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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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Involvement of ETC complex-III in capsaicin mediated ROS generation.Mitochondrial complex-III activity was determined in the pure mitochondria isolated from control and 150 µM capsaicin treated (A) BxPC-3 and (B) AsPC-1 cells for 2, 4 and 24 h. (C) Comparison of complex-III activity in AsPC-1, BxPC-3, BxPC-3 ρ0 and HPDE-6 cells treated with 150 µM capsaicin for 24 h. (D) Capsaicin mediated decrease of complex-III activity was attenuated by pre-treatment of BxPC-3 cells with catalase (2000 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Results are expressed over control 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. (E) complex-III protein expression was determined by immunobloting using pure mitochondrial protein isolated from control and 150 µM capsaicin treated BxPC-3 cells for indicated time periods or (F) Pretreatment with catalase (2000 u/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Expression of complex-III protein was determined by immunoblotting from isolated pure mitochondria as described in the method. Each blot was stripped and reprobed with anti-Cox-IV antibody to ensure equal protein loading. These experiments were performed three times independently with similar result obtained in each experiment.
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pone-0020151-g004: Involvement of ETC complex-III in capsaicin mediated ROS generation.Mitochondrial complex-III activity was determined in the pure mitochondria isolated from control and 150 µM capsaicin treated (A) BxPC-3 and (B) AsPC-1 cells for 2, 4 and 24 h. (C) Comparison of complex-III activity in AsPC-1, BxPC-3, BxPC-3 ρ0 and HPDE-6 cells treated with 150 µM capsaicin for 24 h. (D) Capsaicin mediated decrease of complex-III activity was attenuated by pre-treatment of BxPC-3 cells with catalase (2000 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Results are expressed over control 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. (E) complex-III protein expression was determined by immunobloting using pure mitochondrial protein isolated from control and 150 µM capsaicin treated BxPC-3 cells for indicated time periods or (F) Pretreatment with catalase (2000 u/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Expression of complex-III protein was determined by immunoblotting from isolated pure mitochondria as described in the method. Each blot was stripped and reprobed with anti-Cox-IV antibody to ensure equal protein loading. These experiments were performed three times independently with similar result obtained in each experiment.

Mentions: Mitochondrial ETC complexes are the major generators of ROS in cells and tissues. Since we observed ROS generation by capsaicin, we wanted to see if mitochondria are involved in this process. We therefore determined the enzymatic activities and expression of mitochondrial complex-I, complex-II, complex-III and complex-IV in capsaicin treated BxPC-3, AsPC-1, HPDE-6 and BxPC-3 ρ0 cells. Capsaicin treatment inhibits complex-I activity by about 5–20% in BxPC-3 and 2.5–9% in AsPC-1 cells respectively as compared to respective controls (Fig. 3A–B). On the other hand, as expected, capsaicin failed to inhibit complex-I activity in BxPC-3 ρ0 cells (which lack mitochondrial DNA) and normal HPDE-6 cells (Fig. 3C). Next, we wanted to investigate whether this decrease in complex-I activity can be attenuated by anti-oxidants. Our results reveal that pretreatment of cells with catalase or EUK-134 substantially blocked the decreases in complex-I activity by capsaicin (Fig. 3D). Further capsaicin treatment significantly decreased the protein levels of complex-I protein complex after 4 h of treatment in a time dependent study and catalase or EUK-134 prevented this change (Fig. 3E–F). Similarly, complex-III activity by capsaicin was inhibited by 8–75% in both BxPC-3 and AsPC-1 cells (Fig. 4A–B). Nonetheless, capsaicin failed to decrease complex-III activity in BxPC-3 ρ0 cells (Fig. 4C). A modest decrease in complex III activity was however observed in HPDE-6 cells by capsaicin treatment (Fig. 4C). The decrease in complex-III activity in BxPC-3 cells by capsaicin was attenuated by catalase and EUK-134 (Fig. 4D). In agreement with activity data, expression of complex-III protein complex was drastically reduced in BxPC-3 cells following capsaicin treatment (Fig. 4E). The effect of capsaicin on the protein level of complex-III was abrogated by catalase and EUK-134 (Fig. 4F). Our results show that mitochondrial complex-III is more involved in capsaicin mediated ROS generation as compared to complex-I. Capsaicin had no effect on complex-II and IV (data not shown). Taken together, these results indicate that inhibition of mitochondrial complex I and complex-III by capsaicin cause ROS generation.


