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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-induced Nrf2 activation and HO-1 expression is not dependent on ROS generation. (A) Reduced GSH and GSSG levels were determined after cells were treated with piperlongumine for 3 h. (B) MCF-10A and MCF-7 cells were stimulated with piperlongumine in the absence or presence of either NAC or trolox for 3 h, and the intracellular ROS levels were measured by flow cytometry as described in the Materials and methods section. Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (C, E) Cells were pretreated with either NAC (C) or trolox (E) for 2 h, followed by piperlongumine treatment for 3 h (MCF-10A) or 12 h (MCF-7). Nuclear extracts were subjected to immunoblot analysis for the measurement of Nrf2. Lamin B was used as an equal loading control for normalization. (D, F) MCF-10A and MCF-7 cells were pretreated with either NAC (D) or trolox (F) for 2 h, followed by treatment of piperlongumine for 24 h. Levels of HO-1, cleaved PARP and actin were measured by Western blot analysis.
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f4-molce-38-4-327: Piperlongumine-induced Nrf2 activation and HO-1 expression is not dependent on ROS generation. (A) Reduced GSH and GSSG levels were determined after cells were treated with piperlongumine for 3 h. (B) MCF-10A and MCF-7 cells were stimulated with piperlongumine in the absence or presence of either NAC or trolox for 3 h, and the intracellular ROS levels were measured by flow cytometry as described in the Materials and methods section. Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (C, E) Cells were pretreated with either NAC (C) or trolox (E) for 2 h, followed by piperlongumine treatment for 3 h (MCF-10A) or 12 h (MCF-7). Nuclear extracts were subjected to immunoblot analysis for the measurement of Nrf2. Lamin B was used as an equal loading control for normalization. (D, F) MCF-10A and MCF-7 cells were pretreated with either NAC (D) or trolox (F) for 2 h, followed by treatment of piperlongumine for 24 h. Levels of HO-1, cleaved PARP and actin were measured by Western blot analysis.

Mentions: Because Nrf2 is one of major transcription factors involved in HO-1 regulation (Itoh et al., 1999), we examined whether piperlongumine could activate Nrf2 signaling. Following piperlongumine treatment, Nrf2 was translocated into the nucleus in both MCF-10A and MCF-7 cells (Fig. 3A). To determine whether HO-1 upregulation by piperlongumine was mediated via Nrf2 activation, cells were transiently transfected with siRNA against Nrf2. As shown in Fig 3B, piperlongumine failed to upregulate HO-1 expression in Nrf2 knockdown cells, indicating that activation of Nrf2 signaling is important for piperlongumine-induced HO-1 expression. It has been reported that piperlongumine modulates redox signaling, thereby increasing ROS production in cancer cells but not in normal cells (Raj et al., 2011). In the present study, we also observed that piperlongumine treatment lowered total and GSH levels, while increasing the level of GSSG in MCF-7 cells. However, there were no significant changes in the total GSH level and the GSH/GSSG ratio in piperlongumine-treated MCF-10A cells compared with those in DMSO-treated cells (Fig. 4A). Besides GSH depletion, piperlongumine treatment led to accumulation of intracellular ROS in MCF-7 cells, but this ROS generation was abrogated by pretreatment of NAC or trolox (Fig. 4B). Next, we examined the possible involvement of ROS in piperlongumine-induced Nrf2 activation and HO-1 expression. As shown in Figs. 4C and 4D, piperlongumine-induced nuclear translocation of Nrf2 and upregulation of HO-1 expression were markedly suppressed by NAC treatment in MCF-7 cells. Although piperlongumine did not increase ROS levels in MCF-10A cells, NAC also abolished piperlongumine-induced Nrf2 activation and HO-1 overexpression in MCF-10A cells, indicating that NAC, which can act as a thiol reducing agent, directly inhibited piperlongumine-mediated stimulation of the Nrf2/HO-1 pathway, rather than acting as a ROS scavenger. To confirm the role of ROS in piperlongumine-mediated effects, cells were treated with piperlongumine in the absence or presence of trolox, which also has ROS scavenging activity. Trolox failed to reverse the effect of piperlongumine on Nrf2 activation and HO-1 expression both in MCF-10A and MCF-7 cells (Figs. 4E and 4F). Moreover, the selective killing effect of piperlongumine on MCF-7 cells was not inhibited by trolox treatment (Fig. 4F). These results suggest that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell death do not rely on ROS generation.


