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OBJECTIVE To explore the effect of connexin (Cx) 40-formed gap junctional intercellular communication (GJIC) on Photofrin- photodynamic therapy (PDT) phototoxicity in Cx40- transfected HeLa cells and its potential mechanisms. METHODS HeLa cell line stably transfected to express Cx40 was seeded at high and low cell density, respectively, to assess in vitro photosensitivity using CCK8 assay. Western blot assay was performed to detect the expression of Cx40. The intracellular ROS and Ca2 +concentrations were determined using flow cytometer. 4-HNE and ceramide were measured using ELISA assay. RESULTS Cx40-composed GJ formation at high density enhances the phototoxicity of Photofrin-PDT. When the Cx40 is not expressed or Cx40 channels are blocked, the phototoxicity in high-density cultures substantially reduces, indicating that the enhanced PDT phototoxicity at high density is mediated by Cx40-composed GJIC. The GJIC-mediated increase in PDT phototoxicity was associated with ROS and calcium-mediated stress signaling pathways. CONCLUSION The work uniquely presents the ability of Cx40-composed GJIC to enhance the sensitivity of malignant cells to PDT, and indicates that mainte?nance or increase of Cx40-formed GJIC may be a profitable strategy towards the enhancement of PDT therapeutic efficiency.
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<p><b>Objective</b>To study the effect of Icariin on rat Leydig cells with TGF-β1-induced injury.</p><p><b>METHODS</b>We determined the optimal concentration of Icariin for protecting primarily cultured Leydig cells against TGF-β1-induced injury by methyl thiazolyl tetrazolium assay. We detected the effects of Icariin on the secretion of estradiol (E2) and activity of aromatase in the injured Leydig cells by radioimmunoassay and Tritium water release experiment and its effect on the gap junctional intercellular communication (GJIC) between the Leydig cells by fluorescence distribution after photobleaching.</p><p><b>RESULTS</b>Different concentrations of Icariin showed different degrees of protective effect on the TGF-β1-treated Leydig cells, the effect observed at 20 μg/ml and at its optimum at 160 μg/ml. After treatment of the injured Leydig cells with Icariin at 160 μg/ml, significant improvement was observed in the E2 secretion and aromatase activity (P<0.01) as well as in the GJIC between the Leydig cells (P<0.01).</p><p><b>CONCLUSIONS</b>Icariin can effectively protect rat Leydig cells against TGF-β1-induced injury, which is largely attributed to its effects of increasing E2 synthesis, enhancing aromatase activity, and improving GJIC between Leydig cells.</p>
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Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis.mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (χ2=4.025, P＜0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.
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Objective To investigate the effects of PM2.5 on gap junctional intercellular communication (GJIC) between rat cardiomyocytes. Methods Primary cultured cardiomyocytes were prepared from 1-day-old Sprague-Dawley rats and exposed to PM2.5(1,10,100 ?g/ml)for 24 hours. The GJIC between cardiomyocytes was detected by the scrape loading dye transfer assay. The distribution and density of connexin43(Cx43) in the cells was detected by indirect immunofluorescence and the expression of Cx43 was detected by western blotting. Results The gap junctional intercellular communication between cardiomyocytes was significantly inhibited by PM2.5 in a dose-dependent manner. The fluorescence density of Cx43 was significantly decreased in PM2.5-treated cells,and the expression of Cx43 was also slightly decreased. Conclusion PM2.5 can inhibit GJIC between cardiomyocytes,which may be mediated by the decreased expression and aberrant distribution of Cx43 in PM2.5-treated cells.
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Objective:To investigate Cx43 expression and gap junctional intercellular communication(GJIC) in ectopic and eutopic endometrial stromal cells in endometriosis(EMs),and to explore the influence of aberrant GJIC in stromal cells on pathogenesis of EMs. Methods:The stromal cells were isolated from samples including 24 ectopic endometriotic tissues located in ovaries,41 eutopic endometria with endometriosis and 30 normal endometria. The endometrial stromal cells models were established in vitro by being cultured in manual conditions mimicked with estrogen and progesterone. Laser scanning confocal microscopy(LSCM) was used to determine the expression of Cx43 protein and the function of GJIC in three groups stromal cells. Results:The success rate of isolation and culture of endometriotic stromal cells was 45.8%(11/24);of eutopic endometrial stromal cells with EMs was 92.7(38/41);of normal endometrial stromal cells was 93.3%(28/30). The purities of ectopic and eutopic endometrial stromal cells were 95% and 98% respectively. The level of Cx43 protein and the function of GJIC in stromal cells from ectopic endometrial tissues were much lower than those from the other two groups,the highest level of Cx43 protein and the function of GJIC were observed in normal endometrial stromal cells group,and the differences among these groups were significant (P﹤0.01). Conclusions:(1) It will be helpful to establish models of normal,ectopic and eutopic endometrial stromal cells in vitro simultaneously when investigating the pathgenesis of EMs. (2)Downregulation of Cx43 expression and aberrant function of GJIC are related to pathogenesis of EMs. Regulation of Cx43 or GJIC in endometrial stromal cells is implied to be a potential strategy to treat EMs.
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Objective To examine the effects of different n-6/n-3 polyunsaturated fatty acid(PUFA) ratio in gap junctional intercellular communication (GJIC) in breast cancer cell lines. Methods After MCF-7(ER + )and MDA-MB-231(ER - )cells were treated respectively with n-6/n-3 polyunsaturated fatty acid ratio (1-10), Ki-67 expression in nuclear by immunocytochemistry, connexin 26 and 43 expression in cytoplasm and membrane by Western blotting, and GJIC by scrape-loading dye transfer method were performed. Results Immunocytochemistry showed that pure n-6 and 10∶1 n-6/n-3 increased Ki-67 expression (P