CHAPTER-2shodhganga.inflibnet.ac.in/bitstream/10603/13119/9/09_chapter 2.pdf · CHAPTER-2 Evaluation of anticancer and anti-proliferative potential and underlying mechanism(s) of

CHAPTER-2

Evaluation of anticancer and

anti-proliferative potential

and underlying mechanism(s)

of synthetic dibromotyrosine

analogues using human

prostate cancer celL line DU-

145

Anticancer and antiproliferative potential……… of analogues Chapter 2

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2.1 Introduction

Marine environment is a rich source of novel bioactive chemical products

[Glaser and Mayer, 2009; Molinski et al., 2009; Goodfellow et al., 2009;

Simmons et al., 2005; Blunt et al., 2004]. Complex and highly chiral

structures have been optimized by marine organisms to survive harsh

environmental conditions including high salt concentrations, currents,

high pressure, and hostile natural predators over millions of years, which

confers marine organisms the potential to produce valuable therapeutic

entities [Molinski et al., 2009; Blunt et al., 2004]. Emerging evidence

suggests that marine natural products, especially the secondary

metabolites from marine sponges, are far more likely to yield

anticancer drugs than terrestrial sources [Glaser and Mayer, 2009;

Molinski et al., 2009; Goodfellow et al., 2009;Simmons et al., 2005; Blunt

et al., 2004]. For example, Ara-C (cytarabine, an antileukemic drug) and

trabectedin (Yondelis, ET-743, an agent for treating soft tissue sarcoma)

were developed from marine sources [Molinski et al., 2009; Goodfellow et

al., 2009; Schoffski, 2008]. In the last decade, there has been a dramatic

increase in the number of preclinical anticancer lead compounds

(metabolites) extracted from marine-derived sponges and fungi [Zhang

et al., 2007; Yanagihara et al., 2005; Zhang et al., 2005].

Apoptosis is a normal physiologic process which occurs during

embryonic development as well as in maintenance of tissue homeostasis.

However, resistance to apoptosis is a hallmark of cancer and is

undoubtfully the major cause behind the immortality of cancer cells,

with both the loss of pro-apoptotic signals and the gain of anti-apoptotic

mechanisms contributing to tumorigenesis [Hanahan and Weinberg,

2000]. Several cellular pathways cumulate in the activation of caspases

and apoptosis. Apoptosis is characterized by two major pathways which

are the extrinsic and the intrinsic pathways [Ziegler and Kung, 2008].

The extrinsic pathway is initiated by the interaction between specific


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ligands and surface receptors [Klein et al., 2005],such as

CD95/Fas/Apo1, tumor necrosis factor (TNF) receptor 1 (TNFR1), TNF

receptor 2 (TNFR2) and death receptors 3–6 (DR3–6) [Degterev et al.,

2003], which are able to deliver a death signal from the extracellular

microenvironment to the cytoplasm. Binding of a specific ligand to its

target receptor induces receptor multimerization, binding of Fas-

associated death domain (FADD) adapter protein, formation of the

death-induced signaling complex (DISC) which recruits the initiator

caspases 8 and 10 and subsequently activation of the effector

caspases 3 and 7 [Klein et al.,2005].

The intrinsic pathway is activated by various stimuli, i.e., DNA damage,

hypoxia, cell detachment, cellular distress and cytotoxic drugs, which act

inside the cell [Degterev et al., 2003]. All of these signals converge to

mitochondria, where the propagation of the apoptotic signal is regulated

by the Bcl-2 family members [Danial and Korsmeyer, 2004]. Bcl-2 and

Bcl-xL exert anti-apoptotic effects, while others such as Bid, Bad and

Bim are pro-apoptotic [Debatin, 2004; O’Neill et al., 2004]. An excess of

pro-apoptotic over anti-apoptotic signals initiates mitochondrial outer

membrane permeabilization (MOMP), which leads to the release of

proteins such as cytochrome c and Smac/ Diablo from the mitochondrial

intermembrane space to the cytosol. Once cytochrome c is released, it

binds to Apaf-1 and ATP, which then bind to pro-caspase 9 to create a

protein complex known as apoptosome, which in turn activates the

effector caspase 3 [Green, 2005]. Smac binds to the inhibitor of

apoptosis proteins (IAPs) and deactivates them, preventing the IAPs from

arresting the apoptotic process and therefore allowing apoptosis

to proceed. A third apoptotic pathway, the ‘‘endoplasmic reticulum (ER)

stress’’ pathway has recently been described [Nakagawa and Yuan,

2000].


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It is well documented that the natural product curcumin induces

apoptosis in MCF-7, MDA-MB-231, and HepG2 cells through the

generation of ROS originating from glutathione depletion by buthionine

sulfoximine, thereby further sensitizing the cells to curcumin [Chiu and

Su,2009; Syng-Ai et al., 2004]. In colon cancer cells, curcumin induces

apoptosis via a ROS associated mechanism that converges on JNK

activation, and to a lesser extent via a parallel ceramide-associated

pathway [Moussavi et al., 2006]. Induction of early apoptosis and ROS

generation activity were also observed after curcumin treatment in

human gingival fibroblasts and human submandibular gland carcinoma

cells [Atsumi et al., 2006]. Furthermore, curcumin promoted apoptosis

in human skin cancer cells which is preceded by an increase in

intracellular ROS production, which supports the notion that

mitochondrial respiration and redox tone are pivotal determinants in

apoptosis signaling by curcumin in human skin cancer cells [Numsen,

2008]. Induction of apoptosis by curcumin was also observed in human

breast epithelial cells involving down-regulation of Bcl-2 and up-

regulation of Bax as well as generation of ROS which suggests

redox signaling as a plausible mechanism responsible for curcumin-

induced apoptosis in these cells [Kim et al.,2001].

We have already proved in the previous chapter that the majority of

chemically synthesized marine-derived synthetic dibromotyrosines

developed from parent scaffolds of dibromoverongiaquinol and

aeroplysinin-1 showed no genotoxicity, carcinogenicity, nor mutagenicity

thereby posing indication to be developed as drug like leads. These

promising properties further prompted the evaluation of the anticancer

and anti-proliferative potentials of these synthetic analogues using

human prostate cancer DU-145 cell line.


