Effects Of Casticin On Self-renewal Of Cervical Cancer Sphere-forming Cells Derived From Hela Cell Line

ObjectiveTo investigate whether casticin(CAS) can effectively inhibit the viability ofproliferation and capacity of self-renewal in cervical cancer sphere formingcells(SFCs) from HeLa cell line and explore the mechanism underlying whetherinhibition self-renewal by casticin in cervical cancer SFCs is involved in reducingprotein expression of cell surface marker CD133and stem cell signaling moleculeβ-catenin.MethodsHuman cervical cancer Hela cell line was cultured in vitro. And then the cervicalcancer SFCs were obtained and amplified by suspended culture method with stemcell-conditioned medium. Characteristic of self renewal in cervical cancer SFCs wasidentified by observing sphere-forming capacity of single cell. Compared withparental cells, the protein expression of cell surface marker CD133was detected byflow cytometry(FCM) using phycoerythrin (PE)-conjugated anti-CD133antibodiesand the expression of β-catenin was analyzed using western blot in cervical cancerSFCs. The effect of the different concentration of CAS on the viability in cervical cancerSFCs and parental cells was determined using MTT.The effect of CAS at the differentconcentration on sphere-forming capacity of cervical cancer SFCs including primaryand secondary passages was detected using tumor sphere forming assay. The proteinexpression of cell surface marker CD133in cervical cancer SFCs was analyzed byFCM using PE-conjugated anti-CD133antibodies. And the protein expression ofβ-catenin in cervical cancer SFCs was analyzed using western blot.Results Cervical cancer HeLa cell line cells were plated in6-well ultra low attachmentplates at a density of1000cells/ml and then were cultured in stem cell conditionedmedium for6days. At last, we obtained typical clonal nonadherent3D cervicalcancer spheres which can serial passage to become cervical cancer spheres. And thesecond generation of cervical cancer sphere cells, namely, cervical cancer SFCs wereused to sequential experiments. After cervical cancer SFCs were limiting diluted,single cell in stem-cell conditioned medium was plated in96-well ultra lowattachment plates and it could form new spheres with suspension culture for8days.Compared with parental cells, the protein expression of cell surface marker CD133was increased in cervical cancer SFCs and the expression of β-catenin was elevated aswell.The result of MTT assay demonstrated that CAS preferentially inhibited viabilityof cervical cancer SFCs compared with parental cells. As the tumor sphere formingassay result show, CAS, in a concentration-dependent manner suppressed the capacityof sphere forming in primary and quadratic passage cervical cancer SFCs at theconcentration range from0.1to3.0μmol/L(P<0.05). CAS(0.1,0.3,1.0and3.0μmol/L)decreased the expression of cell surface marker CD133protein, in aconcentration-dependent manner (P<0.05). After the treatment with CAS(0.1,0.3and1.0μM) for24h, the protein expression of β-catenin was down-regulated by CAS, in aconcentration-dependent manner (P<0.05).Conclusion1. Cervical cancer SFCs possess the characteristics of cervical cancer stem-like cells.2. CAS inhibit viability and self renewal of cervical cancer SFCs.3. The effect of CAS on inhibition of self renewal in cervical cancer SFCs isinvolved in down-regulating the protein expression of CD133and β-catenin.