Cx43基因启动子去甲基化与乳腺癌MDA-MB231细胞上皮间质转换的相关性研究

目的:探讨乳腺癌细胞中连接蛋白43(Cx43)基因启动子甲基化与乳腺癌上皮间质转化(EMT)的关系.方法:甲基化PCR检测Cx43启动子甲基化的频度;5-氮杂脱氧胞苷(5-Aza-Dc)培养乳腺癌MD-MBA-231细胞后,反转录PCR(RT-PCR) 乳腺癌细胞中Cx43 Mrna,免疫荧光、蛋白质印迹法检测E-cadherin、波蛋白(vimentin)及Cx43蛋白表达,甲基化PCR检测启动子甲基化的变化.结果:在应用5-Aza-Dc处理前,MD-MBA-231Cx43基因呈甲基化状态,将4.0 μmol/L的5-Aza-Dc作用48 h后,乳腺癌细胞Cx43基因甲基化逆转;Cx43 Mrna表达水平增加,为对照组的6.5倍.蛋白质印迹检测结果显示,Cx43蛋白相对灰度值为4.3±0.2,明显高于对照组.处理组细胞E-cadherin表达增加,vimentin表达下调.结论:5-Aza-Dc能逆转乳腺癌细胞MD-MBA-231的Cx43基因异常甲基化,促进Cx43基因的再表达,在一定程度上逆转乳腺癌MDA-MB231细胞EMT.     

乳腺癌中间隙连接蛋白Cx43和Cx26表达的意义

目的　研究间隙连接蛋白 Cx43和 Cx2 6在乳腺癌中的表达及其与乳腺癌生物学行为的关系。方法　应用 S- P免疫组化方法检测 Cx43和 Cx2 6在 5 0例乳腺癌组织中的表达。结果　间隙连接蛋白 Cx43和 Cx2 6蛋白阳性染色位于乳腺癌细胞的胞膜上和胞浆中 ,阳性表达率分别为 44 .0 %和 32 .0 % ,Cx43和 Cx2 6的阳性表达与乳腺癌恶性程度及转移呈密切相关 (P<0 .0 5 )。结论　间隙连接蛋白表达和细胞间隙连接通讯功能异常 ,可能是乳腺肿瘤发生、发展和恶性进程的一个重要事件 ,也是乳腺肿瘤的生物学行为特征之一     

Can supplementary dietary fibre suppress breast cancer growth?

Case-control studies in diverse populations around the world have reported a lower risk of breast cancer in association with higher intake of dietary fibre and complex carbohydrates. Although this has not been confirmed in prospective studies in the USA, the observations have prompted the hypothesis that prolonged use of dietary fibre supplements might reduce breast cancer risk in high-incidence populations. Several possible mechanisms of action have been suggested, all involving a reduction of bioactive oestrogen levels in the blood. The various mechanisms are not necessarily mutually exclusive. First, a high-fibre diet might reduce circulating oestrogen levels by reducing the enterohepatic recirculation of oestrogen. Second, many plants and vegetables contain isoflavones and lignans capable of conversion in the bowel into weak oestrogens that may compete with oestradiol for target binding-sites. Third, a high-fibre diet is less often associated with obesity, which tends to increase availability of the biologically active 16-alpha metabolites of oestrone. Fourth, a high-fibre diet usually has a lower content of fat and a higher content of antioxidant vitamins, which may protect against breast cancer risk. Finally, diets rich in fibre and complex carbohydrates have been shown to improve insulin sensitivity, with an associated reduction in circulating oestrogen levels. Synergism between these effects offers a possible mechanism by which a high fibre intake might suppress breast cancer growth in women.     

Drug combinations with quercetin: doxorubicin plus quercetin in human breast cancer cells

Purpose  

Doxorubicin is a first-line chemotherapeutic for breast cancer; however, it is associated with severe side effects to non-tumoral tissues. Thus, it is necessary to develop new therapeutic combinations to improve doxorubicin effects at lower concentration of the drug associated with protective effects for non-tumoral cells. In this work, we evaluated whether the plant-derived flavonoid quercetin may represent such an agent.     

In vivo growth of C6 glioma cells transfected with connexin43 cDNA.

