Inhibitory Effect of Melanoma Differentiation Associated Gene-7/Interleukin-24 on Invasion In Vitro of Human Melanoma Cancer Cells

The acquisition of metastasis potential is a critical point for malignant tumors. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a potential tumor suppress gene and frequently down-regulated in malignant tumors. It has been implicated that overexpression of MDA-7 led to proliferation inhibition in many types of human tumor. Invasion is an important process which is potential to promote tumor metastasis. However, the role and potential molecular mechanism of mda-7/IL-24 to inhibit the invasion of human melanoma cancer is not fully clear. In this report, we identified a solid role for mda-7/IL-24 in invasion inhibition of human melanoma cancer LiBr cells, including decreasing of adhesion and invasion in vitro, blocking cell cycle, down-regulating the expression of ICAM-1, MMP-2/9, CDK1, the phosphorylation of ERK and Akt, NF-kappaB and AP-1 transcription activity. Meanwhile, there was an increased expression of PTEN in mda-7/IL-24 over-expression LiBr cells. Our results demonstrated that mda-7/IL-24 is a potential invasion suppress gene, which inhibits the invasion of LiBr cells by the down-regulation of ICAM-1, MMP-2/9, PTEN, and CDK1 expression. The molecular pathways involved were the MAPK/ERK, PI3K-Akt, NF-kappaB, and AP-1. These findings suggest that mda-7/IL-24 may be used as a possible therapeutic strategy for human melanoma cancer.

The effect of oncolyic adenovirus SG600-IL24 expressing human MDA-7/IL-24 on apoptosis of hepatocellular carcinoma cell lines / 中华普通外科杂志

Objective To investigate the effect of oncolytic adenovirus vector SG600-IL24expressing human melanoma differentiation associated gene-7 (mda-7/IL-24) on hepatocellular carcinoma cell lines with different metastatic potential of HepG2, SMMC7721, MHCC97L and normal liver cell line LO2. Methods The oncolytic adenovirus SG600-IL24 which carrying mda-7/IL-24 gene was transfected into hepatocellular carcinoma cell lines and normal liver cell line. The mRNA and protein expression of mda7/IL-24 in HepG2, SMMC7721, MHCC97L and LO2 cell lines was confirmed by RT-PCR,ELISA assay and Western blot respectively. MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst33258 and flow cytometry were studied to indicate the apoptosis effects. Results It was confirmed by RT-PCR, ELISA assay and Western-blot that the exogenous mda-7/IL-24 gene was highly expressed in HepG2, SMMC7721, MHCC97L and LO2 cell lines. MTT and apoptosis detection indicated that MDA-7/IL-24 can induce the growth suppression (the inhibition rate was 75% ±2. 5% ,86% ±3. 5% ,and promotes apoptosis ( the apoptosis rate was 56. 5% ± 4. 0% , 34. 4% ± 2. 0% , 43. 3% ± 2. 5%cell lines at G2/M phase ( the blocking rate was 35. 4% ± 4. 2% , 40. 5% ± 5. 0% , 42. 0% ± 5. 0%metastatic potential hepatocellular carcinoma cell lines but not in normal liver cell line.Conclusions Oncolytic adenovirus vector SG600-IL24 can selectively induce growth suppression, promote apoptosis in hepatocellular carcinoma lines in vitro but not in normal liver cell LO2.

mda-7/IL-24 induces apoptosis of hepatic carcinoma cells through endoplasmic reticulum stress pathway / 第二军医大学学报

