A journey for future anticancer therapy: Hope for disease free survival

Prof. Chitra MandalCancer biology and Inflammatory disorder division

CSIR-Indian Institute of Chemical Biology

Variable Energy Cyclotron Centre, Kolkata

Women's day celebration

5th May, 201545 mins

U. N. Brahmachari developed2nd chemotherapeutic agent(Urea stibamine) in the Worldagainst infectious disease andmade the therapy affordable.It is still in use for thetreatment of Kala-Azar since1922.

Upendranath Brahmachari(1873-1946)

Goal…………

Deliverable low cost affordable healthcare

India’s Journey to Discover New Drugs………..

Ancient Indian Medicine

Ancient India saw great advancements in Medical science depending on natural remedies

Acharya Charak has been crowned as the Father of Medicine.

Acharya CharakaFather of Medicine

In his renowned work (600 BC):"Charak Samhita" describes medicinal qualities and functions of 100,000 herbal plants, that was considered as an encyclopedia of Ayurveda

Global Herbal RemediesCurrently 80% of the world population depends on plant-derived medicine for the first line of primary health care because it has little side effects.

About 25% of pharmaceutical prescriptions in the US contain at least one plant-derived ingredient

In the last century, roughly 121 pharmaceutical products were formulated based on the traditional knowledge.

‘Botanical garden of world’-INDIADry herbs

In the region of Eastern Himalayas, Western Ghats and Andaman & Nicobar Island, ~ 45,000 plant species are found.

Officially documented plants with medicinal potentialare about 3000

Traditional practitioners use more than 6000. Using the 6000 plants, there are estimated to be around 25,000

effective plant-based Formulations used in folk medicine.

CSIR produces an Encyclopedia of India's Raw Material Resources: a 20-volume authoritative reference material

This invaluable information- source for economically important plants, animals, minerals and their applications published in English, French, German, Japanese and Spanish (collaboration with AYUSH)

Today we are facing a new epidemic of life style diseases...of which cancer is a major one.

Cancer scenario

Global Cancer Statistics, 2002Cancer J Clin 2005;55;74-108

Healthy cell

Injured cell

Apoptosis

Apoptoticmalfunction

Uncontrolled cell growth

Tum

our d

evel

opm

ent

Benign tumour

Normal vs Cancer cell division and tumour development

Sustaining proliferativesignaling

Deregulating cellularenergetics

Genome instabilityand mutation

Tumor-promotinginflamation

Avoiding immune destruction

Evading growth supressors

Enabling replicativeimmortality

Activating invasionAnd metastasis

Inducing angiogenesis

Resisting cell death

The Hallmarks of Cancer

Cellular signaling and Cancer

MAPK Signaling

Adhesion

PI3K/Akt Signaling

DNA Damage

TGF-β/Smad Signaling

Wnt signalingNotch

Jak/Stat Pathway

Apoptosis Signaling

Cytoskeletal signaling

Hedgehog

Lipid Signaling

Cell Cycle Checkpoint

Dual function of signaling (Cell survival/apoptosis)

Cell death signaling Cell survival signaling Stem cell signaling

Color Code

NF-κB Signaling

Signaling pathways involved in chemotherapyinduced cell death

Drug Target Therapeutic use

Imatinib Bcr-Abl CML,GI tract tumour

DasatinibTyrosine kinase

inhibitor

CMLNilotinib

Sunitinib Renal cellcarcinoma

Bortezomib Multiple myeloma, mantle cell lymphoma

Fulvestrant Estrogen receptor Breast cancerTamoxifen

Gemcitabine Pancreatic cancerTrastuzumab HER-2 Gastro-esophageal adenocarcinom, breast cancer

Lapatinib HER-2 Metastatic breast tumour

Gefitinib EGFR NSCLC

Erlotinib EGFR NSCLC, Pancreatic cancer

Cetuximab EGFR HNSCC, Colorectal cancer

Targeted therapeutics in cancer

Node properties

Colour- Proteomic expression status

Up regulatedDown regulatedNo changeNot identified

Size- Betweenness

Edge properties

Colour- Present in z≥1 orz≥3 network

Width- Co-expression statusof the gene pairs

Present in z≥1 &z≥3Present only in z≥1

GBM specific network

Differential function of ROS in the cell and the position of cancer cell in the ROS level diagram

Adaptation to stressHomeostasis Damaged induced

Redox adaptation in cancer development and drug resistance

Dysfunction of mitochondrial respiratory chain

Effects of ROS in intracellular environment

Enhancement

Inhibition

Our goal…………….

