SCREENING AND BIOLOGICAL CHARACTERIZATION OF A NATURAL AND SYNTHETIC ANTICANCER MOLECULE

Abstract

The present thesis entitled "Screening And Biological Characterization Of A Natural And Synthetic Anticancer Molecule" deals with anticancer properties and the mechanism of action of a novel Schiff base copper complex and pterostilbene in breast cancer treatments. Breast cancer is the commonest malignancy in women. Since the past two decades, rates from breast cancer have increased at an alarming rate. Cancer is a silent disease which develops through years of undiagnosed symptoms. The use of hormone replacement therapy (HRT) for the cure and management of several diseases related to menopause and age has caused advert effect and aided the development of breast cancers (Campagnoli et al., 2009). Moreover, already available chemotherapeutic drugs which are commonly used have not given a cure to the disease and have a lot of side effects. Hence is the requirement for the search of new molecules with better efficacy and low toxicity. The objectives of present study are to initially screen both novel synthetic compounds and plant extracts to find an effective compound from each category then understanding each of their molecular mechanisms as to elucidate how they can be inhibitory to breast cancer cell proliferation. The first stage biochemical and microscopic analysis led to the study the molecular pathways mainly by reverse transcriptase polymerase chain reaction and western blot analysis. Interactions with major proteins are also confirmed by few transactivation studies. Not only the efficacy of the compounds is checked in in vitro cell based systems, but their effect was validated in the breast cancer tumor models developed with MNU (Thomson and Adlakha, 1991). At the beginning, Chapter 1 introduces briefly the present scenario of the chemotherapeutic drugs available to the patients. It also deals with the key factors present in cancer cells that need to be targeted. These cancer cell characteristics can serve as major points to guide the screening of novel molecule for their respective efficacy. Finally detailed goal to be attained in the study is specified here. Followed by this, Chapter 2 presents (i) a detailed review of general genetic abnormalities related to cancer and the recent molecular targets; (ii) the characteristic anomalies of breast cancer; (iii) a vivid description of the till date discoveries for designing of synthetic chemicals which guides the selection of the Schiff base copper complexes among others; and XIII (iv) recent research detailing benefits of phytochemicals and their characteristics to target multiple pathways in cancer therapy; and finally (v) the hypothetic idea behind the present thesis to manipulate the cancer cells towards death. Each of these hypotheses is explored in subsequent chapters of the thesis. In Chapter 3, the basic principles of anticancer screening assays adapted to preliminary shortlist molecules that bear cytotoxic properties are specified. Whether cytotoxicity is related to apoptotic death or any other form of cell death is then studied. Cancer cells are highly proliferating cells where the mitotic index is very high with chromosomal anomalies (Kops et al., 2005). Although a cell can die in a number of processes, the main form of cell death that is targeted in cancer cells is a sequestration of a sequence of events, rightly called as programmed cell death or apoptosis (Kerr et al., 1972). In this concern, this chapter contains the overall principles of all the experimental assays that were performed to identify a molecule to be anticancer compound. This is explained as stage I, the biochemical parameters like MTT screening, chromatin body formation, DNA fragmentation patterns, activation of caspases are studied initially to select a novel drug; and in stage II, the principles of the biological mechanisms are studied at transcriptional and translational expression levels of various genes to understand how a molecule acts intracellularly, mainly the assays which show the basic determination of the underlying pathways. Investigating the above features in Chapter 4 the anti-cancer properties of a series of copper centered synthetic molecules and crude extracts of medicinal plants are investigated. Copper is a biologically relevant metal due its association with various biomolecules related to essential physiological activities. Anticancer compounds having a copper as a metal center is hypothesized to be less toxic and more potent. The series, designated as a family of Schiff base copper complexes, is screened and the best compound [Cu(Pyimpy)Cl2] where Pyimpy is a tridentate ligand containing two pyridine and one imine nitrogen donor is selected. [Cu(Pyimpy)Cl2] represented here as CuPl, is found to be cytotoxic at the lowest concentration