ABSTRACT
AIMS: Tumour suppressor ING4 is one of ING family genes, which are involved in cell cycle arrest, gene transcription regulation, DNA repair and apoptosis. ING4 inhibition has been reported in various tumours, including gliomas, breast tumours, and stomach adenocarcinoma. The aim was to evaluate ING4 expression in lung cancers. METHOD AND RESULTS: By immunohistochemistry of 246 lung tumour tissues, reduced ING4 nuclear and cytoplasmic expression were both revealed in lung cancer and associated with tumour grade. Interestingly, compared with normal tissues, we found more tumours with ING4 expression in the cytoplasm higher than in the nucleus. Nuclear ING4 inhibition correlated with the tumour stage and lymph node metastasis. Consistent with these findings, semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting demonstrated decreased ING4 mRNA and expression in 100% (50/50) tumour tissues. Furthermore, ING4 expression was lower in grade III than in grades I-II tumours. Reduced ING4 mRNA correlated with lymph node metastasis. CONCLUSIONS: Our results indicate that overall inhibition of ING4 expression and ING4 expression higher in cytoplasm than in nucleus of tumour cells may be involved in the initiation and progression of lung cancers, and thus, analysis for ING4 expression may be useful as a clinical diagnostic and prognostic tool for lung cancer.
Subject(s)
Cell Cycle Proteins/genetics , Homeodomain Proteins/genetics , Lung Neoplasms/etiology , Lung Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Aged , Base Sequence , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA Primers/genetics , Disease Progression , Down-Regulation , Female , Homeodomain Proteins/metabolism , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lymphatic Metastasis , Male , Prognosis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Proteins/metabolismABSTRACT
BACKGROUND: Genes involved in the TGF-beta signaling pathway are often altered in several types of cancers. The TGF-beta-resistant human colon cancer cell line HT-29 has inactivated TbetaRII and deficient expression of RUNX3 and Smad4, which are involved in the TGF-beta signaling pathway. METHODS: Western blot and immunocytochemistry were performed to confirm gene expression, the MTT assay to detect cell growth, flow cytometry to investigate the cell cycle and the TUNEL to detect cell apoptosis. RESULTS: In the absence of TGF-beta, Bim was upregulated, cell growth was inhibited and apoptosis was induced. TGF-beta treatment did not affect RUNX3 expression; however, the increase in Bim expression was significant and time dependent. Interestingly, Smad4 but not Smad2/3 was also upregulated upon exposure to TGF-beta. This was not the case after TGF-beta treatment of parent HT-29 cells. As expected, TGF-beta further inhibited cell growth and induced apoptosis in HT-29/RUNX3+ cells. CONCLUSION: Our data demonstrate that RUNX3 is involved in TGF-beta-dependent and -independent cell growth inhibition and apoptosis induction pathways.
Subject(s)
Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Core Binding Factor Alpha 3 Subunit/genetics , Core Binding Factor Alpha 3 Subunit/metabolism , Transforming Growth Factor beta/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Gene Expression/drug effects , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Signal Transduction , Smad4 Protein/metabolismABSTRACT
ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down-regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT-PCR, real-time RT-PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame-shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma.
Subject(s)
Adenocarcinoma/genetics , Alternative Splicing , Cell Cycle Proteins/genetics , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/genetics , Stomach Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Adenocarcinoma/metabolism , Amino Acid Sequence , Base Sequence , Cell Cycle Proteins/analysis , Cell Line, Tumor , Female , Gene Expression Profiling , Homeodomain Proteins/analysis , Humans , Immunohistochemistry , Male , Middle Aged , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Stomach Neoplasms/metabolism , Tumor Suppressor Proteins/analysisABSTRACT
OBJECTIVE: To evaluate the anti-angiogenetic effect of the combination of low-dose cyclophosphamide(CTX) and ginsenoside Rg3 in mice with Lewis lung carcinoma, and to observe the anti-tumor effect, toxicity, adverse reaction of the treatment and survival time of the tumor bearing mice. METHODS: Holland C57/ BL6 Lewis lung carcinoma mice were taken as the model and randomly divided into 5 groups, i.e. the low-dose CTX (LDCTX) group, the maximum tolerable dose CTX (MTDCTX) group, the ginsenoside Rg3 (Rg3) group, the low-dose CTX combined with ginsenoside Rg3 group (LDCTX + Rg3), and the model group. Tumor volume, weight of mice, peripheral white blood cell counts and survival time of mice were observed, tumor microvascular density (MVD) and vascular endothelial growth factor (VEGF) gene expression were determined during the therapeutic course. RESULTS: In the LDCTX group, tumor grew comparatively slow, no significant decrease in body weight or peripheral white blood cells, and survival time was prolonged. In the LDCTX + Rg3 group, the tumor inhibitory effect was more persistent and steady without any increase of toxicity or adverse reaction. Besides, the survival time of mice was prolonged (P < 0.01). MVD was lower in the LDCTX group than that in the MTDCTX group (P< 0. 05). Compared with the model group, MVD and VEGF expression were lower in the LDCTX and the Rg3 group, and the lowering action was more significant when the two drugs were used in combination (P < 0.05). CONCLUSION: The combination of low-dose CTX and Rg3 has obvious synergetic action of anti-angiogenesis, it shows significant and persistent tumor inhibitory effect, with less toxic and adverse reaction, and could induce longer survival time than treatment of CTX or Rg3 alone.
