Pattern of cell‐to‐cell transfer of microRNA by gap junction and its effect on the proliferation of glioma cells

MicroRNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of microRNA, which exerts biological effects on tumor cells. However, the length of microRNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to microRNAs consisting of 18‐27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the microRNAs were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and shRNA significantly decreased the transfer of these microRNAs. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by microRNA‐34a. Additionally, these effects of microRNA‐34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of microRNA by transfer of microRNA to neighboring cells. Our data are the first to clarify the pattern of microRNA transmission through gap junctions and provide novel insights to show that antitumor microRNAs should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.     

Cigarette smoke components inhibited intercellular communication and differentiation in human pancreatic ductal epithelial cells

Smoking is a well-documented risk factor for the development of pancreatic adenocarcinoma. Although the most abundant polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke are methylated anthracenes and phenanthrenes, the epigenetic toxicity of these compounds has not been extensively studied. We previously showed that methylanthracenes, which possess a bay-like structure, affect epigenetic events such as an induced release of arachidonic acid, inhibition of gap junctional intercellular communication (GJIC) and induction of mitogen-activated protein kinases in a pluripotent rat liver epithelial stem cell line. Anthracenes with no bay-like structures were inactive. These biological effects are all molecular events associated with the promotional phase of cancer. A human immortalized, nontumorigenic pancreatic ductal epithelial cell line, H6c7, was examined to study the epigenetic toxicity of PAHs related to pancreatic cancer by using scrape-loading dye transfer, immunostaining, RT-PCR and telomerase assay methods. H6c7 cells were GJIC-incompetent and exhibited high telomerase activity when grown in growth factor and hormone-supplemented medium. In the presence of the cAMP elevating drugs (forskolin and IBMX) the cells became GJIC competent and expressed connexins. Telomerase activity was also decreased by cAMP elevating drug treatment. After induction of cAMP, 1-methylanthracene with bay-like structures inhibited GJIC, whereas the 2-methylanthracene lacking a bay-like structure had no effect on GJIC. Telomerase activity remained high in 1-methylanthracene treatment but not with 2-methylanthracene. These results indicate that a prominent component of cigarette smoke, namely methylanthracenes with distinct structural configurations, could be a potential etiological agent contributing to the epigenetic events of pancreatic cancer.     

转染连接蛋白基因对人脑胶质瘤细胞间隙连接通讯及其生长变化的研究

目的 研究连接蛋白（Cx)基因对人脑胶质瘤细胞的细胞间隙连接通讯（GJIC）及其增殖的抑制作用,探索以Cx43基因治疗胶质瘤的可行性。方法 将含Cx43cDNA的质粒,以脂质体介导转染Cx43表达缺失的TJ905人胶质母细胞瘤细胞,通过Northern印染杂交、原位杂交及免疫组化染色检测Cx43mRNA及蛋白表达,划痕标记荧光染料示踪技术（SLDT）检测GJIC,MTT法测定细胞增殖率,核仁组成区嗜银蛋白（AgNOR)染色检测细胞增殖活性,TUNEL法检测细胞凋亡。结果 转染后TJ905细胞有不同程度的Cx43mRNA和蛋白表达及GJIC恢复。Cx43表达水平高的克隆细胞增殖明显下降,细胞凋亡并未增加。结论 Cx43基因及GJIC在恶性胶质瘤的发生发展过程中起重要作用,可能成为恶性胶质瘤基因治疗的优选靶的之一。     

依托咪酯对神经胶质瘤细胞缝隙连接通讯的影响

背景与目的：细胞缝隙连接(gap junction,GJ)通讯能增强放疗或化疗药物对肿瘤细胞的细胞毒性作用。以往研究发现部分麻醉药物能够改变GJ功能从而影响肿瘤细胞对放疗的敏感性。该研究拟观察依托咪酯对神经胶质瘤细胞由缝隙连接蛋白Cx43组成的缝隙连接功能的影响,为麻醉药对化疗敏感性影响的机制研究提供线索。方法：采用磺酰罗丹明B法观察依托咪酯对神经胶质瘤细胞的抑制作用;细胞接种荧光法观察依托咪酯对神经胶质瘤细胞GJ功能的影响。结果：0.1、0.5、1和5μmol/L依托咪酯在作用4 h时间内均对细胞生长无抑制作用,将不影响细胞缝隙的数量;取药物浓度接近血药浓度的依托咪酯作用细胞4 h,与对照组相比,0.1μmol/L依托咪酯作用细胞4 h后,神经胶质瘤细胞GJ通讯的荧光传递功能无明显变化;而0.5和1μmol/L依托咪酯作用细胞4 h后,神经胶质瘤细胞的荧光传递功能明显减弱。结论：依托咪酯能抑制神经胶质瘤细胞Cx43组成的GJ功能。     