In order to examine the possible role of intercellular communication via gap junctions in the control of tumor growth, we have transfected C6 glioma cells with connexin43 cDNA. We obtained several clones with variable expression of connexin43. The growth rate of these clones in culture was inversely related to the degree of expression of the transfected cDNA. To examine the growth of these transfected cells in vivo, cells were grown in spinner culture flasks to form spheroids 250-300 microns in diameter. Spheroids of nontransfected C6 cells produced large gliomas. Immunohistochemical and in situ hybridization analyses revealed relatively high levels of connexin43 protein and mRNA in the host tissue, while little of this protein was detected in the glioma. In contrast, spheroids of connexin43-transfected cells grew more slowly and exhibited elevated levels of connexin43 protein and mRNA. These findings suggest that the expression of connexin43 may be associated with the control of brain tumor growth in vivo.     

过表达间隙连接蛋白43表达对宫颈癌细胞恶性生物学行为的影响

目的探讨过表达间隙连接蛋白43(Cx43)对宫颈癌细胞恶性生物学行为的影响。方法选取2015年1月至2017年12月间深圳市龙岗区第三人民医院收治的31例宫颈癌患者,手术切除的经病理诊断明确为宫颈癌的组织及配对癌旁组织,采用RT-qPCR法检测宫颈癌组织及癌旁组织中Cx43的表达水平。设计过表达Cx43的慢病毒载体及阴性对照,采用RT-qPCR法检测宫颈癌细胞中Cx43的表达水平,CCK-8实验检测细胞在不同时间点的增殖能力,划痕实验检测细胞的迁移能力,Transwell实验检测细胞的侵袭能力,Caspase-3检测细胞的凋亡。结果 RT-qPCR显示,与癌旁组织相比,Cx43在宫颈癌中表达降低,差异有统计学意义(P <0. 01)。宫颈癌细胞过表达组的Cx43表达均增加,差异均有统计学意义(均P <0. 01)。Cx43表达上调后,宫颈癌细胞的增殖、迁移及侵袭能力明显下降,细胞凋亡增加,差异均有统计学意义(均P <0. 01)。结论相较于癌旁组织,Cx43在宫颈癌组织中低表达。Cx43在宫颈癌中起到抑癌作用,可作为宫颈癌的治疗靶点。     

Clinical significance of vascular endothelial growth factor and connexin43 for predicting pancreatic cancer clinicopathologic parameters

Vasiformation is essential for the growth and metastasis of tumor. Vascular endothelial growth factor (VEGF) and connexin43 (Cx43) are important regulatory factors of vasiformation. This study aimed to find out the expression features of VEGF and Cx43 and their significance in pancreatic cancer. The expression levels of VEGF and Cx43 protein in the samples, which came from 100 patients of human pancreatic cancer tissues and adjacent normal pancreatic tissues, were examined by using immunohistochemical streptavidin–peroxidase (S–P) method, Western-blotting, and RT–PCR analyses. Compared with adjacent normal pancreatic tissues, RT–PCR showed that the expression of VEGF mRNA was significantly higher in pancreatic cancer tissues (0.788 ± 0.290, P < 0.01) and Cx43 mRNA was significantly lower in pancreatic cancer tissues (0.403 ± 0.204, P < 0.01). The expression of VEGF protein was higher in pancreatic cancer tissue (0.745 ± 0.254, P < 0.01) by Western-blot, and Cx43 protein was obviously lower in pancreatic cancer tissues (0.373 ± 0.164, P < 0.01). The immunohistochemical S–P method showed as follows: the positive expression rate of VEGF and Cx43 protein was 77 and 48% in pancreatic cancer tissues and 15 and 100% in adjacent normal pancreatic tissues; expressions of VEGF were related to tumor size, TNM stage, and lymph node metastasis (P < 0.05); there was a close relation between the expression of Cx43 and histological grades, TNM stage, and lymph node metastasis (P < 0.05). This present study suggests that VEGF is overexpressed and the expression of Cx43 is lower in pancreatic cancer. The expression of VEGF and Cx43 is significantly correlated with TNM stage and lymph node metastasis. Furthermore, VEGF and Cx43 may play an important role in the occurrence, development, and metastasis of pancreatic cancer. To examine VEGF and Cx43 may be of value in judging the malignancy degree and the prognosis of pancreatic cancer.     