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2.2 Materials and Methods

2.2.1 Collection of chemically synthesized analogues of

dibromotyrosine: The analogues used in this study were procured and

used as described in the previous chapter [Sallam et al., 2010].

2.2.2 Collection and maintenance of prostate cancer cell line DU-

145: Human prostate carcinoma cell line DU-145 was purchased

from American Type Culture Collection (Manassas, VA, USA). DU-

145 was maintained in RPMI 1640 (Gibco, NY, USA), supplemented

with 10% heat-inactivated FBS (Sigma), 2 mM L-glutamine (Life

Technologies, Inc., Bethesda, MD, USA), and 1% penicillin. All cells

were grown at 37°C in a humidified atmosphere consisting of 5% CO2

and 95% air.

Analogues (dissolved in DMSO at desired concentration) were employed

for the treatment of cells. DU-145 cells (1x104) were seeded in a 96 well

plate in triplicate. On the next day when the confluency of 70% to 80%

was achieved, the media was taken out and the cells were treated with

50µM and 100µM concentration of all the analogues for 24 and 48 hrs in

complete cell culture medium. Cells that were used as controls were

incubated with 20µl of DMSO along with 20µl of culture media only. In

50µM analogue concentration, each well contained 20µl media, 10µl

analogue and 10µl of DMSO. While for 100µM of analogue concentration

the well contained 20µl media, 20µl analogue only.

2.2.3 Antiproliferative activity assay (MTT assay): Cell viability was

measured by MTT assay to assess the chemosensitivity of tumor cells.

Cell suspension was collected into sterile 96-well flat-bottomed microtiter

plates (1 × 104 cells/per well) with or without dibromotyrosine analogues.

The testing concentrations of all the synthesized analogues used were 50

µM and 100 µM. Each analogue was tested in triplicate. The plates were

then incubated at 37℃ in a humidified atmosphere containing 5% CO2

and 95% air at 24 and 48 hrs time points, respectively. Microtiter wells


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containing DU-145 prostate cancer cells but without treatment were

used to control cell viability, in which the total number prostate cancer

cells were equivalent to that in the test wells, and wells containing only a

complete medium were used as vehicle controls for nonspecific dye

reduction. After incubation, 100ul MTT solution was added to each well

at a final concentration (5 mg/mL in PBS) and the plates were incubated

at 37℃ for another 4 hr. Then from the mixture containing the medium

and the analogues, the unconverted MTT was removed. 100µL of DMSO

was added to each well to dissolve the formazan and absorbance was

read at 570 nm using a spectrophotometric microplate reader

(Labsystems, Finland).

2.2.4 DNA double-strand breaks by Comet assay: This assay was

performed according to the technique of Singh [Singh, 2003].

Immediately after the incubation period of confluent DU-145 cells with

100μM of analogue 12, a single cell suspension was made by using

pipette. From the suspension, 10µl of suspension was mixed with 0.2

ml, 0.7% agarose. Agarose was suspended in phosphate buffered saline

with 3:1 agarose higher resolution and kept at 37°C to maintain

physiological conditions [Singh, 2003]. The mixture was pipetted out

and poured onto a fully frosted slide, immediately covered with

coverglass (24×60 mm). These slides were kept in an ice-cold steel tray

on ice for 1 min to allow the agarose to gel. Again, a layer was made over

the gel with 100µl of agarose as before, after removing the coverglass

[Singh, 2003; Tice et al., 2000]. These slides were immersed in ice-cold

lysing solution and kept for 2 hours at 4 °C. After lysing, the slides were

removed and placed in a horizontal slab of an electrophoresis assembly.

One litre of electrophoresis buffer was gently poured into the assembly.

After 20 min to allow for unwinding, electrophoresis was started at 250

mA (12 V) for 30 min. The slides were removed from the electrophoresis

apparatus and placed in coplin jar containing neutralizing buffer. After


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30 min, the slides were transferred to another jar of neutralizing

solution. After one more change of 30 min, the slides were left vertical at

room temperature to dry and stained with ethidium bromide (EtBr of

0.05 mg/ml) covered with a 24×60-mm cover glass. Microscopic slides

were prepared for each individual analogue separately. Images were

taken at 100X magnification using a charge coupled device camera

GW525X (Genwac, Orangeburg, NY, USA) attached to Leica DMLB

fluorescence microscope (Leica, Wetzlar, Germany) with an excitation

filter of 490 nm, a 500-nm dichroic filter, and an emission filter of 515

nm. The images of double strand DNA break in DU-145 prostate cancer

cell line were recorded with fluorescence microscope.

2.2.4.1 Comet scoring: Slides were assayed for double-strand DNA

breaks. Twenty cells were selected from each slide and hence 40 cells

(two slides) were scored. Head and tail length (mm) and tail movement

(mm) from the beginning of the nuclear area to the last five pixels of DNA

perpendicular to the direction of migration at the leading edge were

measured. The scoring of comet assay was done by using software (IV

4.2 version software, perceptive Instrument).

2.2.5 Apoptosis using Annexin V FITC kit and FACS (Fluorescence

activated cell sorting): DU-145 prostate cancer cells (1x106) were

incubated with analogue 12 at a concentration of 50µM and 100µM for

48 hrs before being trypsinized and washed thrice with PBS. The cells

were then subsequently stained with PI and FITC labeled Annexin V by

using the Annexin V-FITC apoptosis detection kit (BD Biosciences, USA)

to assess cellular integrity and the externalization of phosphatidylserine

(PS) [Pathak and Chauhan, 2011; Balzan et al., 2004]. The cells were

washed twice with cold PBS and then resuspend cells in 1X Binding

Buffer at a concentration of 1 x 106 cells/ml. The 100 µl of the solution

(1 x 105 cells) was transferred to a 5 ml culture tube. To this, 5 µl of

FITC Annexin V and 5 µl PI were added. Cells were gently vortexed and


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incubated for 15 min at 25°C in the dark. The 400 µl of 1X binding

buffer was added to each tube. The cells were analyzed by using FACS at

488 nm excitation and a 515 nm band pass filter for FITC detection and

wavelength >560 nm for PI detection. A total of 10,000 events were

counted at the flow rate. Data analysis was performed using cell Quest

software (Becton–Dickinson Immunocytometry System) [Hwang et al.,

2011].

