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Selective killing of K-ras-transformed pancreatic cancer cells by targeting NAD(P)H oxidase.

Wang P, Sun YC, Lu WH, Huang P, Hu Y - Chin J Cancer (2015)

Bottom Line: Importantly, capsaicin preferentially inhibited the enzyme activity of NOX and induced severe ROS accumulation in K-ras-transformed cells compared with parental E6E7 cells.Furthermore, capsaicin effectively inhibited cell proliferation, prevented invasiveness of K-ras-transformed pancreatic cancer cells, and caused minimum toxicity to parental E6E7 cells.Our study provides a basis for developing a novel therapeutic strategy to effectively kill K-ras-transformed cells through a redox-mediated mechanism.

View Article: PubMed Central - PubMed

Affiliation: Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, 510060, P.R. China. pengwangsysu@foxmail.com.

ABSTRACT

Introduction: Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and plays a critical role in the pathogenesis of this malignancy. Increase of reactive oxygen species (ROS) has also been observed in a wide spectrum of cancers. This study aimed to investigate the mechanistic association between K-ras-induced transformation and increased ROS stress and its therapeutic implications in pancreatic cancer.

Methods: ROS level, NADPH oxidase (NOX) activity and expression, and cell invasion were examined in human pancreatic duct epithelial E6E7 cells transfected with K-ras (G12V) compared with parental E6E7 cells. The cytotoxic effect and antitumor effect of capsaicin, a NOX inhibitor, were also tested in vitro and in vivo.

Results: K-ras transfection caused activation of the membrane-associated redox enzyme NOX and elevated ROS generation through the phosphatidylinositol 3'-kinase (PI3K) pathway. Importantly, capsaicin preferentially inhibited the enzyme activity of NOX and induced severe ROS accumulation in K-ras-transformed cells compared with parental E6E7 cells. Furthermore, capsaicin effectively inhibited cell proliferation, prevented invasiveness of K-ras-transformed pancreatic cancer cells, and caused minimum toxicity to parental E6E7 cells. In vivo, capsaicin exhibited antitumor activity against pancreatic cancer and showed oxidative damage to the xenograft tumor cells.

Conclusions: K-ras oncogenic signaling causes increased ROS stress through NOX, and abnormal ROS stress can selectively kill tumor cells by using NOX inhibitors. Our study provides a basis for developing a novel therapeutic strategy to effectively kill K-ras-transformed cells through a redox-mediated mechanism.

No MeSH data available.


Related in: MedlinePlus

Selective killing and invasion inhibition ofK-ras–transformed cells by capsaicin. A, parental and K-ras–transformed E6E7 cells were incubated with or without 10 μmol/L capsaicin for 4 days in chamber slides. The cells were then stained with hematoxylin and eosin, which show the selective cytotoxicity of capsaicin to K-ras–transformed pancreatic cancer cells (magnification, ×100 and × 400). B, inhibition of cell proliferation by capsaicin in parental and K-ras–transformed E6E7 cells. Cell growth inhibition was measured by long-term MTT assay (mean ± SD of 3 experiments; *P < 0.05). C, measurement of ATP generation after treatment of 1 μmol/L diphenyleneiodonium (DPI) in parental and K-ras–transformed E6E7 cells at various time points as indicated (mean ± SD of 3 experiments). D, measurement results of ATP generation after treatment of 100 nmol/L DPI in normal fibroblasts and naturally occurring pancreatic cancer cells at various time points as indicated (mean ± SD of 3 experiments). E, parental and K-ras–transformed E6E7 cells were seeded onto the chamber insert with a layer of Matrigel matrix, with or without 10 μmol/L capsaicin. Then, 24 hour later, the cells that had digested the Matrigel and migrated to the lower surface of the insert were stained and photographed (magnification, ×100). F, the invasion of K-ras–transformed cells was assessed by counting the number of cells that had migrated onto the lower surface of the insert. The results are presented as the mean ± SD of the numbers from 3 microscopic fields (magnification, ×100). **, P < 0.01.
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Fig3: Selective killing and invasion inhibition ofK-ras–transformed cells by capsaicin. A, parental and K-ras–transformed E6E7 cells were incubated with or without 10 μmol/L capsaicin for 4 days in chamber slides. The cells were then stained with hematoxylin and eosin, which show the selective cytotoxicity of capsaicin to K-ras–transformed pancreatic cancer cells (magnification, ×100 and × 400). B, inhibition of cell proliferation by capsaicin in parental and K-ras–transformed E6E7 cells. Cell growth inhibition was measured by long-term MTT assay (mean ± SD of 3 experiments; *P < 0.05). C, measurement of ATP generation after treatment of 1 μmol/L diphenyleneiodonium (DPI) in parental and K-ras–transformed E6E7 cells at various time points as indicated (mean ± SD of 3 experiments). D, measurement results of ATP generation after treatment of 100 nmol/L DPI in normal fibroblasts and naturally occurring pancreatic cancer cells at various time points as indicated (mean ± SD of 3 experiments). E, parental and K-ras–transformed E6E7 cells were seeded onto the chamber insert with a layer of Matrigel matrix, with or without 10 μmol/L capsaicin. Then, 24 hour later, the cells that had digested the Matrigel and migrated to the lower surface of the insert were stained and photographed (magnification, ×100). F, the invasion of K-ras–transformed cells was assessed by counting the number of cells that had migrated onto the lower surface of the insert. The results are presented as the mean ± SD of the numbers from 3 microscopic fields (magnification, ×100). **, P < 0.01.

