Effects Of Baicalein On Inducing Apoptosis And Inhibiting Proliferation In Human Cutaneous Squamous Cell Carcinoma Cell Line SCL-1

IntroductionSkin squamous cell carcinoma,as a kind of skin malignant cancers,may metastasize more easy than other skin tumors,such as basal cell carcinoma.At present the treatment of most SCC paitients is surgical removal via operation,for example, Mohs' operation.But some special SCC cases are not suitable to skin surgical therapy.It is important and urgent to choose effective medicines.Thus,the development of new effective anticancer agents to treat skin squamous cell carcinoma seems to be an attractive perspective.Baicalein(5,6,7-trihydroxyflavone)is a flavonoid derived from the root of Scutellaria baicalensis or Huang Qin,a widely used Chinese herbal medicine,which has used historically in anti-inflammatory therapy,and has been confirmed to inhibit the activity of 12-lipoxygenase(12-LOX)that is overexpressed in tumor tissues such as prostate cancer,breast cancer and so on.It has been confirmed that Baicalein,can induce apoptosis and inhibit proliferation of many human cancer cells,such as human hepatoma cell lines,prostaye cancer cells,human lung squamous carcinoma CH27 cells,human gastric cancer SGC-790 cells and human breast cancer cells.It was also confirmed that the effect of baicalein on apoptosis of some kind tumor cells was dose-effective.Rightnow there is no clear evidence which show Baicalein's effect on human Cutaneous Squamous Cell Carcinoma Cell Line SCL-1.In this study we try to examine whether Baicalein has effect on apoptosis and proliferation of human Cutaneous Squamous Cell Carcinoma Cell Line SCL-1.we choose HaCaT cells and all-trans retinoic acid as control.We hope our study could give you some information on finding a more effective treatment measure to some SCC patients.Materials and Methods1.Cell cultureSCL-1 cells(obtained from the Free University of Berlin,Germany by Franklin Meidical center)and HaCaT cells(the Free University of Berlin,Germany by Dr.Norbert E.Fusenig)were cultured in RPMI-1640 medium supplemented with 10%fetal calf serum(FCS),in 5%CO₂,at 37℃saturation humidity box.They were stimulated with Baicalein or all-trans retinoic acid for 12,24,48hours and then be collected.2.MTT assaySCL-1 cells were divided into eight groups with different concentrations of Baicalein(purchased from Sigma)at 12.5,25,50,100μg/ml,all-trans retinoic acid at 2×10^-5mol/L,respectively,which contrast to the culture medium cells with PBS.SCL-1 cells were seeded onto 96-well plates.Each well add MTT.solution reagent (5mg/mL)20μL,to culture 4h,at 37℃,and stop culture.Each well add DMSO 200mL,concusing 10min,using ELISA reader to detect the OD at wavelength 570 Nm of each well.Restrain rate(%)=(OD of the contrast team-OD of the disposal team)/OD of the contrast team×100%3.Cell morphology observationWith the culture fluid in which the concentration of Baicalein is 25ug/mg and all-trans retinoic acid is 2×10^-5mol/L,observing the cell morphology and taking photos with inverted phase contrast microscrope after 48h culture.4.Detection by flow cytometryThe earlier period apoptosis was measured by flow cytometry analysis.Incubation SCL-1 cells,after12h,24h,48h culture with the concentration of Baicalein 0μg/ml, 25μg/ml and 50μg/ml,washing in PBS for twice,and then add 5μLAnnexin V/FITC and 10μL PI solution at 20μg/ml,reaction for 10min avoding light,detection the apoptosis cells by flow cytometry.Analyse the result with software(Cell Quest).5.Observation cell apoptosis with AO stainSCL-1 cells were incubated with Baicalein at 0μg/ml and 50μg/ml for 48h,washing with PBS once,add 5μL AO into 95μL cell suspension(1×10⁷/ml).Take one drop sample on the surface of a clean glass slide and observed with fluorescence microscope at 490nM.Results1.Inhibition effect on the growth of SCL-1 and HaCaT cellsBaicalein at certern concentration has inhibition effect on the growth of SCL-1 cell.This effect is dose-dependent.However,Baicalein's effect on HaCaT cells was not as obvious as SCL-1 cells.All-trans retinoic acid at 2×10^-5mol/L show certen inhibition effect on the growth of SCL-1 cell.The growth inhibition rates of SCL-1 cells reached 88.92%when 100μg/ml baicalein acted 48h.2.Cell morphology observationThe shape of SCL-1 cell was not regular,became volume smaller,by Baicalein at 25μg/ml after 48 hours.But The shape of HaCaT cell didn't changed much.Observation cell apoptosis with AO stain:Observation with fluorescence microscope at 490nM,the later stage apoptosis of SCL-1 cell treated with 50μg/ml Baicalein for 48h is obvious.3.Detection the earlier period apoptosis by flow cytometryIncubating with Baicalein at 50μg/ml for 24h,SCL-1 cell early stage apoptosis is obvious.Conclusion1.Baicalein can inhibit proliferation of SCL-1 cell and induce apoptosis. 2.It's effect on HaCaT cells isn't obvious.All-trans retinoic acid at certern concentration can also inhibit proliferation of SCL-1 cell.3.There is synergism effect on Baicalein and all-trans retinoic acid to SCL-1 cell.