Quantitative Structure-activity Relationship Studies on the Antioxidant Activity and Gap Junctional Communication of Carotenoids

The antioxidant and gap junctional communication（GJC） activities of carotenoids are known to be the two main anticancer mechanisms.Quantitative structure-activity relationship（QSAR） models of the two activities were developed using stepwise regression and multilayer perceptron neural network based on the calculated descriptors of quantum chemistry.The results showed that the significant molecular descriptor related to the antioxidant activity of carotenoids was the HOMO-LUMO energy gap（EHL） and the molecular descriptor related to the GJC was the lowest unoccupied molecular orbital energy（ELUMO）.The two models of antioxidant activity both showed good predictive power,but the predictive power of the neural network QSAR model of antioxidant activity was better.In addition,the two GJC models have similar,moderate predictive power.The possible mechanisms of antioxidant activity and GJC of carotenoids were discussed.

Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

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 elu- cidate 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 re- verse-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 malig- nant 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. As- sessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was ab- normally located and markedly diminished as compared with normal prostatic epithelial ones, dis- playing 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 indi- cated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein par- ticipated 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.

THE EFFECT OF ALL-TRANS RETINOIC ACID ON GAP JUNCTIONAL INTERCELLULARCOMMUNICATION AND CONNEXIN 43 GENE EXPRESSION IN GLIOMA CELLS

To illuminate the regulating effect of all trans retinoic acid (ATRA ) on gap junctional intercellular communication (GJIC) and connexin 43 (Cx43) ge ne expression in glioma cells, which is tissue and organ specific. Method. Rat C6 glioma cells were exposed to ATRA at a concentration of 1, 10, 10 0 μmol/L respectively, and the GJIC function of the cells was examined with scr ape loading dye transfer assay 24 hours, 48 hours and 72 hours after ATRA treat ment. The effect of ATRA on Cx43 gene expression was measured with semiquantitat ive reverse transcription polymerase chain reaction (RT PCR) 24 hours after ATR A exposure. Results. The GJIC function of C6 glioma cells was significantly increased by ATR A at each concentration applied. The dye passed 4 to 5 rows of cells from the sc raping edge in ATRA treated cells, but only 1 or 2 rows in the control. The augm ent effect was observed 24 hours after each concentration ATRA treatment, and la sted till 72 hours after treatment with 1μmol/L and 10μmol/L ATRA. Forty eigh t hours after exposed to 100μmol/L ATRA, the enhancement of GJIC was less obvi ous. There was no significant increase induced by ATRA on the transcription of C x43 gene, as demonstrated by semiquantitative RT PCR. Conclusion. ATRA turned out to be a potent enhancer on GJIC function in C6 gliom a cells, and the enhancement effect was most probable at post transcriptional l evel.

STUDIES ON THE GAP JUNCTIONAL INTERCELLULARCOMMUNICATION OF HUMAN NASOPHARYNGEALCARCINOMA CELLS AND THE EFFECT OF RII

Human nasopharyngeal carcinoma(NPC) cell line,CNE-2Z, and its clones(L2, H2, L4) with various invasive and metastatic potentials were examined for their gap junctions(GJ), gap junctional intercellular communication(GJIC) and the concentration of cytosolic free calcium(Ca2+). Only a few intermediate junction(IJ)but no GJ structures were observed under electron microscope(EM). CNE-2Z cells showed marked JGIC,while its variants lacked this function using the scraploading dye-transfer technique(SLDT). There was lower concentration of[Ca2+]. in L2 cells(a variant with high invasive and metastatic Potential) compared to that in H2 and L4 cells(variants with medium and low invasive and metastatic Potentials, respectively). These data suggested that high invasive and metastatic potentials might be correlated with the levcl of[Ca2+]i in NPC cells.The effect of RII(4-hydroxycarbophenyl retinamide) on NPC cells also investigated, After 3-7 d of RII(10-5 M) treatment, there was no change in the number of gap junctions and other kind of intercellular junctions in NPC cells observed under EM. The JGIC of CNE-2Z weaked and then disappeared finally with prolonging of RII treatment. However. there was no influence on its variants. The level of[Ca2+], in NPC cells apparently fell after 6 h of RII treatment, and rose to original level with persisting of RII treatment. Whether the fluctuating of[Ca2+]i level is related to the inhibitory effect of RII treatment on growth and invasion of NPC cells needs to be further studied.

Changes of gap junctional intercellular communication in detrusor instability

Objective: To demonstrate the functional changes of gap junctional mediation of intercellular communication in detrusor instability (DI) and its mechanisms. Methods: The function of gap junctional intercellular communication in the cultured bladder detrusor cells was detected by fluorescence redistribution after photobleaching. Results: At the fourth minute after bleaching, the mean fluorescences recovery rates of DI group bladder detrusor cells were (35 791±0 836)%, that of control group (8 645±0 673)%. The mean fluorescence recovery rates of DI group were significantly higher than those of control group ( P <0 01). Conclusion: It shows that the increase of intercellular excitatory communication is one of the important reasons of pathogenesis of DI.

