Kinase gene expression and subcellular protein expression pattern of protein kinase C isoforms in curcumin-treated human hepatocellular carcinoma Hep 3B cells.

Curcumin, a yellow component of turmeric or curry powder, has been demonstrated to exhibit anti-carcinogenic effects in vitro, in vivo, and in human clinical trials. One of its molecular targets is protein kinase C (PKC) which has been reported to play essential roles in apoptosis, cell proliferation, and carcinogenesis. In this study, PKC mRNA expression was significantly inhibited in curcumin-treated human hepatocellular carcinoma (HCC) Hep 3B cells identified using a kinase cDNA microarray. Furthermore, curcumin decreased total protein expression of all PKCs in a time-related manner by immunoblotting of whole cell lysates, nuclear, membrane, and cytosolic fractions. In cytosolic fraction, the expression of PKC-α was totally inhibited by curcumin. In contrast, the expression levels of PKC-ζ and -µ were dramatically increased. Increases in expression of PKC-δ and PKC-ζ in the membrane and nucleus, and PKC-ι in the membrane were detected. In summary, the changes in expression and distribution of subcellular PKC isoforms in curcumin-treated Hep 3B cells suggest possible PKC-associated anti-tumor mechanisms of curcumin and provide alternative therapies for human HCC.

Androgen depletion up-regulates cadherin-11 expression in prostate cancer.

Men with castration-resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin-11 (also known as OB-cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen-deprivation therapy up-regulates cadherin-11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22/26 (85%) primary PCa tumours from men with castration-resistant PCa expressed cadherin-11. In contrast, only 7/50 (14%) androgen-dependent PCa tumours expressed cadherin-11. In the MDA-PCa-2b xenograft animal model, cadherin-11 was expressed in the recurrent tumours following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin-11 and androgen receptor (AR), and cadherin-11 is expressed in very low levels or not expressed in AR-positive cell lines, including LNCaP, C4-2B4 and VCaP cells. We showed that AR likely regulates cadherin-11 expression in PCa through an indirect mechanism. Although re-expression of AR in the AR-negative PC3 cells led to the inhibition of cadherin-11 expression, depletion of androgen from the culture medium or down-regulation of AR by RNA interference in the C4-2B4 cells or VCaP cells only produced a modest increase of cadherin-11 expression. Promoter analysis indicated that cadherin-11 promoter does not contain a typical AR-binding element, and AR elicits a modest inhibition of cadherin-11 promoter activity, suggesting that AR does not regulate cadherin-11 expression directly. Together, these results suggest that androgen deprivation up-regulates cadherin-11 expression in prostate cancer, and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin-11 expression or function may be considered when applying androgen-ablation therapy.

Genomic structure, gene expression, and promoter analysis of human multidrug resistance-associated protein 7.

The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5'-flanking region at -1,780-1,287 bp, and at -611 to -208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226% by genotoxic 2-acetylaminofluorene and 347% by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.

cDNA cloning and genomic organization of the murine MRP7, a new ATP-binding cassette transporter.

Cellular resistance to cytotoxic drugs is a major obstacle to the treatment of disseminated cancers. Multidrug resistance protein (MRP) subfamily is a member of the ATP-binding cassette transporters which has been shown to cause multidrug resistance, except for P-glycoprotein. A new MRP subfamily gene, mrp7A (Abcc10), and its splicing variant, mrp7B, were isolated from mouse. The lengths of the open reading frames of mouse mrp7A and mrp7B are 4383 and 4506 bp, respectively. Estimated polypeptide sequences of mrp7A and mrp7B are 1460 and 1501 amino acids. The mouse mrp7 gene consists of at least 21 exons and 20 introns spanning around 20 kb that is almost the same as the one in human MRP7 gene, but different with the other MRP subfamily genes. The promoter region was isolated from the genomic clone and shown to support the luciferase activity seven fold over the promoterless negative control and two fold activity higher than the positive control of SV40 promoter. The analysis of tissue expression of mrp7A and mrp7B showed that these two transcripts express differentially in specific tissues.