
The animal model used to conduct the experiments was the immunodepressed NUDE
mice (Swiss Crl:NU (Ico)-Foxn1 nu; Charles River laboratories, France), aged between 4-6
weeks and with an approximated 25 grams of weight.
31
These animals, due to the lack of thymus and, consequently, their immunodepression
state, were maintained always under sterile conditions, kept on plastic cages located on
a sterile closet (EHRET, Labor_U_Pharmatechnik, Deutschland) with a constant
temperature of 28ºC. The cabinet was kept in a room with a 12 hour light/12 hour dark
schedule and a relative humidity of 40-60%. All the work or manipulation of the animals
was conducted in every moment under sterile conditions on BSL-2 flow cabinets.
3.7.2 Determination of the toxicity of the compounds TGM4 and TGM5.
To study the toxicity and to establish the higher but safer dose of each compound, a
toxicity study was designed and performed. Different and increasing doses of TGM4 and
TGM5 were administered to BALB/ NUDE heterozygous mice by oral cannulation. The
doses were: 50 mg/kg, 100 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg. The
compounds were mixed with soy oil when necessary to get the desired concentration.
Each dose was tested on 3 animals for 15 days, during which the animals were treated
and weighed daily. The weight of the animals was used as the first indicator of toxicity.
The behavior of the animals was also observed as an indicator of toxicity.
The diet during the 15 days of treatment was a standard one based on food pellets and
water ad libitum. After the treatment, all the animals were euthanized by decapitation
and dissected to collect different organs (brain, spleen, lung, heart, kidneys and liver) to
anatomical pathology or compound distribution studies.
3.7.3 Antitumor studies
3.7.3.1. Subcutaneous inoculation of tumor cells
To study the in vivo effect of the chosen compounds, a xenotransplant model was used.
The cell line Mia-PaCa-2 was expanded in 15 cm plates. Once achieved the desired
number of plates or cells, the plates were washed with PBS and the cells were trypsinized
and collected in DMEM medium. Then, the viable cells density of the suspension was
determined by counting them on a Bürker camera. Once the concentration of cells was
calculated, they were centrifuged 5 minutes at 600 x g to discard the complete DMEM
medium and they were re-suspended in DMEM medium without fetal bovine serum, to
avoid immune reactions even possible on immunodepressed animals.
A total of 7.5 x 106 cells were injected subcutaneously with a 25G caliber needle, in a
volume of 150 µl. 1 week after inoculation, the tumors had an approximated size of 5
mm and the treatments began.
32
During the treatment the tumors size was measured once a week with a digital caliper.
We also measured once a week the weight of all the animals as an indicator of toxicity.
The tumor volumes were calculated by the formula: Volume (mm3) = (2W x L) / 2, where
W represents the width and L represents the length of the tumor (Barbacci et al. 2003).
3.7.3.2 Animals treatments
To investigate the in vivo efficacy of the chosen compounds, TGM4 and TGM5, the
animals were divided into 4 groups: treated with TGM4, TGM5 or Gemcitabine and
control animals. A total of 8 animals (4 males and 4 females) was used for each condition.
The treated animals received daily 1000 mg/kg of TGM4 or TGM5, both by cannulation.
The control animals received nothing or 100 mg/kg of Gemcitabine twice a week.
Gemcitabine is the standard of care for the treatment of pancreatic cancer and it was
administrated through intraperitoneal injection. The duration of the experiment was 43
days.
After the treatments, the animals were euthanized by decapitation. Immediately, the
animals were dissected and the blood, the tumor, and several organs (brain, spleen, lung,
heart, colon, kidneys and liver) were collected to possible future studies. The blood was
stored at 4ºC and then centrifuged at 4ºC and 1000 x g to collect the plasma fraction.
The tumor and the organs were divided into two parts, one was frozen in liquid nitrogen
and the other one was preserved in formalin 10%.
All the protocols and procedures were revised and approved by the Comité Institucional
de Investigación Animal (Comisión de Bioética de la Universitat de les Illes Balears)
3.8 Data analysis
All data shown in the graphs correspond to the mean values ± standard error of mean
(SEM) of at least 2 independent in vitro experiments (each duplicated) or cellular
experiments. For animal studies there is indicated in the graphs the number of animals
used (N).
The statistical analysis was performed by the average t-student, configured as unpaired,
two-tailed test, with a confidence intervals of 95%. For the statistical analysis of the
animal studies, the non-parametric Mann-Whitney test was used. All statistical analysis
were performed using GraphPad Prism 5.0 program.
The differences between experimental groups were considered statistically significant at
p <0.5. The different significances were represented as: *, p <0.05, ** p <0.01, *** p
<0.001.