细胞间隙连接蛋白在乳腺癌中的作用

早期研究一致认为细胞间隙连接蛋白（connexin）基因是抑癌基因,在乳腺癌中起抑制肿瘤发生发展的作用,但近年研究发现在很多转移性乳腺癌中connexin表达反而升高,connexin基因可能促进乳腺癌的远处转移.故推测在乳腺癌发生发展、侵袭转移中,connexin通过多种作用机制扮演了不同的角色.在乳腺癌治疗中合理应用connexin将改善患者的疗效.     

Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner

Many lines of evidence support the hypothesis that connexins form a family of tumor-suppressor genes. Transfection of connexin43 (Cx43) into rat C6 glioma cells have revealed that Cx43 functions as a growth- and tumor-suppressor in C6 cells. In previous studies, we and others have reported that several mutant connexins can inhibit gap junctional intercellular communication (GJIC) realized by the wild type in a dominant-negative manner. We have now examined dominant-negative effects of Cx43 mutants on cell growth control exerted by wild-type Cx43 in C6 cells. When 2 Cx43 mutants (L160M and A253V) were transfected into Cx43-transfected C6 cells, they restored anchorage-independent growth capacity and reinforced the tumorigenicity of these cells, meaning that these 2 mutants can inhibit growth-suppressive function of wild-type Cx43 in a dominant-negative manner. Neither of the mutants appeared to affect phosphorylation states and subcellular localization of Cx43 proteins. Intriguingly, the mutant A253V did not suppress GJIC capacity, implying a growth-suppressive pathway mediated by Cx43 may not be related to GJIC.Int. J. Cancer 78:446–453, 1998. © 1998 Wiley-Liss, Inc.     

Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells

Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in human cancer and play an important role in survival signaling. In addition to its involvement with cell survival, VEGFR-3 is a primary factor in lymphatic angiogenesis. Because FAK function is regulated by its COOH terminus (FAK-CD), we used FAK-CD as a target to identify binding partners. We isolated a peptide from a phage library that bound to FAK-CD, specifically the focal adhesion targeting domain of FAK and was homologous to VEGFR-3, suggesting these two tyrosine kinases physically interact. We have also shown that VEGFR-3 is overexpressed in human breast tumors and cancer cell lines. For the first time, we have shown the physical association of FAK and VEGFR-3. The association between the NH(2) terminus of VEGFR-3, containing the peptide identified by phage display, and the COOH terminus of FAK was detected by in vitro and in vivo binding studies. We then coupled a 12-amino-acid VEGFR-3 peptide, AV3, to a TAT cellular penetration sequence and showed that AV3 and not control-scrambled peptide caused specific displacement of FAK from the focal adhesions and affected colocalization of FAK and VEGFR-3. In addition, AV3 peptide decreased proliferation and caused cell detachment and apoptosis in breast cancer cell lines but not in normal breast cells. Thus, the FAK/VEGFR-3 interaction may have a potential use to develop novel molecular therapeutics to target the signaling between FAK and VEGFR-3 in human tumors.     

Exogenous CXCL12 activates protein kinase C to phosphorylate connexin 43 for gap junctional intercellular communication among confluent breast cancer cells

Despite ongoing attempts to improve the overall breast cancer (BC) survival rate, BC cells’ (BCCs) predilection for metastasizing to the bone marrow has enabled BCCs to not only remain dormant, but also evade detection. BCCs are able to acquire quiescence by establishing gap junctional intercellular communication (GJIC) with the stroma through the assembly of connexins (Cxs). The chemoattractant CXCL12 also appears to play a role in GJIC based on its tendency to decrease when GJIC is formed between BCCs and bone marrow stroma. This study investigates the role CXCL12 has on Cx43 expression and PKC-mediated Cx43 phosphorylation. Cx43 gene reporter assays revealed that as the BCCs come in contact with each other and establish GJIC, there is an inverse relationship between CXCL12 level and Cx43 expression. Immunoblot analyses confirmed this relationship at the level of protein, showing decreased Cx43 and reduced Cx43 phosphorylation at higher CXCL12 concentrations. However, real-time PCR studies revealed little change in Cx43 mRNA levels, despite stimulation with different concentrations of CXCL12, indicating CXCL12’s effect on Cx43 is post-translational, through phosphorylation. Immunoblot analyses and functional dye exchange studies showed activation of PKC by exogenous CXCL12 in the phosphorylation, which in turn, increased intercellular communication. These findings elucidate the importance of considering the microenvironment’s role in micrometastasis in clinical studies pertaining to prospective breast cancer treatment.     

