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1.
Urol Oncol ; 30(4): 502-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-20864363

RESUMO

OBJECTIVE: To investigate the relationship between the expression of the cancer metastasis suppressor gene KAI1 and MMP-2 and MMP-9 in human bladder cancer cell lines that express variable levels of KAI1. MATERIALS AND METHODS: Five bladder cancer cell lines (BL-28/0, BL-13/0, BL-17/0/×1, B10, and D2) were grown in standard culture conditions. Gelatinase activities in serum-free conditioned medium were assessed using gelatin zymography. Whole cell lysates were prepared and Western blotting used to detect the protein expression of MMP-9, MMP-2, TIMP-1, TIMP-2, and KAI1. Semiquantitative RT-PCR was performed to analyze the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, and KAI1. RESULTS: Western blotting analysis confirmed that KAI1 was expressed in BL-28/0 and Bl-13/0 but not in D2, B10 and BL-17/0/×1 cell lines. This was consistent with in vitro invasion assays reported previously which showed that cell lines lacking KAI1 expression were 2× to 10× more invasive than cell lines that expressed KAI1. MMP-2 protein was detected in BL-28/0, BL-13/0. and BL-17/0/×1 only. Very low levels of MMP-9 were present in BL-28/0, BL-13/0, B10, and BL-17/0/×1 but not D2, whilst very low levels of TIMP-1 were present in all cell lines. No TIMP-2 was detected. Gelatin zymography showed detectable MMP-2 expression in conditioned medium from BL-28/0 and BL-13/0. Very weak MMP-9 was detected in BL-28/0 conditioned medium only. mRNA expression of MMP-2 was only detectable in BL-28/0 and BL-13/0 cell lines. MMP-9 mRNA levels were extremely low in all lines and not detectable in D2 cells. TIMP-1 and TIMP-2 mRNA were detected in all lines. CONCLUSION: We found that KAI1 expression in bladder cancer cell lines is related to a poor invasive potential and expression of latent MMP-2 but not MMP-9. These results are unexpected given other studies showing high levels of MMP-2 and MMP-9 protein expression in patients with invasive bladder cancer. This may reflect differences in the regulation and secretion of MMP-2 and MMP-9 in vitro compared with the in vivo situation, where tumor cells interact with the surrounding environment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína Kangai-1/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Proteína Kangai-1/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
2.
Neoplasia ; 10(12): 1421-32, following 1432, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19048121

RESUMO

Down-regulation of the KAI1 (CD82) metastasis suppressor is common in advanced human cancer, but underlying mechanism(s) regulating KAI1 expression are only now being elucidated. Recent data provide evidence that low levels of KAI1 mRNA in LNCaP cells are caused by binding of beta-catenin/Reptin complexes to a specific motif in the proximal promoter, which prevents binding of Tip60/Pontin activator complexes to the same motif, thus inhibiting transcription. Here, we explored a pathway by which phorbol 12-myristate 13-acetate (PMA) up-regulates KAI1 transcription in LNCaP prostate cancer cells. Pretreatment with specific inhibitors showed that induction of KAI1 by PMA uses classic isoforms of protein kinase C (cPKC), is independent of Ras and Raf, and requires activation of MEK1/2 and ERK1/2, but does not involve p38MAPK. Induction of KAI1 transcription by PMA was associated with enhanced overall acetylation of histones H3 and H4, but only acetylation of H3 was blocked by a PKC inhibitor. Chromatin immunoprecipitation showed that PMA induces recruitment of Tip60/Pontin activator complexes to NFkappaB-p50 motifs in the proximal promoter, and this was blocked by a PKC inhibitor. These changes were not associated with differences in overall levels of Tip60, Pontin, beta-catenin, or Reptin protein expression but with PMA-induced nuclear translocation of Tip60.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Proteína Kangai-1/biossíntese , Ésteres de Forbol/farmacologia , Transcrição Gênica , ATPases Associadas a Diversas Atividades Celulares , Transporte Ativo do Núcleo Celular , Carcinógenos , Linhagem Celular Tumoral , Histonas/metabolismo , Humanos , Lisina Acetiltransferase 5 , Metástase Neoplásica , Acetato de Tetradecanoilforbol , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
Oncol Rep ; 18(6): 1357-63, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17982617

