低氧下丹参酮ⅡA对人胃癌SGC7901细胞HIF-1α与c-Met表达的影响

目的 观察低氧下丹参酮ⅡA(Tan ⅡA)对人胃癌SGC7901细胞c-Met蛋白表达的影响及其与低氧诱导因子-1α(HIF-1α)表达的关系.方法 用氯化钴(CoCl2)创建低氧模型,设常氧对照组、低氧对照组和低氧加不同浓度Tan ⅡA组.分别取0.5、2.0、10.0mg/L Tan ⅡA干预低氧胃癌细胞48 h后,免疫细胞化学二步法检测HIF-1α和c-Met蛋白表达的变化.结果 免疫细胞化学法显示,TanⅡA呈剂量依赖性地抑制低氧诱导的HIF-1α和c-Met蛋白表达,且二者呈高度正相关(n=4,r=0.996,P<0.01).结论 低氧条件下,Tan ⅡA可能通过抑制HIF-1α的表达进而抑制c-Met蛋白的表达,提示Tan ⅡA可能对防治低氧导致的肿瘤侵袭转移具有重要作用.     

低氧下丹参酮ⅡA对人胃癌SGC7901细胞HIF—1α与c—Met表达的影响

目的观察低氧下丹参酮ⅡA（Tan ⅡA）对人胃癌SGC7901细胞c—Met蛋白表达的影响及其与低氧诱导因子-1α（HIF-1α）表达的关系。方法用氯化钴（CoCl2）创建低氧模型,设常氧对照组、低氧对照组和低氧加不同浓度Tan ⅡA组。分别取0．5、2．0、10．0mg／L Tan ⅡA干预低氧胃癌细胞48h后,免疫细胞化学二步法检测HIF-1α和c-Met蛋白表达的变化。结果免疫细胞化学法显示,TanⅡA呈剂量依赖性地抑制低氧诱导的HIF-1α和c-Met蛋白表达,且二者呈高度正相关（n=4,r=0．996,P〈0．01）。结论低氧条件下,TanⅡA可能通过抑制HIF-1α的表达进而抑制c-Met蛋白的表达,提示TanⅡA可能对防治低氧导致的肿瘤侵袭转移具有萤要作用。     

Efficacy of the bystander effect in the herpes simplex virus thymidine kinase-mediated gene therapy is influenced by the expression of connexin43 in the target cells

Tumoricidal "bystander effect" observed in the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy was studied between different rat glioma cell lines (9L and C6 cells) under both in vitro and in vivo conditions. For that purpose, mixed populations of wild-type cells (9Lwt and C6wt) and respective HSVtk gene-transduced cells (9Ltk and C6tk) were examined for their sensitivity to GCV. A potent in vitro bystander effect was observed in 9Lwt/9Ltk and 9Lwt/C6tk combinations but not in C6wt/9Ltk and C6wt/C6tk combinations. In vivo bystander effect studied in a subcutaneous tumor model in athymic nude mice was also potent in 9Lwt/9Ltk and 9Lwt/C6tk combinations. Because the expression of connexin43, a major protein in the connexin family gene products, in 9L cells is much higher than that in C6 cells, the results suggest that the amount of connexin in target (wild-type) cells but not in effector (HSVtk gene-bearing) cells is important for the generation of the bystander effect. This hypothesis was further confirmed by the observation that in vitro bystander effect in C6wt/C6tk combination was potentiated by transduction of the connexin43 gene to the target cells.     

Downregulation of connexin 43 in nasopharyngeal carcinoma cells is related to promoter methylation

Down-regulation of Cx43 expression had been shown to occur in nasopharyngeal carcinoma cells. The present study was undertaken to estimate if methylation of the promoter region in Cx43 gene was responsible for the repression of Cx43 expression in the CNE-1 nasopharyngeal carcinoma cells. Calcein transfer and lucifer yellow transfer were detected to evaluate gap junction intercellular communication (GJIC) in CNE-1 cells. It was found that the control CNE-1 cells showed no fluorescent dye transfer. After treatment with DNA methyltransferase inhibitor 5-aza-CdR, fluorescent dye transfer between cells became obvious. RT-PCR and Western blot were performed to determine the expression of Cx43 gene. The control CNE-1 cells showed a low expression level of Cx43, whereas 5-aza-CdR-treated CNE-1 cells showed an enhanced level of Cx43 expression. Methylation-sensitive restriction enzyme and PCR analysis showed that the methylation of the Cx43 gene promoter region occurred in CNE-1 cells. In addition, treatment with 5-aza-CdR inhibited the growth (including anchorage-independent growth) of CNE-1 cells, and resulted in an accumulation of cells in G0/G1 phase. These results indicate the promoter methylation as an important role in inactivation of Cx43 in CNE-1 cells.     