EFFECTS OF LIMONENE,SALVIA MILTIORRHIZA AND TURMERIC DERIVATIVES ON H-RAS ONCOGENE EXPRESSION AND GAP JUNCTION INTERCELLULAR COMMUNICATION IN HUMAN SOLID TUMOR CELL LINES

Objective: To study gap junction intercellular communication (GJIC), H ras oncogene expression and ras oncogene product (P 21 ras protein) expression in four human solid tumor cell lines, W1-38,CACO 2,A549 and PaCa, and the effects of four compounds, Salvia miltiorrhiza derivative (SMD), d Limonene, Turmeric derivative I (TD I) and Turmeric derivative II (TD II), on them. Methods: The abilities of the four solid tumor cell lines to transfer dye to adjacent cells were examined by the scrape loading/dye transfer technique, and the H ras oncogene expression by Northern blotting and P 21 ras protein expression by Western blotting. Results: The results showed the loss of intercellular coupling in PaCa cells, slight GJIC in A549 and CACO 2 cells, and a good GJIC in W1-38 cells. The four compounds could improve the GJIC of PaCa to different extents. The amount of total and membrane associated P 21 ras in PaCa cells were decreased after treatment with SMD, d Limonene and TD I (2.5 μg/ml) for 48 h. Concomitantly, the growth of PaCa cells decreased in soft agar and had enhanced GJIC. The relative potency was found to be:d Limonene>SMD >TD I=TD II. There was no significant effect of the four compounds on H ras oncogene expression. Conclusion: It was suggested that there was an excellent correlation between loss of Lucifer Yellow dye transfer and ras gene mutation rate in the four solid tumor cell lines (ras gene mutation rate inversely correlated with average cell number coupled, r=0.98) i.e., the high ras gene mutation was closely correlated with loss of GJIC in these malignant human tumor cells; The antitumor effect of the monoterpene d Limonene and the phenol compound, SMD, might be related to inhibition of P 21 ras membrane association and enhancement of GJIC, whilst that of the others may be by a different mechanism; The inhibition of P 21 ras membrane association was directly related to the enhancement of gap junction intercellular com munication.

Genotoxic and Nongenotoxic Effects of Glycidyl Methacrylate on Human Lung Fibroblast Cells

Objective To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro. Methods DNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay. Results Exposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC. Conclusions GMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.

Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca^(2+)-dependent gap junction intercellular communication

Background: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. Methods: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca^(2+)-related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. Results: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca ^(2+ )is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. Conclusions: Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

Connexin 40-formed GJIC increases the phototoxicity of photodynamic therapy through ROS-and calcium-mediated pathways

OBJECTIVE To explore the effect of connexin(Cx)40-formed gap junctional intercellular communication(GJIC)on Photofrin-photodynamic therapy(PDT)phototoxicity in Cx40-transfected He La cells and its potential mechanisms.METHODS He La 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 Ca^(2+) 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 PhotofrinPDT.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 maintenance or increase of Cx40-formed GJIC may be a profitable strategy towards the enhancement of PDT therapeutic efficiency.

Basic Investigations EXPRESSION OF GAP JUNCTION PROTEIN Cx43 IN CULTURED HUMAN NORMAL AND MALIGNANT LUNG CELLS

Gap junctional intercellular communicationexchange of small molecules and ions between contiguous cells through membranous gap junctional channelsis essential for growth control and tissue homecotasis. This work concerns the functional expression of gap junction protein connexin 43 (Cx43) in normal human lung cells and the changes in lung carcinoma cells. By. using Northern blot hybridization analysis and Cx43 immunocytochemical methods, it was otherved that cultured normal human embryonic lung cells expressed a high level of Cx43 in both mRNA and protein levels.The Cx43 immunofluorescence was localized at cell membrane regions corresponding to the location of gap junctions. These normal lung cells were competent of intercellular communication function as detected by Lucifer yellow dye transfer. In contrast to normal celis, Cx43 mRNA and protein was not detectable in the carcinoma PG cell line. These tumor cells were defective of intercellular communication function. These results demonstrate that Cx43 is expressed in normal cultured human embryonic lung cells but not in lung tumor cells. The lack of intercellular communication in the lung tumor cell line correlates with dysfunctional intercellular communication. The suggestive role of Cx as a tumor suppersor gene is discussed.

Protective effects of carbenoxolone are associated with attenuation of oxidative stress in ischemic brain injury

Accumulating evidence has suggested that the gap junction plays an important role in the determination of cerebral ischemia, but the underlying mechanisms remain to be elucidated. In this study, we assessed the effect of a gap-junction blocker, carbenoxolone （CBX）, on ischemia/reperfusion-induced brain injury and the possible mechanisms. By using the transient cerebral ischemia model induced by occlusion of the middle cerebral artery for 30 min followed by reperfusion for 24 h, we found that pre-administration of CBX （25 mg/kg, intracerebroventricular injection, 30 min before cerebral ischemic surgery） diminished the infarction size in rats. And this was associated with a decrease of reactive oxygen species generation and inhibition of the activation of astrocytes and microglia. In PC12 cells, H202 treatment induced more coupling and apoptosis, while CBX partly inhibited the opening of gap junctions and improved the cell viability. These results suggest that cerebral ischemia enhances the opening of gap junctions. Blocking the gap junction with CBX may attenuate the brain injury after cerebral ischemia/reperfusion by partially contributing to amelioration of the oxidative stress and apoptosis.