Functional role and tobacco smoking effects on methylation of CYP1A1 gene in prostate cancer.

Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2'-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment.

CYP1B1 promotes tumorigenesis via altered expression of CDC20 and DAPK1 genes in renal cell carcinoma.

BACKGROUND: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC. METHODS: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses. RESULTS: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p < 0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples. CONCLUSIONS: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.

Functional role of DNA mismatch repair gene PMS2 in prostate cancer cells.

DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.

Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma.

Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells.

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis.

Cytochrome P450 1B1 polymorphisms and risk of renal cell carcinoma in men.

The cytochrome P450 1B1 (CYP1B1) enzyme activates xenobiotics to reactive forms as well as convert estradiol to 4-hydroxy-estradiol that has been shown to play a role in the carcinogenesis process of the kidney in male but not female animals. Prior reports show polymorphic variants of CYP1B1 to alter catalytic activity, and thus, we hypothesize that polymorphisms of the CYP1B1 gene are involved in the malignant transformation of the renal cell in men. The genetic distributions of five CYP1B1 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 480 normal healthy subjects and 403 sporadic renal cell carcinoma cases. All subjects were Caucasian men. The sites evaluated were codons 48 (C → G, Arg → Gly, rs10012), 119 (G → T, Ala → Ser, rs1056827), 432 (C → G, Leu → Val, rs1056836), 449 (C → T, Asp, rs1056837), and 453 (A → G, Asn → Ser, rs1800440). A trend was demonstrated for the 432 Val/Val (χ2, P = 0.06) and 449 T/T (χ2, P = 0.1) genotypes to play a protective role against renal cancer. Odds ratio (95 % confidence interval) for Val/Val compared to Leu/Leu at codon 432 was 0.65 (0.44-0.95) and T/T compared to C/C at codon 449 was 0.67 (0.45-0.99). Codons 432 and 449 were observed to be linked (D = 0.24), and haplotype involving 432 Val and 449 T was significantly reduced in cancer cases (P = 0.04). No association was found, however, when analyzing polymorphic sites with clinical stage of cancer. These results demonstrate polymorphisms of CYP1B1 to be associated with renal carcinogenesis and are of importance in understanding their role in the pathogenesis of renal cell carcinoma.