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  • br ITH inhibits tubulin polymerization in

    2020-08-12


    3.4. ITH-6 inhibits tubulin polymerization in the mitotic phase
    To further elucidate the mechanism by which ITH-6 arrests the colon cancer cells in G2/M phase of the cell cycle, tubulin poly-merization assay was performed according to the manufacturer's pro-tocol. Wortmannin The test drug (ITH-6) was compared against control drugs, pa-clitaxel and colchicine. Our results indicated that paclitaxel (10 μM) stabilizes the microtubule by enhancing the tubulin polymerization for
    Table 2 The effect of synthesized compounds on colon cancer cell lines.
    Compounds Code
    CELL LINES
    Values in tables are representative of at least three independent experiments performed in triplicates. IC50: concentration that inhibits cell survival by 50% (mean ± SD).
    a period of 1 h while colchicine (10 μM) acted as a tubulin poly-merization inhibitor. Interestingly, ITH-6 at 100 μM, it inhibited the tubulin polymerization thus suggesting that ITH-6 acted on the G2/M phase of the Wortmannin by inhibiting the tubulin polymerization activity, an effect similar to colchicine however, less potent than colchicine (Fig. 4).
    3.5. ITH-6 induces apoptosis in colon cancer cells
    To understand the apoptotic effects of ITH-6 on colon cancer cell lines, the cells were treated at different concentrations (0.3, 1, and 3 μM) of ITH-6 for 24 h. In all the three cell lines, most of the cells were viable in the control group and no apoptosis was observed. ITH-6 ex-hibited a concentration dependent increase in the early and late apoptosis of HT-29 (Fig. 5A), COLO 205 (Fig. 5B), and KM 12 (Fig. 5C) cells. Moreover, ITH-6 did not induce any significant necrosis in all the three cell lines (Fig. 5 and Supplementary Fig. 2).
    3.6. ITH-6 elevates ROS production in colon cancer cells
    Since an increase in intracellular ROS is a measure of induction in apoptosis, we investigated the effects of ITH-6 on the intracellular ROS production. The cells were treated at the indicated concentrations for 24 h and the intracellular ROS levels were measured using the flow cytometer. As shown in the Fig. 6, ROS percentage increased from 5.98% (control) to 66.3% (ITH-6 at 3 μM) in HT-29, 1.88% (control) to 71.7% (ITH-6 at 3 μM) in COLO 205, and 4.26% (control) to 69.57% (ITH-6 at 3 μM) in KM 12 cells. These results suggested that ITH-6 elevates intracellular ROS levels and cause apoptosis in colon cancer cells.
    3.7. ITH-6 inhibits GSH levels in colon cancer cells
    Since a decrease in GSH levels is known to induce ROS and in turn induce apoptosis, the effects of ITH- 6 on the intracellular GSH levels
    Fig. 3. Effect of ITH-6 on the cell cycle of HT-29, COLO 205, and KM 12 cell lines after washing the drug out. HT-29 (A), COLO 205 (B), and KM 12 (C) cells were treated with ITH-6 (24 h) in a concentration-dependent manner, incubated in the drug free medium for 24, stained with propidium iodide and then analyzed by flow cytometer. Quantification of the PI staining data is presented as the percentage of distribution through stages of the cell cycle: blue-G0/G1; red- S; green- G2/M (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
    Fig. 4. Effect of ITH-6 on the tubulin polymerization. The tu-bulin polymerization assay was performed as per manufacturer’s protocol. The change in optical density (OD) at 340 nm was plotted against time in min for ITH-6 at 100 μM (green) was compared with control (blue), paclitaxel at 10 μM (orange), and colchicine at 10 μM (yellow). Points with error bars represent the mean ± SD for independent determinations in triplicate. The figures are representative of three independent experiments (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
    were determined at the indicated concentrations. Our results showed
    that ITH-6 exhibits a concentration-dependent decrease in intracellular
    4. Discussion
    Despite of the advances in chemotherapy, the mortality rate of colorectal cancer is quite alarming. Patients with colorectal cancer fall into two categories; ones in which the disease is confined to the primary site of origin (Dukes’A and B) and the other where it spreads to the regional lymph nodes (Dukes’C and D). The first category of patients can be surgically cured while for the later ones, surgery has is only palliative role and survival rate is less than 30% (Hampel et al., 2005).
    The drugs already approved and being used for the treatment of colon cancer include irinotecan, oxaliplatin, capecitabine and the tar-geted drugs include bevacizumab, ramucirumab etc. Irinotecan, ap-proved by the USFDA in 1996, is a prodrug which is converted into its active metabolite, SN-38 inside the body. It has long been used as the first line therapy for patients with recurrent and metastatic colorectal cancer however, the dose related toxicities such as vomiting,