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Role of mitochondrial electron transport chain complexes in capsaicin mediated oxidative stress leading to apoptosis in pancreatic cancer cells.

Pramanik KC, Boreddy SR, Srivastava SK - PLoS ONE (2011)

Bottom Line: On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells.Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells.Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University of Health Sciences Center, Amarillo, Texas, United States of America.

ABSTRACT
We evaluated the mechanism of capsaicin-mediated ROS generation in pancreatic cancer cells. The generation of ROS was about 4-6 fold more as compared to control and as early as 1 h after capsaicin treatment in BxPC-3 and AsPC-1 cells but not in normal HPDE-6 cells. The generation of ROS was inhibited by catalase and EUK-134. To delineate the mechanism of ROS generation, enzymatic activities of mitochondrial complex-I and complex-III were determined in the pure mitochondria. Our results shows that capsaicin inhibits about 2.5-9% and 5-20% of complex-I activity and 8-75% of complex-III activity in BxPC-3 and AsPC-1 cells respectively, which was attenuable by SOD, catalase and EUK-134. On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells. The ATP levels were drastically suppressed by capsaicin treatment in both BxPC-3 and AsPC-1 cells and attenuated by catalase or EUK-134. Oxidation of mitochondria-specific cardiolipin was substantially higher in capsaicin treated cells. BxPC-3 derived ρ(0) cells, which lack mitochondrial DNA, were completely resistant to capsaicin mediated ROS generation and apoptosis. Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells. Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level. Over-expression of catalase by transient transfection protected the cells from capsaicin-mediated ROS generation and apoptosis. Furthermore, tumors from mice orally fed with 2.5 mg/kg capsaicin show decreased SOD activity and an increase in GSSG/GSH levels as compared to controls. Taken together, our results suggest the involvement of mitochondrial complex-I and III in capsaicin-mediated ROS generation and decrease in antioxidant levels resulting in severe mitochondrial damage leading to apoptosis in pancreatic cancer cells.

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Capsaicin causes generation of mitochondrial ROS in pancreatic cancer cells.(A) and (B) BxPC-3 cells were treated with DMSO or 150 µM capsaicin for different time points and stained with HE and DCFDA and analyzed for superoxide radical and hydrogen peroxide respectively by flow cytometery. (C) AsPC-1, (D) HPDE-6, (E) BxPC-3 ρ0 cells were treated with 150 µM capsaicin for 2, 4 and 24 h and analyzed for total ROS generation (superoxide and hydrogen peroxide) by flow cytometer after staining the cells with HE and DCFDA. Results are expressed as mean ± SD (n = 3) from four independent experiments and data represents fold increase of ROS generation over control. *Statistically different when compared with control as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test P<0.05). (F) Effect of antioxidants on capsaicin mediated total ROS generation in BxPC-3 cells. Cells were treated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 2 h. Results are expressed as mean ± SD (n = 3) of four independent experiments. *Statistically different compared with control (P<0.05) and **statistically different when compared with capsaicin treatment (P<0.05), as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test.
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pone-0020151-g002: Capsaicin causes generation of mitochondrial ROS in pancreatic cancer cells.(A) and (B) BxPC-3 cells were treated with DMSO or 150 µM capsaicin for different time points and stained with HE and DCFDA and analyzed for superoxide radical and hydrogen peroxide respectively by flow cytometery. (C) AsPC-1, (D) HPDE-6, (E) BxPC-3 ρ0 cells were treated with 150 µM capsaicin for 2, 4 and 24 h and analyzed for total ROS generation (superoxide and hydrogen peroxide) by flow cytometer after staining the cells with HE and DCFDA. Results are expressed as mean ± SD (n = 3) from four independent experiments and data represents fold increase of ROS generation over control. *Statistically different when compared with control as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test P<0.05). (F) Effect of antioxidants on capsaicin mediated total ROS generation in BxPC-3 cells. Cells were treated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 2 h. Results are expressed as mean ± SD (n = 3) of four independent experiments. *Statistically different compared with control (P<0.05) and **statistically different when compared with capsaicin treatment (P<0.05), as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test.