Mentions: The significant difference between parental and K-ras–transformed E6E7 cells in response to capsaicin-induced ROS accumulation prompted us to compare the cytotoxic effect of this compound in these 2 cell lines. HE staining was used to compare the morphology of these cells before and after treatment. As shown in Figure 3A, 4 days of treatment with 10 μmol/L capsaicin showed minimal effects on parental E6E7 cells. In sharp contrast, after identical treatment, the cell numbers decreased and drastic morphological alterations presented in K-ras–transformed cells as compared with the untreated control cells. Because parental E6E7 cells could not form colonies, an adapted MTT assay was used to compare the effect of capsaicin on long-term cell proliferation in parental and K-ras–transformed E6E7 cells. As shown in Figure 3B, capsaicin exhibited greater inhibition on K-ras–transformed cells after a 14-day period of incubation. To further test the hypothesis that K-ras transformation activates NOX and renders the transformed cells vulnerable to NOX inhibitor, DPI, a potent and specific inhibitor of flavoproteins including NAD(P)H oxidase [22], in pancreatic cancer cells and parental E6E7 cells was compared. As shown in Figure 3C and D, ATP generation levels in K-ras–transformed E6E7 cells and other pancreatic cancer cells were substantially decreased in a time-dependent manner after treatment with 1 μmol/L and 100 nmol/L DPI, and reached approximately 60% inhibition 24 hours after the treatment. In contrast, the same treatment showed no detectable cytotoxic effect on fibroblasts and only 20% inhibition on parental E6E7 cells.Figure 3


Selective killing of K-ras-transformed pancreatic cancer cells by targeting NAD(P)H oxidase.

Wang P, Sun YC, Lu WH, Huang P, Hu Y - Chin J Cancer (2015)

Selective killing and invasion inhibition ofK-ras–transformed cells by capsaicin. A, parental and K-ras–transformed E6E7 cells were incubated with or without 10 μmol/L capsaicin for 4 days in chamber slides. The cells were then stained with hematoxylin and eosin, which show the selective cytotoxicity of capsaicin to K-ras–transformed pancreatic cancer cells (magnification, ×100 and × 400). B, inhibition of cell proliferation by capsaicin in parental and K-ras–transformed E6E7 cells. Cell growth inhibition was measured by long-term MTT assay (mean ± SD of 3 experiments; *P < 0.05). C, measurement of ATP generation after treatment of 1 μmol/L diphenyleneiodonium (DPI) in parental and K-ras–transformed E6E7 cells at various time points as indicated (mean ± SD of 3 experiments). D, measurement results of ATP generation after treatment of 100 nmol/L DPI in normal fibroblasts and naturally occurring pancreatic cancer cells at various time points as indicated (mean ± SD of 3 experiments). E, parental and K-ras–transformed E6E7 cells were seeded onto the chamber insert with a layer of Matrigel matrix, with or without 10 μmol/L capsaicin. Then, 24 hour later, the cells that had digested the Matrigel and migrated to the lower surface of the insert were stained and photographed (magnification, ×100). F, the invasion of K-ras–transformed cells was assessed by counting the number of cells that had migrated onto the lower surface of the insert. The results are presented as the mean ± SD of the numbers from 3 microscopic fields (magnification, ×100). **, P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4593348&req=5