33
4. Results
4.1 Mimetic triglycerides (TGMS) inhibit cell viability and proliferation.
A screening of the antitumor activity of the different TGMs, included in the library of
compounds rationally designed from the structure of the triglycerides, was performed.
The effect of the TGMs on the viability and proliferation of pancreatic cancer cell line
Mia-PaCa-2 was tested through the XTT assay.
34
Figure 11. Effect of different TGMs in cell viability and proliferation of tumor cells Mia-PaCa-2.
Cells were treated with several concentrations of the indicated compounds for 72 hours. To
determine the IC50 the XTT cell viability assay was used. (A) TGM0 (n=12). (B) TGM1 (n=12). (C)
TGM2 (n=12). (D) TGM4 (n=12). (E) TGM5 (n=12). (F) TGM6 (n=12). (G) TGM12 (n=12). (H) TGM16
(n=12). (I) TGM25 (n=12). (J) TGM46 (n=12). (K) TGM146 (N=12).
35
Figure 12. Effect of TGM1 (A) and TGM5 (B) in cell viability and proliferation of tumor cells Mia-
PaCa-2. Cells were treated with several concentrations for 24, 48 and 72 hours. The effect is time-
dependent. (n=12)
Figure 13. Effect of TGM4 (A) and TGM5 (B) in cell viability and proliferation of tumor cells PANC-
1. Cells were treated with several concentrations 72 hours. To determine the IC50 the XTT cell
viability assay was used. (n= 12)
All the TGMs tested proved to have antitumor activity, but there were differences about
their potency (Figure 11). The potency was measured using the IC50, which reflects the
necessary concentration of a compound to have the 50% of the alive cells regarding to
a non-treated control due to inhibition of proliferation or induction of cell death. The
Table 2 resumes all the molecules tested and their correspondent IC50 with a 95%
confidence interval.
36
Molecule 95% Confidence Intervals -
IC50 (µM) Molecule
95% Confidence Intervals -
IC50 (µM)
TGM0 72,75 – 95,64 TGM12 2,94 – 4,25
TGM1 69,84 – 85,65 TGM16 17,20 – 21,52
TGM2 10,57 – 13,48 TGM25 2,40 – 3,52
TGM4 18,59 – 21,86 TGM46 7,94 – 10,05
TGM5 2,60 – 3,16 TGM146 3,60 – 4,71
TGM6 5,43 – 7,50
Table 2. Table summarizing the 95% confidence intervals of the IC50 at 72 hours of all TGMs tested
in vitro with Mia-PaCa-2 cells.
Another factor to consider evaluating the antitumor activity of a compound is its
efficiency. The efficacy is reflected by the ability of the compound to kill all the cells
present in the culture (achieving the 0% of cell survival). With this objective, several TGMs
were tested at a high dose of 200 µM and most of them achieved it, excepting TGM0,
TGM1, TGM2 and TGM6 (Figure 11).
The antitumor effect was shown to be dose-dependent in all cases. From the data
obtained with two compounds at different times of treatment, it can be estimated that
as expected, the effect of TGMs is also dependent on the time, so the longer the
treatment, the greater the effect obtained (Figure 12). The compounds TGM4 and TGM5
were tested on the pancreatic cell line PANC-1 (Figure 13). On PANC-1 both compounds
maintained the antitumor activity.
4.2 Determination of a safe dose to perform in vivo studies with TGM4 and TGM5.
Before performing in vivo studies to test the antitumor activity of TGM4 and TGM5
compounds, a toxicity study was required, to establish the appropriate dose to which the
mice will be treated. Different and increasing doses of TGM4 and TGM5 were
administrated daily to BALB / NUDE heterozygous mice by oral cannulation. Each dose
37
was tested on three animals of mixed sexes for 15 days, during which the animals were
weighed and observed for toxicity indicators daily.
Figure 14. TGM4 effect on weight of BALB/nude mice during 15 days of treatment at different
doses. n=3
Figure 15. TGM5 effect on weight of BALB/nude mice during 15 days of treatment at different
doses. n=3
38
The results showed that the treatment with TGM4 or TGM5 did not affect the weight
(Figure 14 and Figure 15) or the behavior of any animal. Their weight varied during the
15 days without significant differences over the control. The behavior, mainly reflected in
the locomotive activity and variations in normal intake of the animals, was not affected
by the treatment with TGM4 or TGM5.
From the data obtained on these toxicity studies indicated that the highest dose tested
seems to be safe. Owing to that it was used for the antitumor in vivo studies and the
mice were treated at a dose of 1000 mg/kg.
