Dataset Information

Overexpressed PKC? downregulates the expression of PKC? in B16F10 melanoma: induction of apoptosis by PKC? via ceramide generation.

ABSTRACT: In the present study, we observed a marked variation in the expression of PKC? and PKC? isotypes in B16F10 melanoma tumor cells compared to the normal melanocytes. Interestingly, the tumor instructed expression or genetically manipulated overexpression of PKC? isotype resulted in enhanced G1 to S transition. This in turn promoted cellular proliferation by activating PLD1 expression and subsequent AKT phosphorylation, which eventually resulted in suppressed ceramide generation and apoptosis. On the other hand, B16F10 melanoma tumors preferentially blocked the expression of PKC? isotype, which otherwise could exhibit antagonistic effects on PKC?-PLD1-AKT signaling and rendered B16F10 cells more sensitive to apoptosis via generating ceramide and subsequently triggering caspase pathway. Hence our data suggested a reciprocal PKC signaling operational in B16F10 melanoma cells, which regulates ceramide generation and provide important clues to target melanoma cancer by manipulating the PKC?-ceramide axis.

SUBMITTER: Halder K

PROVIDER: S-EPMC3954766 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Overexpressed PKCδ downregulates the expression of PKCα in B16F10 melanoma: induction of apoptosis by PKCδ via ceramide generation.

Halder Kuntal K Banerjee Sayantan S Bose Anamika A Majumder Saikat S Majumdar Subrata S

PloS one 20140314 3

In the present study, we observed a marked variation in the expression of PKCα and PKCδ isotypes in B16F10 melanoma tumor cells compared to the normal melanocytes. Interestingly, the tumor instructed expression or genetically manipulated overexpression of PKCα isotype resulted in enhanced G1 to S transition. This in turn promoted cellular proliferation by activating PLD1 expression and subsequent AKT phosphorylation, which eventually resulted in suppressed ceramide generation and apoptosis. On t ...[more]

PMID: 24632809

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Project description:The diterpene ester PEP005 is a novel anticancer agent that activates PKC and cures subcutaneous murine melanoma by topical application. We now describe the in vitro cytostatic effects of PEP005 and the diterpene ester TPA, observed in 20% of human melanoma cell lines. Primary cultures of normal human neonatal fibroblasts were uniformly resistant to growth arrest, indicating a potential for tumor selectivity. Sensitive cells were induced to senesce and exhibited a G1 and G2/M arrest. There was sustained expression of p21WAF1/CIP1, irreversible dephosphorylation of the retinoblastoma gene product (Rb) and transcriptional silencing of E2F-responsive genes in sensitive cell lines. Activation of MEK1/2 by PKC was required for diterpene ester-induced senescence. Expression profiling revealed that the MAPK inhibitor HREV107 was expressed at a higher transcript level in resistant compared to sensitive cell lines. We propose that activation of PKC over-stimulates the Ras/Raf/MEK/ERK pathway, resulting in sustained induction of p21WAF1/CIP1, dephosphorylation of Rb and transcriptional silencing of E2F-responsive genes required for DNA synthesis and mitosis. To investigate the molecular changes associated with the senescent phenotype, we examined the unique transcriptional changes occurring in sensitive melanoma cell lines treated with TPA or PEP005. Initially a time-course cDNA microarray analysis was conducted on one sensitive and one resistant cell line treated with 1 Âµg/ml of TPA for 6, 24 h and 24 h recovery following 24 h treatment, to determine the earliest time point at which the most significant changes in transcription occurred. The results provided support for conducting array experiments with 24 h treatment, in which three sensitive and four resistant melanoma cell lines were treated with 1 Âµg/ml of either diterpene ester. Our primary objective was to identify those genes which were uniquely up or down-regulated in sensitive or resistant cell lines in response to treatment, which could reflect the phenotypic outcome. Through applying a series of stringent selective criteria (see Material and Methods) we found that the most significant changes occurred in the transcriptional repression of genes required for DNA synthesis and mitosis in cell lines sensitive to treatment (Table 1). To confirm that these changes were reflected at the protein level, western blot analysis was conducted on three sensitive and three resistant cell lines following 6 and 24 h treatment with TPA or PEP005. We also included a 24 h recovery time point to determine the irreversibility of the change.

Dataset Information

Overexpressed PKC? downregulates the expression of PKC? in B16F10 melanoma: induction of apoptosis by PKC? via ceramide generation.

Publications

Overexpressed PKCδ downregulates the expression of PKCα in B16F10 melanoma: induction of apoptosis by PKCδ via ceramide generation.

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