2.2.6 Altered protein expressions of apoptosis markers: Western blot

analysis was carried out for apoptosis markers (Erk1/2, p-P38, p-JNK1/2,

p53, Bax, Bcl-2, caspases 3/9, c-fos, c-jun) in DU-145 cells exposed to

analogue 12 (100µM) for 48 hrs. Following Analogue 12 exposure, cells

were pelleted and lysed using CelLyticTMM Cell Lysis Reagent (Catalog no.

C2978, Sigma, USA) in the presence of protein inhibitor cocktail (Catalog

no. P8340-5ML, Sigma, USA). Protein estimation was done by BCA Protein

Assay Kit (Catalog no.G1002, Lamda Biotech, Inc., St. Louise, MO, USA).

The equal amount (50 mg/well) of denatured proteins was loaded in 10%

tricine-SDS gel and blotted on polyvinylidene fluoride (PVDF) membranes

(Santa Cruz, USA) using wet transfer system. After blocking (2 hrs at

370C), membranes were incubated overnight at 40C with anti-protein

primary antibodies specific for c-fos, c-jun, Erk1/2, p38, p-JNK1/2

(1:500, Chemicon, USA),p53, Bcl-2, Bax, activated caspase-9, activated

caspase-3 (1:1000, CST, USA) and β-Actin (1:2000, Santa Cruz, USA) in

blocking buffer (pH 7.5). The membranes were then re-incubated for 2 hrs

at room temperature with secondary anti-primary immunoglobulin G

(IgG)-conjugated with horseradish peroxidase (Calbiochem, USA). The

blots were developed using luminol (Catalog no. 34080, Thermo

Scientific, USA) and densitometry was done for protein specific

bands in Gel Documentation System (Alpha Innotech, USA) having

AlphaEa-seTM FC StandAlone V. 4.0.0 software. β-Actin was used as

internal control to normalize the data. Analogue 12 induced alterations


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are expressed in relative term fold change in expression by

comparing the data with respective unexposed controls. Autorecovery

pattern of altered protein levels was also studied in a parallel group

exposed to MCP (100µM) for 6 h followed by 18 hrs incubation in fresh

culture medium without analogue 12.

2.2.7 Densitometric analysis and calculation of BAX/Bcl-2 ratio: The

densitometry analysis was performed by analyzing the Western blots in

terms of intensity (INT/mm2) using standard Gel Doc apparatus having

Biorad Quantity One Software and the ratio of BAX and Bcl-2 was

calculated in terms of the ratio of intensity of bands obtained as:-

Overall Densitometry Score Ratio (∆Dratio) = ∆DBAX /∆DBcl-2

If <1.0 means anti-apoptotic and >1.0 means pro-apoptotic

D = Desitometric analysis of western blots expressed as Intensity/mm2

2.2.8 DPPH radical-scavenging assay : Measurement of DPPH radical

scavenging activity was performed according to recommendations by

Nenadis and Tsimidou (2002). Conditions consisted of an approximately

20 min reaction period and a molar ratio between DPPH and antioxidant

that permits 60–80% radical-scavenging activity for the most potent

antioxidant. Briefly, 2,2-diphenyl-1-picrylhydrazyl (DPPH) in ethanol

(100µL of 100µM DPPH + 80µL of 50mM of trisHCL at pH 7.4) was added

in a 200µl reaction mixture in 96 well flat bottomed microtiter plates to

which was added 20 µL of various concentrations of the test analogues.

The final concentrations of the test analogues in the reaction mixtures

were 100 µM and 50 µM. The microtitre plate was then vigorously shaken

and held for 30 min at room temperature in the dark. The decrease in

absorbance of DPPH at 517 nM was measured. Ethanol was used as a

blank solution. DPPH solution (2 ml) in ethanol (2 ml) served as the

control. All tests were performed in triplicate. The radical

scavenging activity of the samples was expressed as % inhibition of

DPPH absorbance:-


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%Inhibition = [(Acontrol -Atest)/Acontrol] x 100

where, A control is the absorbance of the control (DPPH solution without test

sample) and Atest is the absorbance of the test sample (DPPH solution plus

compound). Ascorbic acid and BHT were used as reference standards.

2.2.9 Scanning and transmission electron microscopy (SEM and TEM)

studies of DU-145 prostrate cancer cell line : All treated and un-treated

DU-145 cells (~1×106) were fixed with 2% glutaraldehyde in 0.1%

phosphate buffer for 1hr at room temperature (200C) [Mares, 1989;

Borgers and Cutsen, 1989]. This was followed by washing with 0.1M

phosphate buffer (pH 7.2) and post-fixed with 1% OSO4 in 0.1 M

phosphate buffer for 1 hr at 40C. For SEM, the cells were dehydrated in

acetone and dropped on round glass cover slip with hexam-ethyldisilizane

(HMDS) and dried at room temperature followed by sputter coating with

gold and observed under the SEM (Zeiss EV040). For ultrastructure

study (TEM), samples were dehydrated with graded acetone, cleared with

toluene and infiltrated with toluene and araldite mixture at room

temperature then finally in pure araldite at 500C and imbedded in

Eppendoff tube (1.5 ml) with pure araldite mixture at 600C. Semithin and

ultrathin section cutting was done with ultramicrotome (Ultramicrotome

Lecia EM UC6). Sections were taken on the 3.05 mm diameter and 200

mess copper grid, stained with uranyl acetate and lead acetate before

observing under the TEM (JEOL, JEOL2100F) [Kaneshima et al., 1977].

2.2.10. Statistical analysis : Data are presented as mean ± standard

deviation (SD) of 3 replicates. Statistical analysis of data was done by

employing the two-tailed Student‘t’ test as described by Bennet and

Franklin [1967]. The effect of all the analogues at varied concentrations was

repeated in triplicate. A difference at P < 0.05 was considered statistically

significant.


