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Heme oxygenase-1 determines the differential response of breast cancer and normal cells to piperlongumine.

Lee HN, Jin HO, Park JA, Kim JH, Kim JY, Kim B, Kim W, Hong SE, Lee YH, Chang YH, Hong SI, Hong YJ, Park IC, Surh YJ, Lee JK - Mol. Cells (2015)

Bottom Line: Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1).Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells.Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

View Article: PubMed Central - PubMed

Affiliation: KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, Seoul 139-709, Korea.

ABSTRACT
Piperlongumine, a natural alkaloid isolated from the long pepper, selectively increases reactive oxygen species production and apoptotic cell death in cancer cells but not in normal cells. However, the molecular mechanism underlying piperlongumine-induced selective killing of cancer cells remains unclear. In the present study, we observed that human breast cancer MCF-7 cells are sensitive to piperlongumine-induced apoptosis relative to human MCF-10A breast epithelial cells. Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1). Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells. However, knockdown of HO-1 expression and pharmacological inhibition of its activity abolished the ability of piperlongumine to induce apoptosis in MCF-7 cells, whereas those promoted apoptosis in MCF-10A cells, indicating that HO-1 has anti-tumor functions in cancer cells but cytoprotective functions in normal cells. Moreover, it was found that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell apoptosis are not dependent on the generation of reactive oxygen species. Instead, piperlongumine, which bears electrophilic α,β-unsaturated carbonyl groups, appears to inactivate Kelch-like ECH-associated protein-1 (Keap1) through thiol modification, thereby activating the Nrf2/HO-1 pathway and subsequently upregulating HO-1 expression, which accounts for piperlongumine-induced apoptosis in cancer cells. Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

No MeSH data available.


Related in: MedlinePlus

HO-1 mediates the selective effect of piperlongumine on cancer cell apoptosis. (A-C) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (A, C) and 0, 1 or 5 μM of piperlongumine for 24 h (B). (A, B) Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of HO-1 and Nrf2. Actin was used as an equal loading control for normalization. (C) The expression of HO-1 mRNA was determined by semi-quantitative RT-PCR. The level of GAPDH mRNA was used as an internal control. (D, E) Cells were transfected with scrambled or HO-1 siRNA for 24 h, and then exposed to piperlongumine for additional 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis (D). Cell viability was evaluated by the MTT assay (E). Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (F) MCF-10A and MCF-7 cells were treated with piperlongumine in the absence or presence of ZnPP for 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis.
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f2-molce-38-4-327: HO-1 mediates the selective effect of piperlongumine on cancer cell apoptosis. (A-C) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (A, C) and 0, 1 or 5 μM of piperlongumine for 24 h (B). (A, B) Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of HO-1 and Nrf2. Actin was used as an equal loading control for normalization. (C) The expression of HO-1 mRNA was determined by semi-quantitative RT-PCR. The level of GAPDH mRNA was used as an internal control. (D, E) Cells were transfected with scrambled or HO-1 siRNA for 24 h, and then exposed to piperlongumine for additional 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis (D). Cell viability was evaluated by the MTT assay (E). Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (F) MCF-10A and MCF-7 cells were treated with piperlongumine in the absence or presence of ZnPP for 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis.

Mentions: To elucidate the mechanism underlying the selective killing effect of piperlongumine on MCF-7 cells, we examined the level of cytoprotective proteins including HO-1 and Nrf2 after piperlongumine treatment. In contrast to our expectation, although piperlongumine selectively induced apoptosis in MCF-7 cells, HO-1 and Nrf2 expression levels were upregulated by piperlongumine in both MCF-10A and MCF-7 cells (Figs. 2A and 2B). Piperlongumine upregulated the expression of HO-1 and Nrf2 in MCF-10A cells at an earlier time compared to MCF-7 cells. In parallel with the elevated protein level of HO-1, the expression of its mRNA transcript was also increased in both MCF-10A and MCF-7 cells after piperlongumine treatment (Fig. 2C). To investigate whether HO-1 plays a role in determining the differential effects of piperlongumine, we utilized siRNA against HO-1. As shown in Fig. 2D, piperlongumine increased the level of cleaved PARP in MCF-10A cells transfected with HO-1 siRNA, indicating that HO-1 protects MCF-10A cells from piperlongumine-induced apoptosis. In contrast to MCF-10A cells, MCF-7 cells became less sensitive to piperlongumine-induced apoptosis when HO-1 was silenced (Figs. 2D and 2E), suggesting that piperlongumine triggers apoptosis of MCF-7 cells via HO-1 induction. The different role of HO-1 in piperlongumine-treated MCF-10A and MCF-7 cells was also confirmed by ZnPP (a pharmacological inhibitor of HO-1 activity) treatment. Similar to HO-1 siRNA, ZnPP abrogated piperlongumine-induced apoptosis in MCF-7 cells, but stimulated apoptosis of MCF-10A cells in response to piperlongumine (Fig. 2F). These findings suggest that HO-1 is the key protein involved in determining the selective effect of piperlongumine on cancer cell apoptosis. HO-1 protects MCF-10A cells from piperlongumine-induced apoptosis while amplifying the cytotoxicity of piperlongumine in MCF-7 cells.


Heme oxygenase-1 determines the differential response of breast cancer and normal cells to piperlongumine.

