Oncolytic adenovirus-mediated MDA-7/IL-24 overexpression enhances antitumor activity in hepatocellular carcinoma cell lines

BACKGROUND:Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24(IL-24)is a novel tumor suppressor gene,which has suppressor activity in a broad spectrum of human cancer cells.We investigated the effect of the replication-competent oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24,both expressing human MDA-7/IL-24 on the hepatocellular carcinoma cell lines HepG2,Hep3B,SMMC-7721,HCCLM3,and the normal liver cell line L02. METHODS:Hepatocellular carcinoma cell lines and the normal liver cell line were infected with SG600-IL24 and Ad.IL-24.The mRNA and protein expression of MDA-7/IL-24 in infected cells was confirmed by RT-PCR,ELISA,and Western blotting.MTT assay was used to investigate the proliferation effect.Hoechst staining and Annexin-V and PI staining were performed to study the MDA-7/IL-24 gene expressed in HCC cell lines and the normal liver cell line.Flow cytometry was used to analyse the cell cycle. RESULTS:RT-PCR,ELISA and Western blotting confirmed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24.MTT and apoptosis detection indicated that SG600-IL24 induced growth suppression promoted apoptosis,and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line. CONCLUSIONS:SG600-IL24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma cell lines in vitro but not in the normal liver cell line L02. Compared with Ad.IL-24,SG600-IL24 dramatically enhances antitumor activity in hepatocellular carcinoma cell lines.     

腺病毒介导MDA-7/IL-24选择性促进肝癌细胞的凋亡和增殖阻滞

目的 观察MDA-7／IL-24基因对不同p53状态人肝癌细胞HepG2、MHCC97L以及Hep3B和正常的肝细胞L02的选择性杀伤作用，为肝癌的基因治疗提供理论基础。方法 将携带人MDA-7／IL-24基因的腺病毒Ad．mda-7感染人正常肝细胞L02和不同p53状态的肝癌细胞HepG2，MHCC97L、Hep3B。通过逆转录聚合酶链反应方法观察MDA7／IL24基因的表达，酶联免疫吸附法检测细胞培养上清液中MDA-7／IL-24蛋白的浓度，通过四甲基偶氮唑盐染色法及Hoechst染色观察MDA-7／IL-24对肝癌细胞的生长抑制和杀伤作用，Annexin-V和碘化丙啶双染后流式细胞仪检测细胞的凋亡，应用流式细胞仪检测细胞周期。结果 复制缺陷型腺病毒能介导外源基因MDA-7／IL-24在肝癌细胞株HepG2，MHCC97L和Hep3B以及正常细胞L02中高效表达。细胞培养上清液中有MDA-7／IL-24蛋白表达。MDA-7／IL-24能明显抑制各种肝癌细胞的生长，Hoechst染色提示MDA-7／IL-24促进肝癌细胞的凋亡，流式细胞仪提示MDA-7能选择性杀伤肝癌细胞而对正常的肝细胞无影响，细胞周期分析提示MDA-7／IL-24阻滞肝癌细胞在G2／M期，同时对正常的肝细胞没有促凋亡作用和增殖阻滞作用。结论 复制缺陷型重组腺病毒载体Ad．mda-7能介导MDA-7／IL-24基因在人肝癌细胞中高效表达，选择性地杀伤肝癌细胞HepG2、MHCC97L和Hep3B，促进细胞增殖阻滞及诱导肿瘤细胞凋亡而与肿瘤细胞的P53基因的状态无关，同时对正常的肝细胞L02无任何毒性作用。     