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2.3 Results

Prostate cancer in humans progresses from an androgen-responsive to an

androgen-unresponsive state, and at the time of clinical diagnosis, most

prostate cancers represent a mixture of androgen-responsive and

androgen-unresponsive cells [Gleaven et al., 1998]. Whereas androgen-

responsive cells undergo rapid apoptosis on androgen ablation, androgen-

un-responsive cells evade apoptosis during androgen withdrawal,

although they retain the molecular machinery for apoptosis. Mortality

from prostate cancer generally occurs from the proliferation, invasion,

and metastasis of these androgen-unresponsive cells, which fail to

undergo apoptosis culminating into hormone refractory prostate cancer

for which no cure but only palliative treatment is available [Denmeade et

al., 1996]. Therefore, there is an urgent need to intensify efforts for

a better understanding of this disease and for the discovery and

development of novel mechanism-based approaches and small molecules

for its prevention and treatment [Weisburger,1998]. The above mentioned

facts related to prostate cancer attracted our attention and motivated our

study to see the effect of dibromotyrosine analogues against DU-145

prostate cancer cell line.

2.3.1 Antiproliferative effect using MTT assay: In the first set of

experiments, we evaluated whether concentration and time dependent

analogue treatment imparts any marked difference in anti-proliferative

effects in human prostate cancer cells. Employing the MTT assay,

it was observed that all the analogues were effective in inhibiting the

growth of prostate cancer cells in a dose and time- dependent manner

(figure 2.1). Analogue 12 was proved to be the most potent among all,

such that it inhibited the growth and cell proliferation of DU-145 prostate

cancer cell line with average value of 0.43 ±0.017 leading to 56%

inhibition of growth at 100µM in 24 hrs incubation and average value of

0.35 ±0.001 leading to 65% inhibition of growth with the same


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concentration in 48 hrs incubation. It was also observed that analogue

13 was showing almost neutral effect against DU-145 cells with an

average value of 0.92 ±0.08 and 1.15 ±0.10 at 100µM in 24 hrs and 48

hrs of incubation periods.

To further reconfirm the potency of analogue 12 in inhibiting the growth

and proliferation of prostate cancer cell line DU-145, the MTT assay was

repeated at two different concentrations of 50µM and 100µM for 24 and

48hrs. The prostate cancer cells responded in a similar fashion as

reported earlier with an inhibition average value of 0.71 ±0.007 and 0.56

±0.04 for 24 hrs incubation followed up by average values of 0.66±0.20

and 0.43±0.05 for 48 hrs, respectively with the respective 50 and 100 µM

doses (figure 2.2).

Figure 2.1: Effect of various synthesized dibromotyrosine ether and ester analogues on the proliferation of DU-145 prostate cancer cell line at 100µM concentration at two incubation periods of 24 and 48hrs, respectively. All analogues (except 13) were effective in inhibiting the


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growth and proliferation of prostate cells. Inhibition values are mean ± SE for each concentration of analogue carried out in triplicate. Each value is significantly different from the respective controls at p<0.05.

Figure 2.2: Dose and time-dependent effects of analogue 12 on cell proliferation in DU-145 cell line. Inhibition values are mean ± SE for each concentration of analogue carried out in triplicate. Each value is significantly different from the respective controls at p<0.05.

2.3.2 Double stranded DNA break estimation in DU-145 prostate

cancer cells : In the qualitative picture of DNA double-strand break,

larger tail movement was observed in the exposed DU-145 cells as

compared to the control (figure 2.3). The results of the Comet assay

clearly showed that there was a concentration dependent increase in DNA

double-strand breaks in the DU-145 cells when exposed to 50μM and

100μM of analogue 12. The average values of head length, head intensity,

tail length, tail intensity, tail movement and tail migration of DU-145 cells

exposed at two different concentrations of analogue are plotted in form of

3-dimensional graph (figure 2.4) on the basis of the evaluation of the

various above mentioned parameters by Perspective IV software for comet


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analysis. The results obtained clearly showed that there is a significant

increase in the head, tail length and tail movement of DNA in the

exposed cells as compared to the control ones. Head length of comets

in the control cells were giving average value of 131.22 ±5.09 as compared

to the average values obtained with 50μM and 100μM-treated cells of

110.73 ±4.67 and 100.31 ±7.46, respectively (figure 2.4). Further, with

the analysis of head intensity as compared to the average control value of

93.20 ±2.81, the treated cells were found to give average values of 49.11

±1.37 and 40.06 ±0.93 respectively. Upon further evaluation a very

significant increase in the tail length and intensity was found in the

treated cells as compared to the normal ones, with average values of

262.47 ±16.43 (50μM), 293.68 ±18.63 (100μM) in comparison to control

57.69 ±9.39 for tail length and 50.88 ±1.37 (50μM), 59.93±0.93 (100μM)

and 3.68 ±2.04 (control) for tail intensity. Study of tail movement and tail

migration also yielded significant results in terms of comet formation with

average values of 52.85 ±2.57 (50μM), 61.77 ±3.37 (100μM) and 2.56

±1.04 (control) for tail movement and 202.65 ±14.81 (50μM), 243.52

±17.50 (100μM) and 5.37 ±2.27 (control) for tail migration.


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Figure 2.3: Image showing DNA migration pattern of comets of DU-145 prostate cancer cells when exposed to various concentration of analogue 12 Dye: Ethidium bromide, Magnification: 40X.Comet formation is clearly seen to increase in a concentration dependent manner.


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Figure 2.4: Three-dimensional image showing various parameters of comet assay evaluated by Perspective IV software for complete analysis of comets. Values of various parameters are mean ± SE for each concentration of analogue carried out in triplicate. Each value is significantly different from the respective controls at p<0.05.

