Characterization of the Anticancer Activities of Some Natural and Synthetic Compounds: Molecular Targets and Mechanism

概要

1. Title of the Thesis/Dissertation :
　Characterization of the Anticancer Activities of Some Natural and Synthetic Compounds: Molecular Targets and Mechanism

2. Summary (1,000 - 1,200 words in English)
　Cancer is defined as the uncontrolled and endless mitosis division of cells. After heart disease, it is considered as the second major cause of human mortality in the world. According to the recent 2021 WHO data, it is estimated to have risen to 18.1 million new cases and 9.6 million deaths. While various treatments such as surgery, radiotherapy, surgery with chemotherapy and/or radiotherapy, hormone therapy, immunotherapy, radiation therapy, stem cell transplant, and others are available for cancer, targeted therapy, another treatment strategy, is being developed recently. This type of treatment targets specific oncogenic protein in the cancer. A potential target of this therapy is Mortalin (HSPA9/GRP75/PBP74/mtHsp70), a member of heat shock protein 70 family that is over-expressed in almost all types of cancers. This protein has been shown to contribute to the intricate process of carcinogenesis by multiple mechanism, for instance, inactivation of tumor suppressor p53, deregulation of apoptosis, cell proliferation enhancement, contribution to the phenomenon of cancer cell stemness, pathological cancer cell migration, activation of the Epithelial to Mesenchymal Transition (EMT), angiogenesis and others.

　The aims of my research are (i) exploring molecular mechanisms of human carcinogenesis and its intervention by natural and/or synthetic molecules using a variety of approach, (ii) exploring molecular mechanism of the role of mortalin in human carcinogenesis, and its validation as a therapeutic target (iii) identification of new mortalin inhibitors using abrogation of mortalin-p53 interaction as a target and elucidation of their molecular mechanism of action using p53 wild type and mutant disease models. To achieve the aforementioned aims, I have employed in silico, in vitro, and in vivo methods in this research. The in- silico data is validated by WET lab experiments including short-term and long-term cell viability assays, Western blotting (WB), Immunocytochemistry (ICC), Immunoprecipitation (IP), Luciferase reporter assay systems, Real-Time quantitative Polymerase Chain Reaction (RT-qPCR), PARP-1 activity assays including DNA-Trapping, and in vivo rodent models of tumor growth etc.

　I have been focusing on two natural compounds as follows, Withaferin A (Wi-A) as a withanolide derived from Ashwagandha (Withania somnifera), and CAPE (Caffeic Acid Phenethyl Ester) as an active compound from New Zealand honeybee propolis. Whereas Wi-A was cytotoxic to both cancer and normal cells, high doses of CAPE have been shown to cause selective death of cancer cells. These two active compounds have been known as anti-mortalin compounds, which can inhibit the interaction between stress chaperone protein (Mortalin) and tumor suppressor (p53). With this knowledge, I have been undertaking the bioinformatics and experimental approaches to investigate the effect of Wi-A and CAPE on molecular pathways in cancer cells with specific reference to p53 tumor suppressor pathway. In this study, I am using human ovarian and cervical cancer cells for cell culture model. Control and Wi-A, CAPE and Wi-A+CAPE- treated cells investigated for the molecular signaling by biochemical, imaging and reporter assays with focus to proteins involved in growth arrest, apoptosis, DNA repair, cell migration, invasion and angiogenesis. The in vitro results are supported by in vivo assays using such as nude mice tumor formation and progression assays.

　The result showed that CAPE and Withaferin A were seen to abrogate mortalin- p53 interaction, resulting in nuclear translocation and reactivation of p53 function yielding growth arrest/apoptosis in cancer cells. We developed a combination of Withaferin A and CAPE that showed better potency in in vitro and in vivo models. The results were published (Sari et al, 2020 Cancers:12(5), 1160). Interestingly, our low dose combination of Wi-A and CAPE showed selective and synergistic toxicity towards cancer cells. We reported that the combination inactivated poly ADP-ribose polymerase1 (PARP1); a key regulator of DNA repair and a protein-target for Olaparib (clinically used drug for the treatment of breast, ovarian and cervical cancers). Continuing with this study, I am currently focusing my efforts to dissect the molecular mechanism of action of Wi-A, CAPE and the combination on cancer metastasis, EMT (Epithelial to Mesenchymal Transition), cancer cell stemness and drug resistance by a variety of assays including mRNA, protein, protein-protein interaction analyses. My current results show that sub-toxic dose of Wi-A and CAPE could be used in combination to achieve higher potency and multiple mechanisms to inhibit proliferation and migration of cancer cells. The combination caused effective downregulation of mortalin, and other proteins involved in cancer cell characteristics including EMT, migration, invasion and drug resistance. The combination of Wi-A and CAPE was effective in delaying the migration and anti-angiogenesis of the cancer cells through inactivation of Wnt-ß Catenin and VEGF signaling. It is supported that our low dose combination provides anticancer, anti-metastasis and anti-drug resistance effects and hence may be useful as natural drug for cancer therapy. In scenario of rapidly increasing cancer incidence and high cost of treatment, such results will be of high value.

　In a parallel study, I identified and validated new inhibitors of mortalin using abrogation of mortalin- p53 interaction as a target assay. A chemical library of 12000 compounds (synthetic and natural) were screened using visual assays to isolate compounds that cause nuclear enrichment of p53 and shift of mortalin staining pattern from the perinuclear (typical of cancer cells) to pan-cytoplasmic (typical of normal cells). I focused on a novel synthetic small-molecule (triazole derivative (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]- 1,2,4-triazole),which we named as MortaparibPlus. Osteosarcoma (U2OS) and Luminal-A breast cancer (MCF- 7) cells line possessing a wild-type p53 protein were used in the screening system established to define potential p53-Mortalin disruptors. MortaparibPlus was selected as a potential mortalin-p53 distributor from this screening. Then, to validate the anticancer activity and mechanism of action of MortaparibPlus, we recruited colorectal HCT116 [with wild type p53 (p53WT)] and DLD-1 [with the mutant p53 (p53S241F)] cell lines as a model for this study. The result showed that bioinformatic analysis predicted the ability of MortaparibPlus to competitively prevent the interaction of Mortalin-p53, whereas immunoprecipitation analyses showed that inhibition of mortalin-p53 complex formation occurred in Mortaparibplus-treated cells, arresting cell growth and stimulating apoptosis through activation of p21waf1, BAX and PUMA signallings. Furthermore, Mortaparibplus also induced a cytotoxic effect to cancer cells through a number of other mechanisms relating to carcinogenic properties of cancer cells. These included the inhibition of PARP1, up- regulation of p73 tumor suppressor protein as well as downregulation of mortalin and carf proteins. The results were published (Sari et al, 2021 Cancers:13(4), 853).

　Taken together, my current studies have led to the following conclusions. (i) Combination of Wi-A and CAPE provides a potent natural multimodal anticancer amalgam for treatment of metastatic and aggressive cancers. (ii) MortaparibPlus is a novel small molecule possessing anticancer activity. It inhibits cancer cell proliferation and migration by multiple mechanisms of action including inhibition of mortalin, PARP1 and activation of several tumor suppressor proteins.