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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 triggers apoptosis in pancreatic cancer cells but not in normal HPDE-6 cells.(A) Structure of capsaicin. Apoptosis inducing effects of capsaicin (150 µM, 24 h) in (B) BxPC-3, (C) AsPC-1 and (D) HPDE-6 cells, was determined using annexin-V/FITC and propidium iodide and analyzed by flow cytometery. Results are expressed as mean ± SD (n = 4) of four independent experiments. *Statistically different when compared with control as analyzed by student's t-test (P<0.05). (E) BxPC-3 cells were treated with different concentrations of capsaicin for 24 h and (F) Cell were treated at different time intervals with 150 µM capsaicin and analyzed by immunoblotting for cleavage of caspase-9, caspase-3 and PARP as described in Materials and Methods. Each blot was stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. These experiments were performed three times independently with similar observation made in each experiment.
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pone-0020151-g001: Capsaicin triggers apoptosis in pancreatic cancer cells but not in normal HPDE-6 cells.(A) Structure of capsaicin. Apoptosis inducing effects of capsaicin (150 µM, 24 h) in (B) BxPC-3, (C) AsPC-1 and (D) HPDE-6 cells, was determined using annexin-V/FITC and propidium iodide and analyzed by flow cytometery. Results are expressed as mean ± SD (n = 4) of four independent experiments. *Statistically different when compared with control as analyzed by student's t-test (P<0.05). (E) BxPC-3 cells were treated with different concentrations of capsaicin for 24 h and (F) Cell were treated at different time intervals with 150 µM capsaicin and analyzed by immunoblotting for cleavage of caspase-9, caspase-3 and PARP as described in Materials and Methods. Each blot was stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. These experiments were performed three times independently with similar observation made in each experiment.

Mentions: Capsaicin, a homovanillic acid derivative (N-vanillyl-8-methyl-nonenamide) is an active and spicy component of hot chili pepper (Fig. 1A) [11], [12] and has been used as food additive for long time throughout the world, particularly in South Asian and Latin-American countries [13], [14], [15], [16], [17]. This alkaloid has been used to treat pain and inflammation, associated with a variety of diseases [18], [19], [20], [21]. Several recent studies demonstrated that capsaicin has antiproliferative effect in hepatic [22] gastric [23] prostate [24] colon [25] and leukemic cells [26]. Capsaicin generally exerts its physiologic response by stimulating vanilloid 1 (TRPV-1) receptor, however, receptor independent effects of capsaicin have been observed in cancer cells [25], [26], [27]. Capsaicin suppresses the growth of cancer cells by NF-kB inactivation, ROS generations, cell-cycle arrest and modulating EGFR/HER-2 pathways [28], [29], [30], [31], [32], [33]. The exact molecular mechanism by which capsaicin causes oxidative stress and apoptosis remains vague. We have shown previously that capsaicin induced apoptosis in pancreatic cancer cells was associated with ROS generation and mitochondrial disruption [34]. However the exact mechanism by which capsaicin causes ROS generation and cell death was not clear.


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 triggers apoptosis in pancreatic cancer cells but not in normal HPDE-6 cells.(A) Structure of capsaicin. Apoptosis inducing effects of capsaicin (150 µM, 24 h) in (B) BxPC-3, (C) AsPC-1 and (D) HPDE-6 cells, was determined using annexin-V/FITC and propidium iodide and analyzed by flow cytometery. Results are expressed as mean ± SD (n = 4) of four independent experiments. *Statistically different when compared with control as analyzed by student's t-test (P<0.05). (E) BxPC-3 cells were treated with different concentrations of capsaicin for 24 h and (F) Cell were treated at different time intervals with 150 µM capsaicin and analyzed by immunoblotting for cleavage of caspase-9, caspase-3 and PARP as described in Materials and Methods. Each blot was stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. These experiments were performed three times independently with similar observation made in each experiment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102063&req=5

pone-0020151-g001: Capsaicin triggers apoptosis in pancreatic cancer cells but not in normal HPDE-6 cells.(A) Structure of capsaicin. Apoptosis inducing effects of capsaicin (150 µM, 24 h) in (B) BxPC-3, (C) AsPC-1 and (D) HPDE-6 cells, was determined using annexin-V/FITC and propidium iodide and analyzed by flow cytometery. Results are expressed as mean ± SD (n = 4) of four independent experiments. *Statistically different when compared with control as analyzed by student's t-test (P<0.05). (E) BxPC-3 cells were treated with different concentrations of capsaicin for 24 h and (F) Cell were treated at different time intervals with 150 µM capsaicin and analyzed by immunoblotting for cleavage of caspase-9, caspase-3 and PARP as described in Materials and Methods. Each blot was stripped and reprobed with anti-β-actin antibody to ensure equal protein loading. These experiments were performed three times independently with similar observation made in each experiment.
Mentions: Capsaicin, a homovanillic acid derivative (N-vanillyl-8-methyl-nonenamide) is an active and spicy component of hot chili pepper (Fig. 1A) [11], [12] and has been used as food additive for long time throughout the world, particularly in South Asian and Latin-American countries [13], [14], [15], [16], [17]. This alkaloid has been used to treat pain and inflammation, associated with a variety of diseases [18], [19], [20], [21]. Several recent studies demonstrated that capsaicin has antiproliferative effect in hepatic [22] gastric [23] prostate [24] colon [25] and leukemic cells [26]. Capsaicin generally exerts its physiologic response by stimulating vanilloid 1 (TRPV-1) receptor, however, receptor independent effects of capsaicin have been observed in cancer cells [25], [26], [27]. Capsaicin suppresses the growth of cancer cells by NF-kB inactivation, ROS generations, cell-cycle arrest and modulating EGFR/HER-2 pathways [28], [29], [30], [31], [32], [33]. The exact molecular mechanism by which capsaicin causes oxidative stress and apoptosis remains vague. We have shown previously that capsaicin induced apoptosis in pancreatic cancer cells was associated with ROS generation and mitochondrial disruption [34]. However the exact mechanism by which capsaicin causes ROS generation and cell death was not clear.

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