ANTI-CANCER EFFECT OF NOVEL NATURAL PRODUCTS AND THEIR POSSIBLE MECHANISM OF ACTION

Abstract

Cancer is one of the deadliest diseases in humans. Although the emergence of various new research tools and techniques has improved our knowledge on the disease, its management is becoming complicated due to its molecular heterogeneity. As per the recent reports of the GLOBOCAN published on their website (https://gco.iarc.fr/), the worldwide estimated number of new cases in 2020 is 2261419 for breast cancer, 22306771 for lungs cancer, 1931590 for colorectal cancer, and 1414259 for prostate cancer. These figures show the prevalence of the disease and the need for various therapeutic strategies to manage the condition. The recent increase in the number of cases shows the incidence of breast cancer as the most prevalent, followed by that of the lungs, colorectal and prostate cancer. Even though radiotherapy and chemotherapy are the mainstay therapeutic approaches, cancer management process is often hindered by the severe side effects of the chemotherapy drugs (Spreafico et al., 2021). Neurotoxicity, nephrotoxicity, cardiotoxicity, gastrointestinal toxicity, alopecia, mucositis, and myelosuppression are common side effects of the present-day anti-cancer drugs (Koper et al., 2021). Apart from these physiological alternations, three newly emerged pathways are being recently considered as the major concerns for cancer management. These are: development of drug resistance (Nikolaou et al., 2018), initiation of metastasis (Jones, 2008) and accumulation of cancer stem cells (Medema, 2013). Any of the above conditions can result in the vigorous proliferation of cancer growth and development of the de-novo cancer cells irrespective of their location. Hence, these factors also contribute to the poor treatment modalities of most cancerous conditions. The flora world consists of numerous medicinal plants having various chemical properties. In recent years, the decoction of herbs, extracts and dietary sources are the most common anti-cancer therapeutic regimes under investigation. Complementary and alternative medicine (CAMe) modalities have gained popularity due to their therapeutic relevance and less cytotoxicity (Tagliaferri et al., 2001). Several phyto-derived chemicals exhibit antiproliferative / anti-cancer effects in the cancer cells and tissue either singly or in combination. Hence, the main aim of this study is to find some novel nature-based extracts/products that can help eliminate the growth of cancer cells and target certain recently emerged mechanisms that may help prevent the disease from further spreading. The present thesis entitled “Anti-cancer effect of novel natural products and their possible mechanism of action” deals with experiments that provide new insights into our understanding of the role of selected natural products in terms of alternative medicine and nutraceuticals in the cure and management of various cancers i.e., breast, colon and prostate cancer. In the beginning, Chapter 1 introduces the research gaps in cancer therapy and the need for alternative medicines for cancer management. This chapter mainly presents insights into the potential of nutraceuticals and plant extracts in cancer management. The chapter also imparts a comprehensive idea of the rationale for selecting the compounds/ drugs for this study. The last part of this chapter deals with the research objectives and overall hypothesis of this thesis. Chapter 2 deals with a detailed review of the literature. The first part of this chapter deals with the basic mechanism of cancer progression and the hallmarks. This chapter also describes the mechanisms involved in cell death induced by anti-cancer drugs. The latter part of the chapter describes the role of CAMe in the management of cancer and the role of nutraceuticals and homeopathic medicines in the management of the various form of cancers. All the materials and experimental methods used in this study for achieving our proposed objectives through in vitro and in vivo assays are elaborated precisely in Chapter 3. To start with the current work, we have screened various natural products for their antiproliferative activities in vitro system as mentioned in Chapter 4. Various natural compounds include homeopathic drugs and their dilutions and nutraceuticals like -honey. In Chapter 5, Chimaphila umbellata (CU), a homeopathic mother tincture, was used to evaluate the anti-cancer effect in human breast cancer cells MCF-7. CU is a small perennial plant whose roots and leaves are used in the preparation of homeopathic medicines. In this study, the antiproliferative and apoptotic effects of CU extract, obtained from its homeopathic mother tincture, were evaluated in human breast cancer cell lines. MTT assay was used to evaluate the dose- and time-dependent cytotoxicity of the extract in MCF-7 and HEK293 cells. The anti-proliferative effect of the extract was evaluated using the clonogenic and wound healing assays. The antiangiogenic activity of the drug was evaluated using the chick chorioallantoric membrane assay. The mode of cell death was analyzed using Annexin V and PI staining assays through flow cytometry. In addition, the expression patterns of associated genes were evaluated using immunoblot analysis. CU extracts exerted an anti-proliferative effect in breast cancer cell lines, i.e., MCF-7 cells, by inhibiting their growth and migration. The extract also demonstrated significant anti-angiogenic ability, limiting the de-novo blood vessel growth and development in chick embryos. The extract was found to increase caspase-independent necroptosis by involving RIP1/RIP3 kinases and MLKL proteins. This finding was further confirmed by using NEC-1 (an inhibitor of necroptosis), which significantly abolished the CU extract-induced necroptotic effect. From the above results, it can be deduced that CU extract exhibits great potential in preventing breast cancer and thus warrants further investigations in vivo. In our subsequent studies, in Chapter 6, we have tested the anti-cancer effect of the homeopathic formulations of Conium maculatum (CM) in human prostate cancer cells as well as in