4.3 TGM4 effect on the progression of Mia-PaCa-2 cell line xenograft in nude
immunodepressed mice
The cell studies are always necessary and the basis to test the antitumor activity of new
molecules, but the huge differences in complexity found between a cell culture and a
complex live animal as the rodents are, can make a big difference, therefore, the TGM4
and TGM5 antitumor activity was tested in vivo on mice. A model of human pancreatic
tumor xenograft in immunosuppressed nude mice was used. The tumor was induced by
subcutaneous injection of 7.5 x 106 Mia-PaCa-2 cells per mouse. TGM4 or TGM5 were
administrated orally and daily at a dose of 1000 mg/kg of body weight. Control mice
received nothing or gemcitabine 100 mg/kg i.p. twice a week, the standard of care for
the treatment of pancreatic cancer. The experiment lasted for 43 days, during which the
tumor volume was measured.
Figure 16. Representative images of the size of the pancreatic cancer xenograft in nude mice after
43 days of treatment. 7.5 million cells of the cell line Mia-PaCa-2 were injected subcutaneously in
39
mice immunosuppressed NUDE Swiss Crl: NU (Ico) -Foxn1nu. (A) control mouse non-treated. (B)
mouse treated by intraperitoneal injection with Gemcitabine 100 mg/kg twice a week. (C) mouse
treated orally with 1000 mg/kg of TGM4. (D) mouse treated orally with 1000 mg/kg of TGM5.
The results obtained were visually appreciable at least on the TGM4 treated mice (Figure
16), and their tumors volume was significantly lower than control mice after 43 days of
treatment (Figure 17). The evolution of tumor size throughout the treatment time showed
that from day 12 of treatment, the tumors development of the treated and untreated
mice was different, being the control animal’s tumors always higher than those treated
with TGM4. The effect of the TGM5 was less potent than TGM4 but antitumor activity
was also observed (Figure 17 top). At the end of the treatment, a significant reduction in
tumor volumes of the TGM4 treated animals compared to control animals (Figure 18
bottom) was observed.
40
Mia-Paca-2 xenograft volume at day 43
Contr
ol
Gem
cita
bine
(100
mg/k
g x2/
wee
k)
TGM
4 (1
000
mg/k
g day
)
TGM
5 (1
000
mg/k
g day
)
0
500
1000
1500
n=9
n=3
n=7
n=8
***
Tu
mo
r vo
lum
e (
%)
Figure 17. Effect of TGM4 / TGM5 in xenograft model of pancreatic cancer (Mia-PaCa-2). 7.5 x 106
Mia-PaCa-2 were injected subcutaneously in immunosupressed NUDE Swiss Crl: NU (Ico) -
Foxn1nu mice. Control animals received nothing or subcutaneous injections of 100 mg/kg of
Gemcitabine twice a week. The treated animals received through cannulation 1000 mg/Kg of
TGM4 or TGM5. (Top) Changes in the tumors volumes through 43 days of treatment represented
as the percentage. (Bottom) Percentage of tumors volumes at day 43 of treatment each value
represents the average of the percentages relative to day 0 (taken as 100%). non-parametric
Mann-Whitney test, (*p < 0.05; **p < 0.01).
The lipid compound TGM4 has shown to be at least as efficient as the actual standard of
care for the pancreatic cancer, the Gemcitabine.
4.4 Effect of the TGM4 on the cell cycle.
To establish the basis for the study of TGM4 effect in cell proliferation, first, PANC-1 cells
were treated with TGM4 and the cell cycle was analyzed. Two studies were conducted to
observe how the effect varies in relation to different doses and treatment times.
To study the dose-dependent effects, PANC-1 cells were treated at two different
concentrations, 20 and 40 µM for 48 hours (Figure 18). The results shows a clear dose-
dependency of the TGM4-induced effects. TGM4 treatment at both concentrations
produced a significant increase in the percentage of cells in sub-G1 phase (corresponding
to dead cells) followed by a decrease in the percentage of cells in G2/M phase and an
increase of the percentage of cells in the S phase.
41
Sub-G
1
G0/
G1 S
G2/
M
0
15
30
45
60
Control
TGM4 20 µM
TGM4 40 µM
48hrs
PANC-1
* *
*
**
Cell
perc
en
tag
e (
%)
Figure 18. Effect of the concentration of TGM4 on the cell cycle in PANC-1 cell line. Cells were
treated with TGM4 at two concentrations (20 and 40 µM) for 48 hours and the cell cycle phases
populations were determined. Analysis in percentage of events in the different cell cycle phases
(sub-G1, G0/G1, S and G2/M) .Data represented as mean +- SEM (n=2), Statistical significance was
determined by unpaired two-tailed Student's t test (*P < 0.05)
In other set of experiments, PANC-1 cells were treated at a single concentration, 30 µM,
for 6, 12, 24 and 48 hours, to study the time-dependent effect (Figure 19). As control
treatment the same cells were treated with vehicle for the same time. The results show
a significant increase in the percentage of cells in sub-G1 phase with the treatment of
TGM4, associated to a state of cell death. This increase is accompanied by a significant
decrease of the percentage of cells in G0/G1 phase. The remaining phases were
unaffected by treatment TGM4 at the times studied, but the time-dependence effect and
cell death induction by TGM4 are confirmed.
