TARGETING ANOIKIS RESISTANCE IN TUMOR CELLS USING BIOACTIVE NATURAL COMPOUNDS

Abstract

Cancer has remained one of the greatest health threats globally, contributing to significant mortality, and. in most occasions, life-altering consequences for the patients. Against this background, even with the numerous therapies and drugs applied in such situations, no single treatment works for this lethal disease. One of the primary obstacles that present challenges in cancer treatment and research is the fact that cancer cells become resistant to cell death mechanisms, treatment therapies, drugs, or any other means of treatment that induce cell death. Genetic alterations in normal and neoplastic cells render them resistant to these adverse conditions, thereby leading to their survival, growth, and progression in the body. Normal cells need to communicate with neighbouring cells, adhere to the extracellular matrix, and timely cell death to maintain cellular homeostasis. In contrast, cancer cells circumvent these mechanisms, thereby enabling themselves to survive in conditions that would normally activate death processes and mechanisms. Anoikis is a specialized form of apoptotic cell death resulting from the loss of cell-cell or extracellular-matrix contact. Tumor cells that become resistant to anoikis are termed as anoikis- resistant cancer cells. In such cells, the overexpression and activity of anti-apoptotic proteins inhibit the release of cytochrome c into the cytosol and the consequent activation of the caspase cascade, two events central to apoptosis. Consequently, these anoikis-resistant cells proceed through cell proliferation after escaping cell death and thereby gain the ability to migrate into distant organs for establishment of secondary tumors, hence initiating metastasis. In fact, it was derived from the literature review for this study that key processes, such as epithelial- mesenchymal transition and autophagy, act as major negative regulators of anoikis. These processes facilitate the enhanced migration potential of cancer cells by epithelial-to- mesenchymal transformation and support cell survival without attachment to the ECM or cell neighbours. As such, our research focused on identifying molecular pathways involved in anoikis that would lead to the discovery of common genes between anoikis, apoptosis, EMT, autophagy, and metastasis. Thus, this thesis aims to study the process of anoikis and screen the inhibitors that targeted proteins having a negative regulatory effect on this cellular process. We screened a set of 102 natural compounds with previously described anti tumor properties in order to identify the modulators of anti-apoptotic genes regulating this process of anoikis. A combination of the in-silico methods such as molecular docking and molecular dynamics simulations, differential gene expression analysis, and nanoparticle-based drug delivery was employed to treat anti-tumor activity of one among the several screened bioactive compounds against a cancer cell line. Molecular docking and molecular dynamics simulations enabled us to study interactions occuring between targeted anti-apoptotic (anoikis resistance associated proteins) protein and their ligands by evaluating their binding affinities and behavior in a simulated biological environment. These computational tools have thus served in the screening of hundreds of natural compounds and identification of genes that could be relevant, using data obtained from experiments validated in wet lab environment procured from online databases. Accordingly, these bioactive compounds might be tailored and further therapeutically applied to target anoikis in cell death resistant tumor cells, which would imply a new approach in cancer therapy. As a result, we found out that there are few proteins that are common among anoikis associated pathways of autophagy, apoptosis and EMT. We also screened genes involved in metastasis and core anoikis. Bcl-2, Bax, BIRC3 and IKKα were proteins common among all these pathways, we further studied Bcl-2 and its interaction with natural compounds indicating Dioscin interacts with Bcl-2 by direct binding and hence can be further investigated for cancer therapy. In another approach, we procured genes that were differentially expressed in anoikis resistance, circulating tumor cells (CTC) and metastatic and benign prostate cancer. We extracted common genes in these three data sets and checked how the levels of gene expression 5 of these genes were changed by a set of natural compounds in combination and alone. We employed GEO2R, MS Excel and certain mathematical codes in this methodology. 8 genes out of 53 DEGs including MYLIP, ST7L, SSTR1, ZSCAN12, SKIL, ANXA2, KIF5C, and CAV2 were regulated by all four compounds named nitidine chloride, bilobalide, CDCS (Chenodeoxycholic acid) and gastrodin. This observation suggests potential synergistic effects of natural compounds and alteration of gene expression by multiple compounds proves beneficial in preventing anoikis resistance. Following this we studied the effect of natural compound on differentially expressed anoikis resistance genes at different time intervals. We acquired GEO Dataset GSE158788 which had the gene expression profile of tumor associated genes at different time intervals treated with Gallic acid. We matched this data with the expression of our differentially expressed anoikis resistance genes and found out each of the genes showed fold change in gene expression at 2 hours, 4 hours, 6 hour and 9 hours. Following this, to study the drug delivery of natural compound having potent drug capabilities we conjugated gallic acid with gold nanoparticle. We synthesized gold nanoparticle by citrate reduction method. Gold nanoparticle has been known for its anti-cancerous properties, anti- oxidant property and non-toxic nature. Nanoparticle-gallic acid conjugation was studied by FTIR, UV-Visible spectrophotometry and Size distribution by intensity by Dynamic light scattering. Drug release was studied in PBS and changes in size of the nanoparticle, changes in absorption spectra depicted successful conjugation and drug release. Cytotoxic effect of gallic acid, gold nanoparticle and gallic acid loaded gold nanoparticle was studied by performing MTT Assay. We used Colon cancer cell lines (Col 320) for cell viability assay and studying cytotoxicity by the 3 components (AuNP, GA-AuNP, and GA). After treatment of cells with the different dosage concentrations of AuNP, GA-AuNP and GA at 12 hours, 24 hours and 48 hours and measured absorbance at 570 nm using a microplate reader. The GA-AuNP complex consistently demonstrates superior efficacy in inhibiting cell viability, as evidenced by the lowest IC50 values across all time points. At 12 hours, GA-AuNP (30.27 µg/mL) significantly outperforms Gallic Acid (57.21 µg/mL) and Gold Nanoparticles (70.63 µg/mL). This trend continues at 24 hours, with GA-AuNP (19.23 µg/mL) remaining more effective than Gallic Acid (42.53 µg/mL) and Gold Nanoparticles (59.57 µg/mL). At 48 hours, GA-AuNP (18.35 µg/mL) maintains its superior efficacy compared to Gallic Acid (35.12 µg/mL) and Gold Nanoparticles (48.29 µg/mL). These results highlight the enhanced potential of the GA-AuNP complex in inhibiting cell viability over time. The study has verified potential for targeting anoikis resistance in cancer cells using natural compounds, focusing on the molecular pathways and genes involved in apoptosis, EMT, autophagy, and metastasis. This study elucidates the complex interaction between autophagy, apoptosis, and anoikis in an effort to realize their important roles in cancer prognosis. Common genes and proteins in their pathways could thus be targeted to regulate anoikis, an important process involved in cancer progression. Hence, computational methods using bioactive natural compounds would be a promising way for modulating these regulatory molecules. Dioscin, nitidine chloride, gastrodin, CDCA, bilobalide, and gallic acid were found to have the maximum potential compounds for altering pathways and genes related to anoikis. Additionally, gold nanoparticles offer an effective platform for drug delivery with drug loading and release. Gallic acid showed toxicity towards cancer cells and it showed enhanced efficacy when loaded in gold nanoparticles, as evinced by MTT Assay results. These results point toward a new and effective avenue in cancer therapy by targeting anoikis resistance with natural compounds, particularly in combination and in conjunction with advanced drug delivery systems. Future studies in regard to this shall be based on in vivo studies and clinical trials, with new discovery of other natural compounds and nanoparticles targeted for more infiltration of cancer therapy.