JS-K induces reactive oxygen species-dependent anti-cancer effects by targeting mitochondria respiratory chain complexes in gastric cancer

Abstract

As a nitric oxide (NO) donor prodrug, JS-K inhibits cancer cell proliferation, induces the differentiation of human leukaemia cells, and triggers apoptotic cell death in various cancer models. However, the anti-cancer effect of JS-K in gastric cancer has not been reported. In this study, we found that JS-K inhibited the proliferation of gastric cancer cells in vitro and in vivo and triggered mitochondrial apoptosis. Moreover, JS-K induced a significant accumulation of reactive oxygen species (ROS), and the clearance of ROS by antioxidant reagents reversed JS-K-induced toxicity in gastric cancer cells and subcutaneous xenografts. Although JS-K triggered significant NO release, NO scavenging had no effect on JS-K-induced toxicity in vivo and in vitro. Therefore, ROS, but not NO, mediated the anti-cancer effects of JS-K in gastric cancer. We also explored the potential mechanism of JS-K-induced ROS accumulation and found that JS-K significantly down-regulated the core proteins of mitochondria respiratory chain (MRC) complex I and IV, resulting in the reduction of MRC complex I and IV activity and the subsequent ROS production. Moreover, JS-K inhibited the expression of antioxidant enzymes, including copper-zinc-containing superoxide dismutase (SOD1) and catalase, which contributed to the decrease of antioxidant enzymes activity and the subsequent inhibition of ROS clearance. Therefore, JS-K may target MRC complex I and IV and antioxidant enzymes to exert ROS-dependent anti-cancer function, leading to the potential usage of JS-K in the prevention and treatment of gastric cancer.

Keywords: JS-K; apoptosis; gastric cancer; mitochondria respire chain complex; nitric oxide; reactive oxygen species.

Figure 1

JS‐K inhibits cell proliferation in different gastric cancer cell lines. A, The effect of JS‐K on the proliferation of gastric epithelial cells and gastric cancer cells. GES‐1, SGC7901, MGC803 and HGC27 cells were treated with different JS‐K concentrations for 48 h, and an MTT assay was used to determine cell viability. Cell survival rates were calculated by normalizing cell survivals in different groups with those in the control group. The IC50 values were calculated by using the GraphPad Prism 7 software. B, JS‐K inhibits the clonogenic ability of different gastric cancer cell lines. SGC7901, MGC803 and HGC27 cells were treated with different JS‐K concentrations for 48 h and then cultured with medium without JS‐K for another 7 d. Cells were stained with crystal violet, and the representative plates of three independent experiments are shown. C, JS‐K induces G2‐M phase arrest in SGC7901 cells. Cells were treated with JS‐K at the indicated concentration for 12 h and then collected to determine the cell cycle phases with flow cytometry

Figure 2

JS‐K induces caspase‐dependent apoptosis in SGC7901 cells. A, JS‐K induced apoptosis of SGC7901 cells in a dose‐dependent manner. Cells were treated with JS‐K at the indicated concentration for 24 h, and cell death was measured with flow cytometry. *P < 0.05. **P < 0.01. B, The effect of different caspase inhibitors on JS‐K‐induced cell death. SGC7901 cells were treated with JS‐K in the presence or absence of Z‐VAD (50 μmol/L), Z‐LEHD (50 μmol/L) or Z‐DEVD (50 μmol/L) for 24 h, and cell death was measured with flow cytometry. **P < 0.01. C. JS‐K induces PARP, caspase 9 and caspase 3 cleavage. SGC7901 cells were treated with JS‐K for the indicated time, and Western blotting was used to detect PARP, caspase 9 and caspase 3 cleavage. Actin was used as a loading control. D, JS‐K promotes caspase 9 and caspase 3 activation. SGC7901 cells were treated with JS‐K for 12 h and then harvested to measure the caspase 3 and caspase 9 activities with specific assay kits. More than three independent experiments were performed for each group, and the relative caspase activities were calculated by normalizing the caspase activities of all groups with the activities in a negative control group. *P < 0.05. **P < 0.01

