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  • br dehydration myelosuppression alopecia and diarrhea

    2020-07-30

    
    dehydration, myelosuppression, alopecia, and diarrhea are a serious concern (Rougier et al., 1998). Bevacizumab, a humanized monoclinal antibody was approved by the USFDA in 2004 for the treatment of patients with advanced colorectal cancer. Bevacizumab exhibits some rare serious adverse effects such as bowel perforation, arterial embolic events, and leukoencephalopathy (Glusker et al., 2006; Hurwitz et al., 2004, 2005).
    In the present study, we find that the compound ITH-6 has lower IC50 values on the colon cancer cell lines, HT-29, COLO 205 and KM 12 as compared to the conventional anti-cancer drug, irinotecan. Indanone and its derivatives are well known for their wide range of biological activity (Leoni et al., 2000). Studies done in the past have shown that the indanone derivative are potent anti-inflammatory, analgesic, anti-microbial, anticholinergic, anti-cancer, and antimalarial agents. 3-aryl substituted indanone analog was found significantly active against the HeLa and K562 cell lines (Patil and Patil, 2017).The other derivatives, gallic Protease Inhibitor Cocktail based indanone analogs are cytotoxic (IC50 of 0.01 μM) on breast cancer cell lines MCF-7 and MDA-MB-231 (Saxena et al., 2008). In addition, 2-substituted indanone analogs are active against non-small lung cancer cell line (Charrier et al., 2007) and 5,6-dimethoxy-1-in-danone derivative is significantly cytotoxic on multidrug resistant cell lines, MCF-7/ADR, MES-SA/DX5 and HL-60/ADR (Leoni et al., 2000). The present indanone derivative, ITH-6 exhibited IC50 values of 0.44
    Fig. 5. Effect of ITH-6 on the apoptosis of HT-29, COLO 205, and KM 12 cell lines. HT-29 (A), COLO 205 (B), and KM 12 (C) cells were treated with ITH-6 (24 h) in a concentration-dependent manner, stained with Annexin-V and PI, and analyzed by flow cytometer. The apoptotic cell population was quantified by flow cytometry. Bar graphs in blue represents live cells, in red represents cells undergoing apoptosis, and in green represents cell undergoing necrosis. Bar graphs represents average cell population of three independent experiments and error bars represents SD. * p < 0.05, ** p < 0.001, *** p < 0.0001 by ANOVA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
    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 cell cycle (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.