mda-7 (IL-24) Mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK

Subtraction hybridization identified melanoma differentiation-associated gene-7 (mda-7) as a gene induced during terminal differentiation in human melanoma cells. On the basis of structure, chromosomal localization and cytokine-like properties, mda-7 is classified as IL-24. Administration of mda-7/IL-24 by means of a replication-incompetent adenovirus (Ad.mda-7) induces apoptosis selectively in diverse human cancer cells without inducing harmful effects in normal fibroblast or epithelial cells. The present studies investigated the mechanism underlying this differential apoptotic effect. Infection of melanoma cells, but not normal immortal melanocytes, with Ad.mda-7 induced a time- and dose-dependent increase in expression, mRNA and protein, of a family of growth arrest and DNA damage (GADD)-inducible genes, which correlated with induction of apoptosis. Among the members of the GADD family of genes, GADD153, GADD45 alpha, and GADD34 displayed marked, and GADD45 gamma showed minimal induction. Treatment of melanoma cells with SB203580, a selective inhibitor of the p38 mitogen-activated protein kinase (MAPK) pathway, effectively inhibited Ad.mda-7-induced apoptosis. Additional support for an involvement of the p38 MAPK pathway in Ad.mda-7-mediated apoptosis was documented by using an adenovirus expressing a dominant negative mutant of p38 MAPK. Infection with Ad.mda-7 increased the phosphorylation of p38 MAPK and heat shock protein 27 in melanoma cells but not in normal immortal melanocytes. In addition, SB203580 effectively inhibited Ad.mda-7-mediated induction of the GADD family of genes in a time- and dose-dependent manner, and it effectively blocked Ad.mda-7-mediated down-regulation of the antiapoptotic protein BCL-2. Inhibition of GADD genes by an antisense approach either alone or in combination also effectively blocked Ad.mda-7-induced apoptosis in melanoma cells. These results support the hypothesis that Ad.mda-7 mediates induction of the GADD family of genes by means of the p38 MAPK pathway, thereby resulting in the selective induction of apoptosis in human melanoma cells.     

Apogossypol derivative BI-97C1 (Sabutoclax) targeting Mcl-1 sensitizes prostate cancer cells to mda-7/IL-24-mediated toxicity

Limited options are available for treating patients with advanced prostate cancer (PC). Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an IL-10 family cytokine, exhibits pleiotropic anticancer activities without adversely affecting normal cells. We previously demonstrated that suppression of the prosurvival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), is required for mda-7/IL-24-mediated apoptosis of prostate carcinomas. Here we demonstrate that pharmacological inhibition of Mcl-1 expression with the unique Apogossypol derivative BI-97C1, also called Sabutoclax, is sufficient to sensitize prostate tumors to mda-7/IL-24-induced apoptosis, whereas ABT-737, which lacks efficacy in inhibiting Mcl-1, does not sensitize mda-7/IL-24-mediated cytotoxicity. A combination regimen of tropism-modified adenovirus delivered mda-7/IL-24 (Ad.5/3-mda-7) and BI-97C1 enhances cytotoxicity in human PC cells, including those resistant to mda-7/IL-24 or BI-97C1 alone. The combination regimen causes autophagy that facilitates NOXA- and Bim-induced and Bak/Bax-mediated mitochondrial apoptosis. Treatment with Ad.5/3-mda-7 and BI-97C1 significantly inhibits the growth of human PC xenografts in nude mice and spontaneously induced PC in Hi-myc transgenic mice. Tumor growth inhibition correlated with increased TUNEL staining and decreased Ki-67 expression in both PC xenografts and prostates of Hi-myc mice. These findings demonstrate that pharmacological inhibition of Mcl-1 with the Apogossypol derivative, BI-97C1, sensitizes human PCs to mda-7/IL-24-mediated cytotoxicity, thus potentially augmenting the therapeutic benefit of this combinatorial approach toward PC.     

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (mda-7). Ectopic expression of mda-7 when using a recombinant adenovirus, Ad.mda-7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda-7, pancreatic carcinoma cells are remarkably refractory to Ad.mda-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.mda-7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA-7 and BAX proteins and increases in the ratio of BAX to BCL-2 proteins. Moreover, transfection of mutant K-ras pancreatic carcinoma cells with an antisense K-ras expression vector and infection with Ad.mda-7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.     