2.3.3 Induction of apoptosis or programmed cell death in DU-145

prostate cancer cells: In order to know if the induction of cell death was

either due to necrosis and/or due to apoptosis, we adopted Annexin V-

FITC based apoptosis detection assay [Philips et al., 2003] against DU-145

cell line with the most potent analogue (12) as evident from the earlier

studies at two different time points of 24 and 48 hrs and two

concentrations of 50µM and 100µM, respectively. In a FACS-based

apoptosis detection assay, we monitored the externalization of

phosphatidylserine (PS) to the outer monolayer of the lipid bilayer of the

plasma membrane (PM), which is an early biomarker of apoptosis. A


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concentration-dependent increase in percent apoptosis was seen at 24 hrs

incubation time. As shown in (Figure 2.5) (table 2.1), after 24 hrs of

incubation of cells 15.18% of apoptotic population was observed at 50µM

concentration while at 100µM 24.45% apoptotic population was seen.

Further, after 48 hrs incubation, analogue 12 induced 20.82% apoptosis

in DU-145 cells with 50µM concentration. However, a decreased apoptosis

(15.91%) was observed at 100 µM concentration (figure 2.5 and 2.6)

probably because of the increase in the necrotic population from 10.25%

in 24 hrs to 15.81% in 48 hrs of incubation.


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Figure 2.5: Externalization of phosphatidylserine in DU-145 prostate cancer treated cells. (a) 24 hrs incubation (i) Unstained cell, (ii) cell + PI, (iii) cell + Annexin V-FITC,(iv) cell with Annexin V-FITC + PI,(v) cell with Annexin V-FITC + PI + analogue 12 (50µM concentration),(vi) cell with Annexin V-FITC + PI + analogue 12 (100µM concentration).(b) 48 hrs incubation (i) Unstained cell, (ii) cell + PI, (iii) cell + Annexin V-FITC,(iv) cell with Annexin V-FITC + PI,(v) cell with Annexin V-FITC + PI + analogue 12 (50µM concentration),(vi) cell with Annexin V-FITC + PI + analogue 12 (100µM concentration). Cells were incubated with Analogue 12 for respective time duration and analyzed by flow cytometry. The synthetic marine derived dibromotyrosine analogue 12 is reconfirming its anti-cancer potential.


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Table 2.1: Tabular representation indicating the percentage of DU-145 cells undergoing apoptosis [marked in red (lower right {LR} quadrant)] upon treatment with analogue 12 for 24hrs and 48hrs at 50µM and 100µM concentrations.

Figure 2.6: Comparative study of percentage of viable to apoptotic population of DU-145 cells when treated with analogue 12 at concentrations of 50µM and 100µM at two different time points of 24 and 48 hrs using Annexin V FITC Kit.


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2.3.4 Alteration in the expression of apoptosis markers

2.3.4.1 p53 expression: Upon exposure of DU-145 cell line to analogue

12 treatment, there was an increase in the expression of p53 protein by

two-folds as evident from the densitometric analysis of the obtained

protein band (figure 2.7, table 2.2). Densitometry studies of the protein

bands obtained after Western blot analysis clearly showed that p53 levels

were elevated in prostate cancer cell line DU-145 with the control having

an intensity of 900.5 INT/mm2 as compared to the 100µM treated cells

with an intensity of 2212.23 INT/mm2. The increase in p53 is

synonymous to its well known function to act as a cellular gatekeeper

and trigger the expression of various related genes involved in the

induction of cell cycle arrest and apoptosis.

2.3.4.2 Bax and Bcl-2: The incubation of the prostate cancer cell line

DU-145 with analogue 12 resulted in a 1.5-fold increase in the expression

of pro-apoptotic protein Bax, compared to the level of expression of anti-

apoptotic protein Bcl-2 which fell down even below the basal level (figure

2.7). DU-145 untreated cells showed pro-apoptotic BAX expression

intensity of 2789.24 INT/mm2 in the obtained Western blots as

compared to the 100µM treated cells induced with an increased

expression of BAX with an intensity of 4819.39 INT/mm2. On the

contrary, induction of anti-apoptotic Bcl-2 in control DU-145 cells

revealed an intensity of Bcl-2 as 1927.74 INT/mm2 in comparison with

2046.08 INT/mm2 intensity of cells treated with 100µM of analogue 12.

2.3.4.3 BAX / Bcl-2 ratio: The relevance of BAX/Bcl-2 ratio which

serves as a regulator to determine cell susceptibility to apoptosis as

reported in scientific literature motivated us to reconfirm and calculate

BAX/Bcl-2 ratio synthetic dibromotyrosine analogues with BAX and Bcl-

2, in order to doubly confirm whether these synthetic molecules are

strong apoptotic inducers. The important terminology specific for cancer

cells is that the greater the value of ∆Dratio for a specific molecule, the


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better is its potential to induce apoptosis. This ratio is a more relevant

and reliable strategy to evaluate the programmed cell death-inducing

ability of anticancer drug lead rather than individual values alone.

Analogue 12 was found out to be an apoptotic inducer in terms of their

ability to act as anti-cancer drug lead giving a ∆Dratio of 2.35 much higher

that 1 thereby dictating its apoptosis inducing efficacy (table 2.2).

2.3.4.4 Caspase 3 and Caspase 9: DU-145 prostate cancer cells clearly

indicated (figure 2.7) that they undergo their demise by programming

their death by the process of apoptosis specifically mediated by the

activation of caspase cascade specially caspase 3 and caspase 9 as

evident from the western analysis wherein the expression of these

regulator caspases with analogue 12 in DU-145 prostate cancer cells have

increased significantly.

2.3.4.5 C-fos and C-jun: DU-145 prostate cancer cell line was used to

study the effect of incubation of analogue 12 treatment for a time period

of 48hrs on the expression profile of C-fos and C-jun proteins. The

results obtained clearly indicate that c-fos expression increased, with a

magnitude of 2.87 while similar expression pattern was also observed

with c-jun 2.04 magnitude in comparison to the no treatment control

(figure 2.7).

2.3.4.6 Phosphorylated p38, p38, ERK1/2, phosphorylated ERK1/2

and phosphorylated JNK: The anticancer hit candidacy of analogue 12

was further reconfirmed based on the expression studies of the following

phosphorylated proteins p38, p38, ERK1/2, ERK1/2 and JNK, which

play an active role in the increased cell cycle progression of DU-145

prostate cancer cell line. p38, JNK and ERK are activated by the

phsphorylation mechanism as suggested by the expression analysis

(figure 2.7), analogue 12 treatment led to the blockage of cell cycle,


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inducing programmed of the cell death. Autorecovery was found to be

statistically significant (p < 0.001) for the entire marker proteins studied.

