Effect Of Panax Notoginseng Monomer Saponin On Cytotoxicity Of Cisplatin And Gap Junction

Objective: This study was designed to investigate the effect of panax notoginsengmonomer saponins (Rg1, Rb1, R1, Re) extracted from panax notoginseng saponins onthe gap junction (GJ) and the cytotoxity of cisplatin in Hela cells transfected withconnexin32and connexin26（connexin32/connexin26, Cx32/26）plasmid. We alsoobserved whether the panax notoginseng monomer saponins could enhance thecytotoxity of cisplatin through the gap junction.Methods:1. Cell culture, Transfection and Selection: In this experiment we used Helacells transfected with Cx32/26plasmid. This bidirectional plasmid could expressconnexin32and connexin26under the control of promoter. In the normal medium, theexpression of connexin was suppressed, but when selected with G-418and hygromycinand induced with doxycycline, the connexin protein would express in the Hela cells.This bidirectional expression could prevent the cell damage from the connexinover-expression.2. The effect of panax notoginseng monomer saponins on gapjunctional intercellular communication was detected by “Parachute Dye-CouplingAssay”: The donor cells were double-labeled with Calcein-AM, and treated withdifferent drugs, then allowed to attach to the monolayer receiver cells and formed gapjunctions for4h. Examined the average number of receiver cells containing dye perdonor cell with fluorescence microscope.3. The cytotoxity of cisplatin and panaxnotoginseng monomer saponins were determined by Standard colony-forming assay:The clone cells with or without gap junction channels were established by seeding cellswith different concentration. Using this method we observed whether the effect of panaxnotoginseng monomer saponins on cytotoxity of cisplatin relative with gap junction. Weused the colory forming efficiency, which was called “Surviving Fraction” as index toevaluate the cytotoxicity of cisplatin.4. The expression of Cx32/Cx26of panaxnotoginseng monomer saponins were measured by western blot. Collected protein andused BCA protein excretion. After boiling5min with100°C water, transfered protein on PVDF film with15%polyacrylamide gel electro-phoresis. After overnightincubation of primary antibody and1hour of second antibody, exposured the filmto BIO-RAD. The results were analyzed with Quantity One.Results:1. In the presence of GJ,2-APB could markedly enhance the SurvivingFraction of cisplatin, RA could markedly reduce the Surviving Fraction ofcisplatin(P<0.05). These data indicated that enhancement of GJ could enhance thecytotoxicity of cisplatin, reduction of gap junction reduces the cytotoxicity of cisplatin.2. Rb1(5μM-100μM) and Re(5μM-100μM) had no effect on GJ in Hela cells;5μM and10μM Rg1also had no effect on GJ,(25μM-100μM) Rg1obviously enhanced the gapjunction, the max enhancement rate of100μM was44.04%±8.71%. The enhancementof dye coupling treated with Rg1(100μM) for4h,24h and48h had no statisticsdifference; R1(10μM-100μM) could enhanced the gap junction in dose dependentmanner. But the effect of R1(50μM) was not time-dependent.3. Western blot resultsshowed that treated with Rg1(25-100μM) and R1(25μM-100μM) for4h had no effecton expression of Cx32/26in HeLa cells.4. In the presence of GJ, combination of Rg1and cisplatin significantly decreased the surviving fraction compared to onlycisplatin-treatment group, indicated that Rg1could enhance the cytotoxicity of cisplatin,but in absence of GJ, there was no significantly difference between these two groups;Whenever in the presence of GJ or without GJ, combination of R1and cisplatin had noeffect on the surviving fraction compared to cisplatin group, it indicated that R1couldnot increase the cytotoxicity of cisplatin.Conclusion:1. In the presence of gap junction, inhibition of GJ decreases the cytotoxicity ofcisplatin, while up-regulation enhances it.2. Rb1and Re has no effect on gap junction function composed of Cx32/26; Rg1andR1could obviously enhance GJ of HeLa cells.3. The effect of Rg1and R1on GJ is independence with the expression of Cx32/26.4. Rg1could increase the cytotoxicity of cisplatin through gap junction.