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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

Piperlongumine induces apoptosis in human breast cancer MCF-7 cells relative to human MCF-10A breast epithelial cells. (A) MCF-10A and MCF-7 cells were treated with 0, 1, 5 or 10 μM of piperlongumine for 24 h. Cell viability was evaluated by the MTT assay. (B, C) Cells were treated with 5 μM of piperlongumine for 36 h. Quantification of apoptosis was determined by flow cytometry (B). The proportion of the cells at the sub-G1 phase was also assessed by flow cytometry analysis as described in the Materials and Methods section (C). Means ± S.D. (n=3), *p < 0.05, **p < 0.01, ***p < 0.001. (D, E) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (D) and 0, 1 or 5 μM of piperlongumine for 24 h (E). Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of cleaved PARP. Actin was used as an equal loading control for normalization.
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f1-molce-38-4-327: Piperlongumine induces apoptosis in human breast cancer MCF-7 cells relative to human MCF-10A breast epithelial cells. (A) MCF-10A and MCF-7 cells were treated with 0, 1, 5 or 10 μM of piperlongumine for 24 h. Cell viability was evaluated by the MTT assay. (B, C) Cells were treated with 5 μM of piperlongumine for 36 h. Quantification of apoptosis was determined by flow cytometry (B). The proportion of the cells at the sub-G1 phase was also assessed by flow cytometry analysis as described in the Materials and Methods section (C). Means ± S.D. (n=3), *p < 0.05, **p < 0.01, ***p < 0.001. (D, E) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (D) and 0, 1 or 5 μM of piperlongumine for 24 h (E). Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of cleaved PARP. Actin was used as an equal loading control for normalization.

Mentions: To determine the effects of piperlongumine on the viability of normal and cancer cells, human MCF-10A breast epithelial cells and breast cancer MCF-7 cells were stimulated with varying concentrations of piperlongumine for 24 h. As shown in Fig. 1A, piperlongumine increased MCF-7 cell death in a concentration-dependent manner, but it barely affected the viability of MCF-10A cells. Maximum selective killing effects of piperlongumine on cancer cells were observed at 5 μM. Next, we evaluated apoptosis in MCF-10A and MCF-7 cells after piperlongumine (5 μM) treatment. Through annexin V and PI double staining, it was revealed that piperlongumine-treated MCF-7 cells underwent apoptosis whereas MCF-10A cells were less sensitive to piperlongumine-induced apoptosis. The apoptosis indices were 21.9% in MCF-7 cells and 6.7% in MCF-10A cells, respectively, after treatment of piperlongumine for 36 h (Fig. 1B). Moreover, piperlongumine significantly increased the proportion of MCF-7 cells in the sub-G1 stage, but not in MCF-10A cells (Fig. 1C). We then examined PARP cleavage, a hallmark of apoptosis. Following piperlongumine treatment, cleaved PARP levels increased in time- (Fig. 1D) and dose-dependent (Fig. 1E) manners in MCF-7 cells, but not in MCF-10A cells. These data clearly show that piperlongumine has a cancer cell-selective killing effect.


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)

Piperlongumine induces apoptosis in human breast cancer MCF-7 cells relative to human MCF-10A breast epithelial cells. (A) MCF-10A and MCF-7 cells were treated with 0, 1, 5 or 10 μM of piperlongumine for 24 h. Cell viability was evaluated by the MTT assay. (B, C) Cells were treated with 5 μM of piperlongumine for 36 h. Quantification of apoptosis was determined by flow cytometry (B). The proportion of the cells at the sub-G1 phase was also assessed by flow cytometry analysis as described in the Materials and Methods section (C). Means ± S.D. (n=3), *p < 0.05, **p < 0.01, ***p < 0.001. (D, E) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (D) and 0, 1 or 5 μM of piperlongumine for 24 h (E). Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of cleaved PARP. Actin was used as an equal loading control for normalization.
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Related In: Results  -  Collection

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f1-molce-38-4-327: Piperlongumine induces apoptosis in human breast cancer MCF-7 cells relative to human MCF-10A breast epithelial cells. (A) MCF-10A and MCF-7 cells were treated with 0, 1, 5 or 10 μM of piperlongumine for 24 h. Cell viability was evaluated by the MTT assay. (B, C) Cells were treated with 5 μM of piperlongumine for 36 h. Quantification of apoptosis was determined by flow cytometry (B). The proportion of the cells at the sub-G1 phase was also assessed by flow cytometry analysis as described in the Materials and Methods section (C). Means ± S.D. (n=3), *p < 0.05, **p < 0.01, ***p < 0.001. (D, E) MCF-10A and MCF-7 cells were treated with 5 μM of piperlongumine for the indicated time periods (D) and 0, 1 or 5 μM of piperlongumine for 24 h (E). Total protein isolated from cell lysates was subjected to immunoblot analysis for the measurement of cleaved PARP. Actin was used as an equal loading control for normalization.
Mentions: To determine the effects of piperlongumine on the viability of normal and cancer cells, human MCF-10A breast epithelial cells and breast cancer MCF-7 cells were stimulated with varying concentrations of piperlongumine for 24 h. As shown in Fig. 1A, piperlongumine increased MCF-7 cell death in a concentration-dependent manner, but it barely affected the viability of MCF-10A cells. Maximum selective killing effects of piperlongumine on cancer cells were observed at 5 μM. Next, we evaluated apoptosis in MCF-10A and MCF-7 cells after piperlongumine (5 μM) treatment. Through annexin V and PI double staining, it was revealed that piperlongumine-treated MCF-7 cells underwent apoptosis whereas MCF-10A cells were less sensitive to piperlongumine-induced apoptosis. The apoptosis indices were 21.9% in MCF-7 cells and 6.7% in MCF-10A cells, respectively, after treatment of piperlongumine for 36 h (Fig. 1B). Moreover, piperlongumine significantly increased the proportion of MCF-7 cells in the sub-G1 stage, but not in MCF-10A cells (Fig. 1C). We then examined PARP cleavage, a hallmark of apoptosis. Following piperlongumine treatment, cleaved PARP levels increased in time- (Fig. 1D) and dose-dependent (Fig. 1E) manners in MCF-7 cells, but not in MCF-10A cells. These data clearly show that piperlongumine has a cancer cell-selective killing effect.

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