Objective: To study the effects of mda-7/IL-24 on the growth, proliferation, apoptosis of different hepatic carcinoma cell lines and the related mechanisms. Methods: A recombinant adenovirus Ad-mda-7 was constructed and was used to transfect human hepatic carcinoma cell lines (HepG2, Hep3B and PLC/ PRF/5) and normal liver cell line L02. MTT assay and FACS were employed to assess the growth and apoptosis of cells; the expression of related protein expression was examined by Western blotting. The cells were treated with calpastatin I (ALLN,25 μmol/L) for 30 min to block the endoplasmic reticulum stress (ER-stress) and the above indices were examined again. Results: Treatment with Ad-mda-7 resulted in selective inhibition of cell proliferation and induced apoptosis, especially in HepG2 cells; Ad-mda-7 showed no influence on normal cells. Pretreatment with ALLN partially inhibited the above effects of Ad-mda-7. Western blotting revealed that Ad-mda-7 induced up-regulation of BiP/GRP7B and Bax protein, activation of caspase-12, caspase-3 and phosphorylation of p38 MAPK in HepG2 cells. Blocking ER-stress with ALLN down-regulated Bax, caspase-12 expression and inhibited activation of caspase-3 and caspase-12, but showed no effect on the expression of BiP/GRP78 or phosphorylation of p38 MAPK. Conclusion: mda-7/IL-24 can cause growth inhibition and promote apoptosis of hepatic carcinoma cells through the ER-stress pathway.

Mechanism of IL-24/mda-7 inhibiting the growth of K562 cells / 白血病·淋巴瘤

Objective To explore the mechanism of the cell-cycle arrest induced by human melanoma differentiation associated gene-7 (mda-7/IL-24) in chronic myelocytic leukemia cell line K562. Methods Microarray analysis was performed to determine the genes that were differentially regulated by mda-7/IL-24 in K562 cells, and was validated by realtime PCR. The phosphorylated pRb were detected by Western blotting analysis. Results A microarray analysis showed that G_0/G_1 phase-associated genes p21~(WAF-1) and BCCIP were up-egulated, while cdk6 and Smurf2 were down-regulated. The directional change in the expression of the four genes was successfully validated with real-time quantitative RT-PCR. pRb Set~(795) phosphorylation was observed with no modification of the pRb protein level. Conclusion These results suggest that IL-24/mda-7 may inhibit K562 proliferation and induce G_0/G_1 cell cycle angst by up-regulating p21~(WAF-1) and BCCIP, down-regulating cdk6 and Smurf2.

Effects of IL-24 delE5 on human leukemia cell line K562 / 白血病·淋巴瘤

Objective To investigate the antitumor activity of IL-24 delE5 in human leukemia cell line K562. Methods The expression of mda-7/IL-24 and its splice variant induced by TPA in leukemic cell lines, U937 and HL-60, was evaluated. The effects of IL-24 delE5 in K562 on cell proliferation, colony-forming ability, cell cycle, apoptosis, and tumor growth in vivo by using MTr assay, colony forming assay, flow cytometry, Annexin-V/PI and tumor xenograft models in nude mice were assessed. Meantime, the effects of IL-24 delE-5 and mda-7/IL-2A were compared. Results The expression of IL-24 dciE5 was detected in differentiated U937 and HL-60 cells. Transfection with IL-24 delE5 significantly reduced tumor cell viability, inhibited colony formation. Comparing with the control, G_0/G_1 stage add from (24.46±3.99) % to (42.69±3.04) %, caused cell cycle arrest in G_0/G_1 stage and significantly inhibited the growth of K562 transplantation tumor. No significant differences in the aforementioned antileukemia characteristics between IL-24 delE5 and mda-7/IL-24 was found. Conclusion Similar with mda-7/IL-24, IL-24 delE5 can efficiently inhibit the proliferation of K562 in vitro and in vivo, probably through induction of G_0/G_1 cell cycle arrest.

In Vitro Enhancement of Radiosensitivity by the Combination of Celecoxib and Ad-mda7 in Human Breast Cancer Cells / 한국유방암학회지