Mentions: Intracellular ROS generation by capsaicin was evaluated by flow cytometry using hydroethidine (HE) and DCFDA. As shown in Fig. 2A, in a time dependent study, capsaicin treatment caused about 8–9 folds increase in superoxide radical within 1–2 h which decreased by 24 h as measured by HE fluorescence by flow cytometery. Similarly the generation of hydrogen peroxide upon capsaicin treatment increased by 4–7 folds within 1–2 h and then decreased but maintained levels higher than superoxide by 24 h, as measured by DCF fluorescence by flow cytometery (Fig. 2B). The generation of ROS was as early as 1 h as compared with controls in BxPC-3 cells. In order to see whether antioxidants can block ROS generation, cells were pretreated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or 50 µM EUK −134 (a cell permeable catalase mimetic) prior to capsaicin treatment. PEG-SOD almost completely blocked superoxide radical generation whereas PEG-catalase completely blocked hydrogen peroxide generation as measured by HE and DCF fluorescence respectively by flow cyometery (Fig S1A–B). To confirm the specificity of antioxidants, we used PEG-catalase to block superoxide radical generation. As expected, PEG-catalase completely failed to block superoxide radical generation (Fig S1C). Similarly, PEG-SOD failed to block hydrogen peroxide generation (data not shown). In subsequent experiments, we measured total ROS (superoxide radical+hydrogen peroxide) generation. Similarly, capsaicin treatment increased total ROS generation by about 2.5–4.5 fold in AsPC-1 cells with maximum at 2 h of treatment (Fig. 2C). Capsaicin treatment did not cause any significant ROS generation in normal HPDE-6 cells, suggesting that normal cells are resistant to the effects of capsaicin (Fig. 2D). In a combination treatment, our results indicate that PEG-SOD, PEG-catalase and EUK-134 substantially blocked capsaicin mediated total ROS generation in BxPC-3 cells (Fig. 2F).


Role of mitochondrial electron transport chain complexes in capsaicin mediated oxidative stress leading to apoptosis in pancreatic cancer cells.

Pramanik KC, Boreddy SR, Srivastava SK - PLoS ONE (2011)

Capsaicin causes generation of mitochondrial ROS in pancreatic cancer cells.(A) and (B) BxPC-3 cells were treated with DMSO or 150 µM capsaicin for different time points and stained with HE and DCFDA and analyzed for superoxide radical and hydrogen peroxide respectively by flow cytometery. (C) AsPC-1, (D) HPDE-6, (E) BxPC-3 ρ0 cells were treated with 150 µM capsaicin for 2, 4 and 24 h and analyzed for total ROS generation (superoxide and hydrogen peroxide) by flow cytometer after staining the cells with HE and DCFDA. Results are expressed as mean ± SD (n = 3) from four independent experiments and data represents fold increase of ROS generation over control. *Statistically different when compared with control as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test P<0.05). (F) Effect of antioxidants on capsaicin mediated total ROS generation in BxPC-3 cells. Cells were treated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 2 h. Results are expressed as mean ± SD (n = 3) of four independent experiments. *Statistically different compared with control (P<0.05) and **statistically different when compared with capsaicin treatment (P<0.05), as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102063&req=5