Figure 3

JS‐K‐induced cytotoxicity was mediated by reactive oxygen species (ROS) accumulation but not nitric oxide (NO) release. A, JS‐K induces ROS accumulation and NO release in a dose‐dependent manner. SGC7901 cells were treated with JS‐K at the indicated concentration for 3 h and then harvested to measure the ROS and NO levels with flow cytometry. Three independent experiments were performed for each group. The relative ROS or NO levels were calculated by normalizing the ROS or NO levels in all the groups with those in a control group. *P < 0.05. **P < 0.01. B, JS‐K‐induced cytotoxicity was reversed by a ROS clearance reagent but not a NO scavenger. SGC7901 cells were treated with JS‐K in the presence or absence of carboxy‐PTIO (100 μmol/L), N‐acetyl‐L‐cysteine (NAC) (500 μmol/L) and Z‐VAD (50 μmol/L) for 24 h, and cell survival was measured with flow cytometry. **P < 0.01. C, NAC, but not carboxy‐PTIO, inhibited the PARP, caspase 3 and caspase 9 cleavage induced by JS‐K. SGC7901 cells were treated with JS‐K in the presence or absence of NAC, carboxy‐PTIO or Z‐VAD for 12 h, and Western blot analysis was used to detect PARP, caspase 3 and caspase 9 cleavage. Actin was used as a loading control. D, NAC suppresses JS‐K‐induced caspase 3 and caspase 9 activation. SGC7901 cells were treated with JS‐K in the presence or absence of NAC or Z‐VAD for 12 h and then harvested to measure caspase 9 and caspase 3 activities with specific assay kits. The relative caspase activities were calculated by normalizing the caspase activities of all the groups with the activities of a negative control group. *P < 0.05. E, The effect of NAC on ROS accumulation and nitric oxide release induced by JS‐K. SGC7901 cells were treated with JS‐K in the presence or absence of NAC for 12 h and then harvested to measure the ROS or nitric oxide levels with flow cytometry. The relative ROS and nitric oxide levels were calculated by normalizing the level of ROS and nitric oxide in all the groups with those in a negative control group. **P < 0.01

Figure 4

JS‐K induces reactive oxygen species‐dependent cytotoxicity by activating the mitochondria apoptosis pathway. A, NAC inhibits the depolarization of mitochondria induced by JS‐K. SGC7901 cells were treated with JS‐K in the presence or absence of NAC for 24 h, stained with JC‐1 and analysed with flow cytometry. The JC‐1 red/green fluorescence intensity ratio was normalized by comparing the data with the control group and is represented as relative mitochondrial membrane potential. Each experiment was performed in triplicate, and the representative measurements are shown. **P < 0.01. B, JS‐K induces the cytoplasmic release of Cytochrome c (Cyto‐C). SGC7901 cells were treated with 30 μmol/L JS‐K for the indicated time, and then harvested to isolate mitochondria and cytoplasm. Western blot analysis was used to detect the Cyto‐C levels in the mitochondria and cytoplasm. GAPDH and VADC were used as loading controls. C, The effect of NAC and carboxy‐PTIO on JS‐K‐induced Cyto‐C release. SGC7901 cells were treated with JS‐K in the presence or absence of NAC or carboxy‐PTIO for 12 h and then harvested to isolate mitochondria and cytoplasm. Western blot analysis was used to detect Cyto‐C levels in the mitochondria and cytoplasm. Actin and VADC were used as loading controls. D, The effect of Cyto‐C knockdown on JS‐K‐induced cell death. SGC7901 cells were transfected with Cyto‐C siRNA and a negative control siRNA for 24 h and then treated with or without JS‐K for another 24 h. Cells were stained with annexin V‐FITC and PI and analysed by flow cytometry. Western blot analysis was used to evaluate the efficiency of Cyto‐C knockdown. *P < 0.05. E, JS‐K‐induced apoptosis was inhibited by ectopic expression of Bcl‐2 or Bcl‐xL. SGC7901 cells were transfected with Bcl‐2, Bcl‐xL or negative control plasmids for 24 h, and then treated with 30 μmol/L JS‐K for another 24 h. Cells were stained with annexin V‐FITC and PI, and then analysed by flow cytometry. Western blot analysis was used to determine the Bcl‐2 and Bcl‐xL levels. GAPDH was used as a loading control. **P < 0.01

