br ITH arrests the colon cancer cells in
3.3. ITH-6 arrests the colon cancer cells in the G2/M phase of the cell cycle
In order to investigate the mechanism by which ITH-6 inhibits the proliferation of colon cancer cells, its eﬀects on the progression of Digitonin were studied. On treatment with ITH-6 (0.3, 1, and 3 μM), a concentration dependent increase in the percentage of cells in G2/M phase of the cell cycle of all the three cell lines was observed. The concentrations were selected based on the IC50 values. ITH-6 increased the percentage of cells from 37.5% to 72.1% in HT-29 (Fig. 2A), 15.1% to 33.4% in COLO 205 (Fig. 2B), and 24.1% to 77.8% in KM 12 cells (Fig. 2C). These results suggest that ITH-6 arrests the cells in G2/M phase with negligible eﬀect on other phases of cell cycle in all the three cell lines. To study the permanent cytotoxicity of ITH-6, the experiment was performed 24 h after incubating the cells in drug free medium following a 24 h ITH-6 treatment (0.3, 1, and 3 μM). The results showed there was no eﬀect on G2/M phase (Fig. 3A–C).
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. 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.
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 cell cycle 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).