Genistein sensitizes inhibitory effect of tamoxifen on the growth of estrogen receptor-positive and HER2-overexpressing human breast cancer cells

Although tamoxifen (TAM) is used for the front-line treatment and prevention of estrogen receptor-positive (ER+) breast tumors, nearly 40% of estrogen-dependent breast tumors do not respond to TAM treatment. Moreover, the positive response is usually of short duration, and most tumors eventually develop TAM-resistance. Overexpression of HER2 gene is associated with TAM-resistance of breast tumor, and suppression of HER2 expression enhances the TAM activity. Soy isoflavone genistein has been shown to have anti-cancer activities and suppress expression of HER2 and ERalpha. The objective of this study was to test the hypothesis that genistein may sensitize the response of ER+ and HER2-overexpressing breast cancer cells to TAM treatment. The combination treatment of TAM and genistein inhibited the growth of ER+/HER2-overexpressing BT-474 human breast cancer cells in a synergistic manner in vitro. Determination of cellular markers indicated that this synergistic inhibitory effect might be contributed in part from combined effects on cell-cycle arrest at G(1) phase and on induction of apoptosis. Further determination of the molecular markers showed that TAM and genistein combination synergistically induced BT-474 cell apoptosis in part by synergistic downregulation of the expression of survivin, one of the apoptotic effectors, and downregulation of EGFR, HER2, and ERalpha expression. Our research may provide a novel approach for the prevention and/or treatment of TAM insensitive/resistant human breast cancer, and warrants further in vivo studies to verify the efficacy of genistein and TAM combination on the growth of ER+/HER2-overexpressing breast tumors and to elucidate the in vivo mechanisms of synergistic actions.     

