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. 2015 Apr;38(4):327-35.
doi: 10.14348/molcells.2015.2235. Epub 2015 Mar 20.

Heme oxygenase-1 Determines the Differential Response of Breast Cancer and Normal Cells to Piperlongumine

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Free PMC article

Heme oxygenase-1 Determines the Differential Response of Breast Cancer and Normal Cells to Piperlongumine

Ha-Na Lee et al. Mol Cells. .
Free PMC article

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.

Keywords: HO-1; Nrf2; apoptosis; breast cancer; piperlongumine.

Figures

Fig. 1.
Fig. 1.
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.
Fig. 2.
Fig. 2.
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.
Fig. 3.
Fig. 3.
Piperlongumine upregulates HO-1 expression through Nrf2 activation. (A) Cytosolic and nuclear extracts were prepared from MCF-10A and MCF-7 cells treated with or without piperlongumine (5 μM) for 3 h (MCF-10A) or 12 h (MCF-7). The protein levels of Nrf2 were measured by Western blot analysis. α-Tubulin and Lamin B1 was used as an equal loading control for normalization. (B) Cells were transfected with scrambled or Nrf2 siRNA for 24 h, and then treated with piperlongumine for additional 3 h (MCF-10A) or 12 h (MCF-7). The protein levels of HO-1, Nrf2 and actin were determined by Western blot analysis.
Fig. 4.
Fig. 4.
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.
Fig. 5.
Fig. 5.
Interaction of piperlongumine with cysteine residues within Keap1 activates Nrf2/HO-1 pathway. (A) The structure of piperlongu-mine. Electrophilic carbons containing α,β-unsaturated carbonyl moiety are denoted by asterisks. (B) Cells were treated with 5 μM of piperlongumine in the absence or presence of DTT 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. (C) MCF-10A and MCF-7 cells were exposed to piperlongumine for 24 h. Levels of HO-1, cleaved PARP and actin were measured by Western blot analysis. (D) Cell lysates were incubated with piperlongumine-conjugated Sepharose 4B beads (PL-Sepharose 4B) or Sepharose 4B beads alone, and then the pulled-down Keap1 was detected by immunoblot analysis.
Fig. 6.
Fig. 6.
The proposed mechanism underlying a cancer cell-selective killing effect of piperlongumine. Through direct binding, piperlongumine induces thiol modification of Keap1, thereby allowing nuclear translocation of Nrf2 and subsequent upregulation of HO-1 expression in normal and cancer cells. HO-1 determines the differential response of breast normal cells and cancer cells to piperlongumine, resulting in selective killing of cancer cells.

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