BIOLOGICAL EVALUATION OF A NOVEL PTEROSTILBENE-DERIVATIVE AS POTENT ANTICANCER AGENT

Abstract

Cancer is defined as uncontrolled growth and spread of cells which can affect almost any part of the body. The growths often invade surrounding tissue and can also metastasize to distant sites. Many cancers can be prevented by avoiding exposure to common risk factors, such as tobacco smoke. In addition, a significant proportion of cancers can be cured, by surgery, radiotherapy or chemotherapy, especially if they are detected early. Over the years, the design of cancer chemotherapy has become increasingly sophisticated. Yet there is no cancer treatment that is 100% effective against disseminated cancer. At present cancer therapy interfering with a single biological molecule or pathway has been successfully utilized. Still the problem of drug resistance and a general belief that agents modulating more than one target could have superior efficacy compared to single target drugs has led to the search for molecules modulating multiple targets. The present thesis entitled “Biological evaluation of a novel pterostilbene-derivative as potent anticancer agent” deals with synthesis followed by evaluating the anticancer properties of a novel pterostilbene-isothiocyanate conjugate and further understanding it’s mode of action in preventing breast and prostate cancer progression At the beginning, Chapter 1 introduces briefly the present scenario of the chemotherapeutic drugs available to prevent steroid related cancer (breast and prostate cancer). It also deals with the key factors and the major signaling pathway present in these cancer cells that need to be targeted for its prevention. These cancer cell characteristics can serve as major points to guide the screening of novel molecule for their respective efficacy. Finally detailed objective to be attained in this study is specified here. Followed by this, Chapter 2 presents (i) a detailed review of set of characteristics that defines cancer - Hallmarks of cancer; (ii) a vivid description on endocrine related cancer with special emphasis on breast and prostate cancer; (iii) Understanding the role of inflammation and angiogenesis with major focus on molecular pathways and their therapeutic targets; (iv) recent research detailing benefits of phytochemical and their characteristics to target multiple pathways in cancer therapy; (v) detailed account of the design strategies employed for the synthesis of anticancer agents via molecular hybridization techniques and finally (v) the hypothetic idea behind the present thesis to manipulate the cancer cells towards death. Each of these hypotheses was further explored in subsequent chapters of the thesis. The main objective of the present work is synthesis and biological evaluation of novel pterostilbene conjugate as anticancer agent and understanding its mode of action. There are _____________________________________________________________________Abstract ii several in vitro assays which are already established to confirm a molecule to be anti-cancerous. All these parameters are elaborated in Chapter 3 of this thesis. In this concern, this chapter contains the overall principles and methodology of all the experimental assays that were performed to identify a molecule to be anticancer compound. The stage I includes biochemical parameters like MTT assay, FACS analysis, cell cycle analysis, DNA fragmentation patterns, activation of caspase which were used initially to check for anticancer effect; and in stage II, the principles of the biological mechanisms are studied at transcriptional and translational levels of various genes to understand how a molecule acts intracellularly, mainly the assays which show the basic determination of the underlying pathways. Further several cell lines used for the study have been discussed in detail. Cancer is a multifactorial disease, thus agents that concomitantly address more than one biological target for cancer treatment is preferred choice. Over the last few years, molecular hybridization strategy has emerged as a novel approach that involves conglomeration of two or more pharmacophores in one molecular scaffold to develop hybrid multifunctional molecules. The latter have multiple biological activities, modified selectivity profile, different or dual modes of action and/or reduced undesired side effects due to mixing of pharmacophores in one molecule. Using this approach we have synthesized and further characterized a novel compound (Pterostilbene-isothiocyanate conjugate) by appending an isothiocyanate moiety on pterostilbene (PTER) backbone which is described in Chapter 4. Moreover, we have tested the anticancer properties of the above said compound on different cancer cell lines in comparison to its parent compound i.e. PTER. Our results showed that all the cancer cell lines as tested by us were more sensitive to conjugate treatment as compared to PTER. Next, in Chapter 5, after the initial screening finally the pathways leading to apoptosis were checked biochemically and then at the transcriptional and translational level in hormone-dependent breast cancer cell line (MCF-7) in vitro. Our result confirmed that the pro-apoptotic effect of conjugate is mediated through the activation of caspases, and is correlated with the blockade of the AKT and ERK signaling pathways in