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CENTRE FOR BIOTECHNOLOGY

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1 CENTRE FOR BIOTECHNOLOGY
ANNA UNIVERSITY CENTRE FOR BIOTECHNOLOGY Screening of biological properties of Medicinal plants for anticancer Activity using In vitro techniques - An approach towards Anticancer Drug Development. Senthil.V.*, Jis.C.Joy, Keerthivasan.A, Giridharan.P and Balakrishnan.A Centre for Biotechnology, Anna University, Chennai , India.

2 INTRODUCTION Medicinal plants are the most exclusive source of life saving drugs for the majority of the world’s population. Medicinal herbs have been widely used for treatment of diseases in traditional way for several generations. An interaction between traditional medicine and modern biotechnological tools is to be established towards New Drug development. The interface between cell biology, in vitro assays and structural chemistry will be the best way forward to obtain valuable leads.

3 Structure Elucidation
OVERVIEW Plant powder Non polar B I O A S Y Hexane S E E X Q T U R E A N C T T I I A O L N Active Extract DCM Chromatography Ethyl acetate Methanol Active Fraction Water Chromatography Polar Pure fraction Structure Elucidation

4 OBJECTIVE Traditional medicine
Medicinal Plants Lead molecule Bioassays Confirmation of apoptosis Confirmation of mechanism using analysis of apoptotic markers p53 Tumor suppressor Bcl-2 Anti-apoptotic Bax Pro-apoptotic Cytochrome c Pro-apoptotic Caspase 8 Apoptosis initiator Caspase 3 Apoptosis executioner Preliminary - Thymidine Incorporation assay DNA Fragmentation

5 MAJOR APOPTOTIC PATHWAYS Death Receptor Pathway
FasL TNF Mitochondrial Pathway oxidants ceramide others DNA damage D D D D Bcl-2 D Procaspase 8 dATP Apaf -1 BID Procaspase 9 Cytochrome c Caspase 8 Procaspase 3 dATP Apaf -1 Caspase 9 Cellular targets Caspase 3 apoptosome Apoptosis

6 Andrographis paniculata
Structure of Andrographis paniculata pure compound 3,19- Dihydroxy-11-oxo-8(17),13-labdadien-15, 16-olide Ref: Balmain.A , et al, J.Chem.Soc, Perkin Trans 1,1973,1247

7 THYMIDINE INCORPORATION ASSAY
Thymidine incorporation studies showed a significant anti-proliferative activity of the Crude and Pure compounds isolated from Andrographis paniculata at a dose concentration of Crude 25µg/ml and Pure 12.5 µg/ml at a time point of 24 and 48 hours

8 TRANSCRIPTIONAL EXPRESSION PROFILE
388bp bcl 2 p53 514 bp 364 bp BAX RT-PCR studies reveal the activation of p53 followed by the activation of bax and downregulation of bcl-2 in HeLa cells treated with Crude extract and Pure compound of the Andrographis paniculata at a time point of 12 hours. 100bp ladder Control Positive control A.paniculata Crude A.paniculata Pure Negative control 597 bp GAPDH

9 TRANSLATIONAL EXPRESSION PROFILE
Actin Actin 24 kDa Bcl-2 14 kDa CYT C CAS 3 32 kDa 28 kDa 17 kDa ACTIN 42 kDa The western blot analysis confirmed the down regulation of the anti-apoptotic protein Bcl-2 leading to Cytochrome c release and activation of Caspase 3. Control Positive control A. paniculata Crude A. paniculata Pure Actin control

10 DNA FRAGMENTATION ASSAY
Lane 1: Lane 2: Lane 3: Lane 4: Lane 5: 100bp ladder Control Positive control A. paniculata Crude extract A. paniculata Pure extract Apoptosis is confirmed by DNA fragmentation, observed in cells treated with crude extract and pure compound of Andrographis paniculata

11 RESULTS AND CONCLUSION
Apoptosis or programmed cell death is a genetically regulated process occurring naturally in a response to a variety of signals. We found that the lead molecule obtained from Andrographis paniculata leaf, was able to induce apoptosis in human HeLa cells. In this work the extraction, purification and elucidation of active molecular lead based on the bioactivity directed screening is described. Induction of apoptosis was confirmed by assessing cellular markers involved in apoptotic signals. This work is an example of a natural product with interesting anti- proliferative activity, in which the basic skeleton can be further, used as a template for the production of New Chemical Entity.

12 MECHANISM OF ACTION Activated p53 Suppressed Bcl-2 expression
Up-regulation of pro-apoptotic protein Bax Cytochrome c release from mitochondria Activation of Caspase-3 DNA fragmentation APOPTOSIS

13 Caspase 9 Caspase 3 Cyt C Cyt C Bcl2 Bax p 53 p 53 Apaf 1 APOPTOSIS
(DNA Fragmentation) Bcl2 Bax p 53 p 53

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