Identification of novel redox based chemotherapeutic agents

Threshold level

Normal Cell Cancer Cell

ROS

leve

lsCell death

Exogenous ROS stress

High ROS production

Redox adaptation

Redox

BalancedIntracellular ROS level

Exogenous ROS stress

Could the natural productscome to our rescue?

Immunomodulation

Plant based molecules

Immunotherapy

Chemoprevention

Combinatorial chemotherapy

Chemotherapy

Surh et al, Nature Reviews Cancer 2003;3:768-780

Dietary molecules used for chemoprevention

CM-5

Indian spices

Dehydroxy CM-5

USA patent

Leaves of (Murraya koenigii)Lead molecule: Carbazole Alkaloid (CM-5)

Patent ref: Mandal et. al. USA, IndiaSamanta et.al. (2014) Journal Medicinal Chemistry

Suman

R1 R2 CompoundsOH H CM-5

OC(O)Me H Ac-CM-5O-Me H Me-CM-5

H H Dehydroxy-CM-5H Me Me-Dehydroxy-CM-5

CM-5 & its derivatives

A549HCT116Panc1K562T98G

CM-5 Me-CM-5 Ac-CM-5 Dehydroxy-CM-5

Me-Dehydroxy-CM-5

0

50

100

150

20048 hrs

IC50

(µM

)

CM-5 showed maximum cytotoxic activity on cancer cells

Samanta et al (2013) J Med Chem

(Glioma)

(lung)(colon)

7-OH is essential for enhanced cytotoxicity of CM-5 on cancer cells

DNA fragmentation by TUNEL assay

Samanta et al (2013) J Med Chem

Mitochondrial transmembrane

depolarization by JC-1 assay

(Glioma)

Blockage of 7-OH and 9-NH by methylationreduced cytotoxicity of CM-5

Samanta et al (2013) J Med Chem

Comet assay

7-OH and 9-NH groups are important for DNA minor grove binding through H-bonding

Devawati

CM-5 induces apoptosis in different cancer cells

i) Active against leukemias (Myeloid andLymphocytic), glioblastoma, pancreaticcarcinoma, melanoma, lung, colon & cervicalcancers

ii) Act as an pro-oxidant agent

iii) Anti-diabetic agent & active against prostate cancer

CM-5 induces ROS HOW………..

Superoxide O2•‾

Hydroxyl radical •OH

Organic radical R•

Peroxyl radical ROO•

Alkoxyl radical RO•

Thiyl radical RS•

Sulphonyl radical ROS•

Thiyl peroxylradical

RSOO•

complex I (NADH dehydrogenase),complex II (succinatedehydrogenase)complex III (ubiquinol-cytochrome c reductase), complex IV (cytochrome c oxidase)complex V (ATP synthase).

Generation of ROS by Mitochondrial Electron Transport chain (ETC)

Mito+αKG

AD

P

Rot

enon

e

Succ

inat

e

Mal

onat

e

Du

roqu

inon

e

CM

-5TM

PD

Azi

de

5.2 3.29.6

2.85.8 2.940.5

8.4

0Time (min)

1 2 3 4 5 6 7 8 9 10

Fold

of

rate

kin

etic

s of

com

plex

III

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

ControlCM-5 (25 µM)CM-5(50 µM)CM-5(100 µM)Antimycin A (10 µg/ml)

Bhattacharya et al (2014) Am J Cancer Res

α-keto glutarate ( Complex I substrate)

