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Protein kinase C? and caspase-3 modulate TRAIL-induced apoptosis in breast tumor cells.

ABSTRACT: This report describes that protein kinase C delta (PKC?) overexpression prevents TRAIL-induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL-induced apoptosis was significantly inhibited in PKC? overexpressing MCF-7 (MCF7/PKC?) cells. Our data reveal that PKC? inhibits caspase-8 activation, a first step in TRAIL-induced apoptosis, thus preventing TRAIL-induced apoptosis. Inhibition of PKC? using rottlerin or PKC? siRNA reverses the inhibitory effect of PKC? on caspase-8 activation leading to TRAIL-induced apoptosis. To determine if caspase-3-induced PKC? cleavage reverses its inhibition on caspase-8, we developed stable cell lines that either expresses wild-type PKC? (MCF-7/cas-3/PKC?) or caspase-3 cleavage-resistant PKC? mutant (MCF-7/cas-3/PKC? mut) utilizing MCF-7 cells expressing caspase-3. Cells that overexpress caspase-3 cleavage-resistant PKC? mutant (MCF-7/cas-3/PKC?mut) significantly inhibited TRAIL-induced apoptosis when compared to wild-type PKC? (MCF-7/cas-3/PKC?) expressing cells. In MCF-7/cas-3/PKC?mut cells, TRAIL-induced caspase-8 activation was blocked leading to inhibition of apoptosis when compared to wild-type PKC? (MCF-7/cas-3/PKC?) expressing cells. Together, these results strongly suggest that overexpression of PKC? inhibits caspase-8 activation leading to inhibition of TRAIL-induced apoptosis and its inhibition by rottlerin, siRNA, or cleavage by caspase-3 sensitizes cells to TRAIL-induced apoptosis. Clinically, PKC? overexpressing tumors can be treated with a combination of PKC? inhibitor(s) and TRAIL as a new treatment strategy.

SUBMITTER: Yin S

PROVIDER: S-EPMC3885240 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Protein kinase Cδ and caspase-3 modulate TRAIL-induced apoptosis in breast tumor cells.

Yin Shuping S Sethi Seema S Reddy Kaladhar B KB

Journal of cellular biochemistry 20101101 4

This report describes that protein kinase C delta (PKCδ) overexpression prevents TRAIL-induced apoptosis in breast tumor cells; however, the regulatory mechanism(s) involved in this phenomenon is(are) incompletely understood. In this study, we have shown that TRAIL-induced apoptosis was significantly inhibited in PKCδ overexpressing MCF-7 (MCF7/PKCδ) cells. Our data reveal that PKCδ inhibits caspase-8 activation, a first step in TRAIL-induced apoptosis, thus preventing TRAIL-induced apoptosis. I ...[more]

PMID: 20665667

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Project description:IntroductionTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells.MethodsWe conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors.ResultsThe primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity.ConclusionsThese data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.

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Protein kinase C? and caspase-3 modulate TRAIL-induced apoptosis in breast tumor cells.

Publications

Protein kinase Cδ and caspase-3 modulate TRAIL-induced apoptosis in breast tumor cells.

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