阿托伐他汀增加乳腺癌细胞对多柔比星敏感性的研究

目的探讨他汀类药物增加乳腺癌细胞对多柔比星敏感性的机制。方法(1)用不同浓度的多柔比星(0、0.01、0.02、0.04、0.08、0.16、0.32、0.64、1.28μg/ml)与0、2μmol/L的阿托伐他汀联合处理MDA-MB-231细胞,通过细胞计数检测试剂盒(CCK-8)检测450 nm波长下的吸光度值,从而计算细胞活力。(2)分别用0.3μg/ml多柔比星、2μmol/L阿托伐他汀单药及两药联合处理MDA-MB-231细胞,并以未经任何药物处理的细胞作为对照组,通过Hoechst染色检测MDA-MB-231细胞凋亡情况,通过细胞划痕实验、Transwell实验检测细胞迁移及侵袭能力,通过Western blot检测MDA-MB-231细胞中caspase 3、caspase 9、3-羟基-3-甲基戊二酰辅酶A还原酶(HMGCR)、甾醇调节元件结合蛋白转录因子2(SREBP2)及低密度脂蛋白受体(LDLR)的表达。细胞活力比较采用析因分析,未凋亡细胞数、划痕面积百分比、穿膜细胞数及不同蛋白表达等指标的多组比较采用单因素方差分析,两两比较采用LSD法。结果(1)与多柔比星单药相比,阿托伐他汀与多柔比星联合应用可以显著抑制MDA-MB-231细胞活力(F=243.043,P<0.001);不同浓度多柔比星组细胞活力比较,差异有统计学意义(F=1803.617,P<0.001);两个因素存在交互作用(F=21.030,P<0.001)。(2)各组的未凋亡细胞数比较,差异具有统计学意义(对照组:94.00±4.24:阿托伐他汀组:57.00±1.41,多柔比星组:34.50±2.12,阿托伐他汀+多柔比星组:19.00±2.83,F=261.021,P<0.001),两两比较结果显示,组间差异均有统计学意义(P均<0.050)。各组间的划痕面积百分比比较,差异具有统计学意义[对照组:(28.94±3.59)%,阿托伐他汀组:(31.00±2.99)%,多柔比星组:(40.16±2.38)%,阿托伐他汀+多柔比星组:(60.86±3.60)%,F=42.080,P<0.050]。与对照组、阿托伐他汀组及多柔比星组相比,阿托伐他汀+多柔比星组划痕面积均增加(P均<0.050)。各组穿膜细胞数比较,差异具有统计学意义(对照组:101.20±14.55,阿托伐他汀组:75.80±7.33,多柔比星组:32.40±4.78,阿托伐他汀+多柔比星组:8.80±2.50,F=118.031,P<0.001),两两比较结果显示差异均具有统计学意义(P均<0.050)。各组MDA-MB-231细胞中凋亡相关蛋白caspase 3、caspase 9的表达比较,差异均有统计学意义(F=128.854、247.530,P均<0.001)。与多柔比星组相比,阿托伐他汀+多柔比星组caspase 3、caspase 9蛋白的表达显著增加(P均<0.050)。4组MDA-MB-231细胞中HMGCR、SREBP2、LDLR蛋白的表达量比较,差异均具有统计学意义(F=183.193、227.470、586.087,P均<0.001)。两两比较结果显示,与对照组比较,阿托伐他汀+多柔比星组中HMGCR表达明显升高,SREBP2、LDLR表达明显降低(P均<0.050);与对照组相比,多柔比星组HMGCR的表达无明显改变(P>0.050),SREBP2、LDLR的表达明显降低(P均<0.050),阿托伐他汀组HMGCR表达高于对照组(P<0.050);与多柔比星组相比,阿托伐他汀+多柔比星组HMGCR表达增加(P<0.050),SREBP2的表达降低(P<0.050),LDLR的表达无明显变化(P>0.050)。结论阿托伐他汀通过抑制HMGCR表达,减少细胞内胆固醇的合成,使细胞更依赖于外源性胆固醇的摄取;多柔比星通过抑制LDLR表达减少外源性胆固醇的摄取;因此,当两药联用后由于细胞内胆固醇合成减少,使细胞更依赖于外源性胆固醇的摄取,对多柔比星更加敏感。     

间隙连接蛋白Cx43、Cx45在鼻咽癌组织中的表达

背景与目的：间隙连接在细胞间的营养物质,离子和细胞调节因子的交换中起重要作用。间隙连接异常,细胞间的物质交换障碍,往往导致细胞分裂失控,在有些癌组织和癌细胞中存在间隙连接的功能异常。恢复这些癌细胞的间隙连接功能,它们表现出正常细胞的生物学表型,因此,探讨细胞间隙连接蛋白在鼻咽癌中的表达,将有可能为临床诊断提供方法。为鼻咽癌的发病机理提供新思路。方法;采用免疫组化技术检测间障连接蛋白Cx43,Cx45在鼻咽组织中的表达。结果：（1）Cx43,Cx45在鼻咽慢性炎症组织和鼻咽癌组织中有表达差异,在鼻咽癌中,Cx43,Cx45阳性率为44．8％,46．6％,与鼻咽慢性炎症柱状上皮细胞阳性率为85％和100％比较,显著性下降（P＜0．01）,（2）鼻咽慢性炎症组织比鼻咽癌组织含鳞状细胞的百分率低（P＜0．01）,分别为29．7％和56．9％,（3）Cx43,Cx45在鼻咽癌旁柱状上皮细胞中的表达低于癌旁鳞状上皮细胞（P＜0．001）,高于癌细胞（P＜0．01）,结论：Cx43,Cx45在鼻咽组织中的表达异常,可能与鼻咽组织鳞化和癌变有关。     

Anti-HER2 antibody and heregulin suppress growth of HER2-overexpressing human breast cancer cells through different mechanisms.

