Several studies have indicated the presence of cancer stem cells (CSC) in the proliferating cancer cell population. CSC,s although present as a minority of total cancer population, have immense self renewal capability and are thought to be the major source of tumor cell renewal. Presence of CSCs is a major obstacle for successful cancer chemotherapy because these cells regenerate new pool of rapidly proliferating cells and are often escaped from the conventional chemotherapeutic agents. Thus targeting CSCs is thought to be a powerful strategy for complete eradication of cancer. While CSCs constitute a mere 1% of total tumor, methods have been developed to enrich them ex vivo in culture. I am investigating the role of ginger extracted compound, 6- shogaol in its ability to target cancer stem cells in breast cancer cells and aim to determine the underlying mechanism in its anticancer activity. The project has been funded by DST, India under the Women Scientist A (WOS-A) scheme .

DAT1 [4-amino-5-benzoyl-2(4-methoxyphenylamino) thiazole] was selected in our laboratory as a cytotoxic agent in a preliminary screen based on the cytotoxic activities of a series of synthetic diaminoaroylthiazole analogues of dendrodoine. It binds to the colchicine binding site of tubulin, causing mitotic arrest and disrupting the spindle morphology. It has an average cytotoxicity of 300 nM towards a number of cell lines. The apoptosis inducing activity of DAT1 and the apoptosis pathways activated have been studied. Both the intrinsic and extrinsic pathways are activated following DAT1 treatment. We will further study the effect of DAT1 on the proteins involved in the apoptotic pathway. In addition, DAT1 was found to have cytotoxicity in p53 deficient cancer cells. We would look further into this aspect and extend these studies in animal models.

Microtubule assembly is a long studied process because of its importance in mitotic division, and thus in cancer. Microtubules are complex polymers of tubulin, which itself is a heterodimer of ? and ? tubulin. The maintenance of equilibrium between tubulin monomers and polymers is essential for the proper functioning of the cell. Nucleation or the formation of oligomers of tubulin is the rate determining stage of microtubule assembly. Immense importance lies on the understanding of the nucleation stage and how it mediates the assembly process. Many antimitotic drugs influence the nucleation stage of microtubule assembly. Over the last few years, gamma tubulin (a 50kDa protein) has been identified as the nucleating agent in the cell, although the mechanism is not well known. The mechanism of action of gamma tubulin to nucleate microtubules and the controlling factors in the cell are very much important to know the process of mitosis. Study of the nucleation stage by the gamma tubulin complex as well as other nucleating agents would help us to understand the mechanism of microtubule formation through nucleation in normal cells as well as in abnormal situations like cancer, which in turn would throw light on how these agents control microtubule assembly. This would help in the improvement of their use as cancer chemotherapeutic agents

Microtubule nucleation in eukaryotic cells occurs from the centrosome. The newer member of the tubulin superfamily, gamma-tubulin, is known to mediate microtubule nucleation from the centrosome with aid of some other proteins. The major amount of gamma tubulin complex is believed to be located in the centrosome before the onset of mitotic division. However, a considerable amount is found in the cytoplasm in the form of a complex, the function of which is not known. Cytoplasmic gamma tubulin ring complex was purified from brain in our laboratory and it was found to nucleate microtubules in vitro. Non-erythroid spectrin or fodrin, a 235 kDa protein was found to be associated with the brain gamma-tubulin ring complex. This novel association is found only in brain gamma-tubulin complex and has been reported for the first time by our lab. I am now working on the functional and structural significance of non-erythroid spectrin’s association in the brain gamma-tubulin ring complex.

The p53 gene and its protein product have become the center of intensive study ever since it has been discovered that slightly more than 50% of human cancers contains mutation in this gene. p53 is a tumor suppressor gene and most of its tumor suppressor activity is due to its ability to function as a transcription factor. p53 can integrate various cellular stress signals from the cell and arrest the cell division or commit it to apoptosis. Since p53 gene is mutated in most cancers any drug which is active in p53 mutated cancers will be of interest since it would be more tumor specific. There are many contradictory observations on the action of anti mitotic agents in p53 null cancer cell lines. Some anti mitotic agents are found to be more active in p53 mutated cell lines while some are not. Initial studies shows that DAT1, a synthetic diaminothiazole identified in our lab, is active in p53 null cell lines but the IC50 is three times more showing that it has a p53 dependent mode of action. This scenario needs to be checked both in vitro as well as in in vivo tumor models. I am also checking the status of other proteins in the p53 pathway to elucidate the mechanism of action of DAT1.This will throw light on the mechanism of killing of tumor cells by other anti mitotic agents also. In vitro studies of DAT1 revealed that it is a potent anticancer agent and it activates both extrinsic as well as intrinsic pathway of apoptosis. In vivo studies are needed to confirm this.

Microtubules are dynamic polymers that play vital roles in a large number of cellular functions. Their pivotal role in mitosis makes them a target for the development of antimitotic drugs. Microtubules serves as a scaffold for many signalling mechanisms that regulate apoptosis.

Antimitotic drugs disrupt the normal function of the mitotic spindle and cause spindle checkpoint mediated mitotic arrest. I am interested to study the involvement of spindle check point proteins in anti mitotic drug mediated cell death. A better understanding of the processes coupling to spindle checkpoint mediated mitotic arrest to the onset of the cell death will reveal sites of potential intervention in cancer chemotherapy.

Chemotherapy is being widely used in the treatment of cancer patients, yet it is limited by the development of resistance in cancer cells to multiple chemotherapeutic agents. This is due to various changes in cancer cells such as altered membrane transport involving P-glycoprotein over expression, enhanced expression of heat shock proteins, altered target, decreased drug activation, increased drug degradation and failure to apoptosis. Antimitotic drugs occupy an important place in cancer chemotherapy as some of them are quite successful in the treatment of different cancers and many are in different stages of clinical trials. However, they also face the problem of being resistant after continued use.
In vitro drug resistant models serve as useful tools to understand the mechanisms of drug resistance and also the mode of action of anti-cancer agents. In our laboratory we have developed a paclitaxel resistant colon cancer sub line. My work is mainly focused in characterizing this cell line and understanding the mechanism of action of various antimitotic drugs on such cell lines.