pone-0020151-g002: Capsaicin causes generation of mitochondrial ROS in pancreatic cancer cells.(A) and (B) BxPC-3 cells were treated with DMSO or 150 µM capsaicin for different time points and stained with HE and DCFDA and analyzed for superoxide radical and hydrogen peroxide respectively by flow cytometery. (C) AsPC-1, (D) HPDE-6, (E) BxPC-3 ρ0 cells were treated with 150 µM capsaicin for 2, 4 and 24 h and analyzed for total ROS generation (superoxide and hydrogen peroxide) by flow cytometer after staining the cells with HE and DCFDA. Results are expressed as mean ± SD (n = 3) from four independent experiments and data represents fold increase of ROS generation over control. *Statistically different when compared with control as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test P<0.05). (F) Effect of antioxidants on capsaicin mediated total ROS generation in BxPC-3 cells. Cells were treated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 2 h. Results are expressed as mean ± SD (n = 3) of four independent experiments. *Statistically different compared with control (P<0.05) and **statistically different when compared with capsaicin treatment (P<0.05), as analyzed by one-way ANOVA followed by Bonferroni's post-hoc test.
Mentions: Intracellular ROS generation by capsaicin was evaluated by flow cytometry using hydroethidine (HE) and DCFDA. As shown in Fig. 2A, in a time dependent study, capsaicin treatment caused about 8–9 folds increase in superoxide radical within 1–2 h which decreased by 24 h as measured by HE fluorescence by flow cytometery. Similarly the generation of hydrogen peroxide upon capsaicin treatment increased by 4–7 folds within 1–2 h and then decreased but maintained levels higher than superoxide by 24 h, as measured by DCF fluorescence by flow cytometery (Fig. 2B). The generation of ROS was as early as 1 h as compared with controls in BxPC-3 cells. In order to see whether antioxidants can block ROS generation, cells were pretreated with PEG-SOD (100 U/ml), PEG-catalase (500 U/ml) or 50 µM EUK −134 (a cell permeable catalase mimetic) prior to capsaicin treatment. PEG-SOD almost completely blocked superoxide radical generation whereas PEG-catalase completely blocked hydrogen peroxide generation as measured by HE and DCF fluorescence respectively by flow cyometery (Fig S1A–B). To confirm the specificity of antioxidants, we used PEG-catalase to block superoxide radical generation. As expected, PEG-catalase completely failed to block superoxide radical generation (Fig S1C). Similarly, PEG-SOD failed to block hydrogen peroxide generation (data not shown). In subsequent experiments, we measured total ROS (superoxide radical+hydrogen peroxide) generation. Similarly, capsaicin treatment increased total ROS generation by about 2.5–4.5 fold in AsPC-1 cells with maximum at 2 h of treatment (Fig. 2C). Capsaicin treatment did not cause any significant ROS generation in normal HPDE-6 cells, suggesting that normal cells are resistant to the effects of capsaicin (Fig. 2D). In a combination treatment, our results indicate that PEG-SOD, PEG-catalase and EUK-134 substantially blocked capsaicin mediated total ROS generation in BxPC-3 cells (Fig. 2F).

Bottom Line: On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells.Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells.Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University of Health Sciences Center, Amarillo, Texas, United States of America.

ABSTRACT
We evaluated the mechanism of capsaicin-mediated ROS generation in pancreatic cancer cells. The generation of ROS was about 4-6 fold more as compared to control and as early as 1 h after capsaicin treatment in BxPC-3 and AsPC-1 cells but not in normal HPDE-6 cells. The generation of ROS was inhibited by catalase and EUK-134. To delineate the mechanism of ROS generation, enzymatic activities of mitochondrial complex-I and complex-III were determined in the pure mitochondria. Our results shows that capsaicin inhibits about 2.5-9% and 5-20% of complex-I activity and 8-75% of complex-III activity in BxPC-3 and AsPC-1 cells respectively, which was attenuable by SOD, catalase and EUK-134. On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells. The ATP levels were drastically suppressed by capsaicin treatment in both BxPC-3 and AsPC-1 cells and attenuated by catalase or EUK-134. Oxidation of mitochondria-specific cardiolipin was substantially higher in capsaicin treated cells. BxPC-3 derived ρ(0) cells, which lack mitochondrial DNA, were completely resistant to capsaicin mediated ROS generation and apoptosis. Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells. Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level. Over-expression of catalase by transient transfection protected the cells from capsaicin-mediated ROS generation and apoptosis. Furthermore, tumors from mice orally fed with 2.5 mg/kg capsaicin show decreased SOD activity and an increase in GSSG/GSH levels as compared to controls. Taken together, our results suggest the involvement of mitochondrial complex-I and III in capsaicin-mediated ROS generation and decrease in antioxidant levels resulting in severe mitochondrial damage leading to apoptosis in pancreatic cancer cells.

Show MeSH
Related in: MedlinePlus