Exploration Of The Role Of Cisplatin In Transform Tomor Cells In Laryngeal Carcinoma To Stem-like Cancer Cells

Laryngeal carcinoma is one of the most common malignancies of the headand neck region. The morbidity was dominant increased so far,accounting for5.7%-7.6%of a systemic malignancy.Its etiopathogenesis is not clearly yet,generally believed that it involved with smoking、drinking、air pollution、viral infections and sex hormones and its receptors.Resent years，following the comprehensive treatment of minimally invasive surgery,radiotherapy,chemotherapy,concurrentchemoradiotherapy and biological therapy,the survival rate and quality of patient’s life have been improved.At the same time,patients often have poor prognosis because of the ralapse,metastasis and resistance of chemoradiotherapy.Many reports testified that maybe due to cancer stem cells(CSCs). CSCs is a small subpopulation cells that possess capability of self-renewal and multipoential differentiation in tumor, and are responsible for tumorigenesis, progression, metastasis, and recurrence. Side population cells,one of the methods in isolation and identification of CSCs, have the ability to efflux the DNA binding dye--Hoechst33342, and are highly enriched for stem cells,was used to isolation and identification of CSCs.By virtue of their typical profiles in Hoechst red versus Hoechstblue bivariate fluorescent-activated cell sorting dot plots stimulate by UV, it can be observed on the side of Main Population(MP)[1,2]. Part Ⅰ: The optimizing conditions in sorting of side populationin cell line Hep-2Objective To investigate the optimizing conditions in isolation of the sidepopulation in laryngeal carcinoma cell line Hep-2.Methods Cells were detached from the culture flask with0.25%trypsin-EDTA(Invitrogen),and then suspended in RPMI1640supplemented with2%fetal bovineserum(FBS) at at a concentration of1×106cells/ml.①The trail Samples wereincubated with Hoechst33342(Sigma-Aldrich) at a concentration of5μg/ml、9μg/ml、10μg/ml、11μg/ml for90minutes at37℃.②The were incubated withHoechst for50、70、90、110、130min in water bath individually.③The single-cellsuspension with were incubated Hoechst in water bath and in thermostat each.④Thetwo different density of cells were harvested,which were100%and70%,and thendigest into single-cell suspension.They all incubated in water bath.Once incubationfinished,samples were immediately put on ice to stop dye efflux.Following cells werewashed, suspended in phosphate buffered saline (PBS)containing2%FBS at1×106cells/ml and kept at4℃.All groups were incubated with Propidium iodide(Pl) atconcentration of1μg/ml for5minutes before flow cytometry.Among allgroups,Verapamil hydrochloride was added to the control samples at concentration of150μmol/L,and incubated at37℃for30minutes,the other condition were say thesame with their trial groups.Results①The percentage of Hoechst-negative cells in trial group was（39.96±0.240）%、（26.23±0.385）%、（18.79±0.023）%、（19.01±0.138)%at theconcentration of5μg/ml、9μg/ml、10μg/ml、11μg/ml respectively， when thePI-positive cells were （30.45±0.634)%、(49.9±0.42)%、(50.12±0.68)%、(64.16±0.387)%separately.②There was a typical FACS pattern and SP%was(17.37±1.423)%when the incubation for90min.③Compare to water bath，SP%was more than in thermostat,the SP%was（18.67±0.448）%、（22.6±0.497）%respectively；④SP%decreased from （19.075±0.464)%to (16.073±0.519)%,whenthe cell density is more than90%and70%. SPSS16.0was used in statistical analysis,the groups were compared using T-Test.P <0.05was considered statisticallysignificant.Conclusion The optimum concentration and duration of incubation ofHoechst33342in isolation of the side population cells in laryngeal carcinoma cell lineHep-2is10μg/ml and90min.Incubated in water bath is better than in thermostat.Thebest staining cell density is around80-90%.Part Ⅱ: Isolation and characterization in vitro of sidepopulation cells in laryngeal carcinoma cell lineObjectives To explore a feasible way to detect the tumor stem-like cells in laryngealcarcinoma cell-line Hep-2，and to an1aIyze the properties of the side population(SP)cells being sorted.Methods After the single-cell suspension of Hep-2was prepared,SP andnon-SP(NSP) cells were sorted by fluorescence-active cell sorting(FACS）usingHoechst33342staining. Then measured the percentage of SP cells, and the twosub-population cells were observed by electron microscopy. Their cell-cycle、abilityof proliferation were detected after sorting．The ABCG2mRNA in SP and NSP cellswere detected using quantitive realtime polymerase chain reaction(qPCR),thenanalysed with2-Δ(ΔCt).Results SP cells were isolated from Hep-2cells in a proportion of (13.17±1.85)％with respect to the whole cell population．Cell growth curve indicated thatproliferative capacity of the SP cells was better than the NSP cells(P<0.05).TheS-phase and proliferation index of the two sub-populations are similar. The qPCRshows that he ABCG2had higher expression in SP cells than in NSP cells.Conclusion Human Hep-2cell line contains a subpopulation of cells excludingHoechst33342dye.contained．The SP cells has better proliferative capacity in vitroand stronger tumorigenicity than the NSP cells.Meanwhile,the ABCG2had higher expression in SP cells.Part Ⅲ: Exploration of the role of cisplatin on transformation oflaryngeal non-side population cells to stem-like cancer cellsObjective If the Non-side population cells（NSP） in Hep2can be induced intostem-like cancer cells by cisplatin，this paper was to explore the conceivablemechanism.Methods Hep-2cells was sorted by fluorescence-actived cell sorting,the acquiredNSP cells in trail group was co-cultured with cisplatin while the control group wasco-cultured with normal saline（NS）,for more than48hours.Then identified thepercentage of the side population(SP)cells by Flow Cytometer.The β-catenin、notch-1mRNA in trial and control group were detected using quantitative realtimePCR,theβ-catenin、notch-1protein in two groups were compared by western blot.Results The percentage of side population cells are (17.16±0.18)%、(10.05±1.20)%,respectively.There were significant difference between twogroups(t=5.844，P=0.004).The expression ofβ-catenin、notch-1is higher in trail groupwith Quantitative realtime PCR(qRT-PCR);the NSP cells cocultured with cisplatinwere found to have increased protein levels of β-catenin、notch-1compared with NSPcells without in western blot(t=5.155，P=0.031；t=5.977，P=0.004）.Statistical analysisshowed significant difference between two groups(P<0.05).Conclusion Non-side population cells can be differentiated into stem-like cancercells after being treating with cisplatin.The supposed mechanism is throughwnt/β-catenin、notch signaling.