Ovarian Cancer Stem Cells And Cisplatin Induced Chemoresistence Influenced By The Effective Components Of Lcssy

Part 1Objectives:The purpose of this study was to investigate the existence of Ovarian Cancer Stem Cells(OCSCs)in SKOV3 cell line and enrichment of OCSCs by cisplatin,and to identify the impact of Lichong Shengsui Yin(LCSSY)effective component on SKOV3 cell line and OCSCs.Methods:SKOV3 cells were cultured in vitro,IC50 was identified by CCK8 after 24 hours treatment with cisplatin.Flow Cytometry was employed to observe the percentages of CD44+CD117+,CD133,CD44,CD117 cells after 24 hours treatment with eight different interventions,apoptosis and cycle of SKOV3 cells was detected after 24 and 48 hours treatment with interventions mentioned above.Results:1.Percentages of CD44+CD117+,CD133,CD44 cells with cisplatin treatment increased(P<0.05)and CD117 cells decreased compared with control group(P<0.05).Middle does of LCSSY effective component combined with cisplatin could decline percentages of CD44+CD117+,CD133,cells compared with control and cisplatin group(P<0.05).Interventions with middle does of LCSSY effective component don’t change percentages of OCSCs in SKOV3 cell line compared with control group(P>0.05)。2.Early apoptosis rate and total apoptosis rate of SKOV3 cells with cisplatin were time-dependent(P<0.05).It was time and dose-dependent for the treatment of low,middle,high does with LCSSY effective component.The longer and higher dose of intervention,the higher early apoptosis rate.LCSSY effective component could help cisplatin to improve early apoptosis and total apoptosis rate compared with cisplatin(P<0.05).3.G1 and S phase were influenced by LCSSY effective component and cisplatin.Cells decresed in G1 phase and increased in S phase except for interventions with middle does of LCSSY effective component combined with cisplatin compared with control group with 24 hours treatment(P<0.05).Treatment with LCSSY effective component+DDP shown obviously effect in decreasing G1 phase and increasing S phase fraction compared with cisplatin(P>0.05.Cells decresed in G1 phase and increased in S phase for interventions with low,middle,high does of LCSSY effective component combined with cisplatin compared with control group with 48 hours treatment(P<0.05).S phase fraction was highest in treatment with LCSSY effective component+DDP for 48 hours.Conclusions:1.There are OCSCs such as CD44+CD117+、CD133 cells in ovarian cancer SKOV3 cell line.Cisplatin can enrich OCSCs,while the clinical dose of LCSSY effective component dosen’t.2.LCSSY effective component combined with cisplatin could decrese percentages of OCSCs,and help reduce enrichment of OCSCs indused by cisplatin.3.The apoptosis of ovarian cancer SKOV3 cells indused by LCSSY effective component was time and dose-dependent,and could help improve apoptosis of cisplatin.4.LCSSY effective component may improve cisplatin sensitivity through improving S phase fraction.Part 2Objectives:This study aimed to examine the effect of Lichong Shengsui Yin effective component on Ovarian cancer stem cells regulation related genes and cisplatin resistance.Methods:Establishing subcutaneous xenotransplanted tumor models of human SKOV3 cells in BALB/C nude mouse.48 mice were randomly divided into control group,DDP gurop,Lichong Shengsui Yin effective component gurop and Lichong Shengsui Yin effective component combined with DDP gurop.Each group with 12 mice treat for 18 days.Measure the tumor size every three and draw the tumor growth curve.Immunofluorescence identified morphologies of CD44+CD117+and CD133cells.The mRNA amd protein progression of Nanog、Oct4、ABCC1 were assayed with RT-PCR and immunohistochemistry.Results:1.Tumor with the treatment of Lichong Shengsui Yin effective component and Lichong Shengsui Yin effective component combined with DDP growed slow and had the trend to shrank,and LCSSY Lichong Shengsui Yin effective component combined with DDP had a better effect.Tumor with the control and DDP group increased and tumor size was bigger than the other two groups,and DDP group growed slower than control group.2.CD44+CD117+,CD133 cells were identified in nude mose tumors.These cells account for a small proportion in ovarian cancer,and have a bigger size in cell volume and nucleus than non-cancer stem cells.3.Nanog and POU5F1 mRNA expression was upregulated in the treatment groups compared with control group.ABCC1 mRNA expression was upregulated in the DDP and Lichong Shengsui Yin effective component gurop(P>0.05),and was downregulated in Lichong Shengsui Yin effective component combined with DDP gurop compared with control group(P<0.05)4.Immunohistochemistry detected decrease expression of Nanog in treatment groups compared with control group,of which treatment with DDP gurop and Lichong Shengsui Yin effective component combined with DDP gurop showed significant decrease(P<0.05),and these two interventions had no statistically difference(P>0.05).Compared with control group,expression of POU5F1 decreased in Lichong Shengsui Yin effective component group(P<0.05),DDP gurop and Lichong Shengsui Yin effective component combined with DDP gurop showed no significant decrease(P>0.05).Expression of ABCC1 decreased in Lichong Shengsui Yin effective component group and Lichong Shengsui Yin effective component combined with DDP gurop(P<0.01),but increased in DDP gurop(P>0.05)when compared with control group.Conclusions:1.CD44+CD117+、CD 133 cells account for a small proportion in ovarian cancer,and have a bigger size in cell volume and nucleus.2.Lichong Shengsui Yin effective component could inhibit the growth of tumor and improve the anti-tumor effect of DDP in vivo。3.Cisplatin indused chemoresistence may be influenced by Lichong Shengsui Yin effective component through targeting stem cell properties and cisplatin sensitivity.