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Autophagy is differentially induced in prostate cancer LNCaP, DU145 and PC-3 cells via distinct splicing profiles of ATG5.

ABSTRACT: Autophagic responses to chemotherapeutic agents may vary greatly among different prostate cancer cells and have not been well characterized. In this study, we showed that valproic acid (VPA) induced conversion of LC3-I to LC3-II and formation of LC3 puncta, the typical markers of autophagy, in LNCaP and PC-3 cells. However, these markers were undetectable in DU145 cells upon autophagic stimulation, indicating a defect of autophagy in this cell line. Among several critical autophagy-related proteins, ATG5 and ATG12-ATG5 conjugates, which are essential for autophagy induction, were absent in DU145 cells. No canonical transcripts for full-length ATG5 but only two alternatively spliced ATG5 transcripts were identified in DU145 cells. These alternative transcripts lack one or two exons, leading to premature termination of ATG5 translation. Transfection of the wild-type ATG5 gene into DU145 cells rescued the production of ATG5 and ATG12-ATG5 conjugates, resulting in formation of LC3-II conjugates and LC3 puncta. Moreover, the levels of the SQSTM1 protein, which should be degradable as an autophagy adaptor, were much higher in DU145 than in LNCaP and PC-3 cells, but were significantly decreased after ATG5 restoration in DU145 cells. However, expression of wild-type ATG5 in DU145 or knockdown of ATG5 in LNCaP and PC-3 cells did not change the inhibitory effects of VPA on these cells. Collectively, these results indicated that VPA-induced autophagy in prostate cancer cells depended on ATG5 and more importantly, that the autophagy pathway was genetically impaired in DU145 cells, suggesting caution in interpreting autophagic responses in this cell line.

SUBMITTER: Ouyang DY

PROVIDER: S-EPMC3542215 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Autophagy is differentially induced in prostate cancer LNCaP, DU145 and PC-3 cells via distinct splicing profiles of ATG5.

Ouyang Dong-Yun DY Xu Li-Hui LH He Xian-Hui XH Zhang Yan-Ting YT Zeng Long-Hui LH Cai Ji-Ye JY Ren Shuai S

Autophagy 20121017 1

Autophagic responses to chemotherapeutic agents may vary greatly among different prostate cancer cells and have not been well characterized. In this study, we showed that valproic acid (VPA) induced conversion of LC3-I to LC3-II and formation of LC3 puncta, the typical markers of autophagy, in LNCaP and PC-3 cells. However, these markers were undetectable in DU145 cells upon autophagic stimulation, indicating a defect of autophagy in this cell line. Among several critical autophagy-related prote ...[more]

PMID: 23075929

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Project description:DNA methylation is a DNA methyltransferase-mediated epigenetic modification affecting gene expression. This process is involved in the initiation and development of malignant disease. 5-Aza-2'-deoxycytidine (5-Aza), a classic DNA methyltransferase inhibitor, possesses antitumor proliferation activity. However, whether 5-Aza induces cytotoxicity in solid tumors warrants further investigated. In this study, human prostate cancer (CaP) cells were treated with 5-Aza and subjected to cell viability and cytotoxicity analysis. Reverse transcription-polymerase chain reaction and methylation-specific polymerase chain reaction assay were utilized to test the gene expression and methylation status of the p53 and p21 gene promoters. The results showed that 5-Aza differentially inhibited spontaneous proliferation, arrested the cell cycle at S phase in DU145, at G1 phase in 22RV1 and LNCaP cells, and G2 phase in normal RWPE-1 cells, as well as induced the expression of phospho-H2A.X and tumor suppressive mammary serine protease inhibitor (maspin) in all three types of CaP cells. 5-Aza also increased p53 and p21 transcription through promoter demethylation, and decreased the expression of oncogene c-Myc in 22RV1 and LNCaP cells. Western blotting analysis showed that the poly (ADP-ribose) polymerase cleavage was detected in DU145 and 22RV1 cells. Moreover, there were no significant changes in p53, p21 and c-Myc expression in DU145 cells following treatment with 5-Aza. Thus, in responsible for its apoptotic induction and DNA damage, the mechanism of the antitumor activities of 5-Aza may involve in an increase of tumor suppressive maspin, upregulation of wild type p53-mediated p21 expression and a decrease of oncogene c-Myc level in 22RV1 and LNCaP cells, and enhancing the tumor suppressive maspin expression in DU145 cells. These results enriched our understanding of the multifaceted antitumor activity of 5-Aza, and provided the expression basis of biomarkers for its possible clinical application in prostate cancer.

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Dataset Information

Autophagy is differentially induced in prostate cancer LNCaP, DU145 and PC-3 cells via distinct splicing profiles of ATG5.

Publications

Autophagy is differentially induced in prostate cancer LNCaP, DU145 and PC-3 cells via distinct splicing profiles of ATG5.

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