Preliminary Study On Screening Of Human Tongue Cancer Cell Line CAL27 Cancer Stem Cells With Doxorubicin

Objective: To explore the appropriate concentration and time of doxorubicin intervention on human tongue cancer cell line CAL27,in order to screen out more cancer stem cells and provide an optimal method for subsequent sorting and enrichment of tongue cancer stem cells.Methods:(1)Fluorescence imaging of human tongue cancer cell line CAL27 before and after doxorubicin intervention with different concentrations(0.3 μg/m L、0.6 μg/m L、0.9μg/m L、1.2 μg/m L、1.5 μg/m L)is observed under bright field and dark field by inverted fluorescence microscope.(2)Real time fluorescence quantitative Reverse Transcription Polymerase Chain Reaction is used.The expression levels of CD44,ALDH1 and ABCG2 of cancer stem cells are detected by RT-q PCR 24 hours after doxorubicin treatment in CAL27 cells to determine the appropriate concentration of doxorubicin.(3)Fluorescence imaging of CAL27 cells treated with doxorubicin at different treatment times(6 hours,12 hours,24 hours)is observed,and the expression levels of CD44,a dry marker of cancer stem cells,and p53,the apoptotic gene,are detected by RT-q PCR to determine the appropriate time of doxorubicin.(4)CCK-8 method was used to detect the cell survival rate of tongue cancer cell line CAL27 after of low doxorubicin intervention.Results:(1)The higher the concentration of doxorubicin,the more suspended dead cells are observed under inverted fluorescence microscope.No fluorescence staining is observed in cells without doxorubicin treatment in dark field.After doxorubicin treatment,the cells showed red fluorescence imaging,and the fluorescence intensity increased with the increase of drug concentration.When the concentration is 0.6μg/m L,there is a small area without fluorescence staining under the dark field.(2)The m RNA expression levels of CD44,ALDH1 and ABCG2 are up-regulated after doxorubicin treatment.When doxorubicin concentration is0.6 μg/m L,the expression levels of CD44 and ALDH1 m RNA were the highest(P<0.05),while ABCG2 expression had no significant difference(P>0.05).(3)The fluorescence intensity of cells increased with the increase of doxorubicin treatment time,the treatment time is 12 hours,and there was a part of non-fluorescence staining area under the dark field.Compared with the control group,the m RNA expression levels of CD44 and p53 are up-regulated from 6 hours to 12 hours,and the m RNA expression levels are the highest at 12hours(P<0.01).Compared with 12 hours,the expression of CD44 gene is down-regulated at 6hours and 24 hours,with no statistical significance(P>0.05).P53 gene expression is down-regulated at 6 hours and 24 hours,with statistically significant differences(P<0.01).(4)The results of CCK-8 showed that the median inhibitory concentration of doxorubicin is0.005884 μg/m L for 12 hours,and CAL27 cells remained active at 0.6 μg/m L.Conclusion:(1)The appropriate concentration and action time of doxorubicin for preliminary screening of human tongue squamous cell carcinoma CAL27 cancer stem cells were 0.6 μg/m L and 12 h,respectively.(2)Early intervention of doxorubicin at low concentration is beneficial to the enrichment of tongue cancer stem cells.