| Objectives:1. To investigate the influence of gap junction composed of Cx32onantineoplastic effect of etoposide in vitro.2. To investigate the influence of ellagic acid and its mechanism on theantineoplastic effect of etoposide in vitro.Methods: Hela cells that were transfected with and stably expressed Cx32gene wereinduced by1μg/ml doxycycline.1."Parachute assay"was used to assay the gap junction intercellularcommunication mediated by Cx32in Hela cells and its functional modulation by thepharmacological agents (oleamide,18-α-GA, retinoid acid and ellagic acid).2."Standard colony-forming assay" was applied to determine the cellgrowth-inhibiting effect of etoposide in Hela cells with functional modulation of thegap junction. The effects of ellagic acid on the cytotoxicity of etoposide on cells withor without gap junction channels were assayed by seeding high density (confluent,GJformed) and low density (preconfluent, no GJ formed) cells.3."Hoechst33258staining" and "Annexin V/PI double staining, flowcytometry" were used separately to assess the changes in etoposide-induced apoptosisof Hela cells with the gap junction functions modulation.4."Western blotting" was applied to detected expression of Cx32;"Immunofluorescence assay" was used to detected expression of Cx32located on themembrane in Hela cells.Results:1．Influence of gap junction composed of Cx32on antineoplastic effect ofetoposide 1.1"Parachute assay" results showed that the intercellular dye coupling throughgap junction was decreased when the cells exposed to25μM oleamide or10μM18-α-GA, while it was increased when the cells exposed to10μM RA compared withcontrols. The inhibition rate of colony formation in high-density Hela cells exposed toetoposide was (0.42±0.02) much higher, compared with these in the group oflow-density cells (6.03%±0.75%). The antineoplastic effect of etoposide wasreduced when cells were pretreated with oleamide or18-α-GA, and surviving fractionwas substantially increased at high-density cells (P<0.01). While the antineoplasticeffect of etoposide was increased when cells were pretreated with RA, and survivingfraction was substantially decreased in high-density cells (P<0.01); however, at lowcell density, there was very little effect of oleamide,18-α-GA or RA on etoposideresponses.1.2The results of "Hoechst33258staining"showed that the apoptosis rates ofcells exposed to30μM etoposide for24hours were increased significantly comparingwith control group.The apoptosis rates of cells exposed to30μM etoposide inhigh-density cells(confluent, GJ formed) were (12.13±1.34%) much higher,compared with these in low-density cells (no GJ group,6.03±0.75%). The resultsdemonstrated that the formation of gap junction significantly increased the apoptosisrate induced by etoposide.2． Influence of ellagic acid on the antineoplastic effect of etoposide2.1The results from "standard colony-forming assay" showed that pretreatmentof cells with5μM etoposide for1hour and with8μM ellagic acid for24hours couldsignificantly reduced the surviving fraction of cells compared with etoposide group athigh cell density (confluent, GJ formed), however, at low cell density (preconfluent,no GJ formed), there was very little effect of EA on etoposide responses. The resultssuggested that EA could increase the antineoplastic effect of etoposide by gapjunction function.2.2The results of" Annexin V/PI double staining" showed that the apoptosisrates of cells exposed to30μM etoposide combined with8μM ellagic acid for24hours were (12.61±0.30%) much higher, compared with etoposide group (8.04± 0.04%). The results of "Hoechst33258staining" showed that the apoptosis rates ofcells exposed to30μM etoposide combined with8μM ellagic acid for24hours were(17.04±0.56%) much higher, compared with etoposide group (12.13±0.30%). Theresults demonstrated that ellagic acid could increase the apoptosis rate of Hela cellsinduced by etoposide through gap junction.2.3The cells expressing Cx32was pretreated1μg/ml doxycline combined with8μM ellagic acid for24hours and48hours, compared with control group,"western blotting" showed that Cx32total protein was increased;Immunofluorescence showed the Cx32expression located on the membrane in Helacells was increased.2.4"Parachute assay" results showed that the cells exposed to8μM ellagic acidfor4hours, the intercellular dye coupling through gap junction was no different withcontrol group; while the cells exposed to8μM ellagic acid for24hours and36hours,the intercellular dye coupling through gap junction was increased comparing withcontrol group.Conclusion:1. The antineoplastic effect of etoposide was enhanced in Hela cells with anincreased gap junction intercellular communication mediated by Cx32and wasreduced in cells with a decreased gap junction intercellular communication;2. At high-density Hela cells (confluent, GJ formed), ellagic acid could enhancedthe antineoplastic effect of etoposide in vitro;3. Antineoplastic effect of etoposide in vitro increased with ellagic acid wasrelated to the expression of Cx32total protein and located on the membrane;4. Antineoplastic effect of etoposide in vitro increased with Ellagic acid wasrelated to the function of gap junction intercellular communication mediated by Cx32. |
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