Inhibition of intrinsic gap-junction intercellular communication and enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant-negative connexin 43 mutant

The tumor-suppressive property of the connexin gap-junction proteins was postulated from the fact that their function of cell coupling is impaired in most cancer cells. However, in conflict with this notion, certain cancer cells are able to communicate through gap junctions despite their malignancy. To explain this phenomenon, we studied by using a dominant-negative strategy the effect on tumorigenicity of loss of intrinsic gap-junction intercellular communication (GJIC) in the rat bladder carcinoma cell line BC31, which shows both expression of connexin 43 (Cx43) and intercellular communication. In cells transfected with a mutant Cx43 with seven residues deleted from the internal loop at positions 130–136 (Cx43Δ), transport of the resulting connexin protein to the plasma membrane occurred normally, but the GJIC of the cells was effectively abolished at the level of permeability of established gap junctions. Dominant-negative inhibition of GJIC by Cx43Δ accelerated growth of BC31 cells in nude mice. In contrast, when GJIC in BC31 cells was artificially enforced by transfection of wild-type Cx43, the cells lost the capacity to grow in vivo. Decreased phosphorylation of Cx43Δ suggested close interaction of the internal loop of connexin with its commonly phosphorylated domains in the C-terminal tail and involvement of this interaction in gap-junction permeability. Therefore, we conclude that the intrinsic GJIC observed in cancer cells should be considered a tumor-suppressor factor and that its level may influence malignant growth capacity. Mol. Carcinog. 23:254–261, 1998. © 1998 Wiley-Liss, Inc.     

人脑胶质瘤组织高表达nNav1.5促进肿瘤细胞的迁移和侵袭

目的：观察电压-门控钠离子通道（voltage-gated sodium channel, VGSC）亚型nNav1.5在人脑胶质瘤组织中的表达,并探讨其对脑胶质瘤U251细胞迁移及侵袭的影响。方法：收集中国医科大学附属第一医院神经外科于2011年10月至2012年10月手术切除并经病理证实的脑胶质瘤组织标本68例,应用免疫组织化学S-P法检测脑胶质瘤组织中nNav1.5的表达。设计并化学合成nNav1.5基因特异性小干扰RNA（nNav1.5-siRNA）,用脂质体介导转染胶质瘤U251细胞,应用Real-time PCR和Western blotting法分别检测U251细胞中nNav1.5 mRNA和蛋白的表达水平,并采用细胞划痕实验和Transwell侵袭实验检测U251细胞迁移和侵袭能力的变化。结果：nNav1.5在人脑胶质瘤组织中表达的阳性率显著高于正常组织（72.6% vs 23.0%,P<0.01）,并且其在高级别胶质瘤（WHO Ⅲ～Ⅳ级）组织中的阳性率明显高于低级别胶质瘤（WHOⅠ～Ⅱ级）组织（85.8% vs 52.9%,P<0.01）。nNav1.5-siRNA转染可显著抑制U251细胞中nNav1.5 mRNA和蛋白的表达（P<0.01）;转染后U251细胞的迁移距离明显小于未转染细胞\[(0.019±0.015) vs（0.223±0.031）mm,P<0.01\],且其侵袭指数明显低于未转染细胞\[(2.99±0.15)% vs（6.77±0.26）%,P<0.01\]。〖JP2〗结论：nNav1.5在人脑胶质瘤组织中高表达,干扰nNav1.5表达可显著抑制胶质瘤细胞的迁移和侵袭能力,nNav1.5是胶质瘤恶性侵袭的调控因子并有望成为胶质瘤的新标志物和治疗靶点。     

Suppressed gap junctional intercellular communication in carcinogenesis of endometrium.

