Hypoxia induces metabolic alteration in cancer cells by stabilizing hypoxia-inducible factor 1α (HIF-1α (HIF1A)), which regulates the bioenergetic genes of glycolysis and lipid metabolic pathways. However, the target genes of hypoxia-induced metabolic alterations in the prostate remain uncertain. Mitochondrial aconitase (mACON) (ACONM) is an enzyme that is central to carbohydrate and energy metabolism and is responsible for the interconversion of citrate to isocitrate as part of the citric acid cycle in the human prostate. We evaluated the effects of the molecular mechanisms of hypoxia on mACON gene expression in PC-3 and LNCaP human prostate carcinoma cells. Immunoblotting assays revealed that hypoxia modulated mACON and lactate dehydrogenase A (LDHA) protein expression, while these effects were attenuated when HIF-1α was knocked down. Hypoxia induced fatty acid synthase (FASN) in PC-3 cells while hypoxia blocked FASN gene expression in LNCaP cells after 24-h incubation. Results of real-time RT-qPCR, immunoblotting, and transient gene expression assays revealed that hypoxia treatment or co-transfection with H IF-1α expression vector enhanced gene expression of mACON, implying that hypoxia modulated mACON at the transcriptional level. Hypoxia-induced mACON promoter activity is dependent on the DNA fragment located at −1013 to −842 upstream of the translation initiation site. l-mimosine, an iron chelator, stabilized HIF-1α but downregulated mACON gene expression, suggesting that iron chelation blocked the hypoxia-induced mACON gene expression. These results suggest that hypoxia dysregulates the expressions of LDHA, FASN, and mACON genes, and the hypoxia-induced mACON gene expression is via the HIF-1α-dependent and iron-dependent pathways in prostate carcinoma cells.
Ke-Hung Tsui, Li-Chuan Chung, Shyi-Wu Wang, Tsui-Hsia Feng, Phei-Lang Chang, and Horng-Heng Juang
See-Tong Pang, Wen-Chi Hsieh, Cheng-Keng Chuang, Chun-Hsiang Chao, Wen-Hui Weng, and Horng-Heng Juang
Thioredoxin-interacting protein (TXNIP), also known as vitamin-D3 upregulated protein 1, interacts with reduced thioredoxin. This protein modulates the cellular redox state and plays a role in stress-induced cellular apoptosis. This study examined TXNIP gene expression in prostate cancer cells. In vitro studies by immunoblot assay have shown that elevated glucose levels (1–15 mM) upregulate TXNIP gene expression two- to fourfold in human prostate carcinoma cells (LNCaP) and hepatocellular carcinoma cells (HepG2). Transient gene expression assays reveal that the promoter activity of the TXNIP gene is upregulated by glucose, 3-O-methylglucose, and maltose, but not by mannitol. These results suggest that glucose and 3-O-methylglucose induce TXNIP expression through both glucose metabolism-dependent and -independent pathways. Cotransfection of a plasmid expression carbohydrate response element-binding protein (ChREBP) with a TXNIP reporter vector into LNCaP cells dramatically enhances reporter activity in a low glucose (1 mM) condition. The effects of glucose are apparently mediated in a region located −341 to −324 bp upstream of the translational starting point of the TXNIP gene as indicated by 5′-deletion and site-directed mutagenesis reporter assays. Mutation of the putative carbohydrate response element (ChoRE) from CACGAGGGCAGCACGAG to TTTGAGGGCAGCACGAG abolishes glucose upregulation of TXNIP promoter activity. The present study demonstrates that TXNIP is transcription induced in both LNCaP and HepG2 cells in an increased glucose metabolism-dependent or -independent response, and a putative glucose regulatory system including ChREBP and ChoRE is needed for glucose-induced TXNIP gene in human prostate carcinoma cells.
Ke-Hung Tsui, Ying-Ling Chang, Tsui-Hsia Feng, Li-Chuan Chung, Tzu-Yi Lee, Phei-Lang Chang, and Horng-Heng Juang
Growth differentiation factor-15 (GDF15), a member of the transforming growth factor-β superfamily, is associated with human cancer progress. We evaluated the role GDF15 plays in tumorigenesis of prostate carcinoma PC-3 cells. Results from real-time RT-PCR and ELISA revealed that expression of GDF15 was approximately threefold higher in LNCaP cells than in PC-3 cells. Other prostate cell lines (PZ-HPV-7, CA-HPV-10, and DU145 cells) expressed extremely low levels of GDF15. Stable overexpression of GDF15 in PC-3 cells enhanced the degree of cell proliferation and invasion as shown in the 3H-thymidine incorporation assay and in the Matrigel invasion assay respectively. Soft agar assays and xenograft animal studies indicated that overexpression of GDF15 in PC-3 cells increased tumorigenesis in vitro and in vivo. Results from RT-PCR, immunoblot, and reporter assays revealed that overexpression of GDF15 resulted in decreased expression of maspin and upregulation of interleukin-6 (IL6), matriptase, and N-myc downstream-regulated gene 1 (NDRG1) expression. Further studies revealed that overexpression of IL6 enhanced GDF15 expression in LNCaP cells while knockdown of IL6 blocked the expression of GDF15 in PC-3 cells, suggesting that expression of GDF15 is upregulated by IL6. This study demonstrated that expression of GDF15 induces cell proliferation, invasion, and tumorigenesis of prostate carcinoma PC-3 cells. The enhancement of tumorigenesis and invasiveness of prostate carcinoma cells that stably overexpress GDF15 may be caused by the dysregulation of maspin, matriptase, and IL6 gene expression. The expression of GDF15 and IL6 is controlled via a positive feedback loop in PC-3 cells.