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1.
Anticancer Res ; 34(9): 4767-73, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25202056

ABSTRACT

A previously established arsenite-resistant cell line, KAS, is also resistant to a variety of anticancer drugs. In order to understand responsible molecules for the multidrug resistance phenotype of KAS cells, we examined the expressions of ATP-binding cassette (ABC) transporters and found that the ABCB6 and ABCC1/ multidrug resistance protein 1 (ABCC1/MRP1) were increased. ABCC1/MRP1 was not completely responsible for the drug resistance spectrum of KAS cells and several reports have suggested that ABCB6 is related to anticancer drug and metal resistance. We, therefore, established and examined ABCB6-expressing KB cells and ABCB6-knockdown KAS cells. ABCB6 expression enhanced resistance to 5-fluorouracil (5-FU), SN-38 and vincristine (Vcr) but not to arsenite. Conversely, down-regulation of ABCB6 in KAS cells increased the sensitivity of KAS cells to 5-FU, SN-38 and Vcr, but not to arsenite. Our findings suggest that ABCB6 is involved in 5-FU, SN-38 and Vcr resistance.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Camptothecin/analogs & derivatives , Drug Resistance, Neoplasm/genetics , Fluorouracil/pharmacology , Gene Expression , Vincristine/pharmacology , ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents/pharmacology , Camptothecin/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Irinotecan , KB Cells , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Transfection
2.
Int J Oncol ; 40(4): 951-9, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22179486

ABSTRACT

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 miR­574­3p-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 miR­574­3p 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.


Subject(s)
MicroRNAs/genetics , Molecular Chaperones/genetics , Urinary Bladder Neoplasms/genetics , Aged , Apoptosis/genetics , Cell Growth Processes/genetics , Cell Line, Tumor , Cell Movement/genetics , Down-Regulation , Gene Expression , Humans , Male , Microarray Analysis , RNA, Messenger/genetics , Transfection , Urinary Bladder Neoplasms/pathology
3.
Eur J Cancer ; 48(6): 827-36, 2012 Apr.
Article in English | MEDLINE | ID: mdl-21745735

ABSTRACT

PURPOSE: The aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. METHODS: We checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N=40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. RESULTS: The expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. CONCLUSIONS: Our data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.


Subject(s)
Carcinoma, Renal Cell/metabolism , Kidney Neoplasms/metabolism , MicroRNAs/metabolism , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis/drug effects , Blotting, Western , Cell Cycle/drug effects , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation , Humans , Male , Middle Aged , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction
4.
Biochem Biophys Res Commun ; 417(1): 588-93, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22178073

ABSTRACT

We have previously found that restoration of tumor suppressive microRNA-1 (miR-1), induced cell apoptosis in bladder cancer (BC) cell lines. However, the apoptosis mechanism induced by miR-1 was not fully elucidated. Alternative splicing of mRNA precursors provides cancer cells with opportunities to translate many oncogenic protein variants, which promote cell proliferation and survival under unpreferable condition for cancer development. Serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splicing, plays a critical role for regulating apoptosis by splicing apoptosis-related genes. However, transcriptional regulation of SR proteins, themselves, has not been elucidated. In this study, we focused on splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) on the basis of our previous genome-wide gene expression analysis using miR-1-transfected BC cell lines because putative target sites of miR-1 are existed in 3'-untranslated region (UTR) of SRSF9 mRNA. The expression levels of mRNA of SRSF9 were extremely reduced in the miR-1 transfectants. A luciferase activity significantly decreased in the transfectants suggesting that actual binding occurred between miR-1 and 3'UTR of SRSF9 mRNA. Loss-of-function assays demonstrated that significant inhibitions of cell proliferation, migration, and invasion were observed in the si-SRSF9 transfectants. Apoptosis assays demonstrated that cell apoptosis fraction increased and that caspase-3/7 was activated in the si-SRSF9 transfectants. Our data indicated that tumor suppressive miR-1 induces apoptosis through direct inhibition of SRSF9 in BC. The identification of molecular mechanisms between miRNAs and SR proteins could provide novel apoptosis pathways and their epigenetic regulations and offer new strategies for BC treatment.


Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , MicroRNAs/metabolism , Nuclear Proteins/genetics , RNA-Binding Proteins/genetics , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , Caspase 3/biosynthesis , Caspase 7/biosynthesis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Knockdown Techniques , Humans , MicroRNAs/genetics , Neoplasm Invasiveness , Nuclear Proteins/antagonists & inhibitors , RNA-Binding Proteins/antagonists & inhibitors , Serine-Arginine Splicing Factors
5.
Adv Urol ; 2010: 943073, 2010.
Article in English | MEDLINE | ID: mdl-21197426

ABSTRACT

We report a case of a rapidly progressing giant retroperitoneal liposarcoma weighing 22 kg in a 41-year-old Japanese man, successfully treated with surgical excision. To our knowledge, this is the largest liposarcoma in the Japanese population reported in the literature.

6.
Oncol Rep ; 18(3): 721-7, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17671726

ABSTRACT

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.


Subject(s)
Arsenites/pharmacology , Sodium Compounds/pharmacology , Carcinoma, Squamous Cell , Cell Division/drug effects , Cell Membrane/physiology , DNA Probes , Drug Resistance, Neoplasm , Humans , KB Cells/drug effects , Multidrug Resistance-Associated Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction
7.
Biochemistry ; 46(33): 9443-52, 2007 Aug 21.
Article in English | MEDLINE | ID: mdl-17661442

ABSTRACT

Expression of the ATP-binding cassette transporter ABCB6 has been associated with multiple cellular functions, including resistance to several cytotoxic agents, iron homeostasis, and porphyrin transport. To further elucidate its physiological function and/or role in drug resistance, we determined the subcellular location of ABCB6. Using three novel ABCB6-specific antibodies, Western blot analysis of cells expressing cDNA-derived or endogenous ABCB6 revealed two distinct molecular weight forms. Confocal microscopy indicates that the protein localizes to both mitochondria and the plasma membrane. Differential centrifugation revealed that the lower molecular weight form predominantly resides in the mitochondria, while the larger protein form is more abundant in the plasma membrane. Preliminary studies indicate that ABCB6 is functionally relevant in the plasma membrane, where its expression prevents the accumulation of specific porphyrins in the cell. Digitonin solubilization of mitochondria demonstrated that ABCB6 is present in the outer mitochondrial membrane, while back-titration assays with the ABCB6-specific antibodies reveal that the nucleotide binding domain of ABCB6 is cytoplasmic. These studies are the first to demonstrate that ABCB6 exists in two molecular weight forms, is localized to both the outer mitochondrial membrane and the plasma membrane, and plays a functional role in the plasma membrane.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cell Membrane/metabolism , Mitochondrial Membranes/metabolism , ATP-Binding Cassette Transporters/analysis , Cell Line, Tumor , Cell Membrane/chemistry , Glycosylation , Humans , Mitochondrial Membranes/chemistry , Protein Binding
8.
J Urol ; 178(1): 301-7, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17499794

ABSTRACT

PURPOSE: SKP2 and CKS1 promote aggressive tumor behavior via the regulation of p27 degradation. Our previous DNA microarray analysis of human urothelial carcinoma and normal urothelial epithelium showed that in urothelial carcinoma the 2 most highly up-regulated genes among SKP2-p27 interaction related genes are SKP2 (4.7-fold) and CKS1 (2.2-fold). We hypothesized that SKP2 and CKS1 gene expression is associated with urothelial carcinoma invasiveness and prognosis. MATERIALS AND METHODS: A total of 84 urothelial carcinoma specimens from patients with bladder (71) and upper urinary tract (13) cancer were examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemical study. RESULTS: Real-time reverse transcriptase-polymerase chain reaction showed that the average mRNA expression level of SKP2 and CKS1 significantly correlated with tumor stage, that is superficial vs invasive urothelial carcinoma (SKP2 and CKS1, p<0.001 and 0.006) and grade (p<0.001 and 0.009, respectively). Of the superficial urothelial carcinomas examined the SKP2 and CKS1 expression level was significantly higher in pT1 than in pTa samples (p=0.005 and 0.017, respectively). Immunohistochemical expression patterns of SKP2 and CKS1 also significantly correlated with tumor stage (p<0.001 and 0.048) and grade (p=0.003 and 0.025, respectively). In contrast, p27 expression inversely correlated with tumor stage and grade (p<0.001 and 0.011, respectively). Logistic regression analysis revealed that while SKP2 mRNA expression was a significant dependent predictor of p27 expression (p=0.021), there was no correlation between CKS1 mRNA expression and p27 (p=0.748). Kaplan-Meier curves and log rank tests revealed that the high mRNA expression levels of SKP2 and CKS1 had a significant adverse effect on prognosis (p=0.043 and 0.003, respectively). CONCLUSIONS: Our results suggest that SKP2 has a major role in the regulation of p27 degradation and CKS1 has a supporting role for SKP2 function in human urothelial carcinoma.