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)

Involvement of ETC complex-III in capsaicin mediated ROS generation.Mitochondrial complex-III activity was determined in the pure mitochondria isolated from control and 150 µM capsaicin treated (A) BxPC-3 and (B) AsPC-1 cells for 2, 4 and 24 h. (C) Comparison of complex-III activity in AsPC-1, BxPC-3, BxPC-3 ρ0 and HPDE-6 cells treated with 150 µM capsaicin for 24 h. (D) Capsaicin mediated decrease of complex-III activity was attenuated by pre-treatment of BxPC-3 cells with catalase (2000 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Results are expressed over control 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. (E) complex-III protein expression was determined by immunobloting using pure mitochondrial protein isolated from control and 150 µM capsaicin treated BxPC-3 cells for indicated time periods or (F) Pretreatment with catalase (2000 u/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Expression of complex-III protein was determined by immunoblotting from isolated pure mitochondria as described in the method. Each blot was stripped and reprobed with anti-Cox-IV antibody to ensure equal protein loading. These experiments were performed three times independently with similar result obtained in each experiment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102063&req=5

pone-0020151-g004: Involvement of ETC complex-III in capsaicin mediated ROS generation.Mitochondrial complex-III activity was determined in the pure mitochondria isolated from control and 150 µM capsaicin treated (A) BxPC-3 and (B) AsPC-1 cells for 2, 4 and 24 h. (C) Comparison of complex-III activity in AsPC-1, BxPC-3, BxPC-3 ρ0 and HPDE-6 cells treated with 150 µM capsaicin for 24 h. (D) Capsaicin mediated decrease of complex-III activity was attenuated by pre-treatment of BxPC-3 cells with catalase (2000 U/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Results are expressed over control 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. (E) complex-III protein expression was determined by immunobloting using pure mitochondrial protein isolated from control and 150 µM capsaicin treated BxPC-3 cells for indicated time periods or (F) Pretreatment with catalase (2000 u/ml) or EUK-134 (50 µM) for 1 h followed by 150 µM capsaicin for 24 h. Expression of complex-III protein was determined by immunoblotting from isolated pure mitochondria as described in the method. Each blot was stripped and reprobed with anti-Cox-IV antibody to ensure equal protein loading. These experiments were performed three times independently with similar result obtained in each experiment.
Mentions: Mitochondrial ETC complexes are the major generators of ROS in cells and tissues. Since we observed ROS generation by capsaicin, we wanted to see if mitochondria are involved in this process. We therefore determined the enzymatic activities and expression of mitochondrial complex-I, complex-II, complex-III and complex-IV in capsaicin treated BxPC-3, AsPC-1, HPDE-6 and BxPC-3 ρ0 cells. Capsaicin treatment inhibits complex-I activity by about 5–20% in BxPC-3 and 2.5–9% in AsPC-1 cells respectively as compared to respective controls (Fig. 3A–B). On the other hand, as expected, capsaicin failed to inhibit complex-I activity in BxPC-3 ρ0 cells (which lack mitochondrial DNA) and normal HPDE-6 cells (Fig. 3C). Next, we wanted to investigate whether this decrease in complex-I activity can be attenuated by anti-oxidants. Our results reveal that pretreatment of cells with catalase or EUK-134 substantially blocked the decreases in complex-I activity by capsaicin (Fig. 3D). Further capsaicin treatment significantly decreased the protein levels of complex-I protein complex after 4 h of treatment in a time dependent study and catalase or EUK-134 prevented this change (Fig. 3E–F). Similarly, complex-III activity by capsaicin was inhibited by 8–75% in both BxPC-3 and AsPC-1 cells (Fig. 4A–B). Nonetheless, capsaicin failed to decrease complex-III activity in BxPC-3 ρ0 cells (Fig. 4C). A modest decrease in complex III activity was however observed in HPDE-6 cells by capsaicin treatment (Fig. 4C). The decrease in complex-III activity in BxPC-3 cells by capsaicin was attenuated by catalase and EUK-134 (Fig. 4D). In agreement with activity data, expression of complex-III protein complex was drastically reduced in BxPC-3 cells following capsaicin treatment (Fig. 4E). The effect of capsaicin on the protein level of complex-III was abrogated by catalase and EUK-134 (Fig. 4F). Our results show that mitochondrial complex-III is more involved in capsaicin mediated ROS generation as compared to complex-I. Capsaicin had no effect on complex-II and IV (data not shown). Taken together, these results indicate that inhibition of mitochondrial complex I and complex-III by capsaicin cause ROS generation.

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