Succinate (complex-II substrate)

Malonate (complex-II inhibitor)

10 um10 um10 um10 um

10 um10 um10 um10 um

10 um10 um10 um10 um

10 um10 um 10 um 10 um

DAPI H2DCFDA Mito-tracker Merge

U87

MG

U87

MGv

III

Mahanine

0 µM

15 µM

0 µM

15 µM

Fluorescence imaging of mahanine-mediated intracellular total ROS and mitochondrial ROS generation

10 um10 um10 um10 um

10 um10 um10 um10 um

10 um10 um10 um10 um

10 um10 um10 um10 um

DAPI MitiSOX Mito-tracker MergeMahanine

U87

MG

U87

MG

ρ0

0 µM

15 µM

0 µM

15 µM

Fluorescence imaging of mahanine mediated mitochondrial ROS in functional and non-functional ETC

CM-5 is mitochondrial ETC complex-III

inhibitor by which it can produce

oxidative stress

CM-5 induces apoptosis in leukemias

Bhattacharya et.al. (2010) Biochemical Pharmacology

MOLT-324 hr

10 μM mahanine 15 μM mahanine 20 μM mahanine

Annexin V

K56224 hr

0 μM mahanine

0.34

0.000.00 0.18 1.88

12.57 17.04

3.050.48 1.94 21.28

27.54

0.00 0.02

0.26 20.62

2.070.30

25.12

3.480.62 2.9656.76

15.31

CM-5 induced apoptosis of leukemic cells

Annexin V-PI staining assay…………

Cytochrome c

Caspase 9 activation

Caspase 3, 7 activation and PARP cleavage

DNA fragmentation

Apoptosis

Cytochrome c

Caspase 9 activation

Caspase 3, 7, 8 activation and PARP cleavage

DNA fragmentation

Apoptosis

Fas

FasL

FADDPro caspase 8

Active caspase 8

Bid

tBid

Bcl2 Bax Bcl2 Bax

No caspase 8 activation

No Bid cleavage

(Fas, FasL WT cell) MOLT-3 K562 (Fas, FasL null cell)

CM-5

Bhattacharya et al (2010) Biochem Pharmacol

Pancreatic cancer

Stage Possible treatments Stage of diagnosis

5 yr survival

rate

0 Not needed

7% 20%I

SurgeryChemotherapy

Radiation

IISurgery

Chemotherapy (Gemzar)Radiation

Clinical trail therapy 26% 8.2%

III Chemotherapy (Gemzar)Pain treatment

IV Chemotherapy (Gemzar)Pain treatment

Clinical trail therapy

52% 1.8%

Recurrent Cancer

,, N/A <1%

Gemcitabine

According to global figures,

2,32,000 people diagnosed with pancreatic cancer in 2002

2,27,000 died by 2010

Neuroendocrine tumourSteve Jobes had extremely rare form.

5% of people diagnosed with PaCa

Some Facts and Features

Adenocarcinoma Ralph M. Steinman had the type that is

usually fatal within a year after diagnosis

Sedentary lifestyle Smoking High alcohol intakePeople suffering fromdiabetes and chronicpancreatic inflammation

Causes:

CM5 shows anti-proliferative activity in array of pancreatic cancer cell lines

CELL LINES

Grade, Site

MIAPaCa-2

IV, Pancrea

s

AsPC-1 IV, Ascites

Panc10.05

II, Pancrea

s

Panc-1 II, Pancrea

s

IC50 (µM) 16.5 17.2 18.5 13.9

48 hrs

CM-5

CM-5

Sarkar et al (2013) Int J Cancer

oligomerization

CM-5-induced ROS is responsible for Hsp90 client proteins degradation in cancer cells

Sarkar et al (2013) International Journal of Cancer

Sayantani

SPR studies show CM-5 also binds

with recombinant Hsp90

CM-5 does not affect the ATP

binding of Hsp90

Hsp90α Hsp90β

90-

Resp

onse

uni

t

Time (S)