Figure 2.7. Alterations in the expression of proteins involved in the induction of apoptosis as studied in DU-145 prostate cancer cells exposed to 100µM of analogue 12 for a time period of 48hrs. β-Actin was used as the internal control to normalize the data. Lane A, untreated control; B, cells exposed to analogue 12 for 48hrs; Molecular weight of protein studied: p53 (53 kDa), Bax (29 kDa), Bcl2 (23 kDa), activated Caspase-9 (35 kDa), activated Caspase-3 (21 kDa), C-fos (47 kDa), C-jun


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(42 kDa), and β-Actin (42 kDa) for normalization. Relative quantification of alterations in the expression of proteins involved in the induction of apoptosis in DU-145 cells exposed to 100µM of analogue 12 for various time periods. Quantification was conducted in Gel Documentation System (Alpha Innotech, USA) using AlphaEase FC StandAlone V.4.0 software. Table 2.2: Densitometric analysis of BAX, Bcl-2 and p53 proteins along with the BAX/Bcl-2 ratio in the western blot analysis of the synthetic dibromotyrosine analogues when incubated with DU-145 prostate cancer cell line.

Synthetic Analogue

Control Band Intensity

(INT/mm2)

Treated Band Intensity (INT/mm2)

Analogue 12 at 100 µM concentration was incubated with DU-145 cell line

BAX 2789.24 4819.39

Bcl-2 1927.74 2046.08

β actin 3380.76 3564.03

p53 900.5 2212.23

β-actin 2888.81 2888.27

∆Dratio = BAX/Bcl-2 4819.39/2046.08 = 2.35

*∆Dratio = ∆DBAX / ∆DBcl-2 D = Desitometric analysis of western blots expressed as Intensity/mm2

2.3.5 DPPH radical-scavenging assay: Comparison of the antioxidant

potential of dibromotyrosine analogues at 50µM and 100µM

concentrations in addition to the standard antioxidants ascorbic acid

and BHT used in the study revealed that most analogues behaved like

neutral compounds, which can imply the fact that they did not act via

the generation of free radicals nor did they act as antioxidants, unlike

their parent natural product dibromoverongiaquinol (figure 2.8). Almost

all analogues were less effective in scavenging the free radicals in

comparison with the controls but they even did not individually generate

any free radicals which can harm living systems by inducing various

stress-borne diseases. Analogues 10 and 12 at 50µM concentration were

found to be weak inducers of ROS with average values of 0.20 ±0.15 and

0.18 ±0.011, compared to standards BHT and ascorbic acid having


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average value of 0.045 ±0.021 and 0.04 ±0.026, respectively. In addition,

when these analogues were re-evaluated at 100µM, the results obtained

were in agreement with those obtained at 50µM with average values of

0.176 ±0.026, 0.164 ±0.002, compared to the same standards BHT and

ascorbic acid with average values of 0.035 ±0.013, 0.051 ±0.026,

respectively. Further the analysis of results indicated that analogue 13

was the lowest in terms of ROS induction at 50 µM concentration with an

average value of 0.114 ±0.025 while at a concentration of 100 µM,

analogues 1 and 13 were the lowest in terms of ROS generation capacity,

with average values of 0.101 ±0.020 and 0.086 ±0.024, respectively.

Figure 2.8: Antioxidant potential of the chemically synthesized marine ether and ester analogues at 50µM and 100µM concentration.


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2.3.6 Electron microscopy studies

2.3.6.1 Scanning electron microscopy studies: To further understand

the mode of action, we investigated the morphological changes in DU-145

prostate cancer cells by scanning electron microscopy (SEM) (figure 2.9).

Cells treated with analogue 12, at their respective concentrations of

50µM and 100µM for 48 hrs, showed marked and evident changes in

their morphological features. After 48 hrs from seeding (figure 2.9), the

human prostatic cancer cells DU-145 were rather well-spread and

attached to the culture plate. It’s possible to notice the polymorphism of

these cells, and the numerous specializations of cell surface in the

form of radial lamellipodia, microvilli and filopodia-like extensions of

variable dimensions and lengths. One of the most distinct

morphologies observed was that the un-treated cells show a tendency to

aggregate and, when they are confluent (figure 2.9A) and appear flat.

Microvilli were lost in those cells that have spread at high extension.

Among all the DU-145 cells treated with 50µM of analogue 12 followed

by 48 hrs of incubation, those whose number in the culture was

decreased, many typical features of apoptosis were visible. The cells

have reduced the normal dimensions and lost their original morphology;

in addition, they present several cell surface blebs (figure 2.9b, 2.9c and

2.9d).

When the DU-145 cells treated with 100 µM of analogue 12 followed up

by 48hrs of incubation were visualized, marked difference in the

morphology was observed, underlying the fact that the changes in the

morphology was concentration-dependent at same time point. Cells were

found to be round up or flattened exposing large round protuberances

and filament’s fringes attached to the substrate (figure 2.9E). The DU-

145 cells showed marked pleomorphism: many morphological alterations

in the form of spheroidal and/or elongated shapes and macro and micro

blebs enriched surfaces (figure 2.9F and 2.9G).


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Figure 2.9: Scanning electron microscopy pictures of DU-145 prostate cancer cell line treated with analogue 12 (FARN) at 50µM and 100µM concentrations for 48 hrs.

2.3.6.2 Transmission electron microscopy studies

Upon examination under the transmission electron microscope (TEM),

the control DU-145 cultures displayed a mixture of elongated and

polygonal cells. DU-145 cells treated with 50µM of analogue 12 followed

by 48 hrs of incubation were found to contain numerous, long

projections on the cell surface and displayed distinct junctional

complexes between overlapping cells (figure 2.10C and 2.10D). The

cytoplasm of these cells contained abundant glycogen and profiles

of rough endoplasmic reticulum (RER).