42
PANC-1
Sub-G
1
G0/
G1 S
G2/
M
Sub-G
1
G0/
G1 S
G2/
M
Sub-G
1
G0/
G1 S
G2/
M
Sub-G
1
G0/
G1 S
G2/
M
0
15
30
45
60
Control
TGM4 30 µM
6 hrs 12 hrs 48 hrs24 hrs
***
*
**
**
Cell
perc
en
tag
e (
%)
Figure 19. Effect of the time treated with TGM4 on the cell cycle in PANC-1 cell line. Cells were
treated with TGM4 30 µM for 6, 12, 24 and 48 hours and analyzed by flow cytometry to determine
the different cell cycle phases populations. Analysis in percentage of events in the different cell
cycle phases (sub-G1, G0/G1, S and G2/M). Data represented as mean +- SEM (n=4), Statistical
significance was determined by unpaired two-tailed Student's t test (*P < 0.05; ***P < 0.001).
Another confirmation of the results shown in the Figure 18 and 19 is the picture obtained
by microscopy of TGM4-treated PANC-1 cells (Figure 20), where the number of cells is
clearly reduced and the morphology is altered in a way that the cells seems to be
damaged and dying, as can be observed for the round shape and the presence of vesicles
in cytoplasm.
Figure 20. Representative pictures (400x) of PANC-1 cells with or without incubate with TGM4 30
µM for 48 h. The reduction of the number of cells and the loose of their epithelial morphology
can be observed in the TGM4-treated cells. No apoptotic bodies or blebbing (characteristic of
apoptotic cells) were observed.
43
4.5 Finding the type of cell death and related proteins behind the TGM4 effect.
In order to know the type of cell death produced by TGM4 treatment and how affects
some of the main oncogenic signaling pathways, protein studies were performed trough
the classic Western Blot technique. PANC-1 cells were treated with TGM4 at a
concentration of 30 µM with different treatment times of 6, 12 and 24 hours. Then, they
were analyzed to determine the protein levels or phosphorylation state of key proteins
in stress and cell death processes, such as apoptosis, endoplasmic reticulum stress and
autophagy, as well as, proliferation induction.
Figure 21. TGM4 effect on different proteins studied. PANC-1 cells were treated with TGM4 30 µM
at different times (6, 12 and 24 hours), lysed, and an immunoblot was performed. In this
representative immunoblot it shows how the TGM4 affects proteins involved in reticular stress
and autophagy (BIP, CHOP and LC3B), the apoptosis reporter protein PARP, and to the protein
used as loading control α-tubulin.
As observed previously by flow cytometry and XTT assay, TGM4 induced cell death in
TGM4, based in that, the levels of key protein markers for apoptosis and autophagy were
studied (Figure 21). The fragmentation of PARP, typical indicator of apoptosis, was not
observed, but there was a clear downregulation of the full form since the 6 hours of
treatment. On the other hand, the increase of BIP indicates reticular stress on the treated
cells. Besides, the stress inducible CHOP was clearly expressed at 12 and 24 hours, in a
time- and dose-dependent manner. In a similar way, the bottom fragment of LC3B (LC3B-
II) is upregulated upon TGM4 treatment. Knowing that LC3B-II is an essential protein for
the formation of autophagosomes and so for autophagy, TGM4 can be considered an
inducer of autophagy in PANC-1 cells.
44
After determining that PANC-1 cells die through autophagy, and taking into account that
TGM4 modified other cell cycle phases populations, the protein levels alteration of critical
proteins for the right cellular proliferation were studied to try determining the sequence
of the events (Figure 22). For example, a downregulation of the levels of DHFR,
dihydrofolate reductase, an enzyme necessary for the synthesis of DNA was observed.
Two proteins involved in the regulation of the cell cycle were studied, Cyclin D3 and p19.
The downregulation of Cyclin D3 is remarkable from the 12 hours, justifying the decrease
of cells in G1/G0 phase, but p19 remained unaltered, corresponding with the cell death
instead of cell cycle arrest. Finally, as important oncogenic signaling pathway the
PI3K/AKT pathway, which contributes to proliferation and survival, was studied through
the protein AKT, and changes in its regulation by phosphorylation and its total protein
were observed, but not a clear upregulation or downregulation. Moreover, the mitogen-
activated protein kinases (MAPK) pathway, also involved in the regulation of proliferation,
Figure 22. TGM4 effect on different proteins studied. PANC-1 cells were treated with TGM4 30 uM
at different times (6, 12 and 24 hours), lysed, and an immunoblot was performed. In this
representative immunoblot it shows how the TGM4 affects proteins of pathways involved in
oncogenesis (Jun, AKT and ERK), regulatory proteins of the cell cycle (CD3 and p19), protein
required for DNA synthesis DHFR, and to the protein used as loading control α-tubulin.
