Abstract
Cancer remains a life-threatening disease that greatly affects millions of people. In most cases, cancer is diagnosed in the advanced stage hence the prognosis remains poor. Despite all the efforts to implement new chemotherapeutic strategies, cancer remains a huge concern worldwide. Synthetic compounds are widely used in drug discovery because they possess various pharmaceutical activities including anticancer. Most chemotherapeutic drugs are designed to inhibit proliferation and induce apoptosis. However, they have limitations. This study aims to explore in vitro anti-proliferative, pro-apoptotic, and anti-metastatic effects of 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole against pancreatic and melanoma cancer cell lines. The study hypothesizes that 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole will exhibit anticancer properties by inhibiting cell proliferation, inducing apoptosis, and inhibiting metastasis in pancreatic and melanoma cancer cells.
To study the anti-proliferation effects of 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole, Alamar blue assay was conducted. The number of viable cells was reduced on both cell lines after treatment. 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole exhibited the highest inhibitory activity as compared to the parent compound 1,3-thiazino [3,2-a] benzimidazole and other three derivatives and was further tested against HEK293 normal cell line and cancer cells (PaCa-2 and A375) using different concentrations, to calculate the IC50. In this study, the ATP level on each cancer cell line was also quantified to determine cell viability. Both PaCa-2 and A375 cells exposed to 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole exhibited lower ATP levels as compared to untreated cancer cells which confirm anti-proliferative activity shown by alamar blue.
The pro-apoptotic effects of 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole were studied using caspase 3/7 activity. Upregulation of caspase 3 and 7 was observed on the PaCa-2 cell line only which suggests a late stage of apoptosis. In the A375 cell line, there was no upregulation of caspase. However, it was reported in previous studies that cell death can be induced in the absence of caspase. The morphological effect of 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole on PaCa-2 and A375 cancer cells was also studied. Both cell lines changed from spindle to round shape after treatment, which is one of the apoptotic features. Fluorescence Hoechst staining was also
ii
conducted to study the effects of the compound on the cell’s nucleus. PaCa-2 cell nuclei were fragmented after treatment which is one of the characteristics of apoptosis. In the A375 cell line, most of the cell’s nucleus shrinks without fragmentation, a morphological feature of caspase-independent cell death. Gene expression was also conducted using conventional PCR to study the expression level of three apoptotic-related genes p53, Bcl-2, Bax, and one housekeeping gene (B-actin). The p53 gene is an imported regulator of cell death and it was upregulated on both focus cell lines. Bax was also upregulated on both PaCa-2 and A375 which suggest an increased cell death. Bcl2 is a Pro-survival gene and it was downregulated on both cell lines.
To study the anti-metastatic activity of 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole, wound healing assay was conducted to measure cell migration. The compound inhibited cell migration in both cell lines, an important step of metastasis. This study concluded that 2-imino-7-methoxy-4-(4-fluorophenyl)-2h-1,3-thiazino [3,2-a] benzimidazole induce both caspase-dependent and caspase-independent cell death for PaCa-2 and A375, respectively. The mechanism of metastasis was also suppressed on both cell lines. Therefore, the molecule studied can potentially be used as a chemotherapy drug in the development of new anti-cancer treatments.
keywords: cancer, synthetic compounds, anti-proliferation, pro-apoptosis, anti-metastatic, gene expression, p53, Bcl-2, Bax, anti-cancer