DU-145 cells treated with 100µM of analogue 12 followed by 48 hrs of

incubation demonstrated short surface projections and a loose

association of overlapping cells, lacking junctional regions. These cells


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were typified by bizarre shaped nuclei with complex nucleoli; abundant

mitochondria, numerous cytoplasmic dense bodies, as well as profiles of

rough endoplasmic reticulum (figure 2.10E and 2.10F). The SEM and

TEM analysis clearly demonstrate distinctive morphological

characteristics associated with androgen un-responsive cells DU-145 at

the desired concentration used.

Figure 2.10: Transmission electron microscopy pictures of DU-145 prostate cancer cell line treated with Analogue 12 (FARN) at 50µM and 100µM concentrations for 48 hrs.

2.4 Discussion

DU-145 cells showed evidence of apoptosis by MTT assay employed to

assess the cytotoxicity/antiproliferative potential of the total synthesized

analogues. Our results clearly indicate that the dibromotyrosine

analogues 1-13 induced metabolic activation primarily due to altered

mitochondrial activity. It is well known and reported that MTT assay is


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related to mitochondrial activity and was found out to be the most

sensitive among the tests employed to determine antiproliferative activity.

Such differences in sensitivity due to organelle specificity of chemicals

have already been reported for different toxicants, even in the same cell

system [Fotakis and Timbrell, 2006]. In general, the MTT assay is used in

studies conducted with organophosphates for cytotoxicity assessment in

primary cultures of mouse brain cerebellar cells [Giordano et al., 2007],

cultured human peripheral blood lymphocytes [Das et al., 2007],

daphnia, fish, and rodents [Benjamin et al.,2006; Wang et al., 2006]. It

was further observed that all the ether analogues [Analogue 12-FARN;

Analogue 10-BnZ; Analogue 13-MPP and Analogue 08- EAC] showed lower

antiproliferative values than any of the ester analogues [Analogue 01–

AAC; Analogue 05-ABNz; Analogue 04-AOME; Analogue 03-ISOB;

Analogue 09-EBNz; Analogue 02-EOME and Analogue 11-ISON]. To

better understand the structure-activity relationship, a study was further

performed on the basis of pharmacophoric and structural features of the

most active analogues and it was found out that chain extension and un-

saturation, as in analogue 12, are associated with a better activity profile

compared to those with shorter saturated chains, as in analogue 13

[Sallam et al., 2010]. Electron donating substituents and longer chain

linking the aromatic ring to the ether oxygen, as in analogue 13, afforded

a better anti-proliferative activity profile versus the simple unsubstituted

benzyl ether analogue 10. Studies have shown that treatment given by

anticancer compounds at low concentration to cancer cells either

induces apoptosis distinguished by cell surface blebbing whereas, at

higher concentrations, caused a second mode of cell death, oncosis,

distinguished by cell surface blistering [Ding et al.,2002; Weerasinghe et

al.,2001(a); Weerasinghe et al.,2001(b); Weerasinghe et al.,2001(c)]. In

order to reconfirm the efficacy of analogue 12 to be the best from the

available rest, we repeated the MTT assay and the results obtained were


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in agreement with the above finding and we came to the conclusion that

analogue 12 has the best antiproliferative activity among all the analogues

used.

ROS generation in several anti-cancer therapies has been reported to

contribute to their anti-cancer activities. Manipulation of ROS generation

in cancer cells has also been reported to be a potential therapeutic

strategy to enhance the cytotoxicity of drugs [Wang and Yi, 2008;

Stadtman and Berlett, 1998]. It was further observed that all the ether

analogues [Analogue 12-FARN; Analogue 10-BnZ ; Analogue 13-MPP and

Analogue 08-EAC] induces the generation of ROS (Figure 2.8) as

compared to the ester analogues [Analogue 01 –AAC; Analogue 05-ABNz;

Analogue 04-AOME; Analogue 03-ISOB; Analogue 09-EBNz ; Analogue

02-EOME and Analogue 11-ISON] which were found out to be neutral in

terms of their effects (figure 2.8). The above mentioned finding was not

found to be theoretically correct as the ethers should be more

metabolically stable in comparison to the esters which can be easily

hydrolyzed, affording the mother phenol which can easily provides ROS

species. The increased oxidative stress has been shown to cause DNA

damage, followed by cell cycle arrest in cancer cells [Wang and Yi, 2008;

Passos et al., 2007; D'Autreaux and Toledano, 2007; Trachootham et al.,

2006 ; Renschler, 2004 ; Stadtman and Berlett, 1998 ] At this stage, we

can say that ether analogues specially analogue 12 may be inducing the

demise of prostate cancer cells by the process of ROS generation which

leads to the loss of membrane permeability thereby resulting in the

release of cytochrome c in the cytosol triggering a cascade of events

mainly involving activation of pro-caspases and subsequently the cells

programming their death.

DNA damage in cells has an important implication on human health.

Normally, DNA is capable of repairing itself efficiently, through a

homeostatic mechanism. Cells maintain a delicate balance between

spontaneous and induced DNA damage. DNA damage accumulates if


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such a balance is altered. Most cells have considerable ability to repair

DNA single-strand breaks. However, DNA double-strand breaks, if not

properly repaired, may lead to cell death (apoptosis) [Lai, 1998].

Different molecular events occur in different types of DNA repair. Among

these are DNA repair mechanism, which can occur after base damage

induced by a genotoxic agent, and DNA single strand break and

double strand break rejoining. DNA repair mechanism is a slower

process, taking as much as 20 hr or longer depending on the amount of

initial damage within the exposed cells. DNA single-strand break

rejoining is very rapid, being close to complete within 2 hr. DNA

rejoining may also take longer [Foray et al.,1996] and not occurs

always, leading to cell death or causing mutation [Kesari et al.,2010].