45
was studied through the protein ERK, and as in the AKT case, both regulation and total
protein were affected in different forms depending of the time. Interestingly, c-jun is a
proto-oncogene involved in proliferation and apoptosis, and its activity was studied
through the protein Jun, in this case only its phosphorylation state was affected by TGM4
treatment indicating an inhibition, being the levels of total Jun unaltered by the TGM4.
4.6 Lipid alterations produced by the treatment with TGM4
When working with lipid compounds that are intended to intercalate into the lipid
membrane and change its structure to produce effects in cell proliferation and survival,
the study of specific alterations produced in the different cellular lipids becomes
important. For this, PANC-1 cells were treated at two concentrations of TGM4 (20 and 30
µM) for 12 or 48 hours, then a TLC was carried to separate the major phospholipids
present in the whole cell.
46
Figure 23 Thin-layer chromatography quantification after treat PANC-1 cells with TGM4 30 µM for
12h (Top) or 48h (Bottom). Data are presented as mean ± SEM. Statistical significance was
determined by unpaired two-tailed Student's t test (*p < 0.05; **p < 0.01). CLP: cardiolipin; PE:
phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine; PC:
phosphatidylcholine; SM: Sphingomyelin.
A significant increase of cardiolipin was found after 12 hours of treatment with TGM4
(Figure 23 Top). Intriguingly, this increase disappeared after 48 hours of treatment (Figure
23 Bottom). There was also a little but significant downregulation of Phosphatidylcholine
after 12 hours of incubation with TGM4 (Figure 23 Top). The rest of the studied lipids
remained constant for all the conditions.
47
5. Discussion
5.1 The TGMs, a novel library of compounds, are effective antiproliferative drugs.
The pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death
in the United States, sixth in Europe, despite of the relative short incidence of 10-
12:100.00.
The high mortality due to their aggressive nature, the difficulty of an early detection and
thus its high metastatic rate, besides the lack of effective treatments for control the tumor
if surgical resection fails, make of this cancer one of the cancers with more clinical
significance nowadays. Of all pancreatic tumor cell lines we chose working with Mia-
PaCa-2 for growth studies and with PANC-1 line for molecular studies, because the last
line was obtained from ductal cells and it represents better at molecular level the cancer
that mostly affects humans.
The library of compounds named TGMs are rationally designed from different natural
triglycerides. The hypothesis of this study is that all the molecules studied due to their
lipid character will act on the lipid membrane, regulating the composition and membrane
fluidity. Once in the membrane they will affect the interaction and activity of peripheral
membrane proteins involved in several signaling pathways that are eventually related to
cellular membrane and the regulation of vital cellular processes for oncogenesis. In
summary, the changes produced by the action of these molecules will cause several
changes in cell signaling pathways that ultimately end up producing changes in the
processes of cell proliferation and survival.
In vitro studies carried out for this master thesis showed that the use of the molecules
tested decreases the number of pancreatic tumor cells, and that the number of cells
decreases in proportion to the concentration and time.
It is noteworthy that the differences in the IC50s of the molecules are not significantly
different and generally most of them are considered to show good antitumor potency.
The most potent molecules, those who have a lower IC50 were TGM5, TGM12 and
TGM25, but for selecting a molecule to develop and study deeply there are other factors
to take into account as its structure. That is the reason why at the end TGM4 was
highlighted, the free fatty acids that are part of its structure are promising antitumor
candidates by themselves.
Another factor to consider evaluating the antitumor activity of a compound is its efficacy.
The potency of a compound is measured mainly from its IC50, so a compound has with
48
a lower IC50 has greater potency. The efficacy here is reflected by the ability of the
compound to kill, its maximal therapeutic effect. Most of the TGMs showed to kill all the
cells of the culture.
The benefits associated with great antitumor efficacy and potency (low concentrations
needed) are remarkable, because at low concentration decrease the possibility that
appear secondary or unwanted side effects. For that reason we chose TGM5 to test its
effect on in vivo models, together with the TGM4 that we chose for structural reasons.
The study of the mixed TGMs, though very interesting, was postponed for future
experiments.