Dual cancer-specific targeting strategy cures primary and distant breast carcinomas in nude mice

Limitations of current viral-based gene therapies for malignant tumors include lack of cancer-specific targeting and insufficient tumor delivery. To ameliorate these problems and develop a truly effective adenovirus gene-based therapy for cancer, we constructed a conditionally replication competent adenovirus (CRCA) manifesting the unique properties of tumor-specific virus replication in combination with production of a cancer-selective cytotoxic cytokine, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), which embodies potent bystander antitumor activity. Cancer cell selective tropism was ensured by engineering the expression of the adenoviral E1A protein, necessary for viral replication, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells. In the E3 region of this CRCA, we introduced the mda-7/IL-24 gene, thereby mediating robust production of this cytokine as a function of adenovirus replication. Infection of this CRCA (designated Ad.PEG-E1A-mda-7) in normal mammary epithelial cells and breast cancer cells confirmed cancer cell selective adenoviral replication, mda-7/IL-24 expression, growth inhibition, and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 into human breast cancer xenografts in athymic nude mice completely eradicated not only the primary tumor but also distant tumors (established on the opposite flank of the animal) thereby implementing a cure. This dual cancer-specific targeting strategy provides an effective approach for treating breast and other human neoplasms with the potential for eradicating both primary tumors and metastatic disease. Additionally, these studies support the potential use of mda-7/IL-24 in the therapy of malignant cancers.     

mda-7/IL-24 inhibits the proliferation of hematopoietic malignancies in vitro and in vivo

OBJECTIVE: Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) has been consistently shown to exert growth inhibitory effects on various tumor types. However, the majority of these reports were limited to solid tumors. The purpose of this study was to investigate the antitumor activity of mda-7/IL-24 and the underlying mechanism in hematopoietic malignancies. MATERIALS AND METHODS: We determined the expression of mda-7/IL24 and its heterodimeric receptors in hematopoietic tumor cell lines and then stably transfected mda-7/IL-24 into K562 (leukemia) and Namalwa (lymphoma) cell lines to assess the effects of mda-7/IL-24 on cell proliferation, cell cycle, apoptosis, colony-forming ability, and tumor growth in vivo. Microarray analysis was performed to determine the genes that were differentially regulated by mda-7/IL-24 in K562 cells. RESULTS: Expression of mda-7/IL-24 or its intact receptor pairs was not detected in the 11 cell lines tested. Ectopic expression of mda-7/IL-24 induced significant (p < 0.05) inhibition of cell growth and colony formation in both K562 and Namalwa cells, and the growth inhibition in K562 cells was associated with G(0)/G(1) cell-cycle arrest. Results of in vivo studies showed good correlation with in vitro inhibition of tumor cell proliferation in both the cell lines. We also showed that the increase in p21(WAF-1) and BCCIP and decrease in cdk6, smurf2, and phosphorylated pRb, which are regulators of cell-cycle progression, might account for G(0)/G(1) cell-cycle arrest in K562 cells. CONCLUSIONS: The present study demonstrated for the first time the potential antitumor activity of mda-7/IL-24 in chronic myelogenous leukemia and lymphoma.     

重组腺病毒载体携带多药耐药基因1反义RNA靶向逆转肝癌细胞多药耐药

目的 探讨携带多药耐药基因1(multidrug resistance gene 1,mdr1)反义RNA的重组腺病毒载体靶向逆转甲胎蛋白阳性(AFP+)的肝癌多药耐药细胞HepG2R的疗效及作用机制.方法 分别构建携带AFP启动子和mdr1基因反义核苷酸片段的重组腺病毒载体Adeno-asmdr及携带AFP启动子和增强绿色荧光蛋白基因的重组腺病毒载体Adeno-EGFP,将Adeno-EGFP转染人正常肝细胞L02(AFP-),人官颈癌细胞HeLa(AFP-)及HepG2(AFP+)细胞,检测增强绿色荧光蛋白基因在各细胞的转录水平;将Adeno-asmdr转染HepG2R细胞,Western blot检测不同时间P-gp170的表达,末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测HepG2R细胞凋亡,流式细胞术检测HepG2R细胞在不同药物作用下细胞周期、凋亡率.结果 增强绿色荧光蛋白基因在AFP阳性的HepG2细胞可得到显著转录,而在L02细胞和HeLa细胞,其转录减少,显示了该载体的良好转录活性以及靶向特异性.Adeno-asmdr转染HepG2R细胞后,HepG2R细胞P-gp170表达明显减弱,HepG2R细胞凋亡增加,HepG2R细胞对多种化疗药物的耐受能力明显下降,细胞出现显著的周期阻滞,大量细胞被阻滞于S期和G0/M期,凋亡细胞比例增加.结论 实验构建的Adneo-asmdr重组腺病毒载体可在AFP阳性HepG2R细胞内特异靶向性表达目的 基因,并可有效降低mdrl基因产物P-gp170的表达,从而达到对HepG2R细胞多药耐药的逆转作用.     

