The ROS generation after 6?h was immoderate after 6?h of treatment and had no discrimination; thus, we evaluated that 6?h was the most effective point to analyze ROS level in acetylshikonin-treated colorectal cancer cells
The ROS generation after 6?h was immoderate after 6?h of treatment and had no discrimination; thus, we evaluated that 6?h was the most effective point to analyze ROS level in acetylshikonin-treated colorectal cancer cells. of subG1 phase in cell cycle arrest assay and early/late apoptotic rates in annexin V/propidium iodide (PI) double staining assay was observed, which indicates an apoptotic potential of acetylshikonin against colorectal cancer cells. 2,7-Dichlorofluorescin diacetate (DCF-DA) staining was used to evaluate reactive oxygen species (ROS) generation in acetylshikonin-treated colorectal cancer KW-2478 cells. Fluorescence-activated cell sorting (FACS) analysis showed that acetylshikonin induced an increase in reactive oxygen species (ROS) levels and apoptotic rate in a dose- and time-dependent manner in HCT-15 and LoVo cells. In contrast, cotreatment with N-acetyl cysteine (NAC) has reduced ROS generation and antiproliferative effects in colorectal cancer cells. Western blotting analysis showed that acetylshikonin treatment induced increase of cleaved PARP, value of KW-2478 0.05 was considered statistically significant. 3. Results 3.1. Acetylshikonin Inhibited Cell Viability and Proliferation in HCT-15 and LoVo Cells Cell viability was analyzed by MTT assay. Dose- and time-dependent inhibition of cell viability was observed in acetylshikonin-treated cells. The result shows that acetylshikonin has a significant ( 0.05) inhibitory effect from 1? 0.05 and ?? 0.01, respectively). 3.2. Acetylshikonin Induced Apoptosis in HCT-15 and LoVo Cells To investigate acetylshikonin-induced apoptosis, annexin V/PI double staining assay and cell cycle arrest assay were performed using flow cytometry. The apoptotic rates of HCT-15 cells at 24?h were increased to 7.71%, 20.69%, and 28.69% (= 0.0001), and at 48?h, they were increased to 7.95%, 13.39%, and 49.92% (= 0.0006), respectively (Figures 2(a) and 2(c)). In LoVo cells, the apoptotic rates were increased to 8.7%, 24.31%, and 42% (= 0.0003) at 24?h and they rose to 13.48%, 41.93%, and 66.9% (= 0.0006) at 48?h (Figures 2(b) and 2(d)). In the cell cycle arrest assay, the result showed that this portions of subG1 phase were increased (4.76%, 5.44%, 13.06%, and 19.19% in HCT-15 and 2.49%, 5.37%, 7.84%, and 17.14% in LoVo after 24?h of treatment; 4.72%, 9.77%, 23.56%, and 27.01% in HCT15 and 2.37%, 2.79%, 9.63%, and 26.22% in LoVo after 48?h of treatment) as the concentration of acetylshikonin was increased (Figures 3(a) and 3(b)). TUNEL assay visualized Rabbit Polyclonal to OR8K3 the DNA damage via enzymatic labeling of free 3-end of DNA, which is one of the features of apoptosis; it is observed that the number of TUNEL-positive cells was significantly increased in acetylshikonin-treated cells (Figures 4(a) and 4(b)). These results show that the apoptotic rates of colorectal cancer cells treated with acetylshikonin were increased in a dose- and time-dependent manner and significantly in 5? 0.05 and ?? 0.01, respectively). Open in a separate window Figure 3 Cell cycle progression analysis of acetylshikonin-treated HCT-15 and LoVo cells. (a) Cells were treated with 0, 1.25, 2.5, and 5? 0.05) increase of intracellular ROS generation in HCT-15 and LoVo cells. ROS level was increased by 24.1%, 55.7%, and 72.6% (= 0.0002) in HCT-15 cells and 105.1%, 188.8%, and 197.7% (= 0.011) in LoVo cells, after 6?h of treatment with 1.25, 2.5, and 5?= 0.0127) in HCT-15 cells and 123.3% (= 0.0094) in LoVo cells compared to cells only treated with acetylshikonin, when cotreated with NAC for 6?h KW-2478 (Figures 5(c) and 5(d)). The ROS generation after 6?h was immoderate after 6?h of treatment and had no discrimination; thus, we evaluated that 6?h was the most effective point to analyze ROS level in acetylshikonin-treated colorectal cancer cells. In NAC-treated cells, acetylshikonin-induced ROS generation in both cells was significantly ( 0.05) abolished. Open in a separate window Figure 5 ROS generation in colorectal cancer HCT-15 and LoVo cells treated with acetylshikonin. (a, c) Intracellular ROS generation in acetylshikonin-treated HCT-15 and LoVo cells and those cotreated with NAC was measured by using DCFH-DA (10? 0.05 KW-2478 and ?? 0.01, respectively). The data represent the mean SD of three independent experiments. Number sign indicates a significant difference of NAC-treated cells from acetylshikonin-treated cells (# 0.05). 3.4. NAC Inhibited ROS-Mediated Apoptosis by Acetylshikonin in HCT-15 and LoVo Cells The cell viability was increased when acetylshikonin-treated cells were cotreated with NAC. The viability was increased by 5.31% (= 0.4232), 45.47% (= 0.0019), and 60.74% (= 0.0014) in HCT-15 cells and 25.71% (= 0.002), 55.65% (= 0.0074), and 75.65% (= 0.005) in LoVo cells at 12?h, 24?h, and 48?h, respectively (Figure 6(a)). In the cell cycle arrest assay, the result showed that portions of subG1 phase were decreased in cells cotreated with NAC. Portions of subG1 phase were reduced by 8.66% in HCT-15 and 10.01% in LoVo compared to those cells treated only with acetylshikonin. Slight increases in G2/M phases occur, but no significant distinction was observed between cells treated only with NAC or DMSO (control) (Figure 6(b)). In the.