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  • br that ITH exhibited lower IC as compared

    2022-05-19


    that ITH-6 exhibited lower IC50 as compared to the newer drugs, re-gorafenib and irinotecan. The difference in response to different colon cancer cell lines are due to their establishment from different origin and p53 mutation status Barretina, 2012).
    Inhibition of the cell proliferation has long been known to be as-sociated with the changes in the DHEA (Gupta et al., 2016b). The alterations in the cell cycle progression cause tumor growth and pro-liferation. It has been stated that anti-cancer drugs can arrest the cells in various phases of cell cycle and inhibit the tumor growth (Malumbres et al., 2008). Our cell cycle results indicate that ITH-6 arrest the cells in G2/M phase and the maximum effect is at high concentration (3 μM) and there is no significant effect on other phases of cell cycle. These cell cycle results show that the test compound is G2/M phase specific. This instigated the idea to investigate the effects of ITH-6 on tubulin poly-merization and mitotic spindle formation, two processes that take place in G2/M phases of cell cycle. The tubulin polymerization assay results show that ITH-6 at 100 μM inhibits tubulin polymerization for 1 h. Paclitaxel (Taxol), a well-known anti-cancer drug, stabilizes the mi-crotubule against depolymerization, and is hence known as poly-merization enhancer (Arnal and Wade, 1995). Colchicine on the other hand, inhibits the microtubule polymerization and is thus known as a polymerization inhibitor (Hastie, 1991). We compared the tubulin polymerization effects of ITH-6 to that of paclitaxel and colchicine and found that, similar to colchicine, ITH-6 inhibited the tubulin poly-merization. However, the extent of inhibition was not significantly comparable.
    Since the cell cycle arrest is related to apoptosis, an apoptotic analysis was carried out using HT-29, COLO 205 and KM 12 cell lines. In all the three cell lines, a substantial number of apoptotic cells were observed in the lower and upper right quadrants, which are the re-presentatives of early and late apoptosis. The results showed an in-crease in early and late apoptosis in these cell lines with maximum apoptosis seen at the highest concentration of 3 μM. Cellular studies 
    have shown that an increase in the level of ROS causes an oxidative stress which results in oxidative damage to the cellular components (Ngamchuea et al., 2017). It enters into the cells, gets converted into the fluorescent (5-chloromethyl-20-7′-dichlorofluorescein (DCF)) pro-duct by the action of intracellular peroxides, hence, the ROS analysis is conducted in all the cell lines (Circu and Aw, 2010; Matés and Sánchez-Jiménez, 2000). We found that ITH-6 at the highest concentration (3 μM) induced intracellular ROS production in HT-29, COLO 205 and KM 12 cell lines. The mitochondrial GSH maintains the integrity of mi-tochondrial proteins and lipids and modulates ROS production. Oxi-dative damage is associated with an increase in mitochondrial ROS production and a decrease in GSH which in turn triggers apoptosis (Circu and Aw, 2008). Therefore, intracellular GSH assay was per-formed in all the three colon cancer cell lines. A significant decrease in GSH levels was also observed with compound ITH-6 in all the three cell lines with the maximum decrease at the highest concentration of 3 μM.
    5. Conclusion
    Anti-cancer drug discovery and development are considered as the grand challenges for the pharmaceutical industry. Extremely dynamic mitotic-spindle microtubules indeed remain the most successful and promising targets for anti-cancer therapy. Microtubule-stabilizing agents are continually playing an important role in anti-cancer drug discovery and development. In this study, we have shown that ITH-6 is an effective cytotoxic agent against colon cancer cells and exhibits a better cytotoxic effect compared to other drugs approved for colon cancer. Mechanistically, ITH-6 inhibits tubulin polymerization, alters the cell cycle progression and induces apoptosis by elevating the intra