PURPOSE: Celecoxib and Ad-mda7 have shown its ability to enhance radiosensitivity in various cancer cells in vitro. We expected to synergistically enhance radiosensitivity by combing celecoxib and Ad-mda7 in breast cancer cells in vitro. METHODS: MDA-MB-436 and MDA-MB-468 human breast cancer cells were exposed to different doses (0, 2, 4, and 6 Gy) of radiation with or without pretreatment with either Ad-mda7 or celecoxib alone, or with the combination for three days prior to irradiation. Clonogenic cell survival assay was used to compare the radiosensitizing effect. Fluorescence activated cell sorting analysis was performed to assess cell cycle changes and the subdiploid cell population. We determined the prostaglandin E2 (PGE2) concentration before and after the irradiation (2 Gy, 24 hours). We performed western blot analysis of Akt, phosphorylated Akt, beta-catenin, and cyclooxygenase-2 (COX-2). RESULTS: At the sublethal dose of celecoxib and Ad-mda7, the combination showed significantly enhanced radiosensitivity. The enhancement factor for the combination treatment was 1.44 in MDA-MB-468 cells and 1.75 in MDA-MB-436 cells. There were an increased percentage of apoptotic cells in the combination therapy group as compared to the controls, but this was not statistically significant. Cell cycle analysis demonstrated an increase in the G2/M phase of the cell cycle in the combination group compared with controls. The concentration of PGE2 was significantly decreased after the irradiation in both cell lines compared to the controls. Western blot analysis confirmed that this combination treatment effectively suppress the expression of Akt, phosphorylated Akt, and COX-2 in those cell lines, except beta-catenin. CONCLUSION: Cotreatment of Ad-mda7 plus celecoxib definitely showed radioenhancing effect. We presumed that this effect may be the arrest of the cells at the radiosensitive G2/M phase of the cell cycle.

In vitro Efficacy of mda-7 Gene for Hepatocellular Carcinoma Gene Therapy Mediated by Human Ribosomal DNA Targeting Vector / 生物化学与生物物理进展

Human ribosomal DNA (hrDNA) targeting vector pHr is a homologous recombinant plasmid for human genome which developed in the State Key Laboratory of Medical Genetics. pHr was used to construct a recombinant plasmid pHr-CMG expressing mda-7/GFP fusion gene and its efficacy in the hepatocellular carcinoma cell line Bel-7402 was investigated. The expression of mda-7/GFP fusion gene was detected by fluorescent microscope, RT-PCR and Western blotting, and its function was detected by cell-cycle analyses, MTT assay and Hoechst33258 staining. The results demonstrated that pHr-CMG vector could express MDA-7/GFP fusion protein effectually and the mda-7 gene could induce cell apoptosis and proliferation suppression in Bel-7402 cell line, which might be caused by the G2/M cell cycle arrest. These results also suggested that human ribosomal DNA targeting vector system and the pHr-CMG vector may be applied in further gene therapy researches for hepatocellular carcinoma.

A Novel Therapeutic Approach to Breast Cancer using a Selective Cyclooxygenase 2 Inhibitor and Adenovirus-mediated Delivery of the Melanoma Differentiationassociated Gene-7 (Ad-mda7) / 한국유방암학회지

Recently, many preclinical and clinical researches have focused on the possible roles of new therapeutic modalities to enhance current treatment efficacy or to extend the current limitations against breast cancer treatment. Th melanoma differentiation-associated gene-7 (mda-7 ), now classified as a member of the interleukin (IL)-10 gene family, has attracted attentions from several investigators for its unique ability to act against various cancers including breast cancer. In addition to mda-7, highly selective cyclooxygenase-2 (Cox-2) inhibitors, have continuously demonstrated possible anticancer effects against various cancers even though theray with many of the inhibitors has resulted in major set backs due to complications after long-term use. However, few have performed to demonstrate the synergistic effects of these two efficient treatment options or to demonstrate preventive measures to reduce the size of tumors. We summarize important results and our experience related to the use of a selective cyclooxygenase 2 inhibitor and adenovirus-mediated delivery of mda-7.

Replication-incompetent Adenovirus Vector-mediated MDA-7/IL-24 Selectively Induces Growth Suppression and Apoptosis of Hepatoma Cell Line SMMC-7721 / 华中科技大学学报（医学）（英德文版）

In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture supernatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were meas- ured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the ex- ogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL24 proteins in the culture supernatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the G2/M phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.