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

AIM： To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin （ADFMChR） on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved.METHODS： HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry （FCM） using propidium iodide （PI） fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ （PPARγ）, NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting.RESULTS： MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose- dependent manner, with little effect on growth of L-02 cells, and when ICs0 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil （5-FU, ICso was 9.27 μmol/L）. The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 （191.55/8.45）, higher than 5-FU （SI was 7.05 （65.37/9.27）. FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR （16.0%） （P 〈 0.05） and were similar to those obtained with 30.0 μmol/L 5-FU（41.0%）. DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 pmol/1 GW9662, a blocker of PPARy. Western blotting analysis revealed that aEer 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFMChR, PPARy and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARy and NF-KB protein expression in HepG2 cells.CONCLUSION： ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.     

The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.

Cancer is a disease characterized by defects in growth control, and tumor cells often display abnormal patterns of cellular differentiation. The combination of recombinant human fibroblast interferon and the antileukemic agent mezerein corrects these abnormalities in cultured human melanoma cells resulting in irreversible growth arrest and terminal differentiation. Subtraction hybridization identifies a melanoma differentiation associated gene (mda-7) with elevated expression in growth arrested and terminally differentiated human melanoma cells. Colony formation decreases when mda-7 is transfected into human tumor cells of diverse origin and with multiple genetic defects. In contrast, the effects of mda-7 on growth and colony formation in transient transfection assays with normal cells, including human mammary epithelial, human skin fibroblast, and rat embryo fibroblast, is quantitatively less than that found with cancer cells. Tumor cells expressing elevated mda-7 display suppression in monolayer growth and anchorage independence. Infection with a recombinant type 5 adenovirus expressing antisense mda-7 eliminates mda-7 suppression of the in vitro growth and transformed phenotype. The ability of mda-7 to suppress growth in cancer cells not expressing or containing defects in both the retinoblastoma (RB) and p53 genes indicates a lack of involvement of these critical tumor suppressor elements in mediating mda-7-induced growth inhibition. The lack of protein homology of mda-7 with previously described growth suppressing genes and the differential effect of this gene on normal versus cancer cells suggests that mda-7 may represent a new class of cancer growth suppressing genes with antitumor activity.     

Conditionally replicative adenovirus-based mda-7/IL-24 expression enhances sensitivity of colon cancer cells to 5-fluorouracil and doxorubicin

Background

Multiple drug resistance (MDR) greatly limits the efficacy of chemotherapy for colon cancer. An adenovirus armed with Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24; abbreviated to ‘IL-24’ here) was shown to reverse the MDR of colon cancer cells to oxaliplatin and doxorubicin. However, the relatively low expression level of IL-24 mediated by a replication-deficient adenoviral vector hindered its clinical application.

Methods

To enhance IL-24-dependentreversion of the MDR phenotype, we utilized a conditionally replicative adenoviral vector, AdBB-IL24, to express IL-24 at a high level for more efficient MDR reversion.

Results

An enzyme-linked immunosorbent assay (ELISA) suggested conditionally replicative adenoviral vector-mediated IL-24 expression was elevated in comparison with that of a replication-deficient adenoviral vector, Ad-IL24. AdBB-IL24 was shown to reverse MDR in colon cancer cells more potently than Ad-IL24. The AdBB-IL24-induced MDR reversion was linked to reduced P-glycoprotein (Pgp) and breast cancer resistance protein 1 (BCRP1) expression. Consistently, 5-fluorouracil and doxorubicin induced more apoptosis in AdBB-IL24-infected colon cancer cells compared with that in the Ad-IL24-infected cells. A cell viability assay showed that AdBB-IL24 could enhance the growth-inhibitory effect of 5-fluorouracil and doxorubicin on colon cancer cells more effectively than Ad-IL24 in vitro. In a mouse model, we also found that the combination of 5-fluorouracil and doxorubicin with AdBB-IL24 completely inhibited the growth of colon cancer cells.