Previous reports have shown that certain anti-HER2 antibodies and heregulin can inhibit clonogenic growth of breast and ovarian cancers that overexpress HER2. Anti-HER2 antibodies bind to HER2 directly, whereas heregulin does not bind to HER2 alone, but rather interacts with HER2 through the formation of heterodimers with HER3 or HER4. The purpose of the present study was to elucidate the mechanisms by which anti-HER2 antibody and heregulin inhibit tumor growth. The anti-HER2 monoclonal antibody (mAb) ID5 was found to block G1-S progression of the cell cycle, whereas heregulin inhibited passage through G2-M. Compatible with the effects on the cell cycle, treatment with mAb ID5 decreased levels of cyclin-dependent kinase (CDK) 2, cyclin E, and CDK6 proteins and reduced cyclin E-CDK2-associated kinase activity; mAb HD5-treated cells had increased p27Kip1 expression and an increased association of p27Kip1 with CDK2. In contrast, treatment with heregulin increased protein levels of CDK2, CDK6, CDC2, and cyclin B1. More Retinoblastoma protein was found in the hypophosphorylated state in the cells treated with mAb ID5, whereas more retinoblastoma protein was in the hyperphosphorylated state in heregulin-treated cells. Heregulin was able to induce cell differentiation as assessed by Oil Red O staining and apoptosis as assessed by sub-G1 peak on flow cytometry and the presence of DNA fragmentation in ApopTag histochemistry staining. Neither differentiation nor apoptosis was observed in the cells treated with mAb ID5. We conclude that anti-HER-2 mAb ID5 and heregulin exert growth inhibition through different mechanisms. In mammary cells overexpressing HER2, anti-HER2 mAb ID5 induces G1 arrest, whereas heregulin induces G2-M arrest, cell differentiation, and apoptosis.     

Combination of non-viral connexin 43 gene therapy and docetaxel inhibits the growth of human prostate cancer in mice

Docetaxel (DTX) is used for the treatment of advanced hormone refractory prostate cancer. Connexin 43 (Cx43) is a tumor suppressor gene, and transfection of the Cx43 gene increases sensitivity to several chemotherapeutic agents. The objective of this study was to evaluate the effectiveness of combination therapy of Cx43-expressing plasmid DNA (pCMV-Cx43) and DTX both in vitro and in vivo using a non-viral vector in human prostate cancer PC-3 cells. Transfection of pCMV-Cx43 into the cells neither inhibited tumor growth nor increased gap junctional intercellular communication; however, combination therapy of pCMV-Cx43 and DTX significantly inhibited cell growth. Forced expression of Cx43 in the cells induced apoptotic cells by down-regulation of Bcl-2 expression and significantly more up-regulation of caspase-3 activity than either treatment alone. The combination of repeated intratumoral injection of pCMV-Cx43 (10 microg/tumor) with non-viral vector and a single intravenous injection of DTX (15 mg/kg) was compared with a repeated injection of Cx43 alone and a single injection of DTX alone on PC-3 tumor xenografts. Significant antitumoral effects were observed in mice receiving combined treatment, compared with DTX alone. The data presented here provide a rational strategy for treating patients with advanced hormone refractory prostate cancer.     

Vitamin D analogues suppress IGF-I signalling and promote apoptosis in breast cancer cells

Survival factors are known to promote cell viability, and factor deprivation can be a potent apoptotic signal. Insulin-like growth factors are potent mitogens and inhibitors of apoptosis for many normal and neoplastic cells with insulin-like growth factor-I (IGF-I) being the most effective in many breast cancer cell lines. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues inhibit IGF-I-stimulated growth of MCF-7 human breast cancer cells. The aim of this study was to determine the relationship between inhibition of IGF-I responsiveness and induction of apoptosis by vitamin D analogues in breast cancer cells. Vitamin D analogues EB1089 and CB1093 inhibited autonomous and IGF-I-stimulated growth of MCF-7 and T47D cells and autonomous growth of IGF-I-insensitive Hs578T cells. In MCF-7 cells, IGF-I alone (4 nM) protected against apoptosis mediated by serum deprivation. Co-treatment with vitamin D analogues prevented the anti-apoptotic effects of IGF-I. In T47D cells, IGF-I treatment provided only partial protection against apoptosis induced by serum deprivation and co-incubation of serum-deprived cells with 100 nM CB1093 and IGF-I abrogated this partial protection. In Hs578T cells, addition of IGF-I did not prevent apoptosis induced by serum deprivation. However, treatment with CB1093 attenuated the protective effect of the serum in these cells. Our findings suggest that vitamin D analogues inhibit IGF-I signalling pathways to promote apoptosis in breast cancer cells.