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Objective To investigate the relationship between the expression of ING4 and clinical primary breast cancer.Methods Tissue and peripheral blood samples of primary breast cancer patients were collected and the expression levels of ING4 and NF-κB in tissue samples were detected with immunohistochemistry.The expression of ING4 in the peripheral blood was detected with ELISA.Results The positive rates of expression of ING4 and NF-κB in breast cancer tissues were 100.00 ％ (30/30),which were significantly higher than that in the adjacent tissues 12.50 ％ (3/24) and 93.75 ％ (45/48) compared to 6.67 ％ (1/15) respectively.Compared with the preoperative,the ING4 level in peripheral blood from the postoperative breast cancer patients was significantly reduced (P =0.044).Conclusions The expression of ING4 does not decrease in the primary breast cancer patients.The increasing is perhaps due to the body's stress response against tumor development in early stage by secreting more ING4 protein.
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Objective To explore the inhibitor of growth family member 4 (ING4) and hypoxia inducible factor-1α(HIF-1α)expression in colorectal cancer and the prognostic significance. Methods Immunohistochemistry was used to detect the ING4 and HIF-1α expression in 133 cases of colorectal cancer tissues and 76 cases of normal rectal tissues. Survival analysis was performed on the following data. Results ING4 in colorectal cancer tissues with positive rate (53.4%) was significantly lower than normal rectal tissue (85.5%) and the difference was statistically significant (P < 0.01); HIF-1α in colorectal cancer tissues with positive rate (69.9%) is higher than normal rectal tissue (42.1%), and the difference was statistically significant (P < 0.01); ING4 and HIF-1αexpression was related with tumor differentiation, Dukes stage and lymph node metastasis (P < 0.05); colorectal cancer tissues ING4 and HIF-1α expression was negatively correlated (r = -0.317, P < 0.001); By multivariate analysis, tumor differentiation, Dukes stage, lymph node metastasis, ING4 expression of HIF-1α expression has independent prognostic significance. Conclusion ING4 and HIF-1α may be involved in the occurrence and development of colorectal cancer , and combined detection could help determine the prognosis of colorectal cancer.
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Objective To investigate the inhibitory effect of inhibitor 4 of growth(ING4)delivered by adenovirus on human breast carcinoma cell MDA-MB-231.Methods MDA-MB-231 human breast carcinoma cells were irfected with Ad-ING4.The expression level of ING4 gene was detected by RT-PCR and Western blot;The growth inhibition,cell-cycle alteration,and apoptosis were detected by MTT,Flowcytometry and Hochest33258 staining.respectively.RT-PCR was used to detect the transcription of Bax,Bc1-2,Survivin genes;The expression level of Ang-1 gene was detected by ELISA;Ad-ING4 was given intratumorally in athymic nude mice beating MDA-MB.231 tumors.and then tumor growth was monitored;The expression of Bc1-2,Bax and Caspase-3 was analyzed by immumohistochemistry.Results ING4 was successfully transcribed and translated iU MDA-MB-231 cells:Ad-ING4 significantly inhibited the proliferation and induced G_2/M phase arrest to(24.86±1.24)% and cell apoptosis of MDAMB-231.Intratumoral injection of Ad-ING4 suppressed the tumor growth obviously with a inhibitory rate of 49%;Immumohistochemistry showed that the expression of Bax,Caspase-3 were up-regulated and the expression of Bc1-2.Survivin,CD34 were down-regulated by Ad-ING4.Conclusions Ad-ING4 can inhibit the growth of MDA-MB-231 cells and induce apoptosis in vitro and in vivo.
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Objective:To study the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the MG-63 osteosarcoma xenografts in nude mice.Methods:Ad-ING4 was transfected into QBI-293 cells and harvested.15 nude mice of the subcutaneous tumor models were established with MG-63 osteosarcoma cells and were randomly divided into PBS,Ad-GFP and Ad-ING4 groups.Then PBS(100 μl),Ad-GFP(100 μl,10~9pfu/ml) and Ad-ING4 (100 μl,10~9pfu/ml) for each one were given respectively QOD for 5 times,with intratumor injections.Tumor volume changes were monitored;and the 15 mice were sacrificed 2 weeks after treatment,the tumors were removed,weighed and ratios of tumor-suppression were calculated.The morphological changes of apoptotic tumor cells were observed under microscope.Bcl-2,Bax,Caspase-3,VEGF,CD34 expression was tested by immumohistochemistry.Results:High titer(10~9pfu/ml)adenoviral vector of ING4 gene were obtained.In nude mice bearing MG-63 osteosarcoma xenografts,the growth of MG-63 tumors treated by intratumoral injecting of Ad-ING4 was significantly suppressed,compared with PBS group and Ad-GFP group.The ratios of tumor weight-suppression of Ad-ING4 group was 59.3%(P＜0.05).Immumohistochemistry displayed that the expression of Bax,Caspase-3 was up-regulated and the expression of Bcl-2,VEGF,CD34 was down-regulated by Ad-ING4.Conclusion:Ad-ING4 can inhibit the growth of MG-63 osteosarcoma xenografts in nude mice,which may be via activating the apoptosis pathway and inhibiting tumor angiogenesis.