To examine whether and at which stage of endometrial carcinogenesis decreased connexin expression occurs, we investigated changes in the expression of the gap junction proteins, connexin 26 (Cx26), Cx32 and Cx43, in human endometrial hyperplasia and cancer samples. Forty-eight endometrial tissue samples (15 endometrial hyperplasias and 33 endometrial cancers) were subjected to immunofluorescence and RT-PCR analysis. In endometrial hyperplasia, Cx26 was aberrantly expressed in all samples as revealed immunohistochemically. There was weak or negative expression in 12 samples (80.0%) and diffuse expression in cytoplasm in 3 samples (20.0%). Cx32 expression in those samples was similar to that of Cx26; there was weak or negative expression in 11 samples (73.3%) and diffuse expression in 4 samples (26.7%). In endometrial cancer, Cx26 was expressed weakly or negatively in 25 samples (75.8%), diffusely in 6 samples (18.2%) and normally in 2 samples (6.1%), while Cx32 was expressed weakly or negatively in 26 samples (78.8%), diffusely in 5 samples (15.2%) and normally in 2 samples (6.1%). It was confirmed that weak staining of Cx26 and Cx32 was due to poor expression of their mRNA. All samples showed weak Cx43 protein expression as revealed by immunohistochemical analysis. In the majority of samples, concomitant expression levels of Cx26 and Cx32 protein were observed, confirming our long-term hypothesis that Cx26 and Cx32 are both abnormally regulated in a coordinated fashion in the endometrium. Our results indicate that during endometrial carcinogenesis, loss of gap junctional intercellular communication (GJIC) may occur due to the suppressed expression and the aberrant localization of connexin at relatively early stages.     

The phosphorylation of ephrin‐B2 ligand promotes glioma cell migration and invasion

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin‐B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin‐B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin‐B1 and ‐B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan–Meier analysis demonstrated ephrin‐B2, but not ephrin‐B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin‐B2 were high in GBM. Immunohistochemistry demonstrated ephrin‐B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin‐B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin‐B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin‐B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin‐B2. These results demonstrate that high expression of ephrin‐B2 is a strong predictor of short‐term survival and that ephrin‐B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.     

Human giant larvae-1 promotes migration and invasion of malignant glioma cells by regulating N-cadherin

Human giant larvae-1 (Hugl-1) is a human homologue of Drosophila tumor suppressor lethal (2)-giant larvae and has been reported to be involved in the development of human malignancies. Previous studies performed by our group demonstrated that Hugl-1 inhibits glioma cell proliferation in an intracranial model of nude mice. However, the exact molecular mechanisms underlying the participation of Hugl-1 in glioma invasion and migration, and in the depolarizing process remain largely unknown. Utilizing the U251-MG cells with stable expression of Hugl-1, the present study used wound healing, Transwell invasion and western blot assays to explore the role and specific mechanism of Hugl-1 in glioma invasion and migration. The results of the present study demonstrated that overexpression of Hugl-1 decreased cell-cell adhesion and increased cell-cell extracellular matrix adhesion. In addition, overexpression of Hugl-1 promoted pseudopodia formation, glioma cell migration and invasion. The molecular mechanism of action involved the negative regulation of N-cadherin protein levels by Hugl-1. Overexpression or knockdown of N-cadherin partially suppressed or enhanced the effects of Hugl-1 on glioma cell migration and invasion, respectively. Furthermore, Hugl-1 inhibited cell proliferation, while promoting cell migration, which suggests that it may serve a two-sided biological role in cellular processes. Taken together, these results suggest that Hugl-1 promotes the migration and invasion of malignant glioma cells by decreasing N-cadherin expression. Thus, Hugl-1 may be applied in the development of targeted and personalized treatment.     

