Search

Our previous studies suggested that microRNA (miR)-574-3p is a candidate tumor suppressor microRNA (miRNA) in human bladder cancer (BC). Among 17 down-regulated miRNAs, miR-574-3p is located on chromosome 4p14 where we had identified a chromosomal loss region by array-CGH in BC cell lines. MiR-574-3p expression was down-regulated in BC cell lines. Gain-of-function analysis revealed that cell proliferation, migration and invasion were significantly inhibited in miR‑574‑3p-transfected BC cell lines. Flow cytometry analysis showed that cell apoptosis was induced in miR-574-3p transfectants. Oligo microarray analysis suggested that the mesoderm development candidate 1 (MESDC1) gene was a target gene in miR-574-3p transfectants. Luciferase assays revealed that miR‑574‑3p was directly bound to MESDC1 mRNA. MESDC1 is predicted to be a novel actin-binding protein located on chromosome 15q13. Although the gene is conserved among many species, its functional role is still unknown in both human malignancies and normal tissues. Loss-of-function studies demonstrated that cell proliferation, migration and invasion were significantly inhibited in si-MESDC1-transfected BC cell lines. Flow cytometry analysis showed that apoptosis was induced in si-MESDC1 transfectants. We are the first to demonstrate that miR-574-3p is a miRNA with tumor suppressor function and that MESDC1 (which has a potential oncogenic function in BC) may be targeted by miR-574-3p.

Arsenic trioxide (As2O3) has been used with success in the treatment of acute promyelocytic leukemia. However, resistance to arsenite agents reduces their efficacy. We have isolated arsenite-resistant human epidermoid carcinoma KB cells, termed KAS. KAS cells were resistant to sodium arsenite (22-fold) and showed a reduced accumulation of arsenite as a result of an active efflux mechanism. Further analysis indicated that resistance of KAS cells extended to other drugs including cisplatin (17-fold), antimony potassium tartrate (11-fold) and doxorubicin (27-fold). Although increased expression of multidrug resistance protein 1 (MRP1) in KAS cells was confirmed by quantitative RT-PCR and immunoblot analysis, specific inhibitors of MRP1 did not completely eliminate arsenite resistance. The level of glutathione (GSH) in KAS cells was 3-fold higher than that in KB-3-1 cells, and the inhibition of GSH synthesis by buthionine sulfoximine (BSO) considerably increased the cytotoxic effect of arsenite on KAS cells. A pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridine-carboxylate P oxide (PAK-104P), partially reversed the arsenite resistance and increased the arsenite accumulation in KAS cells. We suggest that the increased level of GSH is involved in arsenite resistance and an as yet unidentified arsenite transporter is expressed in the arsenite-resistant KAS cells.

Large-scale gene expression profiling is an effective strategy for understanding the progression of bladder cancer (BC). The aim of this study was to identify genes that are expressed differently in the course of BC progression and to establish new biomarkers for BC. Specimens from 21 patients with pathologically confirmed superficial (n = 10) or invasive (n = 11) BC and 4 normal bladder samples were studied; samples from 14 of the 21 BC samples were subjected to microarray analysis. The validity of the microarray results was verified by real-time RT-PCR. Of the 136 up-regulated genes we detected, 21 were present in all 14 BCs examined (100%), 44 in 13 (92.9%), and the other 71 in 12 BCs (85.7%). Of 69 down-regulated genes, 25 were found in all 14 BCs (100%), 22 in 13 (92.9%), and the other 22 in 12 BCs (85.7%). Functional annotation revealed that of the up-regulated genes, 36% were involved in metabolism and 14% in transcription and processing; 25% of the down-regulated genes were linked to cell adhesion/surface and 21% to cytoskeleton/cell membrane. Real-time RT-PCR confirmed the microarray results obtained for the 6 most highly up- and the 2 most highly down-regulated genes. Among the 6 most highly up-regulated genes, CKS2 was the only gene with a significantly greater level of up-regulation in invasive than in superficial BC (p = 0.04). To confirm this result, we subjected all 21 BC samples to real-time PCR assay for CKS2. We found a considerable difference between superficial and invasive BC (p = 0.001). Interestingly, there was a considerable difference between the normal bladder and invasive BC (p = 0.001) and less difference between the normal bladder and superficial BC (p = 0.005). We identified several genes as promising candidates for diagnostic biomarkers of human BC and the CKS2 gene not only as a potential biomarker for diagnosing, but also for staging human BC. This is the first report demonstrating that CKS2 expression is strongly correlated with the progression of human BC.

Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), has been implicated in bladder cancer angiogenesis and invasion. However, the molecular basis of its role in invasion remains unclear. We investigated the expression of TP and 10 invasion-related genes in bladder cancers from 72 randomly selected patients by real-time two-step RT-PCR assay. We found that the expression levels of TP, MMP-9, uPA, and MMP-2 were significantly higher in invasive tumors than those in superficial tumors. Also, the expression level of TP significantly correlated with that of uPA, MMP-1, MMP-9, PAI-1 and VEGF. KK47/TP cells, bladder cancer cells that overexpress TP, had a higher expression of MMP-7 and MMP-9 than KK/CV cells that express lower level of TP in hypoxic condition. PC/TP cells, prostate cancer cells that overexpress TP, also had a higher expression of MMP-1 and MMP-7 than PC/CV cells that express no detectable TP. Taken together these data indicate that TP enhances the invasion of tumor cells through the induction of invasion-related genes.