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.     

Action of db-cAMP on the bystander effect and chemosensitivity through connexin 43 and Bcl-2-mediated pathways in medulloblastoma cells

Medulloblastoma (MB) is one of the most common malignant brain tumors of childhood and is associated with a poor prognosis. Gap-junctional intercellular communication (GJIC) is an important mode for cell-to-cell communication. Dysfunctional GJIC is exhibited in most cancer cells. There is significant evidence that GJIC is important in at least some prodrug/suicide gene systems by augmenting the bystander effect (BE). GJIC is made up of connexins (Cxs), among which Cx43 is present in most tissues. Bcl-2, an important apoptosis blocker, is closely associated with the sensitivity to anticancer drugs. Our study showed that dibutyryl cyclic adenosine monophosphate (db-cAMP) upregulated the Cx43 expression and GJIC function in Daoy medulloblastoma cells. It directly enhanced the BE using a herpes simplex virus thymidine kinase (HSV?tk)/ganciclovir (GCV) system, which was blocked by a Cx43 inhibitor. In addition, db-cAMP increased the cytotoxicity of temozolomide and teniposide, possibly by downregulating the Bcl-2 expression and inducing apoptosis. Taken together, we demonstrated the beneficial effect of db-cAMP in treating medulloblastoma depending on the upregulation of BE and chemosensitivity through Cx43 and Bcl-2-mediated pathways.     

肝细胞癌中间隙连接蛋白Cx43和E-钙粘附素的表达

目的 研究间隙连接蛋白(connexin43 Cx43)和E-钙粘附素(E-cadherin)在肝细胞癌(hepatocelluar carcinoma, HCC)中的表达及其关系,探讨它们在HCC发生和发展中的作用.方法采用免疫组化技术检测47例HCC中间隙连接蛋白Cx43、E-cadherin的表达.结果 Cx43、E-cadherin阳性率分别为42.55%、46.81%.随着疾病进展(TNM分期),Cx43阳性率降低.肝硬化组Cx43阳性率明显低于无肝硬化组(χ2＝4.7135,P＝0.03).肝内血管瘤栓组E-cadherin的阳性率明显高于无肝内血管瘤栓组(P＝0.028).Cx43、E-cadherin的阳性率在肿瘤大小、癌组织分化程度组无显著差异(P＞0.05).Spearman相关分析未发现Cx43与E-cadherin存在显著相关.结论 Cx43、E-cadherin蛋白的异常表达可能在HCC的发生、进展和肝内转移中发挥作用.检测二者的表达有助于综合判断HCC的生物学行为.     

维甲酸对HeLa细胞间隙连接蛋白基因cx43表达的调节作用

目的：探讨分化诱导剂维甲对肿瘤抑制基因－细胞间隙连接蛋白基因cx４３在人子宫颈癌细胞系ＨeＬa中表达的调节作用。方法：应用核酸原位杂交、流式细胞仪、Ｗesten blot及Ｌucifer Ｙellow划痕标记荧光传输技术,研究维甲酸作用对ＨeＬa细胞cx４３mＲＮＡ及其蛋白表达,以及对ＨeＬa细胞生长和通迅功能找调节作用。结果：ＨeＬa细胞经维甲酸处理后,原位杂交显示,ＨeＬa细胞cx４３ mＲＮＡ水平上调;流式细胞仪分析,Ｃx４３蛋白     

Increased susceptibility to urethane-induced lung tumors in mice with decreased expression of connexin43

Gap junction intercellular communication capacity and connexin expression are reportedly decreased in human lung cancer. The mechanisms by which connexins, the gap junction proteins, act as tumor suppressors are unclear. In order to understand the involvement of connexins in tumorigenesis, we analyzed the effect of the heterologous deletion of Gja1 [the connexin43 (Cx43) gene] on the development of lung adenomas in mice. Heterozygous (Cx43(+/-)) and wild-type mice (Cx43(+/+)) were treated or not with single doses of urethane at 15 and 17 days after birth. Twenty-five weeks later, both the number and size of nodules were increased in Cx43(+/-) mice as compared with Cx43(+/+) mice. Moreover, the lesions were histologically more aggressive in the heterozygous mice. However, no increase in spontaneous lesions was observed in the lungs of untreated Cx43(+/-) mice. Heterozygous mice effectively presented lower expression of Cx43 genes and decreased amounts of Cx43. In conclusion, our results indicate that deletion of one allele of the Cx43 gene clearly favors the carcinogenic effect of urethane administration and results in a higher susceptibility to lung adenoma formation in mice.