Analysis of the Apoptotic Mechanisms of Snake Venom Toxin on Inflammation-induced HaCaT Cell-line

Abstract

Objectives : Inthisstudy,therolesofInterleukin(IL)-4andSignaltransducerandactivatorof transcription6(STAT6),whichhavebeenreportedtoplayaroleinthepathogenesisofinflammationandcancer,wereevaluatedinsnakevenomtoxin(SVT)-inducedapoptosis. Methods : InflammationwasinducedinhumanHaCaTkerationocytes,bylipopolysaccharide (LPS;1μg/mL)ortumornecrosisfactor-α (TNF-α),followedbytreatmentwithSVT(0,1,or 2μg/mL).CellviabilitywasassessedbyMTTassaysafter24h,andtheexpressionoflevels ofIL-4,STAT6,andtheapoptosis-relatedproteinsp53,Bax,andBcl-2wereevaluatedby westernblotting.Electromobilityshiftassays(EMSAs)wereperformedtoevaluatetheDNA bindingcapacityofSTAT6. Results : MTTassaysshowedthatinflammation-inducedgrowthofHaCaTcellsfollowingLPSor TNF-α stimulationwasinhibitedbySVT.Westernblotanalysisshowedthatp53andBax, whichpromoteapoptosis,wereincreased,whereasthatofBcl-2,ananti-apoptoticprotein, wasdecreasedinaconcentration-dependentmannerinLPS-orTNF-α-inducedHaCaTcells followingtreatmentwithSVT.Moreover,followingtreatmentofHaCaTcellswithLPS,IL-4concentrationswereincreased,andtreatmentwithSVTfurtherincreasedIL-4expressionina concentration-dependentmanner.WesternblottingandEMSAsshowedthatthephosphory-latedformofSTAT6wasincreasedinHaCaTcellsinthecontextofLPS-orTNF-α-induced inflammationinaconcentration-dependentmanner,concomitantwithanincreaseintheDNA bindingactivityofSTAT6.