Buffer

500 nm

1 µM 5 µM

10 µM

0 100 200 300

0

-100

300

600

900

CM-5

Molecular modelling reveals interaction of CM-5 with recombinant Hsp90

Hydrogen bondingResidues for hydrophobic interaction

CM5

ATP in ATP binding pocket

ATP binding pocket

CM5 binds on outer side of ATP binding site

Sarkar et al (2013) Int J Cancer

CM-5

CM-5 inhibits pancreatic adenocarcinoma cell haptotaxis

in a dose dependent manner

Krasmu

Kraswt

CM-5

Down regulation of Hsp90 client proteins

Apoptosis of pancreatic cancer cells

NAC

MG 132

Dysfuntion of Hsp90

Restoration of client proteins

Activation of caspases

Accumulation of unfolded protein

Proteasome

Probableinhibition of in vitro migration

Down regulationof MMP9

ROS accumulation

Disruption of Hsp90-Cdc37

complex

Directly binds to Hsp90

Restoration of client proteins

Inhibition of in vivo metastasis in Lung, Liver

and Kidney

direct targeting Hsp90

Cell Cycle progression of eukaryotic cell

Differentiation

Normal cells arehighly differentiatedless proliferated

Cancer cells areless differentiatedhighly proliferated

Due to this,accumulation ofimmature andrapidly proliferatingcells occur in cancermalignancy

2n

4n

2n

4n

2n

G0/G1 phase cell cycle arrest in Glioblastoma multiforme

(GBM)

Arup

Kaushik

Bhattacharya et al (2014) Am J Cancer Res

LN229

U87MG

DNA Area

CM-5 (μM)0 15

G1 G1

G1G1

G1=53.48% G1=68.29%

G1=63.07% G1=84.13%

S

S S

S G2/M

G2/MG2/M

G2/M

24 hr

S=20.79%G2/M=25.13%

S=15.45%G2/M=18.28%

S=7.45%G2/M=8.72%

S=17.54%G2/M=18.34%

CM-5 induced G0/G1 phase cell cycle arrest in cells of Glioblastoma multiforme (GBM)

Bhattacharya et al (2014) Am J Cancer Res

Hypoxic stress-induced higher ROS production enhanced the cell cycle inhibitory activity of CM-5

H2D

CFD

A M

FI (a

. u.)

150

350 U87MG

1% O2

8% O2

350

650

1% O2

8% O2

LN229

10 15 200 10 15 200CM-5 (µM) CM-5 (µM)

1% O2

8% O2 * *

**

U87MG LN2290

15

% A

ccum

ulat

ion

of G

0/G

1 ce

lls in

m

ahan

ine

trea

tmen

t (w

.r.t

cntr

l)

24 hr

1 hr 1 hr

1% O2 Brain tissue hypoxia

8% O2 Brain tissue normoxia

Bhattacharya et al (2014) Am J Cancer Res

Cancer cell Anticancer drug treated cancer cell

Non-differentiated epithelial like brain tumor cell

Well differentiated astrocytes or glial like cells from the undifferentiated brain tumor cells

Bhattacharya et al (2014) Am J Cancer Res

CM-5

CI

CII

CIII

CIV

ATP

synthaseROS

ROS

DNA damage response

Chk1/Chk2

G0/G1 cell cycle arrest

•Inhibition of in vitro migration,•Inhibition of connective tube formation•Inhibition of poor differentiation •Inhibition of in vivo tumor formation

NAC

esiRNA

Untreated

100 mg/kg/day mahanineK562 Xenograft Tumor

MIAPaCa-2 Orthotopic Tumor

CM-5 reduced in vivo tumor

Bhattacharya et al (2010) Biochem Pharmacol

Sarkar et al (2013) Int J Cancer

U87MG Xenograft TumorBhattacharya et al (2014) MS communicated

Control Mahanine

Control Mahanine

B16F12 melanoma Xenograft Tumor

PTEN (Tumor Suppressor Protein ) activation

Ranjita Das

CM-5

5-Fluorouracil Paclitaxel

Cervical cancerColon cancer

Synergistic enhancement of cytotoxicity and 5-8 folds reduction of concentrations of

chemotherapeutics

In combination with known chemotherapeutics

Cisplatin

5-FU (µM)0

40

60

80

100

20

2 4 8 16Mahanine (µM) 3 6 12 24

HCT116, 48 h

Cel

l via

bilit

y (%

)

