MECHANISTIC INSIGHT INTO THE METASTATIC BREAST CANCER AND BONE DISORDER AMELIORATIVE EFFECT OF A CONJUGATE COMPOUND, PTEROSTILBENE-ISOTHIOCYANATE (PTER-ITC)

Abstract

Cancer has become one of the most prominent unmet medical challenges of the current century, unfortunately, becoming a household familiarity. When a disease reaches such a stage, it indicates that serious revisions in its management are of utmost pertinence. Prior to the 1950s, most cancers were treated with surgery and radiation. During the period 1949–1955, the only marketed drugs for the treatment of cancer were mechlorethamine, ethinyl estradiol, triethylenemelamine, mercaptopurine, methotrexate, and busulfan. In 1971, a single piece of legislation changed how we view cancer care forever. The “National Cancer Act” cemented our nation's commitment to science, establishing networks of cancer centers, clinical trials, data collection systems, and advanced research. It is clear that the development of better cancer treatments has improved the prognosis and quality of life for those who hear the words, “You have cancer.” However, despite such decisive measures and a huge amount of scientific research dedicated to various aspects of cancer, such as understanding its pathobiology, identifying potential therapeutic targets, discovering early diagnostic markers for better disease prognosis, etc. the tangible progress is still disproportionate. This indicates that there is an eminent requirement for revising the disease management strategies. These requirements that have shaped the four objectives of this thesis work are briefly introduced in Chapter 1: Introduction. An exhaustive review of literature on breast cancer metastasis, available treatment options, their drawbacks, miR-21-mediated drug resistance, collateral damages caused to bone health by breast cancer pathogenesis and its treatment are provided in Chapter 2: Review of Literature. This chapter closes with the mention that the paradigm shift from the ‘one drug one target’ approach to the ‘one drug multitarget approach’ has taken the centre stage for cancer treatment. Such multitarget potentials are the inherent properties of phytocompounds. Consequently, anti-cancer efficacies of several such phytocompounds are being actively explored, some of which give very promising results, particularly, when converted to hybrid molecules through chemical conjugation of synthetic groups. One such phytocompound in pterostilbene. Our laboratory has engineered a derivative of pterostilbene, by the conjugating Isothiocyanate group to it. The resultant compound is called Pterostilbene-Isothiocyanate (PTER-ITC). Earlier studies from our group showed that PTER-ITC was effective as an anti-cancer agent against different types of cancer cell lines. It was also found that this compound could also affect several breast cancer-associated changes in the body, such as angiogenesis and osteoclastogenesis. However, the ameliorative potentials of PTER-ITC in the above-mentioned aspects of breast cancer remained unexplored, which became the research gaps for this thesis work, which were addressed using a combination of a battery of in silico, in vitro and in vivo approaches detailed in Chapter 2: Materials and Methods. The first objective of this thesis was designed to evaluate the anti-metastatic potential of PTER-ITC against breast cancer and obtain mechanistic insight into its mode-of-action. The observations made from the execution of this objective are reported in Chapter 4, titled “Evaluating the anti-metastatic activity of Pterostilbene-Isothiocyanate (PTER-ITC) in prevention of breast cancer metastasis and understanding its mode of action in breast cancer cell line” of this thesis. PTER-ITC selectively affected breast cancer cells, sparing the normal ones, and achieves this by targeting the cancer-specific NF-B activation pathway. Precisely, PTER- ITC could inhibit the physical interaction between IKK and IKKγ/NEMO, the pivotal step along NF-B activation pathway. The recalcitrance of breast cancer treatments is a compounding factor behind complicating disease management. Converging evidence over the last decade has shown significant involvement of micro-RNAs in cancer pathogenesis, and rapidly growing treatment resistance. This has snowballed the already intractable cancer management situation into a medical crisis on a global scale. The second objective of this thesis has attempted at exploring the potential of PTER-ITC in ameliorating the hormone and chemo-resistivity in breast cancer cells, and the underlying mechanism behind it. The outcomes of this objective are reported in Chapter 5, titled “Assessing the impact of Pterostilbene-Isothiocyanate (PTER-ITC) on overexpressed miR-21 and its downstream gene expression responsible for drug resistance in breast cancer” of this thesis. It was observed that PTER-ITC interfered with the maturation of miR-21 from pre-miR-21, by binding to the Dicer cleavage site of the latter. Consequently, there was a reduction in Dicer binding to pre-miR-21 and concomitantly inhibit the biosynthesis of miR-21. Metastatic breast cancer cells are mainly home to bones, where they induce osteoclastogenesis, thereby, adversely affecting bone remodeling homeostasis. Additionally, prolonged high-dose exposure to glucocorticoids during hormonal therapy of breast cancer leads to osteoporosis in the long run. Thus, breast cancer pathogenesis and its treatment-associated side-effects, both are detrimental to the overall bone health of the patient. Prevention of osteoporosis and regeneration of bone mass is still unmet challenges, and available treatment options are sub-optimal and have serious side-effects. The third objective of this thesis was designed to address this breast cancer-associated effect on bone by exploring the anti-osteoporotic potential of the semi-synthetic compound, Pterostilbene-Isothiocyanate (PTER-ITC) in glucocorticoid-induced osteoporosis (GIOP) rat model and bone formation potential in vitro. Dysregulated bone-remodelling leads to osteoporosis. Osteoporotic amelioration through PTER-ITC treatment was also substantiated in this study. The study also identified that PTER-ITC inhibited the RANK/RANKL mediated TRAF6 activation pathway. These observations are reported and discussed in detail in Chapter 6, titled “Understanding the mechanistic insight into Pterostilbene-Isothiocyanate (PTER-ITC) for its role in maintaining bone homeostasis: inhibition of osteoclastogenesis and facilitation of osteoblastogenesis” of this thesis. The observed multi-faceted anti-breast cancer and bone health restoration potentials of PTER-ITC encourage us to explore the efficacy of this compound against the difficult-to-treat primary bone cancer, osteosarcoma. Chemotherapy is only sub-optimally effective against osteosarcoma and has adverse side-effects. Hence, there is a pressing need for innovative therapeutic approaches. The fourth objective of this thesis aimed at evaluating the anticancer effect of PTER-ITC, on human osteosarcoma MG-63 cells and decipher the underlying mechanism. PTER-ITC exhibited potent anticancer effects against human osteosarcoma cells by abrogating β-Catenin/TCF-4 interaction, suggesting that PTER-ITC may be regarded as a new approach for osteosarcoma treatment. These observations are included in Chapter 7, titled “Deciphering the mode of action of Pterostilbene-Isothiocyanate (PTER-ITC) in prevention of osteosarcoma in MG-63 cell line” of this thesis. The outcomes of this study provide complelling evidence for the multi-faceted anti-cancer effects of PTER-ITC, and are summarized in Chapter 8: Summary.