Even after checking TGM4 and TGM5 lack of toxicity evaluating the survival, no significant
body weight loose and no behavioral effect of mice after their administration, it would
be necessary to test their toxicity and effect in some non-tumor cell lines, for example
MRC-5 lung human fibroblasts, that would allow to determine if we are within the range
of normal cell toxicity. As they has shown to be non-toxic for mice, we hypothesize that
they will have a greater effect on cancer cells than in normal cells, which is a must in
therapies against these diseases. Specific drugs are sought to make these effects will
occur only in cancer cells, thus preventing a large number of unwanted side effects.
For future studies there are remaining few molecules of the TGMs library that, for time
reasons, does not have been tested in this master thesis and based on the results of the
rest of compounds we hypothesize that they will work fine as antiproliferative drugs.
On the other hand there are more pancreatic cancer cell lines and lines from other kind
of important cancers in which the molecules (or at least some of them) should be tested.
If the molecules do not works fine on other cancer cell lines there should be some
specifically characteristic of the pancreatic cancer cells that make them sensitive to the
molecules. If the tested molecules work on several different cancer cell lines will mean its
effect is produced disrupting some essential oncogenic step. Both possibilities will offer
important data to understand how the TGMs works.
5.2 The TGM4 is an effective antitumor drug, tested on Mia-PaCa-2 xenograft in
vivo models of pancreatic cancer.
In vivo studies in nude immunodepressed mice confirmed that the antitumor effect of
the molecule TGM4 against pancreatic cancer. Unfortunately, TGM5 did not have
significant antitumor activity in these studies. Regarding the effect produced by the
TGM4 and the treating conditions, there are remarkable points.
49
When an animal or a human has to be treated with any drug due to cancer disease, the
toxicity of the compound is the factor to take into account for deciding the most effective
dose but with the minimum side effects. From the toxicity studies the dose of 1000 mg/kg
was chosen without any adverse effect that could make us consider that TGM4 could be
dangerous for a potential future patient.
As shown in Figure 17 the effect of the TGM4 at a daily oral dose of 1000 mg/kg is very
similar to that produced by the drug of reference gemcitabine at a dose of 100 mg / kg
given to the mice by intraperitoneal injection twice a week. When considering a drug for
antitumor therapy in humans are taken into account several factors, but two essential are
potency and toxicity. The fact that the effect has been the same even though the TGM4
dose was much higher may suggest that the TGM4 is less effective for the treatment of
pancreatic cancer than gemcitabine, but not necessarily. The possibility of such a high
dose without toxicity effects show that the TGM4 is an antitumor molecule with a low
toxicity profile, which provides a greater therapeutic window, ie, can be administered at
higher doses without the fear of producing the known side effects of most
chemotherapeutic drugs, which may range from mild to very severe.
Furthermore, the fact that the TGM4 can be administered orally maintaining its antitumor
effect provides information about its absorption and distribution. For a triglyceride orally
ingested to reach its destination, it must be absorbed in the intestine. Triglycerides form
a fat emulsion by the action of bile salts and these are processed by digestive lipases that
cleave fatty acids and glycerol to be absorbed separately by intestinal epithelial cells.
Within these enterocytes, triglycerides are reformed and transported, together with
proteins, lymph circulation and blood (Trauner et al. 2010). Once in the blood, triglyceride
can reach the cells that form the tumor, where lipoproteins lipases from the endothelial
cells cleave the triglycerides again and allow the fatty acids and glycerol enter the cell by
the vascular endothelium, penetrating in the tumor cell wherein the fatty acids will
produce their antiproliferative effect.
Oral administration is an advantage over intravenous administration, because apart from
allowing patients avoid continued injections, it has a number of benefits over other
routes of administration. Oral delivery is suitable for the vast majority of patients
regardless of age or physical condition, is a natural way and completely painless. In
addition there are several possible formats for oral administration, whether tablets,
capsules, syrups, emulsions...and these do not require medical attention for their
administration. Finally, we consider that the most important point is that oral
administration is associated with less toxicity due to the fact that the absorption is
prolonged and sustained, allowing better tolerance to side effects of the drug.
50
Additionally and especially in pancreatic cancer, tumors eventually produce resistance to
most drugs after continued treatment time. Thus, for example, for pancreatic cancer the
only therapy that is considered to have curative potential is resection of the affected area
of the pancreas, and other treatments are palliative, because usually the treatment does
not remove 100% of the tumor cells before they produce resistance. The TGM4 by its
lipid nature would be included within the known Membrane-Lipid Therapy, which affect
not only a unique protein as current drugs do. TGM4 would affect current up to several
signaling pathways that are initiated in the membrane. It has been observed the ease of
tumor cells for produce metabolic bypasses so they avoid the proteins that are used as
targets for anti-tumor cell growth. These bypasses would be much more difficult to
perform if several signaling pathways in the initial point are affected, so it is hypothesized
that the development of resistance to the molecules acting through MLT would be much
lower than in conventional chemotherapy.