in the series. It is found to show DNA binding in vitro and caused apoptosis in MCF 7 cells as observed by acridine orange staining assay. In the next part similar cytotoxicity screening assays are performed to select the best of the plant extract tested. The methanolic extracts are only considered for the screening procedures. The basic idea for the screening was to check the effect of polyphenols fractions in these crude extracts. The results are also validated for apoptosis XIV characteristic by acridine orange staining. The phytochemical screening results led to the selection of the methanolic extract of the heartwood of Pterocarpus marsupium as most effective against MCF-7 breast cancer cell lines. Next, in Chapter 5, the biological characteristics of CuPl are further tested. The effect of the synthetic CuPl on DNA content of the MCF-7 is validated by comet assay and DNA fragmentation assay. Not only this, the study also focuses on whether the molecule activates caspases, which is a positive marker of apoptosis. Finally the copper Schiff base compound is tested on rat breast tumor models and the characteristic pathways which lead to the reduction in breast tumor volume is investigated. After the initial screening and selection of the methanolic extract of Pterocarpus marsupium in chapter 4, the present chapter deals with the purification and characterization of the lead compound in the extract for anticancer properties. Maurya et al., (1984) showed that the major content in the heartwood Pterocarpus sp. is mainly stilbene. The pterostilbene, a dimethyl ester derivative of resveratrol is thus isolated in Chapter 6. Thereafter, the isolated pure compound is tested for a similar efficacy in the breast cancer cell lines as was obtained with the extract. Finally the pathways leading to apoptosis are checked biochemically and then at the transcriptional and translational level. Finally the efficacy of the molecule is tested on the rat mammary tumor model. It is worth mentioning here that the involvement of reactive oxygen species is found as a basic mechanism to activate apoptosis by pterostilbene. This effect is somewhat different than the general trend of activation of phytochemicals. Hence the involvement of ROS as an effector agent in the action of pterostilbene is further taken as a matter of study in the following chapter. Because of the fact that mode of action of pterostilbene is found to be mediated by the induction of mitochondrial oxidative stress Chapter 7 initially observes the effect of pterostilbene in presence of ROS scavengers. The results show that the molecule not only apoptosis, low and sub-acute dose of pterostilbene ceases the mitotic and metastatic potential in MCF-7 cell lines although they were still live. This study shows that at low dose and long term treatment pterostilbene leads to a gradual accumulation of neutral lipids in MCF-7 cells and characterize them to differentiate into more of epithelial like morphology. Simultaneously, it shows that there is an induction of autophagic characteristics which are adapted by the breast cancer cells as an alternative form of cell death due to long term exposure to the stilbene XV molecule. Thus this study shows for the first time, that pterostilbene increases in the expression of adipogenic differentiation marker c/EBPa and intracellular accumulation of the oxysterols and induces the phenomenon of autophagy in breast cancer cells. Studies on tamoxifen and its metabolite supports the present findings very strongly (Medina et al., 2009; Payre et al., 2008). In the next section (Chapter 8), the effect of pterostilbene is studied on the progesterone receptor positive breast cancer cell lines. This study is in continuation to the observed fact in chapter 6 that pterostilbene is also effective in the progesterone receptor (PR)-positive cell lines. Progesterone receptor is expressed in breast cancers along with estrogen receptor (Hoskins et al., 2009). This study focuses on the inhibition of the PR mediated pathways by pterostilbene in T47D cells. Pterostilbene inhibits the PR transcription and expression. It also inhibits the progesterone up-regulated breast cancer markers Kallikrein 4 which known to involve in cancer cell metastasis. The results in this chapter also describes that pterostilbene inhibits the localization of PR to the nucleus as seen in the PR-GFP transfection studies and the immunoblot analysis. Finally the PR antagonism of pterostilbene is checked at the in vivo animal models. Finally, Chapter 9 summarizes the complete work in brief and the future prospects that is still to venture. The scientific findings dealt with in this thesis may be of use to the future researchers working in this area. And finally the list of bibliographies which was consulted in course of the present work is presented in Chapter 10.