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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

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.
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f5-molce-38-4-327: 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.

Mentions: It is well established that Keap1 is bound to Nrf2 under physiological conditions and inhibits the nuclear translocation of Nrf2. Oxidative stress or electrophiles can cause oxidation or covalent modification of cysteine residues present in Keap1, thereby activating Nrf2 signaling (Kim et al., 2014). Considering that piperlongumine contains the α,β-unsaturated carbonyl moiety (Fig. 5A), we expected that piperlongumine-induced Nrf2 activation and HO-1 expression is mediated by thiol modification of cysteine residues present in Keap1. In support with our hypothesis, the effects of piperlongumine on the nuclear translocation of Nrf2 and the expression of HO-1 were abrogated when cells were pre-treated with DTT, a thiol reducing agent (Figs. 5B and 5C). To investigate whether piperlongumine induces thiol modification through direct binding to Keap1, pull-down assays were performed using piperlongumine-conjugated Sepharose 4B beads with MCF-10A and MCF-7 cell lysates. As shown in Fig. 5D, piperlongumine directly bound to Keap1 both in MCF-10A and MCF-7 cells. These data imply that piperlongumine induces Nrf2 activation and HO-1 expression through modification of Keap1 cysteine residues.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

f5-molce-38-4-327: 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.
Mentions: It is well established that Keap1 is bound to Nrf2 under physiological conditions and inhibits the nuclear translocation of Nrf2. Oxidative stress or electrophiles can cause oxidation or covalent modification of cysteine residues present in Keap1, thereby activating Nrf2 signaling (Kim et al., 2014). Considering that piperlongumine contains the α,β-unsaturated carbonyl moiety (Fig. 5A), we expected that piperlongumine-induced Nrf2 activation and HO-1 expression is mediated by thiol modification of cysteine residues present in Keap1. In support with our hypothesis, the effects of piperlongumine on the nuclear translocation of Nrf2 and the expression of HO-1 were abrogated when cells were pre-treated with DTT, a thiol reducing agent (Figs. 5B and 5C). To investigate whether piperlongumine induces thiol modification through direct binding to Keap1, pull-down assays were performed using piperlongumine-conjugated Sepharose 4B beads with MCF-10A and MCF-7 cell lysates. As shown in Fig. 5D, piperlongumine directly bound to Keap1 both in MCF-10A and MCF-7 cells. These data imply that piperlongumine induces Nrf2 activation and HO-1 expression through modification of Keap1 cysteine residues.

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