In the study conducted, a clear and significant reduction of the tumor caused by the
effect of TGM4 was observed. We should repeat the experiments to confirm the observed
results. Once confirmed the effect of the TGM4 in vivo, this could be an effective therapy
against pancreatic cancer. Currently and increasingly, it is pointing to the use of
customized and combined with different chemotherapy drugs that allow greater control
over the different signaling pathways targeted therapies. This combination with other
chemotherapy drugs would allow to reduce the dose of chemotherapy and thus reduce
the high toxicity that often appear in patients. Ease of administration and low toxicity of
the TGM4 make this molecule an ideal candidate to offer an alternative as monotherapy
or in combination in patients with pancreatic cancer.
5.3 TGM4 produces an antiproliferative effect and death by autophagy in cancer
cells PANC-1.
The study of the TGM4 mechanism of action of revealed that induces cell death. The cell
cycle analysis of TGM4-treated cells with this drug for 6, 12, 24 and 48 hours showed an
increase of cell death at concentrations near to its IC50 (Figure 19). The cell death was
confirmed by an increase in the percentage of cells found in the sub-G1 phase. As a result
of this increase, a significant decrease in the percentage of cells in the G0 / G1 phase of
the cell cycle.
However, in the studies of dose-dependency (Figure 18) the increase in the percentage
of cells found in the sub-G1 phase was followed by a decrease of the cell population in
the S and G2/M phases, not in in the G0/G1 phase as in figure 19. This studies have to
be repeated in order to confirm the results, but a priori, by the statistical significance of
51
the experiments, we believe that the decrease of the cell population in G0/G1 phase is
more interesting and significant.
The findings in the cell cycle may be related to the alterations found in some cell cycle
regulatory proteins. In the protein studies, it was found that the treatment with TGM4
produced a drop in the cyclin D3 levels. Cyclin D3 acts as a regulatory subunit of a
complex formed with Cdk4 or Cdk6 and its activity is required for the G1/S cell cycle. As
this is a protein necessary for the transition or regulation from the G0/G1 phase to the S
phase and the TGM4 produced a decrease in the population of cells in the G0 / G1 phase,
reduction of this protein levels is expected and supports the results obtained in the flow
cytometry. On the other hand, p19 protein has-been shown to form a stable complex
with CDK4 or CDK6, preventing the activation of the CDK kinases, functioning as a cell
growth regulator that controls the G1 cell cycle progression and induces the arrest in
that cycle. This protein levels were not affected by the treatment with TGM4, in
accordance with the drop of the cell number in G0/G1 and cell death instead of cell cycle
arrest. Other protein important for the cell proliferation included in this study is DHFR.
This protein is required for DNA synthesis and therefore to cell replication, especially if
this is accelerated as in cancer. TGM4 decreases expression of the DHFR protein, which
hampers DNA replication to cancer cells and thus cell replication. Some drugs such as
methotrexate and have as target the expression of DHFR protein but are highly toxic.
Part of the TGM4 effect may be due to its effect in reducing the expression of DHFR.
Other lipid compounds developed in this group have shown that DHFR downregulation
is key for their effect (Lladó et al. 2009)
The study of cell proliferation signaling pathways indicates that the TGM4 affects the
regulation by phosphorylation of the studied proteins AKT, ERK, and c-jun. Unexpectedly,
we found that in the AKT and ERK cases, the stability or expression of the protein was
affected also, because an alteration in the total levels of these proteins (not only in the
phosphorylated protein) were found.
Specifically, TGM4 affects the PI3K/AKT and ERK pathways, showing variations in the
phosphorylation and total levels of AKT and ERK proteins. However, the effect on them
is variable, not a clear dose or time dependent upregulation or downregulation, one
possibility is that AKT is being altered in response of the pathways directly modified by
TGM4, for example, AKT upregulation at 6hrs could be due to an attempt to
counteracting the antiproliferative effects and stress signals of other pathways, such as
JNK pathway or BIP/CHOP induction. This hypothesis would need more exploration and
could be accepted and discarded after we establish a timeline. In the case of c-jun we
found the TGM4 reduced the levels of phosphorylated c-jun, but TGM4 did not affected
the total levels of Jun. The alterations of this pathways are highly involved in the
52
regulation of proliferation and expected when working with an antiproliferative drug.
These results may raise suspicion of the cellular stress that TGM4 produces, a fact that
can be confirmed by the overexpression of the protein BIP treatment cells are found. BIP
is a protein which recognizes misfolded proteins, and protein subunits that are not
assembled into their final oligomer complexes, so the presence of BIP is associated with
a reticular stress.
