TY - JOUR
T1 - Heregulin-induced apoptosis is mediated by down-regulation of Bcl-2 and activation of caspase-7 and is potentiated by impairment of protein kinase C α activity
AU - Le, Xiao Feng
AU - Marcelli, Marco
AU - McWatters, Amanda
AU - Nan, Bicheng
AU - Mills, Gordon B.
AU - O’brian, Catherine A.
AU - Bast, Robert C.
N1 - Funding Information:
We sincerely thank Karen Ramirez at the Flow Cytometry Core Laboratory of the MD Anderson Cancer Center for her expert assistance with flow cytometry analysis. This work was supported in part by a Grant CA39930 from the National Cancer Institute and in part by an Institutional Research Grant IRG3721206 from the University of Texas MD Anderson Cancer Center.
PY - 2001
Y1 - 2001
N2 - Heregulins are a group of growth factors that play diverse and critical roles in the signaling network of the human epidermal growth factor receptor (HER or EGFR) superfamily. Our earlier studies have shown that recombinant heregulinβ1 (HRG) induces apoptosis in SKBr3 breast cancer cells that overexpress HER2. Here we report molecular mechanisms of HRG-induced apoptosis. HRG treatment of SKBr3 cells for 72 h decreased the level of Bcl-2 protein. HRG treatment led to degradation of poly (ADP-ribose) polymerase (PARP) and activated both caspase-9 and caspase-7. No significant activation of caspase-3, -6, or -8 was detected. Expression of exogenous caspase-7 by adenovirus-caspase-7 (Ad-casp-7) in SKBr3 cells resulted in apoptosis, which mimicked the effect of HRG treatment. Expression of exogenous caspase-7 had no impact on Bcl-2 expression, but promoted PARP degradation. Two highly selective inhibitors of protein kinase C (PKC), GF109203X (GF) and Ro318425 (Ro), significantly enhanced HRG-induced apoptosis as determined by flow cytometric analysis and DNA fragmentation assay. Accordingly, the PKC inhibitor GF further decreased the level of Bcl-2 protein and further degraded PARP in HRG-treated cells. Assay of PKC activity indicated that HRG activated PKC in SKBr3 cells, predominantly affecting the PKCα isoform. To confirm which PKC isoform(s) mediated potentiation of HRG-induced apoptosis, the profile of PKC isoforms was measured in SKBr3 cells. Five PKC isoforms, PKCα, PKCι, PKCζ, PKCλ, and PKCδ as well as their receptors (RACK1) were expressed in this cell line. Treatment with PKC inhibitors GF and Ro decreased protein levels of both PKCα and PKCδ at 24 h. PKCα levels were still depressed at 72 h. GF and Ro had little effect on the expression of other PKC isoforms. An inhibitor of classical PKC isoforms (Go6976) enhanced HRG-induced apoptosis, whereas the PKCδ selective inhibitor rottlerin did not. As PKCα was the only classical isoform expressed in SKBr3 cells, the effect of Go6976 on HRG-induced apoptosis largely related to inhibition of PKCα. Constitutive expression of wild-type PKCα attenuated the apoptosis produced by HRG and GF. Consequently, HRG-induced apoptosis in SKBr3 cells appeared to involve down-regulation of Bcl-2 protein, activation of caspase-9 and caspase-7, and degradation of PARP. Inhibition of PKC function enhanced HRG-induced apoptosis, leading to synergistic down-regulation of Bcl-2 expression. Impairment of the PKCα isoform alone was sufficient to potentiate HRG-induced apoptosis.
AB - Heregulins are a group of growth factors that play diverse and critical roles in the signaling network of the human epidermal growth factor receptor (HER or EGFR) superfamily. Our earlier studies have shown that recombinant heregulinβ1 (HRG) induces apoptosis in SKBr3 breast cancer cells that overexpress HER2. Here we report molecular mechanisms of HRG-induced apoptosis. HRG treatment of SKBr3 cells for 72 h decreased the level of Bcl-2 protein. HRG treatment led to degradation of poly (ADP-ribose) polymerase (PARP) and activated both caspase-9 and caspase-7. No significant activation of caspase-3, -6, or -8 was detected. Expression of exogenous caspase-7 by adenovirus-caspase-7 (Ad-casp-7) in SKBr3 cells resulted in apoptosis, which mimicked the effect of HRG treatment. Expression of exogenous caspase-7 had no impact on Bcl-2 expression, but promoted PARP degradation. Two highly selective inhibitors of protein kinase C (PKC), GF109203X (GF) and Ro318425 (Ro), significantly enhanced HRG-induced apoptosis as determined by flow cytometric analysis and DNA fragmentation assay. Accordingly, the PKC inhibitor GF further decreased the level of Bcl-2 protein and further degraded PARP in HRG-treated cells. Assay of PKC activity indicated that HRG activated PKC in SKBr3 cells, predominantly affecting the PKCα isoform. To confirm which PKC isoform(s) mediated potentiation of HRG-induced apoptosis, the profile of PKC isoforms was measured in SKBr3 cells. Five PKC isoforms, PKCα, PKCι, PKCζ, PKCλ, and PKCδ as well as their receptors (RACK1) were expressed in this cell line. Treatment with PKC inhibitors GF and Ro decreased protein levels of both PKCα and PKCδ at 24 h. PKCα levels were still depressed at 72 h. GF and Ro had little effect on the expression of other PKC isoforms. An inhibitor of classical PKC isoforms (Go6976) enhanced HRG-induced apoptosis, whereas the PKCδ selective inhibitor rottlerin did not. As PKCα was the only classical isoform expressed in SKBr3 cells, the effect of Go6976 on HRG-induced apoptosis largely related to inhibition of PKCα. Constitutive expression of wild-type PKCα attenuated the apoptosis produced by HRG and GF. Consequently, HRG-induced apoptosis in SKBr3 cells appeared to involve down-regulation of Bcl-2 protein, activation of caspase-9 and caspase-7, and degradation of PARP. Inhibition of PKC function enhanced HRG-induced apoptosis, leading to synergistic down-regulation of Bcl-2 expression. Impairment of the PKCα isoform alone was sufficient to potentiate HRG-induced apoptosis.
KW - Apoptosis
KW - Bcl-2
KW - Caspase-7
KW - HER2
KW - Heregulin
KW - Protein kinase C
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UR - http://www.scopus.com/inward/citedby.url?scp=0035857113&partnerID=8YFLogxK
U2 - 10.1038/sj.onc.1205039
DO - 10.1038/sj.onc.1205039
M3 - Article
C2 - 11781840
AN - SCOPUS:0035857113
SN - 0950-9232
VL - 20
SP - 8258
EP - 8269
JO - Oncogene
JF - Oncogene
IS - 57
ER -