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Myc down-regulation affects cyclin D1/cdk4 activity and induces apoptosis via Smac/Diablo pathway in an astrocytoma cell line.

ABSTRACT: OBJECTIVES:We investigated the antiproliferative effect of Myc down-regulation via cell proliferation inhibition, cell cycle perturbation and apoptosis in two human astrocytoma models (T98G and ADF) steadily expressing an inducible c-myc Anti-sense RNA. MATERIALS AND METHODS:Cell growth experiments were performed using the trypan blue dye exclusion test and cell cycle analysis was evaluated by flow cytometry. Cell cycle molecules were detected by Western blot analysis, co-immunoprecipitation and reverse transcription-polymerase chain reaction assays. RESULTS:We showed that Myc down-regulation in astrocytoma cells led to G1 accumulation and an inhibition of cell proliferation characterized by S phase delay. Co-immunoprecipitation experiments detected formation of inactive cyclin D1/cdk4 complexes as evaluated by presence of an active unphosphorylated form of retinoblastoma protein, the best characterized target substrate for cyclin D1/cdk4 complex, in ADF pINDc-myc anti-sense 7 cells. We also found that either p57Kip2 "apice" or p27Kip1 "apice" inhibitors bound to cyclin D1/cdk4 complex, thus, suggesting that they cooperated to inhibit the activity of cyclin D1/cdk4. Moreover, c-Myc down-regulation led to activation of the apoptotic mitochondrial pathway, characterized by release of cytochrome c and Smac/Diablo proteins and by reduction of c-IAP levels through activation of proteasome-mediated protein degradation system. CONCLUSIONS:Our results suggest that c-Myc could be considered as a good target for the study of new approaches in anticancer astrocytoma treatment.

SUBMITTER: Amendola D

PROVIDER: S-EPMC6496913 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Myc down-regulation affects cyclin D1/cdk4 activity and induces apoptosis via Smac/Diablo pathway in an astrocytoma cell line.

Amendola D D De Salvo M M Marchese R R Verga Falzacappa C C Stigliano A A Carico E E Brunetti E E Moscarini M M Bucci B B

Cell proliferation 20090201 1

<h4>Objectives</h4>We investigated the antiproliferative effect of Myc down-regulation via cell proliferation inhibition, cell cycle perturbation and apoptosis in two human astrocytoma models (T98G and ADF) steadily expressing an inducible c-myc Anti-sense RNA.<h4>Materials and methods</h4>Cell growth experiments were performed using the trypan blue dye exclusion test and cell cycle analysis was evaluated by flow cytometry. Cell cycle molecules were detected by Western blot analysis, co-immunopr ...[more]

PMID: 19143767

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Project description:BackgroundAs a regulator essential for many cell cycle-related proteins, the robust expression of Cell cycle-Related and Expression-elevated Protein in Tumor (CREPT) implicates a poor diagnosis of endoderm and mesoderm-derived tumors. Whether CREPT plays the same role in the tumorigenesis derived from ectodermal tissues remains elusive.MethodsTo explore the role of CREPT in ectoderm-derived tumors, cells from 7oral squamous cell carcinoma (OSCC) lines and 84clinical OSCC samples were exploited in this study. Quantitative PCR, Western blot assay and immunohistochemistry were applied in the evaluation of CREPT, cyclin D1 and c-Myc expression. Knocking-down of CREPT was performed by lentivirus delivering specific shRNA of CREPT. The effects of CREPT on OSCC were examined by cell proliferation, colony formation, apoptosis, cell migration and xenograft implantation experiments.ResultsCompared with human normal oral keratinocytes, OSCC cell lines showed a significantly elevated expression of CREPT in both mRNA and protein levels. Consistently, samples from OSCC patients also exhibited a noticeably stronger CREPT expression than the noncancerous samples. In contrast, knocking down of CREPT in OSCC cell lines significantly reduced proliferation, colony formation and migration as well as the expression of cyclin D1 and c-Myc, but promoted apoptosis. Statistical analysis also suggested that CREPT expression was significantly correlated with the T and N classification of OSCC. Furthermore, CAL27 mouse xenograft model confirmed that down-regulation of CREPT prohibited cyclin D1 and c-Myc expression, through which decreased the in vivo tumor growth, but increased the survival ratio of hosts.ConclusionIn OSCC cell lines, up-regulated CREPT expression enhanced cell proliferation, migration and cell cycle as well as promoted cyclin D1 and c-Myc expression as it did in endoderm and mesoderm-origin tumors. Our study strongly suggests that CREPT could be used as a marker for the OSCC prognosis and might work as a potential target in future OSCC therapy.

Project description:SMAC/Diablo, a pro-apoptotic protein, yet it is overexpressed in several cancer types. We have described a noncanonical function for SMAC/Diablo as a regulator of lipid synthesis during cancer cell proliferation and development. Here, we explore the molecular mechanism through which SMAC/Diablo regulates phospholipid synthesis. We showed that SMAC/Diablo directly interacts with mitochondrial phosphatidylserine decarboxylase (PSD) and inhibits its catalytic activity during synthesis of phosphatidylethanolamine (PE) from phosphatidylserine (PS). Unlike other phospholipids (PLs), PE is synthesized not only in the endoplasmic reticulum but also in mitochondria. As a result, PSD activity and mitochondrial PE levels were increased in the mitochondria of SMAC/Diablo-deficient cancer cells, with the total amount of cellular PLs and phosphatidylcholine (PC) being lower as compared to SMAC-expressing cancer cells. Moreover, in the absence of SMAC/Diablo, PSD inhibited cancer cell proliferation by catalysing the overproduction of mitochondrial PE and depleting the cellular levels of PC, PE and PS. Additionally, we demonstrated that both SMAC/Diablo and PSD colocalization in the nucleus resulted in increased levels of nuclear PE, that acts as a signalling molecule in regulating several nuclear activities. By using a peptide array composed of 768-peptides derived from 11 SMAC-interacting proteins, we identified six nuclear proteins ARNT, BIRC2, MAML2, NR4A1, BIRC5 and HTRA2 Five of them also interacted with PSD through motifs that are not involved in SMAC binding. Synthetic peptides carrying the PSD-interacting motifs of these proteins could bind purified PSD and inhibit the PSD catalytic activity. When targeted specifically to the mitochondria or the nucleus, these synthetic peptides inhibited cancer cell proliferation. To our knowledge, these are the first reported inhibitors of PSD acting also as inhibitors of cancer cell proliferation. Altogether, we demonstrated that phospholipid metabolism and PE synthesis regulated by the SMAC-PSD interaction are essential for cancer cell proliferation and may be potentially targeted for treating cancer.

Dataset Information

Myc down-regulation affects cyclin D1/cdk4 activity and induces apoptosis via Smac/Diablo pathway in an astrocytoma cell line.

Publications

Myc down-regulation affects cyclin D1/cdk4 activity and induces apoptosis via Smac/Diablo pathway in an astrocytoma cell line.

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