On the other hand we sought to discover what type of cell death is produced by the
TGM4, apoptosis was almost ruled out from the start by the non-presence of apoptotic
bodies in the cultivation nor evidence in cell morphology that indicate apoptosis after
treatment with TGM4 (Figure 20). To discard apoptosis, PARP protein fragmentation was
analyzed because it is considered an indicator of apoptosis. As, fragmentation of PARP
was not observed, the apoptosis via caspases cascade was discarded. However, in
caspase independent apoptosis, a downregulation of full length PARP (116 kDa) is
produced. Since in our experiments this downregulation of the full PARP is observed, it
would be interesting to investigate. Because autophagy and apoptosis are not exclusive.
When analyzing autophagy related proteins, positive results were obtained. Autophagy
is an intracellular bulk degradation system, through which a portion of the cytoplasm is
delivered to lysosomes to be degraded. Aggregation and posttranscriptional
modification of microtubule-associated protein light chain 3 (LC3), a mammalian
homolog of yeast Atg8, has-been used as a specific marker to monitor autophagy (Kuma
et al. 2007). LC3B is first cleaved at the carboxy terminus immediately following synthesis
to yield a cytosolic form LC3B-I. During autophagy LC3B-I is converted to LC3B-II through
lipidation by an ubiquitin-like system involving Apg7 and Apg3 that allows for LC3 to
become associated with autaphagic vesicles (Kabeya et al. 2000; He et al. 2003; Tanida et
al. 2004; Wu et al. 2006; Ichimura et al. 2000). Thus, the presence of LC3 in
autophagosomes as well as the conversion of LC3 to the lower migrating form LC3B-II
have been used as indicators of autophagy. The marked overexpression of LC3B-II after
the treatment with TGM4 indicates that autophagy is triggered in treated cells.
One candidate to be the link between the ER stress and autophagy is CHOP, it encodes
a ubiquitous transcription factor that is one of the most important components in the
network of stress-inducible transcription. In particularly, this factor is known to mediate
cell death in response to stress (B´chir et al. 2014). CHOP promotes IRE1α and autophagy
especially in ER stress conditions. (Shimodaira et al. 2014). Increasing levels of CHOP were
found in the TGM4 treated cells and this corresponds with an increasing of the protein
BIP (associated with reticular stress) and increased LC3B-II (associated with autophagy).
As a part of his mechanism of action, we suggest that TGM4 produces a reticular stress
53
in the cells, being CHOP one of the responsible of promoting autophagy given the
situation of reticular stress caused by TGM4 and confirmed by overexpression of BIP.
All work performed in this master thesis is based on the theory that the compounds used,
for its lipid nature, will be inserted in the membrane and eventually leading to changes
in the activity of peripheral proteins membrane. The study of the alteration produced by
the TGM4 in the composition of the lipid membrane was limited to the phospholipids in
the cell whole extract, as only a reduced number of lipids were studied, among the
hundreds present in a cell. But despite this, a remarkable increasing of the cardiolipin
levels and a little but significant decrease in the phosphatydilcholine levels was found.
Further study of more lipids is needed to establish the relationship between the
mechanism of action showed by TGM4 and the alteration produced in membrane lipids
levels or composition. Indeed, recently, our group has published that TGM1 treatment (a
mimetic triglyceride derived from triolein, not included in this master thesis) in lung
cancer cells modifies the levels of neutral lipids (DAG, TAG and free fatty acids) what
induces an increase of ROS production in the cell, triggering autophagy cell death
(Guardiola-Serrano et al. 2015). Besides, cardiolipin is the most abundant lipid in the
mitochondria and the alteration of its levels/localization could induce mitophagy, other
possible cause of cell death that could be explored.
Generally, more studies are needed to clarify the sequence of events leading to the
confirmed cell death. The possibilities of membrane-lipid therapy are high because
altering the lipid membrane, we find not only a single change to the level of a target
protein as with current treatments. By targeting the membrane, restoring the lipid levels
and distribution to a normal state, a single compound can produce countless changes in
signaling pathways and thus in biological processes associated with proliferation or
apoptosis, which may be decisive for the regulation of oncogenic processes.
54
6. Conclusions
The main conclusions of this master thesis are:
1. The studies performed for this master thesis highlight the idea that the Membrane-
Lipid Therapy can be effective in the treatment of an important disease as cancer
2. The TGMs library exerts a remarkable antiproliferative effect on pancreatic cancer cells
Mia-PaCa-2.
3. TGM4 causes cell death by reticular stress followed by autophagy in PANC-1 cells.
4. The TGM4 affects important signaling pathways, like the SAPK/JNK/c-jun pathway, and
other studied proteins. More studies are needed to clarify the sequence of events leading
to cell death.
5. Studies in vivo: chronic administration of TGM4 reduces the progression of tumors in
nude mice inoculated with Mia-PaCa-2 cells.
55
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