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Objective: To construct a recombinant human inhibitor of growth 4 (hING4) gene and observe its growth inhibition effect on lung adenocarcinoma NCI H460 cells. Methods: The GFP-labeled recombinant adenovirus vector hING4 (Ad-hING4) was constructed using pcDNA3. 0-mING4 plasmid as a template and site-specific mutagenesis technique. RT-PCR and fluorescence microscope were used to detect the expression of hING4 in NCI H460 cells. Bax and Bcl-2 protein expression were detected by Western blotting. Colony formation assay and flow cytometry were used to observe the effect of hING4 on the proliferation and apoptosis of H460 cells. Results: DNA sequence analysis and PCR indicated that Ad-hING4 were successfully constructed. Ad-hING4 was expressed in lung cancer H460 cells and had obvious cytopathic effect (CPE). Western blotting suggested that hING4 gene caused up-regulation of Bax expression and down-regulation of Bcl-2 expression. Colony formation assay and flow cytometry showed that hING4 inhibited the proliferation of H460 cells and induced apoptosis of lung cancer cells. Conclusion: Ad-hING4 gene was successfully constructed. It inhibited the growth of lung cancer H460 cells in vitro.
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Objective To construct the pCDNA3.1-ING4 eukaryotic expression vector and investigate its effect on the proliferation of melanoma cell line M14. Methods The targeted cDNA fragment encoding ING4 was cloned by reverse transcription-PCR with normal gastric mucosa from patients with gastric ulcer, and subcloned into eukaryotie expression vector pcDNA3.1. PCR and DNA sequencing were performed to identify the eukaryotic expression vector pCDNA3.1-ING4, which was then transfected into M14 cells with Lipofectamine 2000 reagent. The expression of ING4 was detected in untransfected, pCDNA3. 1-ING4-transfected and pcDNA3.1-transfected M14 cells by Western blot and immunocyto-chemistry, and cell proliferation by MTT assay. Results A fragment of expected size (750 bp) was amplified by PCR analysis, and DNA sequencing confirmed the correctness of the recombinant plasmid. As shown by immunocytochemistry, the percentage of cells positive for ING4 protein was significantly higher in pCD NA3. 1-ING4-transfected MI4 cells than in non-transfected M14 cells and pcDNA3.1-transfected M14 cells (71.80%±9.88% vs 4.20%±3.35%, P < 0.01). Western blot also revealed an increased expression of ING4 in pCDNA3.1-ING4-transfected cells. Decreased cell viability was observed in ING4-transfected cells compared with nontransfected cells and pCDNA3.1-transfected cells (both P < 0.01). Conclusions The pCDNA3. 1-ING4 eukaryotic expression vector has been constructed successfully, and M14 cells transfected by this recombinant plasmid could effectively express ING4 protein, which may inhibit the cell proliferation of M14 cells.
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The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to regulate brain tumour angiogenesis through transcriptional repression of NF-?B-responsive genes, induce G2/M arrest by the increased p21 expression in a p53-dependent manner, suppress the loss of contact inhibition and represses activation of the hypoxia inducible factor, which plays an important role in the progression of tumorigenesis. However, seldom studies about ING4 inducing tumor cells apoptosis were reported.The C6 cells (mouse glioma cells) were infected respectively with the blank adenovirus carrying GFP (Ad) and the recombinated Ad-hING4-His, then RT-PCR assay was used to detect the transcriptions of hING4, as well Western-blotting assay was ued to detect the expressions of hING4. The effects of hING4 expression upon C6 cells were observed, and the growth curve was drawed and tumor control rates were calculated. The C6 cells, which were affected by blank Ad and Ad-hING4-His, were respectively observed by LSCM (laser scan confocal microscope) and transmission electron microscope (TEM), detected by flow cytometry; and the genomic DNA of both groups were extracted and electrophoresised in agarose gel to examinate the DNA fragments. The results showed hING4 can significantly inhibit the growth of C6 cells by promoting the cell’s apoptosis, which probably is the first one to prove this property of ING4.The experimental and theoretical foundation for gene therapy for gliomas with ING4 in the future was established.
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Objective:To investigate the effect of IGN4 gene trasfection on SGC-7901 cells,and the possible mechanism for its anti-tumor effect .Methods:Ad-ING4 was obtained by gene recombination and packaging technique in vitro.The expression of ING4 mRNA and protein were analyzed by RT-PCR and western-blot respectively.The effect of Ad-ING4 on growth of SGC-7901 cells was detected by MTT.Apoptotic cells were detected by Laser Scan Co-focal Microscope (LSCM).The expression of p21 in SGC-7901 cells was analyzed by western-blot.Results:After infection with Ad-ING4,the expression of ING4 mRNA and protein were showed in SGC-7901 cells detected.The proliferation of SGC-7901 cells was inhibited significantly(P