MahanineCombination

Control5-FU

Das et al (2013) Apoptosis

Das et al (2014) Cancer Letter

Colon cancer

CM-5

Inhibition of Complex III in mitochondrial ETC

Reactive Oxygen Species

Inhibitionof Hsp90

PTEN activation

G0/G1 phase cell cycle arrest

Adjunct therapy

Colon cancer

5-Fluorouracil

Synergistic enhancement of cytotoxicity and 4-5 folds reduction of concentrations of

chemotherapeutics

Identification of Functional Moiety

GliomaPancreatic

Cancer

Establishment of probable high efficacy therapeutics

CisplatinCervicalcancer

Bhattacharya et al (2014) AJCRDas et al Apoptosis 2014

Sarkar et al IJC 2013

Das et al Cancer Lett. 2014

Besides the development of such effective anti-cancer agents……we are far behind..to discover the ‘Magic bullet’..

Rays of hope……..Bhattacharya et.al. (2015) Am J cancerDas et al (2014) Cancer LetterDas et al (2013) ApoptosisSamanta et.al. (2013) Journal of Medicinal ChemistrySarkar et.al. (2013), International Journal of Cancer,Mondal et.al. (2012), Plos OneMondal et al, (2012) Anticancer Agents Medicinal ChemistryMondal et.al. (2012), Adv Expl Med and Biol, 749:295Mondal et.al. (2010), Molecular Cancer, 9:239Bhattacharya et.al. (2010), Biochem Pharmacol, 79:361Mandal et.al. (2008), Apoptosis, 13:1450

CSIR -IICB launched

A Herbal Extract for the Treatment of

Benign Prostate Hyperplasia in market in

2008

Traditional knowledge guiding drug discovery : AN integrative approach

Kaushik Suman Sayantani

Cell Signalling pathways to identify new drug targets

Devawati

RanjitaArup Chandan Susmita

SamarpanDr. ChhabinathMandal

Bis

waj

it

Kau

shik

Dev

awat

iSa

ptar

shi

Sum

anC

hand

an

Ran

jita

Aru

p

Sam

arpa

n

Rita

MY GROUPCancer biology

Bacterial sialobiology

Leishmanialsialobiology

Dr. Suman Kumar Samanta (Chemist)

Devawati Dutta (molecular modelling)

Dr. Manjusha Chakrobarty (nano particle)

Dr. Susmita Mondal (Leukemia)

Dr. Chandan Mandal (leukemia)

Dr. Sayantani Sarkar (pancreatic cancer)

Dr. Kaushik Bhattacharya (Brain cancer)

Arup Kumar Bag ( brain cancer)

Samarpan Maity (brain cancer)

Ranjita Das (colon and cervical)

Shalini Nath (bone cancer)

Susmita Mandal (junior) cancer Stem cell

Biswajit Khatua

Kaustubh Mukherjee

Saptarshi RoyArup Kumar Bag

Jayasree Karmakar

Asish Mullick, Technical Officer

Rita Maity

Dr. Chhabinath MandalNIPER, Kolkata

Dr. Bikas C PalNIPER, Kolkata

Dr. Bibhuti SahaSchool of Tropical Medicine

Kolkata

Dr. Sarmila ChandraPark Clinic, Kolkata

Dr. Uttara ChatterjeePark Clinic, Kolkata

Prof. Paul R CrockerUniversity of Dundee,UK

Dr. Peter WaldenHumboldt University, Germany

Kaustubh DattaUniversity of Nebraska Medical

Center, USA

My Collaborators

FUNDING: J.C. Bose Fellowship, DST, DBT, ICMR. WHO, CSIR