Breast cancer metastatic potential: correlation with increased heterotypic gap junctional intercellular communication between breast cancer cells and osteoblastic cells

The breast cancer metastasis-suppressor gene BRMS1 is downregulated in metastatic breast cancer cells. Previous reports have shown restoration of gap junctional intercellular communication (GJIC) in the metastatic human breast carcinoma cell line MDA-MB-435 (435) transfected with BRMS1 cDNA. Metastasis, to a large extent in most breast cancers, occurs to bone. However, the reason for this preferential metastasis is not known. We explored cell-to-cell communication between 435 carcinoma cells and a human osteoblastic cell line, hFOB1.19, to determine whether carcinoma cells can form gap junctions with bone cells and to explore the role of these heterotypic gap junctions and the BRMS1 gene in breast cancer metastasis to bone. 435 cells displayed greater cell-to-cell communication with hFOB 1.19 cells than with themselves. Transfection of BRMS1 into 435 cells increased homotypic gap junctional communication but did not significantly affect heterotypic communication with hFOBs. However, heterotypic communication of BRMS1 transfectants with hFOB cells was reduced relative to homotypic communication. In contrast, parental 435 cells displayed greater heterotypic communication with hFOBs relative to homotypic communication. Our results suggest that there are differences in the relative homotypic and heterotypic GJIC of metastasis-capable and -suppressed cell lines.     

Leptin促进人脑胶质瘤U87MG细胞的迁移和侵袭

目的:探讨瘦素(leptin)对人脑胶质瘤U87MG细胞迁移及侵袭能力的影响及其机制。方法:Leptin处理U87MG细胞,采用细胞划痕实验检测U87MG细胞的迁移能力,Transwell实验检测U87MG细胞的侵袭能力,RT-PCR及Western blot-ting法检测U87MG细胞中MMP-2及MMP-9 mRNA和蛋白的表达。结果:Leptin明显促进U87MG细胞迁移能力[(152.42±3.29)vs(83.24±2.61)μm,P<0.05]和侵袭能力[(31.78±5.04)vs(17.03±2.41)个细胞,P<0.05],leptin能显著上调U87MG细胞中MMP-2、MMP-9 mRNA[(0.76±0.04)vs(0.35±0.02),(0.84±0.02)vs(0.41±0.06);均P<0.05]及蛋白[(0.79±0.03)vs(0.23±0.01),(0.81±0.05)vs(0.39±0.03);均P<0.05]的表达。MMP抑制剂GM6001(10μmol/ml)可以逆转leptin对U87MG细胞迁移[(82.05±2.98)vs(81.76±3.25)μm,P>0.05]和侵袭能力[(19.23±2.46)vs(18.02±1.98)个细胞,P>0.05]的影响。结论:Leptin可以促进人脑胶质瘤U87MG细胞的侵袭及迁移,其机制可能与上调MMP-2、MMP-9的表达有关。     

Wnt信号调控胶质瘤或胶质瘤干细胞增殖及迁移作用的研究进展

近年研究发现Wnt信号通路与神经胶质瘤及其肿瘤干细胞的细胞增殖及细胞迁移相关,Wnt信号蛋白及其关键家族成员的表达水平与胶质瘤恶性程度密切有关,提示Wnt信号及相关通路的靶干预可能成为恶性胶质瘤治疗的新途径.     

Inducible expression of the gap junction protein connexin43 decreases the neoplastic potential of HT-1080 human fibrosarcoma cells in vitro and in vivo

Numerous studies have demonstrated a correlation between dysregulation/loss of connexin expression or gap junction intercellular communication (GJIC) function and decreased growth control both in human tumors and tumor cell lines. Likewise, restoration of constitutive connexin expression/function is correlated with increased growth control/decreased tumorigenicity. Here, we show for the first time that inducible restoration of connexin43 (Cx43) expression and GJIC function in a human tumor line of mesenchymal origin (HT-1080, fibrosarcoma) resulted in a lowered neoplastic potential. Specifically, HT-1080 cells induced to express Cx43 demonstrated diminished foci formation when in co-culture with normal fibroblasts, decreased colony formation under anchorage-independent conditions, and reduced tumor growth when injected into immunodeficient mice. These results, obtained utilizing an inducible system that helps address issues of clonal heterogeneity, strongly implicate Cx43 as a tumor suppressor in human tissue of mesenchymal origin and GJIC as a regulatory mechanism for cellular growth control both in vitro and in vivo. This study also further supports the hypothesis that loss of Cx43/GJIC in human tumors may play an important role in the dysregulation of normal growth control.     

Gap junctions modulate glioma invasion by direct transfer of microRNA

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.     