Figure 5

The anti‐tumour effects of JS‐K in a gastric cancer xenograft mouse model. BALB/c nude mice were injected subcutaneously with SGC7901 cells to establish the tumour‐bearing model, and JS‐K was administered once every 2 d. A, The in vivo anti‐tumour effect of JS‐K on gastric cancer. Tumour‐bearing mice were administered the indicated dose of JS‐K for 4 wk and then killed to isolate tumour tissue. The tumours were aligned according to size for imaging and then weighed using a microbalance. B, the malondialdehyde (MDA) content in the tumour tissues. The isolated tumour tissues were grinded and lysed, and the MDA level in the tumour tissue lysate was measured with a specific assay kit. **P < 0.01. (C,D) The effect of NAC and carboxy‐PTIO on JS‐K‐induced inhibition of gastric tumour growth. Tumour‐bearing mice were administered JS‐K (3 mg/kg) in the presence or absence of NAC (10 mg/kg) or carboxy‐PTIO (0.5 mg/kg) for 3 wk. The tumours were isolated from the killed mice, aligned according to size for imaging, and then weighed with a microbalance. **P < 0.01. (E,F) The effect of JS‐K on the liver and kidney. Mice were administered with JS‐K (3 mg/kg) once every 2 d for 30 d, and blood was obtained before being killed to isolate serum. The levels of alanine transaminase, aspartate transaminase and creatinine in serum were measured using specific assay kits. After blood collection, the mice were killed to isolate the liver and kidney. The tissues were cut into slices and then stained with haematoxylin and eosin. Four mice in each group were examined independently, and representative images are shown (×200)

Figure 6

JS‐K down‐regulates mitochondria respiratory chain (MRC) complex I and IV core proteins, as well as antioxidant enzymes. A, The effect of JS‐K on MRC complex activities in SGC7901 cells. Cells were treated with JS‐K at the indicated concentration for 12 h and then harvested to measure MRC complex activities using specific assay kits. The relative activities of the MRC complexes were calculated by normalizing the MRC complex activities in all the groups with the activity of a negative control group. **P < 0.01. B, N‐acetyl‐L‐cysteine (NAC) partially reversed the reduction of MRC complex I and IV activity induced by JS‐K. SGC7901 cells were treated with JS‐K in the absence or presence of NAC for 12 h, and the MRC complex I and IV activity was measured using specific kits. **P < 0.01. C, JS‐K decreased MRC complex I and IV activity in gastric cancer xenografts. The isolated gastric tumour tissues were lysed by homogenization and sonication, and the activities of MRC complex I and IV in the lysates were measured with specific assay kits. The relative MRC complex activities were calculated by normalizing the activities of MRC complex I and IV in all the groups with those in a negative control group. **P < 0.01. D, JS‐K down‐regulates Ndufs4 and COX2 in SGC7901 cells. Cells were treated with JS‐K at the indicated time‐points and then collected to determine the protein level of Ndufs4 and COX2 by Western blot. Actin was used as a loading control. E, JS‐K administration suppresses Ndufs4 and COX2 expression in tumour tissues. The tumour tissues isolated from the mice used in Figure 5C were minced and lysed, and Western blot assays were used to determine the protein levels of Ndfus4 and COX2 in the tumour tissue lysates. GAPDH was used as a loading control. F, JS‐K decreases SOD1 and catalase activity in SGC7901 cells. Cells were treated with JS‐K at the indicated concentration for 12 h and collected to measure SOD1 and catalase activity using specific assay kits. The activity in JS‐K‐treated groups was normalized to that in the control group. *P < 0.05, **P < 0.01. G, JS‐K decreases SOD1 and catalase activity in gastric tumour tissues. The lysates of isolated gastric tumour tissues (Figure 5A) were used to measure the activities of SOD1 and catalase using specific assay kits, and the relative activities were normalized by comparing the activities of SOD1 and catalase in all groups with those in a negative control group. **P < 0.01. H, JS‐K down‐regulates SOD1 and catalase in SGC7901 cells. Cells were treated with JS‐K at the indicated concentrations for 12 h and then harvested to detect the protein levels of SOD1 and catalase with Western blot analysis. Actin was used as a loading control. I, JS‐K administration suppresses SOD1 and catalase expression in gastric tumour tissues. Tumour tissues were isolated from the negative control and JS‐K‐treated mice used in Figure 5C and minced and lysed to determine SOD1 and catalase protein levels using Western blot. Actin was used as a loading control

Figure 7

The model of JS‐K‐induced anti‐tumour activity in gastric cancer. As a lead anti‐cancer drug compound, JS‐K significantly suppressed the expression of the core proteins of mitochondria respiratory chain (MRC) complex I and IV, resulting in the reduction of MRC complex I and IV activity and the subsequent reactive oxygen species (ROS) production. In addition, JS‐K down‐regulated SOD1 and catalase, which facilitated the reduction of SOD1 and catalase reducing activity and promoted the inhibition of ROS clearance. The aberrant ROS then induces mitochondria depolarization, caspase signalling pathway activation and subsequent apoptotic cell death. Therefore, MRC complex I and IV or antioxidant enzymes act as novel targets for JS‐K in mediating ROS‐dependent anti‐cancer activity in gastric cancer