br described previously Gupta et al a In brief
described previously (Gupta et al., 2017a). In brief, the cells were harvested and centrifuged at 800 rpm for 5 min. The supernatant was removed and the cell pellet was washed with 1X PBS. The cells were fixed overnight in ice cold 70% ethanol at 4 °C. The fixed cells were stained with 50 μg/ml PI and 100 μg/ml of RNase at 37 °C for 30 min in the dark. The flow cytometric analysis was performed and the percen-tage of cells in diﬀerent phases of Ascorbic acid were determined (G0/G1, S, G2/M).To determine whether the eﬀect of ITH-6 on the cell cycle phases are permanent or not, another flow cytometric experiment was performed, after 24 h incubation in the drug free medium following the 24 h treatment using FL-2 of BD Accuri™ C6 flow cytometer from BD Biosciences, CA (Gupta et al., 2017a).
2.5. Tubulin polymerization assay
The action of the test compound, ITH-6 on the tubulin poly-merization was assessed by tubulin polymerization kit. The preparation of samples and assay protocol was carried out as per manufacturer’s instructions (Schneider et al., 2003). ITH-6 (100 μM) was used a test compound while paclitaxel and colchicine (10 μM) were used as con-trols.
2.6. Apoptosis analysis
The cells were incubated with ITH-6 for 24 h at concentrations of 0.3, 1 and 3 μM. After 24 h, the cells were washed, harvested and stained with FITC-labeled annexin-V and PI at 37 °C for 30 min. The degree of apoptosis was measured at FL-1 and FL-2 of the flow cyt-ometer.
2.7. Intracellular ROS measurement
In order to investigate the eﬀects of ITH-6 on the intracellular levels of ROS, the cells were treated with ITH-6 at diﬀerent concentrations ranging from 0 to 3 μM for 24 h. After 24 h, the cells were washed and harvested. Subsequently, 10 μM of CM-H2DCFDA was added. The CM-H2DCFDA dye enters into the cells, gets converted into the fluorescent (5-chloromethyl-20-7′-dichlorofluorescein (DCF)) product by the action of intracellular peroxides. The cells were incubated in dark at 37 °C for 30 min. Intracellular ROS levels were measured using the flow cyt-ometer.
2.8. Intracellular GSH assay
In order to better understand the inverse relation between oxidative stress and GSH, the colon cancer cell lines were treated with ITH-6 at diﬀerent concentrations 0.3, 1, and 3 μM for 24 h. The intracellular GSH was measured using GSH assay kit and the protocol was carried out as per manufacturer’s instructions (Kim et al., 2016). The samples were prepared and analyzed as per manufacturer’s protocol using FL-1 of flow cytometer. International Journal of Biochemistry and Cell Biology 110 (2019) 21–28
2.9. Statistical analysis
All experiments were repeated at least three times and the diﬀer-ences were determined using a one-way analysis of variance (ANOVA). The statistical significance was determined at p < 0.05, p < 0.001 and p < 0.0001. The post hoc analysis was performed using Tukey’s test. The data were analyzed using GraphPad Prism, version 6.
3.1. Non-cytotoxic eﬀect of ITH-6 on normal cell lines
To determine the cytotoxic eﬀect of ITH-6 on normal healthy cell lines, MTT was done against human embryonic kidney cell line, HEK293 and mouse fibroblast cell, 3T3. ITH-6 did not show any cyto-toxicity on these cell lines and IC50 was more than 30 μM (Table 1).
3.2. ITH-6 inhibits cell proliferation of colon cancer cell lines
In order to determine the cytotoxicity of synthesized compounds on colon cancer cell lines, MTT assay was performed against 7 colon cancer cell lines (as mentioned in cell lines and cell culture). Among all com-pounds, four compounds exhibited remarkable cytotoxic activities against most of the tested colon cell lines (Table 2). For the five types of tested human colorectal adenocarcinoma cells SW620, COLO 205, KM
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 cell cycle 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).