Direct gap junction communication between malignant glioma cells and astrocytes

Gap junctions are intercellular channels that connect the interiors of coupled cells. We sought to determine the extent to which malignant glioma cells form gap junction channels with astrocytes from either adult human brain or rat forebrain. The astrocytic gap junction protein, connexin 43 (Cx43), was identified in immunoreactive plaques at areas of cell-to-cell contact between cocultured glioma cells and astrocytes. These gap junction plaques were composed of functional channels, because extensive dye coupling was evident between the glioma cells and astrocytes from both human and rat brain. Calcium signaling was also readily transmitted from glioma cells to astrocytes and vice versa. In live rat brain, injection of glioma cells prelabeled with the gap junction tracer, dicarboxy-dichlorofluorescein, revealed extensive dye transfer to host cells, demonstrating that malignant glioma cells directly couple with normal brain cells. These observations suggest that intercellular communication via gap junctions may play a role in regulating cellular interactions during tumor invasion. In fact, the presence of gap junctions between astrocytes and glioma cells was sufficient to induce a transformation of astrocytic phenotype. Astrocytes cocultured with C6 glioma cells overexpressing Cx43 were significantly smaller and expressed a lower level of glial fibrillary acidic protein than astrocytes cocultured with otherwise identical mock-transfected, gap junction-deficient C6 cells. Thus, direct cellular coupling with glioma cells result in a phenotypic transformation of astrocytes that may contribute to the susceptibility of surrounding tissue to glioma invasion.     

Resveratrol and X rays affect gap junction intercellular communications in human glioblastoma cells

Resveratrol (3,4',5-trihydroxystilbene) is a polyphenol synthesized by a wide variety of plant species in response to injury, UV irradiation and fungal attack. Many studies have revealed a variety of resveratrol intracellular targets whose modulation gives rise to overlapping responses leading to growth arrest and death. Many authors have reported different human cancer cell lines, treated with resveratrol at micromolar concentrations, arrested their proliferative cycle in the G1/S boundary or in the S phase and this cell cycle arrest was followed by apoptotic death. Less is known about the ability of resveratrol to modify the effect of radiation exposure in normal and cancer cells. Considering that controlled exposure to ionizing radiation is one of the most used treatments in cancer patients and that these schedules are not always effective in medical practice, as in the case of glioma patients, the testing of combined treatment protocols (resveratrol and ionizing radiation) could be of interest, opening the door to future studies which would examine the pharmacological activity of resveratrol. In this study we have looked into whether resveratrol is able to modulate cell cycle progression in human glioblastoma cells and to regulate GJs expression in cancer cells. With this aim in mind we have performed a cytofluorimetric multiparameter assay to quantify the presence of GJs in U87 glioma cells treated with resveratrol and/or X rays. We report that resveratrol induces a delay in cell cycle progression and both alone and in combination with X rays is able to enhance gap junction Intercellular Communications.     

Ubiquitin‐specific peptidase 39 promotes human glioma cells migration and invasion by facilitating ADAM9 mRNA maturation

Glioma cells are characterized by high migration and invasion ability; however, the molecular mechanism behind both processes still remains to be investigated. Several studies have demonstrated that ubiquitin‐specific protease 39 (USP39) plays an oncogenic role in various cancer types. Here, we investigated the expression and function of USP39 in patients with glioma. Oncomine database analysis revealed that high USP39 expression was significantly correlated with poor overall survival in patients with glioma. Knockdown of USP39 in U251 and U87 cell lines significantly inhibited their migration and invasion in vitro. Gene expression profiling of glioma cells transduced with short hairpin RNA (shRNA) against USP39 revealed that disintegrin and metalloproteinase domain‐containing protein 9 (ADAM9), a molecule previously related to tumor cell migration and invasion, was significantly downregulated. Furthermore, USP39 induced ADAM9 messenger RNA (mRNA) maturation and decreased the expression of integrin β1. Additionally, overexpression of ADAM9 inhibited the migration and invasion of glioma cells caused by USP39 depletion in vitro. USP39 promoted the invasion of glioma cells in vivo and reduced the overall survival of the mice. Altogether, our data show that USP39 induces mRNA maturation and elevates the expression of ADAM9 in glioma cells and may thus be considered potential target for treating patients with glioma.