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-induced Nrf2 activation and HO-1 expression is not dependent on ROS generation. (A) Reduced GSH and GSSG levels were determined after cells were treated with piperlongumine for 3 h. (B) MCF-10A and MCF-7 cells were stimulated with piperlongumine in the absence or presence of either NAC or trolox for 3 h, and the intracellular ROS levels were measured by flow cytometry as described in the Materials and methods section. Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (C, E) Cells were pretreated with either NAC (C) or trolox (E) for 2 h, followed by piperlongumine treatment for 3 h (MCF-10A) or 12 h (MCF-7). Nuclear extracts were subjected to immunoblot analysis for the measurement of Nrf2. Lamin B was used as an equal loading control for normalization. (D, F) MCF-10A and MCF-7 cells were pretreated with either NAC (D) or trolox (F) for 2 h, followed by treatment of piperlongumine for 24 h. Levels of HO-1, cleaved PARP and actin were measured by Western blot analysis.
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Related In: Results  -  Collection

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f4-molce-38-4-327: Piperlongumine-induced Nrf2 activation and HO-1 expression is not dependent on ROS generation. (A) Reduced GSH and GSSG levels were determined after cells were treated with piperlongumine for 3 h. (B) MCF-10A and MCF-7 cells were stimulated with piperlongumine in the absence or presence of either NAC or trolox for 3 h, and the intracellular ROS levels were measured by flow cytometry as described in the Materials and methods section. Means ± S.D. (n = 3), **p < 0.01, ***p < 0.001. (C, E) Cells were pretreated with either NAC (C) or trolox (E) for 2 h, followed by piperlongumine treatment for 3 h (MCF-10A) or 12 h (MCF-7). Nuclear extracts were subjected to immunoblot analysis for the measurement of Nrf2. Lamin B was used as an equal loading control for normalization. (D, F) MCF-10A and MCF-7 cells were pretreated with either NAC (D) or trolox (F) for 2 h, followed by treatment of piperlongumine for 24 h. Levels of HO-1, cleaved PARP and actin were measured by Western blot analysis.
Mentions: Because Nrf2 is one of major transcription factors involved in HO-1 regulation (Itoh et al., 1999), we examined whether piperlongumine could activate Nrf2 signaling. Following piperlongumine treatment, Nrf2 was translocated into the nucleus in both MCF-10A and MCF-7 cells (Fig. 3A). To determine whether HO-1 upregulation by piperlongumine was mediated via Nrf2 activation, cells were transiently transfected with siRNA against Nrf2. As shown in Fig 3B, piperlongumine failed to upregulate HO-1 expression in Nrf2 knockdown cells, indicating that activation of Nrf2 signaling is important for piperlongumine-induced HO-1 expression. It has been reported that piperlongumine modulates redox signaling, thereby increasing ROS production in cancer cells but not in normal cells (Raj et al., 2011). In the present study, we also observed that piperlongumine treatment lowered total and GSH levels, while increasing the level of GSSG in MCF-7 cells. However, there were no significant changes in the total GSH level and the GSH/GSSG ratio in piperlongumine-treated MCF-10A cells compared with those in DMSO-treated cells (Fig. 4A). Besides GSH depletion, piperlongumine treatment led to accumulation of intracellular ROS in MCF-7 cells, but this ROS generation was abrogated by pretreatment of NAC or trolox (Fig. 4B). Next, we examined the possible involvement of ROS in piperlongumine-induced Nrf2 activation and HO-1 expression. As shown in Figs. 4C and 4D, piperlongumine-induced nuclear translocation of Nrf2 and upregulation of HO-1 expression were markedly suppressed by NAC treatment in MCF-7 cells. Although piperlongumine did not increase ROS levels in MCF-10A cells, NAC also abolished piperlongumine-induced Nrf2 activation and HO-1 overexpression in MCF-10A cells, indicating that NAC, which can act as a thiol reducing agent, directly inhibited piperlongumine-mediated stimulation of the Nrf2/HO-1 pathway, rather than acting as a ROS scavenger. To confirm the role of ROS in piperlongumine-mediated effects, cells were treated with piperlongumine in the absence or presence of trolox, which also has ROS scavenging activity. Trolox failed to reverse the effect of piperlongumine on Nrf2 activation and HO-1 expression both in MCF-10A and MCF-7 cells (Figs. 4E and 4F). Moreover, the selective killing effect of piperlongumine on MCF-7 cells was not inhibited by trolox treatment (Fig. 4F). These results suggest that piperlongumine-induced Nrf2 activation, HO-1 expression and cancer cell death do not rely on ROS generation.

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