Fig3: Selective killing and invasion inhibition ofK-ras–transformed cells by capsaicin. A, parental and K-ras–transformed E6E7 cells were incubated with or without 10 μmol/L capsaicin for 4 days in chamber slides. The cells were then stained with hematoxylin and eosin, which show the selective cytotoxicity of capsaicin to K-ras–transformed pancreatic cancer cells (magnification, ×100 and × 400). B, inhibition of cell proliferation by capsaicin in parental and K-ras–transformed E6E7 cells. Cell growth inhibition was measured by long-term MTT assay (mean ± SD of 3 experiments; *P < 0.05). C, measurement of ATP generation after treatment of 1 μmol/L diphenyleneiodonium (DPI) in parental and K-ras–transformed E6E7 cells at various time points as indicated (mean ± SD of 3 experiments). D, measurement results of ATP generation after treatment of 100 nmol/L DPI in normal fibroblasts and naturally occurring pancreatic cancer cells at various time points as indicated (mean ± SD of 3 experiments). E, parental and K-ras–transformed E6E7 cells were seeded onto the chamber insert with a layer of Matrigel matrix, with or without 10 μmol/L capsaicin. Then, 24 hour later, the cells that had digested the Matrigel and migrated to the lower surface of the insert were stained and photographed (magnification, ×100). F, the invasion of K-ras–transformed cells was assessed by counting the number of cells that had migrated onto the lower surface of the insert. The results are presented as the mean ± SD of the numbers from 3 microscopic fields (magnification, ×100). **, P < 0.01.
Mentions: The significant difference between parental and K-ras–transformed E6E7 cells in response to capsaicin-induced ROS accumulation prompted us to compare the cytotoxic effect of this compound in these 2 cell lines. HE staining was used to compare the morphology of these cells before and after treatment. As shown in Figure 3A, 4 days of treatment with 10 μmol/L capsaicin showed minimal effects on parental E6E7 cells. In sharp contrast, after identical treatment, the cell numbers decreased and drastic morphological alterations presented in K-ras–transformed cells as compared with the untreated control cells. Because parental E6E7 cells could not form colonies, an adapted MTT assay was used to compare the effect of capsaicin on long-term cell proliferation in parental and K-ras–transformed E6E7 cells. As shown in Figure 3B, capsaicin exhibited greater inhibition on K-ras–transformed cells after a 14-day period of incubation. To further test the hypothesis that K-ras transformation activates NOX and renders the transformed cells vulnerable to NOX inhibitor, DPI, a potent and specific inhibitor of flavoproteins including NAD(P)H oxidase [22], in pancreatic cancer cells and parental E6E7 cells was compared. As shown in Figure 3C and D, ATP generation levels in K-ras–transformed E6E7 cells and other pancreatic cancer cells were substantially decreased in a time-dependent manner after treatment with 1 μmol/L and 100 nmol/L DPI, and reached approximately 60% inhibition 24 hours after the treatment. In contrast, the same treatment showed no detectable cytotoxic effect on fibroblasts and only 20% inhibition on parental E6E7 cells.Figure 3

Bottom Line: Importantly, capsaicin preferentially inhibited the enzyme activity of NOX and induced severe ROS accumulation in K-ras-transformed cells compared with parental E6E7 cells.Furthermore, capsaicin effectively inhibited cell proliferation, prevented invasiveness of K-ras-transformed pancreatic cancer cells, and caused minimum toxicity to parental E6E7 cells.Our study provides a basis for developing a novel therapeutic strategy to effectively kill K-ras-transformed cells through a redox-mediated mechanism.

View Article: PubMed Central - PubMed

Affiliation: Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, 510060, P.R. China. pengwangsysu@foxmail.com.

ABSTRACT

Introduction: Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and plays a critical role in the pathogenesis of this malignancy. Increase of reactive oxygen species (ROS) has also been observed in a wide spectrum of cancers. This study aimed to investigate the mechanistic association between K-ras-induced transformation and increased ROS stress and its therapeutic implications in pancreatic cancer.

Methods: ROS level, NADPH oxidase (NOX) activity and expression, and cell invasion were examined in human pancreatic duct epithelial E6E7 cells transfected with K-ras (G12V) compared with parental E6E7 cells. The cytotoxic effect and antitumor effect of capsaicin, a NOX inhibitor, were also tested in vitro and in vivo.

Results: K-ras transfection caused activation of the membrane-associated redox enzyme NOX and elevated ROS generation through the phosphatidylinositol 3'-kinase (PI3K) pathway. Importantly, capsaicin preferentially inhibited the enzyme activity of NOX and induced severe ROS accumulation in K-ras-transformed cells compared with parental E6E7 cells. Furthermore, capsaicin effectively inhibited cell proliferation, prevented invasiveness of K-ras-transformed pancreatic cancer cells, and caused minimum toxicity to parental E6E7 cells. In vivo, capsaicin exhibited antitumor activity against pancreatic cancer and showed oxidative damage to the xenograft tumor cells.

Conclusions: K-ras oncogenic signaling causes increased ROS stress through NOX, and abnormal ROS stress can selectively kill tumor cells by using NOX inhibitors. Our study provides a basis for developing a novel therapeutic strategy to effectively kill K-ras-transformed cells through a redox-mediated mechanism.

No MeSH data available.


Related in: MedlinePlus