RESUMO

Levels of the KAI1 metastasis suppressor are reduced in advanced stages of many human cancers, leading to the loss of KAI1 function. A recent study has suggested that the loss of KAI1 function may also occur via alternative splicing of KAI1 mRNA which deletes an exon encoding a critical 28 amino acids from the protein. Using PCR, 20 bladder tumours at differing stage and grade, a non-tumourigenic urothelial cell line (SV-HUC-1) and 17 bladder cancer cell lines were examined for expression of this alternatively spliced (AS) KAI1 mRNA. Full-length KAI1 mRNA was present in all tumour samples and low levels of AS KAI1 mRNA in 15/20 samples. There was no association between the presence or absence of AS mRNA and clinicopathological characteristics of these tumours. Low levels of AS KAI1 mRNA were present in SV-HUC-1 and 14/17 bladder cancer cell lines. There was no association between the presence or absence of AS KAI1 mRNA and tumourigenicity, or in vivo invasive abilities of these cell lines. In all cell lines expressing AS KAI1 mRNA, levels were 3- to 5-fold lower than levels of wild-type mRNA, irrespective of wild-type mRNA levels. Low levels of an alternatively spliced form of KAI1 mRNA are present in most bladder cancer tumours and tumour cell lines, but are not associated with invasive behaviour.


Assuntos
Processamento Alternativo , Proteína Kangai-1/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Neoplasias da Bexiga Urinária/genética , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Primers do DNA , Seguimentos , Humanos , Dados de Sequência Molecular , Invasividade Neoplásica , Reação em Cadeia da Polimerase , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia
4.
Neoplasia ; 9(4): 322-31, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17460776

RESUMO

Few treatment options exist for metastatic prostate cancer (PC) that becomes hormone refractory (HRPC). In vitro, plant-derived MINA-05 caused dose-dependent decreases in cell numbers in HRPC cell lines LNCaP-C4-2B and PC-3, and in androgen-sensitive LNCaP-FGC, DuCaP, and LAPC-4, by WST-1 assay. MINA-05 pretreatment significantly decreased clonogenic survival in agar and on plastic at 1 x and 2 x IC50 for PC-3 (P < .05 and P < .001, respectively), and at 1/2 x, 1 x, and 2 x IC50 for LNCaP-FGC cells (P < .001). MINA-05 also induced G2M arrest of LNCaP-FGC and PC-3 cells (by flow cytometry) and caused some apoptosis in LNCaP-FGC (sub-G1 peak on flow, expression of activated caspase-3) but not in PC-3 cells. Western blotting indicated that these cell cycle changes were associated with decreased levels of regulatory proteins cyclin B1 and cdc25C. MINA-05 given daily by gavage for 39 days did not diminish primary orthotopic PC-3 growth in nude mice, but decreased the extent of lymph node invasion at higher doses. We conclude that MINA-05 induces G2M arrest, inhibits cell growth, reduces PC cell regrowth in vitro, and reduces lymph node invasion after orthotopic PC-3 cell implantation in vivo. It has potential as an adjuvant treatment for patients with PC.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Inibidores do Crescimento/uso terapêutico , Linfonodos/patologia , Metástase Linfática/prevenção & controle , Neoplasias da Próstata/prevenção & controle , Schisandra/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Transformada , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Inibidores do Crescimento/química , Inibidores do Crescimento/isolamento & purificação , Humanos , Metástase Linfática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias da Próstata/patologia
5.
Oncol Rep ; 16(6): 1267-72, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17089048

RESUMO

KITENIN is a newly identified binding partner of the KAI1/CD82 metastasis suppressor. Recent studies using a mouse model of colon cancer, have suggested that KITENIN might be a metastasis enhancer whose functions are modulated by an interaction with KAI1/CD82. To begin exploration of the possible importance of KITENIN to human cancer, we examined KITENIN mRNA (by RT-PCR) and protein expression (by Western blotting) in a large series of bladder cancer cell lines, and then compared these levels to the expression of KAI1/CD82 and of previously determined in vitro invasive behaviour of these same cancer cell lines. We report that KITENIN was uniformly expressed in all cancer cell lines, but those lines in which KAI1/CD82 was not detected, had a higher in vitro invasive ability and altered actin organisation (as determined by fluorescence microscopy), than those lines in which KAI1/CD82 was present. Our data suggest that the relationship between KITENIN and KAI1/CD82 may be an important determinant of tumour cell behaviour.


Assuntos
Proteínas de Transporte/biossíntese , Proteína Kangai-1/biossíntese , Proteínas de Membrana/biossíntese , Invasividade Neoplásica/fisiopatologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Western Blotting , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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