Lee HN, Jin HO, Park JA, Kim JH, Kim JY, Kim B, Kim W, Hong SE, Lee YH, Chang YH, Hong SI, Hong YJ, Park IC, Surh YJ, Lee JK - Mol. Cells (2015)

HO-1 mediates the selective effect of piperlongumine on cancer cell apoptosis. (A-C) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (A, C) and 0, 1 or 5 μM of piperlongumine for 24 h (B). (A, B) Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of HO-1 and Nrf2. Actin was used as an equal loading control for normalization. (C) The expression of HO-1 mRNA was determined by semi-quantitative RT-PCR. The level of GAPDH mRNA was used as an internal control. (D, E) Cells were transfected with scrambled or HO-1 siRNA for 24 h, and then exposed to piperlongumine for additional 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis (D). Cell viability was evaluated by the MTT assay (E). Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (F) MCF-10A and MCF-7 cells were treated with piperlongumine in the absence or presence of ZnPP for 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400307&req=5

f2-molce-38-4-327: HO-1 mediates the selective effect of piperlongumine on cancer cell apoptosis. (A-C) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (A, C) and 0, 1 or 5 μM of piperlongumine for 24 h (B). (A, B) Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of HO-1 and Nrf2. Actin was used as an equal loading control for normalization. (C) The expression of HO-1 mRNA was determined by semi-quantitative RT-PCR. The level of GAPDH mRNA was used as an internal control. (D, E) Cells were transfected with scrambled or HO-1 siRNA for 24 h, and then exposed to piperlongumine for additional 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis (D). Cell viability was evaluated by the MTT assay (E). Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (F) MCF-10A and MCF-7 cells were treated with piperlongumine in the absence or presence of ZnPP for 24 h. The protein levels of cleaved PAPR, HO-1 and actin were determined by Western blot analysis.
Mentions: To elucidate the mechanism underlying the selective killing effect of piperlongumine on MCF-7 cells, we examined the level of cytoprotective proteins including HO-1 and Nrf2 after piperlongumine treatment. In contrast to our expectation, although piperlongumine selectively induced apoptosis in MCF-7 cells, HO-1 and Nrf2 expression levels were upregulated by piperlongumine in both MCF-10A and MCF-7 cells (Figs. 2A and 2B). Piperlongumine upregulated the expression of HO-1 and Nrf2 in MCF-10A cells at an earlier time compared to MCF-7 cells. In parallel with the elevated protein level of HO-1, the expression of its mRNA transcript was also increased in both MCF-10A and MCF-7 cells after piperlongumine treatment (Fig. 2C). To investigate whether HO-1 plays a role in determining the differential effects of piperlongumine, we utilized siRNA against HO-1. As shown in Fig. 2D, piperlongumine increased the level of cleaved PARP in MCF-10A cells transfected with HO-1 siRNA, indicating that HO-1 protects MCF-10A cells from piperlongumine-induced apoptosis. In contrast to MCF-10A cells, MCF-7 cells became less sensitive to piperlongumine-induced apoptosis when HO-1 was silenced (Figs. 2D and 2E), suggesting that piperlongumine triggers apoptosis of MCF-7 cells via HO-1 induction. The different role of HO-1 in piperlongumine-treated MCF-10A and MCF-7 cells was also confirmed by ZnPP (a pharmacological inhibitor of HO-1 activity) treatment. Similar to HO-1 siRNA, ZnPP abrogated piperlongumine-induced apoptosis in MCF-7 cells, but stimulated apoptosis of MCF-10A cells in response to piperlongumine (Fig. 2F). These findings suggest that HO-1 is the key protein involved in determining the selective effect of piperlongumine on cancer cell apoptosis. HO-1 protects MCF-10A cells from piperlongumine-induced apoptosis while amplifying the cytotoxicity of piperlongumine in MCF-7 cells.

Bottom Line: Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1).Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells.Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

View Article: PubMed Central - PubMed

Affiliation: KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, Seoul 139-709, Korea.

ABSTRACT
Piperlongumine, a natural alkaloid isolated from the long pepper, selectively increases reactive oxygen species production and apoptotic cell death in cancer cells but not in normal cells. However, the molecular mechanism underlying piperlongumine-induced selective killing of cancer cells remains unclear. In the present study, we observed that human breast cancer MCF-7 cells are sensitive to piperlongumine-induced apoptosis relative to human MCF-10A breast epithelial cells. Interestingly, this opposing effect of piperlongumine appears to be mediated by heme oxygenase-1 (HO-1). Piperlongumine upregulated HO-1 expression through the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2) signaling in both MCF-7 and MCF-10A cells. However, knockdown of HO-1 expression and pharmacological inhibition of its activity abolished the ability of piperlongumine to induce apoptosis in MCF-7 cells, whereas those promoted apoptosis in MCF-10A cells, indicating that HO-1 has anti-tumor functions in cancer cells but cytoprotective functions in normal cells. Moreover, it was found that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell apoptosis are not dependent on the generation of reactive oxygen species. Instead, piperlongumine, which bears electrophilic α,β-unsaturated carbonyl groups, appears to inactivate Kelch-like ECH-associated protein-1 (Keap1) through thiol modification, thereby activating the Nrf2/HO-1 pathway and subsequently upregulating HO-1 expression, which accounts for piperlongumine-induced apoptosis in cancer cells. Taken together, these findings suggest that direct interaction of piperlongumine with Keap1 leads to the upregulation of Nrf2-mediated HO-1 expression, and HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine.

No MeSH data available.


Related in: MedlinePlus