Novel cell based assays for large scale screening of plant derived compounds for anticancer property: Technology Development and preliminary screening

Plant based medicines are becoming an important part of cancer chemotherapy regimens and it forms the foundation of Indian traditional ayurvedic treatments. However, India’s biodiversity treasure and rich traditional ayurvedic medicines are not yet scientifically explored for development of novel bioactive anticancer drugs. Partly this happened because of inadequate assay systems to screen large number of potential candidate drugs for the most desired property. An ideal, sensitive, and simple cell based assay system is expected to provide an adequate screening system thereby one can evaluate large number of potential candidate drugs with short time for its anticancer property.

It is recognized that the most important target in chemotherapy using drugs is stopping of cell division and induction of apoptosis in cancer cells. This form of cell death often renders least damage to the normal cells compared to the cytotoxicity that damage the cancer cells and normal cells equally. A reliable tool to evaluate sensitive markers of apoptosis is imperative for high throughput screening of apoptosis inducing compounds.

During apoptosis, a cytosolic proapoptotic Bax protein undergoes conformational change and translocate to mitochondrial outer membrane forming ion channels through which certain proteins are released in to the cytosol like cytochrome c, Smac, AIF, Omi or HtrA2. In the cytosol, cytochrome c participates in the activation of certain cysteine proteases called caspases that ultimately dismantle the cell. In the death receptor mediated apoptosis, activated caspases cleave a proapoptotic protein Bid that translocates to mitochondria to amplify the release of cytochrome c.

So monitoring of the co ordinated movement of these proteins within the cells can be used as an ideal marker of apoptosis signaling and thereby anticancer property. Work is progressing to develop cells stably expressing these proteins along with fluorescent tag like GFP or Ds Red to be used for screening of antitumor agents.Certain cell lines were already developed and validation is progressing in our lab.

In the coming two years the developed cell lines will be extensively used for screening of apoptosis inducing compounds to be used against cancer. In the second phase more plant derived bioactive compounds will be screened using the assay system and potential lead compounds will be further evaluated for drug development.

(See Fig showing the translocation of cytosolic Bid to mitochondria during cell death induced by antitumor agents in MCF-7 Bid Ds Red cells developed)

Drug resistance in cancer

Cancer chemotherapy has gradually improved with the development of novel antitumor drugs and yielded promising results when applied to many haematologic malignancies, and some solid tumors. However the effectiveness has often been limited by inherent drug resistance in most solid tumors. The thrust area of our research is to understand the molecular mechanisms of chemoresistance in cancer and develop novel approaches for circumventing drug resistance.

Since the induction of apoptosis following chemotherapy is associated with triggering of the death receptor and mitochondrial apoptotic pathway, attenuation of proapoptotic genes or overexpression of antiapoptotic genes causes resistance to apoptosis. Research is centered on the concept that to increase the therapeutic effect of cancer chemotherapy, suppression of antiapoptotic proteins is an ideal approach to deal cancer chemoresistance.

Expression of heat shock proteins like hsp70, hsp27, and hsp90 are reported to have anti-apoptotic functions. They are specifically and abundantly expressed in metastatic tumors compared to the adjacent normal tissues. Recent studies from this laboratory substantiated the molecular mechanism of their anti-apoptotic function in response to anticancer drugs and identified the heat shock proteins as the best target to circumvent chemoresistance in cancer. The preliminary studies show that heat shock protein inhibitors like Geldanamycine, Novobiocine, Radicicol can render chemoresistant cancer cells susceptible to traditional anticancer drugs like Resveratrol and Quercetin. This concept will be further tested and validated in well-designed cellular systems of drug resistance developed in our laboratory, in combination with current therapeutic regimens as well as experimental anti tumor agents.