Subject(s)
Carrier Proteins/genetics , Cyclin-Dependent Kinases/genetics , S-Phase Kinase-Associated Proteins/metabolism , Urinary Bladder Neoplasms/metabolism , Urologic Neoplasms/metabolism , CDC2-CDC28 Kinases , Disease Progression , Gene Expression Profiling , Humans , Immunohistochemistry , Logistic Models , Oligonucleotide Array Sequence Analysis , Prognosis , Proliferating Cell Nuclear Antigen/metabolism , Reverse Transcriptase Polymerase Chain Reaction
9.
Oncol Rep ; 16(3): 521-31, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16865252

ABSTRACT

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.


Subject(s)
Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Genome, Human , Neoplasm Proteins/genetics , Urinary Bladder Neoplasms/genetics , Aged , Aged, 80 and over , Disease Progression , Female , Gene Expression Profiling , Humans , Male , Middle Aged , Neoplasm Invasiveness , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Urinary Bladder Neoplasms/pathology
10.
Int J Mol Med ; 17(6): 1085-91, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16685420

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasm Invasiveness/genetics , Thymidine Phosphorylase/genetics , Transcriptional Activation , Urinary Bladder Neoplasms/enzymology , Urinary Bladder Neoplasms/pathology , Cell Line, Tumor , Female , Genes, Neoplasm , Humans , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Plasminogen Activator Inhibitor 1/genetics , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Urinary Bladder Neoplasms/genetics , Urokinase-Type Plasminogen Activator/genetics
11.
Cancer Lett ; 244(2): 239-46, 2006 Dec 08.
Article in English | MEDLINE | ID: mdl-16457948

ABSTRACT

Thymidine phosphorylase (TP) regulates intracellular thymidine metabolism and can enhance the anti-tumor effectiveness of 5'-deoxy-5-fluorouridine (5'-DFUR) by conversion of the pro-drug 5'-DFUR to 5-fluorouracil (5-FU) in tumor tissues. 5'-DFUR is an effective anti-tumor drug in cells expressing high levels of TP. 3'-Azido 3'-deoxythymidine (AZT) is a thymidine analog that has been proven useful in the treatment of acquired immunodefiency syndrome (AIDS). In this study, we found that AZT induces TP expression and enhances the sensitivity of human myeloid leukemia U937 cells to 5'-DFUR. Both the protein level and the activity of TP in U937 cells were elevated for 48h after exposure to AZT (20, 100 or 300muM). AZT enhanced TP promoter activity in a dose-dependent manner. AZT also increased TP mRNA levels in U937 cells as assayed by Real-time reverse-transcription PCR. AZT enhanced the cytotoxic effect of 5'-DFUR on U937 cells. A TP inhibitor, TPI, abrogated the cytotoxic activity of 5'-DFUR, and attenuated the combined cytotoxicity of AZT and 5'-DFUR. These results suggest that AZT enhances the cytotoxic effect of 5'-DFUR on U937 cells by upregulating TP activity in addition to its inhibition of thymidine kinase (TK) activity and reduction of intracellular dTTP pools.


Subject(s)
Antimetabolites/pharmacology , Cell Survival/drug effects , Drug Synergism , Floxuridine/toxicity , Thymidine Phosphorylase/metabolism , Zidovudine/pharmacology , Fluorouracil/toxicity , Humans , Transfection , U937 Cells/drug effects
12.
Mol Cell Biol ; 22(14): 5212-21, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12077348

ABSTRACT

Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.


Subject(s)
Thymidine Phosphorylase/deficiency , Uridine Phosphorylase/deficiency , Animals , Brain/pathology , Disease Models, Animal , Gene Targeting , Humans , Intestine, Small/enzymology , Liver/enzymology , Mice , Mice, Knockout , Mitochondrial Encephalomyopathies/enzymology , Mitochondrial Encephalomyopathies/genetics , Mitochondrial Encephalomyopathies/pathology , Phenotype , Thymidine Phosphorylase/genetics , Thymidine Phosphorylase/physiology , Uridine Phosphorylase/genetics , Uridine Phosphorylase/physiology
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