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Protein kinase Cδ amplifies ceramide formation via mitochondrial signaling in prostate cancer cells
Makoto Sumitomo, … , Tomohiko Asano, Masamichi Hayakawa
Makoto Sumitomo, … , Tomohiko Asano, Masamichi Hayakawa
Published March 15, 2002
Citation Information: J Clin Invest. 2002;109(6):827-836. https://doi.org/10.1172/JCI14146.
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Protein kinase Cδ amplifies ceramide formation via mitochondrial signaling in prostate cancer cells

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Abstract

We studied the role of protein kinase C isoform PKCδ in ceramide (Cer) formation, as well as in the mitochondrial apoptosis pathway induced by anticancer drugs in prostate cancer (PC) cells. Etoposide and paclitaxel induced Cer formation and apoptosis in PKCδ-positive LNCaP and DU145 cells but not in PKCδ-negative LN-TPA or PC-3 cells. In contrast, these drugs induced mitotic cell cycle arrest in all PC cell lines. Treatment with Rottlerin, a specific PKCδ inhibitor, significantly inhibited drug-induced Cer formation and apoptosis in LNCaP cells, as did overexpression of dominant negative–type PKCδ. Overexpression of wild-type PKCδ had an opposite effect in PC-3 cells. Notably, etoposide induced biphasic Cer formation in LNCaP cells. The early and transient Cer increase resulted from de novo Cer synthesis, while the late and sustained Cer accumulation was derived from sphingomyelin hydrolysis by neutral sphingomyelinase (nSMase). Cer, in turn, induced mitochondrial translocation of PKCδ and stimulated the activity of this kinase, promoting cytochrome c release and caspase-9 activation. Furthermore, the specific caspase-9 inhibitor LEHD-fmk significantly inhibited etoposide-induced nSMase activation, Cer accumulation, and PKCδ mitochondrial translocation. These results indicate that PKCδ plays a crucial role in activating anticancer drug–induced apoptosis signaling by amplifying the Cer-mediated mitochondrial amplification loop.

Authors

Makoto Sumitomo, Motoi Ohba, Junichi Asakuma, Takako Asano, Toshio Kuroki, Tomohiko Asano, Masamichi Hayakawa

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Figure 1

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Correlation of anticancer drug–induced ceramide formation and apoptosis ...
Correlation of anticancer drug–induced ceramide formation and apoptosis induction with PKCδ protein expression in PC cells. (a) Total cell lysates (20 μg) from PC cells were analyzed by Western blot analysis as described in Methods using specific Ab’s. (b) PC cells were treated with or without 10 μM etoposide (upper panel) or 100 nM paclitaxel (lower panel) for 24 hours, and Cer levels were determined as described in Methods. The data are expressed as fold increase relative to each control and are representative of three experiments. (c) DNA flow cytometry histograms of PC cells cultured in media containing 10% FCS treated with or without 10 μM etoposide for the indicated periods. The data are representative of three experiments. Percentage of apoptosis (sub-G1 DNA content) is indicated for each sample. (d) LNCaP and PC-3 cells were treated with 10 μM etoposide or 100 nM paclitaxel for various periods. Sub-G1 DNA content and fragmented DNA in each sample were measured by cell cycle analysis and ELISA. Bars represent SD. Experiments were repeated three times with similar results.

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