Analogue 12 was found to induce DNA damage as evident from the results

of comet assay or single cell gel electrophoresis. G1-phase arrest of cell

cycle progression provides an opportunity for cells to either undergo

repair mechanisms or follow the apoptotic pathway. In the case of

advanced prostate cancer, cancer cells become resistant to apoptosis and

do not respond to the cytotoxic effects of most of the available

chemotherapeutic agents [Pilat et al., 1998-99]. Therefore, identification

of agents that can induce apoptosis in hormone refractory prostate

cancer cells became our high priority. Also the results obtained from

comet assay wherein as per Lai (1998) if the DNA break caused by the

analogue 12 is not repaired then it may lead to apoptosis prompted us to

move forward for the evaluating the ability of analogue 12 to induce

programmed cell death. Apoptosis plays a crucial role in eliminating the

mutated neoplastic and hyperproliferating neoplastic cells from the

system and therefore is considered as a protective mechanism against

cancer progression [Hickman, 1992]. Acquired resistance toward

apoptosis is a hallmark of most and perhaps all types of cancer. As far as

the results obtained for most promising analogue 12, it seems out to be a

potent chemotherapeutic agent for prostate cancer inhibition. Apoptosis


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is tightly regulated by anti-apoptotic and pro-apoptotic effector

molecules, including proteins of the Bcl-2 family, and can be mediated

by several different pathways. As the activity of the regulatory molecules

can be lost in cancer cells or be affected by other chemotherapeutic drugs,

it is important to elucidate the mechanisms by which anti-apoptotic drugs

exert their effects, especially in androgen-unresponsive DU-145 cells.

These facts prompted us to investigate the contribution of Bcl-2 family

proteins to the analogue 12 induced apoptosis of prostate cancer cells

and, in particular, androgen-unresponsive DU-145 cells. The proteins of

the Bcl-2 family either promote cell survival (e.g., Bcl-2 and Bcl-xL) or

induce programmed cell death. The ratio of Bax/Bcl-2 is critical for the

induction of apoptosis and this ratio determines whether cells will

undergo apoptosis [Tang and Porter, 1997; Reed, 1995]. An increase in

the ratio of Bax/Bcl-2 stimulated the release of cytochrome c from

mitochondria into the cytosol. The cytosolic cytochrome c then binds to

Apaf-1, leading to the activation of caspase-3 and poly (ADP-ribose)

polymerase [Yang et al., 1997; Kluck et al., 1997]. We found out that

treatment of DU-145 cells with analogue 12 resulted in an increase in

the expression of Bax protein and a decrease in the expression of Bcl-

2 and Bcl-xL (figure 2.7) which subsequently uplifted the ratio of

Bax/Bcl-2 (table 2.2). This may be responsible for the concomitant

execution phase of apoptosis that we observed, which included

disruption of mitochondrial membrane potential and increased release

of cytochrome c from mitochondria to cytosol (figure 2.7). As the level

of cytochrome c is increased in the cytosol, it interacted with Apaf-1

and ATP forms a complex with procaspase-9 (apoptosome), leading to

activation of procaspase-9 and caspase-3 [Thornberry and

Lazebnik,1998]. Activated caspase-3 is the key executioner of apoptosis

and cleaved caspase-3 leads to cleavage and inactivation of key

cellular proteins, such as poly(ADP-ribose) polymerase [Thornberry

and Lazebnik,1998;Wolf and Green,1999]. We found out that analogue


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12 treatment to DU-145 cells resulted in the activation of caspase-

9,caspase-3,C-fos and C-jun.The involvement of analogue 12 induced

increase in caspase-3 and its effect on apoptosis were further

confirmed by measuring its activity and induction of apoptosis by

flow cytometry [figure 2.5 and table 2.1].

Many studies have shown that certain exogenous stimuli may result in a

p53-dependent and p53-independent induction of p21/ WAF1, which

in turn may trigger a series of events, ultimately resulting in a cell

cycle arrest and/or apoptosis protein kinase complexes. Each complex

is composed minimally of cyclins (regulatory subunit) that bind to cdks

(catalytic subunit) to form active cyclin-cdk complexes. These complexes

are activated at various checkpoints after specific intervals during the

cell cycle and can also be regulated by several exogenous factors

[Kastan et al.,1992]. Our data is possibly showing an induction of p21 by

analogue 12 to be p53 dependent in DU-145 cells (with wild-type

p53) because analogue 12 treatment to these prostate cancer cells was

found to result in an increase in protein levels of p53 (figure 2.7).

We also identified ROS generation as a major mediator of p53 activation

and apoptosis.

DU-145 cells as accessed by SEM, extend numerous, long filopodial

processes in culture that contact adjacent cells. The cells tend to

aggregate with sheet-like and filopodial processes in contact. These

contacting regions are the sites of formation of desmosomal junctions. By

TEM, the cytoplasm of the DU-145 cells exhibited large stores of

cytoplasmic glycogen, and abundant rough endoplasmic reticulum,

features frequently observed in the well-differentiated tumor cells

[Feuchter et al., 1980]. In the current study, DU-145 cells exhibited

bizarre nuclei, numerous mitochondria, lipid inclusions, and cytoplasmic

dense bodies.


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2.5 Conclusions

We have presented the first definitive evidence to suggest that

among all the synthetic analogues used in this study, analogue 12

(farnesyl ether) is effective in preventing/inhibiting the growth of

androgen insensitive prostate carcinoma DU-145 cells by inducing

apoptosis through generation of ROS and DNA double strand break as

evident from comet assay and loss of mitochondrial activity as evaluated

by MTT assay resulting in the release of cytochrome c in the cytosol from

the mitochondria subsequently leading to the activation of cascade of

events and expression of apoptotic related proteins. Our results hold

significance because they suggest that the most potent, analogue 12,

limits the growth of prostate cancer lesions by p53 mediated pathways

which are unresponsive to conventional therapies. Furthermore, this is

the first study to document the ability of a synthetic analogue derived

from marine parent compounds aeroplysinin-1 and

dibromoverongiaquinol which in turn inhibits tumor cell proliferation

through induction of apoptosis and morphological changes as also

evident from the SEM and TEM studies. We speculate that analogue 12

influences prostate carcinoma cell viability by lowering the expression of

anti-apoptotic Bcl-2 and elevating the expression of p53, bax, caspase 9,

caspase 3, c-fos, c-jun, phosphorylated ERK, JNK and p38 protein levels.

Based on the observations that this dibromotyrosine analogue has

significant anticancer activity in cell culture, it deserves a hit status and

further evaluation of its potential preventative or therapeutic utility in

vivo.

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