solid tumor mice models. This chapter also proposed the energy theory of homeopathic drugs and the involved mechanisms therein. Homeopathy is one of the most popular alternative forms of medicine practiced across the globe. It is gaining popularity in developing nations due to its natural origin and low cost of treatment. The working mechanism and dilution/ potentization of homeopathic drugs have always been debated in the scientific arena. Although there are several scientific efforts, more scientific validations are warranted to establish the above proposition. CM, a homeopathic drug formulation, is used to treat several prostate and urinary disorders. In the first phase of the chapter, the working mechanism of CM drugs is deciphered by analyzing the physicochemical characterization using transmission electron microscopy (TEM) and atomic force microscope (AFM). By these, I attempted to establish the nanoparticulate nature of the drugs and energy calculations by using molecular simulations. Next, the anti-cancer potential of CM in prostate cancer cell lines (DU145) was validated through assays like MTT, AnnexinV/PI staining, cell cycle arrest, and gene expression analysis. CM was found to induce apoptosis in DU145 cell lines by acting on the p53 via NF-κB dependent axis. Moreover, the formulation also restricted solid tumor growth by targeting angiogenic factors like VEGF and TNF-α. Overall, this study demonstrated that CM is a potent anti-cancer agent and capable of targeting various cancer-specific targets. In the next phase of the thesis, an attempt was made to decipher the anti-cancer effect of another homeopathic formulation within the cancer cells. Androgen deprivation therapy (ADT) is a principal method for managing prostate cancer (PCa). Repeated dosage of ADT drugs and evolution of androgen-independent mechanism cause sustained tumor progression and metastasis. Castration resistance to PCa (CRPC) and metastasis are two significant factors that cause higher mortality in PCa. Chapter-7 deals with the anti-cancer effects of homeopathic formulation Sabal serulata (SS) against PCa cells and solid tumor animal models. SS mother tincture inhibited DU145 cell proliferation by inducing nuclear fragmentation and apoptosis. SS also arrested the cell cycle at G2/M phase. Our study identified that SS attenuated the mesenchymal markers in DU145 and increased the drug sensitivity of AR-based drugs in AR negative cell line DU145 by targeting the transcription factor FOXC2. Collectively, our study suggested that SS homeopathic formulation can increase drug sensitivity to the ADT drug, enzalutamide, and reverse the mesenchymal nature of the DU145 cells. Moreover, SS can help in arresting tumor growth by altering the BAX/Bcl2 ratio. Hence, SS may be used as an alternative or a complementary form of medicine along with conventional ADT drugs to address the problem of CRPC and can result in cell proliferation by attenuating the effect of mesenchymal markers. Colon cancer is the most prevalent cause of death from cancer across the globe. Although chemotherapy drugs are predominantly used, the toxicity involved in these drugs always remains a question. As an alternative to synthetic drugs, natural compounds or nutraceuticals are comparatively less toxic. Honey is widely used across different cultures as an alternative form of medicine. It represents a prominent source of plant-phenolic compounds, and its anti-oxidant, as well as anti-microbial activities, have been well demonstrated. The next chapter of this thesis (Chapter-8) aims to investigate the anti-proliferative effect of Indian honeys and their mechanism of action in the management of colon cancer. In order to establish the compositionactivity relationship, we evaluated the bio-active components present in the selected honey samples by GC-MS and HPLC analysis. Indian honey samples showed a significant inhibitory impact on cell growth by restricting cell proliferation, causing apoptosis, and restricting the cell cycle in the G2/M phase, specifically to colon cancer cells. The apoptotic activities as imparted by the honey samples were established by AnnexinV/PI staining, real-time PCR and immunoblot analysis. The treated cells showed increased expressions of p53, caspase 3, 8 and 9, thus indicating the involvement of both extrinsic and intrinsic apoptotic pathways. The honey samples were also found to inhibit the β-catenin/ Wnt pathway. We further demonstrated the efficacy of these honey samples in colon carcinoma-induced SD rats. Overall, these findings demonstrate that the selected Indian honeys can be established as effective nutraceuticals for the prevention as well as cure of colon cancer. In the next chapter (Chapter-9) of the thesis, the role of Indian mustard honey was deciphered in the higher grade of colon cancer, i.e., colonospheres and the 5-Flurouracil resistance colon cancer cell lines. Heterogeneity in the colon cancer microenvironment has become a critical factor that hinders successful therapeutic outcomes against the disease. The existence of a cancer stem cell niche, metastasis, and drug resistance are vital domains that orchestrate cell death escape during the treatment regimen. 5-Fluorouracil, conventionally used as the first line ofto the development of resistance, resulting in poor clinical outcomes. This chapter shows LGR- 5 as a potential target for combating metastasis and drug resistance in colon cancer. Here, we report, for the first time, the therapeutic efficacy of Indian mustard honey in outwitting the mesenchymal nature of colonospheres and enhancing drug sensitivity in drug-resistant cell lines. Finally, Chapter 10 summarizes all the major findings, demonstrating the effects of several phytochemicals in the cure and management of various forms of cancer in invitro as well as invivo models. The scientific findings dealt within this thesis may be of use to the future researchers conducting research in this area. Last but not least, Chapter 11 of this thesis lists the bibliography that was referred to in the course of the present work. defense against colorectal cancer, has a great therapeutic value. However, repeated dosage leads