Raav-PIEG-MDA-7 inhibits the growth of hepatocellular carcinoma in vivo / 中华普通外科杂志

Objective To investigate the anti-tumor effect of the recombined adeno-associated virus encoding melanoma differentiation -associated gene-7 (MDA-7) regulated by progression-elevated gene (PEG) promotor on human hepatocellular carcinoma (HCC) in nude mice. Methods A nude mouse model of subcutaneously implanted HCC cell line HepG2 tumor was established. AAV-PEG-MDA-7 was injected from the tail vain after tumor cell innoculation. RT-PCR, Western blot and immunohistochemical analysis were employed to detect MDA-7 expression in mice; MDA-7 plasma concentration was detected by ELISA assay. Tumor growth was observed, tumor cell apoptosis and angiogenesis in tumor tissues were measured by TUNEL and immunohistochemical analysis. Results Seven days after tumor cell innoculation RT-PCR, Western blot, and immunohistochemistry showed that MDA-7 was only expressed in the liver. ELISA assay showed that the concentration of MDA-7 in plasma was gradually increased to reach the plateau (200 ng/ml). Tumor growth was significantly inhibited in mice injected with rAAV-PEG-MDA-7, and the tumor growth-inhibiting rate was 62%. TUNEL and immunohistochemical analysis demonstrated significant induction of tumor cell specific apoptosis and reduction of vascular formation in tumor tissues. Conclusions rAAV-PEG-MDA-7 exhibits tumor-specific cytotoxicity and liver tendency, inhibiting tumor growth possibly by tumor cell apoptosis-induciug effect and antiangiogenesis.

Inhibition of human mda-7/IL-24 gene on the proliferation of human lung cancer cells / 医学研究生学报

Objective:To analyze the role of human mda-7/IL-24 gene on the proliferation of human non-small cell lung cancer cell line (H460). Methods:The mda-7/IL-24 cDNA was subcloned into expression vector pcDNA3 and transient transfected in human lung carcinoma cell H460. The overexpressed mda-7/IL-24 was identified by RT-PCR using SP6 primer and mda-7/IL-24 upstream primer. The role of human mda-7/IL-24 on the proliferation of H460 cells was analyzed with MTT after transfection of (pcDNA3)-mda-7/IL-24 for 72h. Results:The mda-7/IL-24 cDNA was subcloned into pcDNA3 successfully. RT-PCR study showed that mda-7/IL-24 could express in H460 cells at different ratio of DNA and lipofectamine (1 ?g ∶ 2?l-1?g ∶ 4?l). Compared with H460 cells and H460 cells transfected with (pcDNA3), pcDNA3-mda -7/IL-24 could inhibit the proliferation of H460 cells. The inhibition rate was (18.4%). Conclusion:The mda-7/IL-24 can inhibit the proliferation of human non-small-cell lung carcinoma cell H460.

Adenovirus mediated MDA-7/IL-24 gene transfer selectively kills hepatocellular carcinoma lines HepG2 / 中国普通外科杂志

Objective To investigate the selective killing effect of MDA/IL-24 on human hepatocellular carcinoma line HepG2 in vitro and provide a theoretical basis for gene therapy of hepatocellular carcinoma.Methods The MDA-7/IL-24 gene was transfected into human hepatocullular carcinoma cell line HepG2 and normal liver cell line L02 with a replication-incompetent adenovirus vector.The mRNA and protein expression of MDA7/IL-24 in HepG2 and L02 cells was examined by RT-PCR and ELISA assay respectively.MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro.Hoechst and Annexin-V and PI staining were studied to indicate the apoptosis.Results RT-PCR confirmed that the exogenous MDA-7/IL-24 gene was expressed in HepG2 and L02 cells.The protein product was confirmed by assay of the supernatant with ELISA.MTT and apoptosis test indicated MDA-7/IL-24 induced growth suppression and cell apoptosis of the HepG2 cell in vitro but not in cell line L02,and cell cycle test revealed MDA-7/IL-24 could block HepG2 cell in G2/M but not in L02.Conclusions MDA-7/IL-24 selectively induces growth suppression and apoptosis in lines HepG2 in vitro but not in L02 cell,which indicates that adenovirus mediated MDA-7/IL-24 can be an excellent tool for gene therapy in hepatocellular carcinoma.