Conclusion

We here provide evidence supporting conditionally replicative adenoviral vector-based gene therapy as a powerful strategy to enhance mda7/IL-24-dependent MDR reversion of colon cancer cells.     

Overexpression of the homeobox gene DLX-7 inhibits apoptosis by induced expression of intercellular adhesion molecule-1

OBJECTIVE: DLX genes constitute a subfamily of divergent homeobox genes. We have previously reported that inhibition of DLX-7 expression by an antisense oligonucleotide caused apoptosis in the K562 erythroleukemia cell line, which highly expresses DLX-7. In this study, we have constructed an expression vector encoding human DLX-7, and examined the effects of overexpression of DLX-7 in the IL-3-dependent lymphoid precursor cell line Ba/F3. METHODS: DLX-7 expression vector was electroporated into Ba/F3 cells, and generate a DLX-7 expressing Ba/F3 cells. Northern blot analysis was performed to determine DLX-7 gene expression. WST-1 assay was used to cell proliferation assay. To detect apoptosis, we performed TUNEL assay. Expression of cell surface adhesion molecules was examined by FACS analysis. RESULTS: Growth properties of DLX-7-transfected Ba/F3 cells in the presence of IL-3, did not differ from those of control Ba/F3 cells. However, in the absence of IL-3, DLX-7-transfected cells abrogated growth dependence on cytokines due to inhibition of apoptosis. Because adhesion properties of DLX-7-transfected cells increase, we examined expression of adhesion molecules in these cells. Expression of intercellular adhesion molecule (ICAM)-1 and ICAM-2 were markedly upregulated in DLX-7-transfected cells. Both anti-ICAM-1 antibody and anti-LFA-1 antibody blocked the aggregation of DLX-7-transfected cells. Moreover, in the absence of IL-3, cytokine-independent cell growth was blocked by anti-ICAM-1 antibody. CONCLUSION: These results indicate that DLX-7 overexpression blocks apoptosis and that ICAM-1 expression induced by DLX-7 contributes to this antiapoptotic effect.     

没药甾酮对人肝癌细胞HepG2增殖和凋亡的影响

目的研究没药甾酮对人肝癌细胞HepG2增殖和凋亡的影响。方法以正常人肝细胞L-02作为对照,采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐法观察不同浓度没药甾酮（5～100μmol/L）对人肝癌细胞HepG2和L-02细胞增殖的影响并观察细胞形态的变化;应用流式细胞术检测细胞周期变化和凋亡发生。结果不同浓度没药甾酮均可显著抑制人肝癌细胞HepG2生长,并呈时间、剂量依赖性,最大抑制率可达81.9%±1.92%（100μmol/L）;没药甾酮可使G0/G1期细胞比例增多,G2/M期细胞比例下降,可将细胞阻滞于G0/G1期;没药甾酮诱导人肝癌细胞HepG2发生凋亡,50μmol/L和75μmol/L没药甾酮早期细胞凋亡率分别为24.91%±2.41%、53.03%±2.28%,与对照组相比,差异均具有统计学意义（P〈0.05）。结论没药甾酮可抑制人肝癌细胞HepG2增殖并诱导凋亡,其作用可能与干扰细胞周期有关。     

人遗传印记基因PEG10重组真核表达质粒的构建及其在转染细胞株中的表达

目的构建人遗传印记基因PEG10的全长表达基因质粒,观察PEG10的过表达对人正常肝细胞及其非肝脏来源的对照细胞的作用。方法将PEG10基因全长cDNA直接连入真核细胞表达质粒pcDNA3．1hisC中,以脂质体介导的方法将该表达质粒pcDNA3．1hisC-PEG10转染入人正常肝细胞及其对照细胞,采用RT-PCR、Western blot、MTT、TUNEL等方法分析PEG10基因在正常人肝细胞及其对照细胞中的表达和功能。结果限制性酶切及DNA测序结果均说明所构建质粒为PEG 10全长表达质粒。该质粒经过稳定筛选后稳定表达于细胞内,促进人肝细胞的生长,抑制其凋亡,但对非肝脏来源的对照组细胞没有明显影响。结论PEG10全长表达质粒的构建为进一步研究PEG10基因的效应和机制提供了有利的工具,研究结果显示PEG10基因可以促进人正常胚肝细胞增殖并抑制其凋亡,但对非肝脏来源的对照组细胞人胚肾细胞株293细胞没有明显效应。     

Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells

AIM: To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin(IL)-24 in hepatocellular carcinoma(HCC) cells.METHODS: γ-secretase inhibitors(GSIs) were used to down-regulate Notch1.Hep G2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h.Cell viability was measured by MTT assay.The cellular and nuclear morphology was observed under a fluorescence microscope.To further verify the apoptotic phenotype,cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining.The expression of Notch1,SNAIL1,SNAIL2,E-cadherin,IL-24,XIAP and VEGF was detected by Western blot.The invasion and migration capacities of HCC cells were detected by wound healing assays.Notch1 and Snail were downregulated by RNA interference,and the target proteins were analyzed by Western blot.To investigate the mechanism of apoptosis,we analyzed Hep G2 cells treated with si Notch1 or si CON plus IL-24 or not for 48h by caspase-3/7 activity luminescent assay.RESULTS: GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in Hep G2 cells.The addition of 50 ng/m L IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%,respectively.Treatment with IL-24 alone did not induce any cytotoxic effect.In SMMC7721 cells with the addition of IL-24 to GSI-I(2.5 μmol/L),the reduction of cell viability was only about 25%.Following GSI-I/IL-24 combined treatment for 6 h,the apoptotic rate of Hep G2 cells was 47.2%,while no significant effect was observed in cells treated with the compounds employed separately.Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in Hep G2 cells.Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I.Furthermore,the increased GSI-I and IL-24 in Hep G2 cell was associated with downregulation of MMP-2,XIAP and VEGF.In the absence of treatment,Hep G2 cells could migrate into the scratched space in 24 h.With IL-24 or GSI-I treatment,the wound was still open after 24 h.And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I.Treatment of Notch-1 silenced Hep G2 cells with 50 ng/m L IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination.Caspase-3/7 activity was increased in the presence of si Notch1 plus IL-24 treatment.CONCLUSION: Down-regulation of Notch1 by GSI-I or si RNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of Hep G2 cells.     

Molecular therapy for peritoneal dissemination of xenotransplanted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transfer

BACKGROUND: Gene therapy is an innovative therapeutic approach for cancer. An adenoviral vector expressing the tumour suppressor p53 gene (Ad/p53) is currently under clinical evaluation for various cancers. We recently developed a binary adenoviral vector system that can express the strong proapoptotic gene Bax (Ad/PGK-GV16+Ad/GT-Bax: Ad/Bax). AIMS: To evaluate the potential of Bax gene therapy for gastric cancer, we assessed its antitumour effect in comparison with that of p53. METHODS: The human gastric cancer cell lines MKN-1, MKN-7, MKN-28, and MKN-45 were treated with Ad/Bax or Ad/p53, and cell viability, transgene expression, and caspase activation were assessed in vitro. To compare the antitumour effects of Ad/Bax and Ad/p53 treatment in vivo, subcutaneous tumours and peritoneal dissemination of MKN-45 cells were generated in nude mice. Each mouse underwent intratumoral or intraperitoneal administration of viruses and the growth of implanted tumours was observed after treatment. RESULTS: Treatment with Ad/Bax and Ad/p53 resulted in marked Bax and p53 protein expression and effective apoptosis induction in MKN-1, MKN-7, and MKN-28 cells in vitro. In contrast, MKN-45 cells showed resistance to Ad/p53 and only treatment with Ad/Bax resulted in activation of caspase 3 expression and massive apoptosis. Ad/Bax treatment was more effective in suppressing both subcutaneous and peritoneally disseminated MKN-45 tumours compared with Ad/p53 treatment. CONCLUSION: Ad/Bax treatment significantly inhibited the growth of even p53 resistant gastric cancer in vitro and in vivo. Therefore, adenovirus mediated Bax gene transfer may be useful in gene therapy for gastric cancers.