MCF-7 cells. Finally the compound was tested on Ehrlich ascitic cell induced mouse tumor models and the characteristic pathways which lead to the reduction in tumor volume was investigated. PPARγ is a nuclear receptor which is widely expressed in many tumors and cell lines, and has become a promising target for anticancer therapy. This molecule is considered as the most promising target for metabolic syndrome, anti-inflammation and cancer in the 21st _____________________________________________________________________Abstract iii Century. Furthermore this receptor has a critical role in breast cancer proliferation, survival, invasion, and metastasis. Hence in Chapter 6 we tested whether PTER-ITC mediates its anti-proliferative and pro-apoptotic effects in breast cancer cells through activation of the PPARγ signaling cascade. Our results showed that PTER-ITC activated PPARγ expression in a dose-dependent manner, followed by downregulation of anti-apoptotic genes (Bcl-2 and survivin) to induce noteworthy levels of apoptosis in hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancer cells. Androgen and AR functions play a pivotal role in carcinogenesis and progression of prostate cancer (PCa), as well as in normal prostate development. Hence in the next section (Chapter 7), the effect of PTER-ITC on regulation of AR signaling in PCa was studied on both androgen dependent (LNCaP) and androgen independent (PC-3) cell lines. Our study showed that PTER-ITC effectively inhibited proliferation of both these PCa cells by inducing apoptosis and causing cell cycle arrest. Both PI3K/AKT and MAPK/ERK pathways played an important and differential role in PTER-ITC-induced apoptosis of these PCa cells. Further PTER-ITC inhibited the AR transcription and translation and also inhibited the localization of AR to the nucleus as seen in the AR-GFP transfection studies suggesting its AR antagonist nature. Breast cancer as a heterogeneous disease is characterized by activation of multiple signal pathways which stimulate tumor growth, proliferation, inhibit apoptosis, and promote the formation of new blood vessels (angiogenesis), as well as invasion and metastasis. Angiogenesis is essential for tumor progression by supplying sufficient oxygen and nutrients required for tumor growth and metastasis . Targeting angiogenesis is an exciting and attractive area in the treatment of cancer. Human umbilical vein endothelial cells (HUVECs) are cells derived from the endothelium of veins from the umbilical cord and are used as a laboratory model system for the study of the function and pathology of endothelial cells including angiogenesis. Chapter 8 of this thesis described the isolation and characterization of HUVEC cells from human umbilical cord. In the later part we examined the anti-angiogenic activity of PTER-ITC using a combination of in vitro and in vivo angiogenesis assays. This was followed by understanding the pathway targeted for preventing angiogenesis. Chronic inflammation has been linked to various steps involved in tumorigenesis, including cellular transformation, promotion, survival, proliferation, invasion, angiogenesis, and metastasis. Numerous evidence clearly shows that inflammatory pathways are critical targets in both prevention and therapy of cancer. Therefore, identification of agents/drugs that _____________________________________________________________________Abstract iv can suppress these pathways has enormous potential in cancer prevention. In Chapter 9 of this thesis we further attempted to investigate the anti-inflammatory potential of PTER-ITC followed by understanding its probable mode of action in lipopolysaccharide (LPS) stimulated RAW264.7 macrophages. In addition, the in vitro study was followed by in vivo studies on λ-carrageenan-induced rat paw edema model. Our data showed that non-toxic doses of PTER-ITC could inhibit inflammatory responses against LPS-stimulated RAW264.7 cells through down regulation of both IKK/NFκB and MAPK/AP-1 signaling Inflammation and tumors contribute to osteolysis causing bone pain and debilitating skeletal instability. Skeletal integrity depends on bone homeostasis which is achieved by balanced function of bone cells. Bone formation by osteoblasts and bone resorption by osteoclasts are lifelong events delicately balanced in healthy individuals. This homeostasis is compromised under pathologic conditions such as metabolic and inflammatory diseases including osteoporosis, inflammatory osteolysis, and skeletal tumor metastases, wherein heightened osteoclast activity leads in most cases to increased bone loss. Gene targeting studies have shown that the transcription factor nuclear factor-κB (NFκB) has a crucial role in osteoclast differentiation, and blocking NFκB is a potential strategy for preventing inflammatory bone resorption. The Chapter 10 deals with understanding the role PTER-ITC on preventing RANKL induced osteoclast differentiation in RAW264.7 and understanding the underlying mechanism. Finally, the Chapter 11 summarizes the primary contributions of this research and provides suggestions for future work in this area. The scientific findings dealt with in this thesis may be of use to the future researchers working in this area. Last but not the least, the Chapter 12, listed the bibliography which was consulted in course of the present work.