Effects Of Camptothecin On Autophagy And Apoptosis In HaCaT Cells And Human Primary Keratinocytes

Objective To investigate the effect of camptothecin(CPT)on autophayg and apoptosis Ha Ca T cells and human primay keratinocytes.Discuss the potentially therapeutic mechanism of topical CPT in treating psoriasis.Methods(1)Some Ha Ca T cells were divided into several experimental groups treated with CPT at concentrations of 5?10?25?50?100?200 nmol/L,respectively and a control group treated with 0.1% dimethyl sulfoxid(DMSO).All the groups were cultured for 24 or 48 hours,a cell counting kit-8(CCK-8)assay was conducted to estimate the proliferation of Ha Ca T cells;cell apoptosis was detected by flow cytometry after 24 h culture;western blot was performed to measure the expression of autophagic-associated proteins,including microtubule-associated protein 1 light chain 3(LC3)and p62;indirect immunofluorescence was used to detect location of LC3 and transmission electron microscopy was used to evaluate the formation of autophagsomes and to validate the induction of autophagy after treated with 10 nmol/L CPT for 24 h.(2)The foreskin tissue was obtain from teenagers who recepted the circumcision,primary keratinocytes was extracted and cultured,the third generation cells were used for each experiment.Primary keratinocytes were divided into several experimental groups treated with CPT at concentrations of 200 nmol/L?2 ?mol/L?6 ?mol/L,respectively and a control group treated with 0.1% DMSO.All the groups were cultured for 24 or 48 hours,CCK-8 assay was conducted to estimate the proliferation of primary keratinocytes;cell apoptosis was detected by flow cytometry after 24 h culture;western blot was performed to measure the expression of autophagic-associated proteins,including LC3 and p62;indirect immunofluorescence was used to detect location of LC3 and transmission electron microscopy was used to evaluate the formation of autophagsomes and to validate the induction of autophagy after treated with 2 ?mol/L CPT for 24h;immunohistochemistry was used to observe the LC3 expression of the psoriasis lesions and normal epidermis.(3)Statistical analysis was done by Levene,s Test,one-way ANOVA analysis,Dunnett-t test,independent-t test using the SPSS 17.0 software.Results(1)The low concentrations(5?10?25 nmol/L)of CPT have no significant effect on the proliferation and apoptosis of the Ha Ca T cells.The cell proliferation rates were inhibited by(31.23±1.00)%,(54.21±8.10)%,(66.75±10.70)% respectively in Ha Ca T cells after 24 h treatment with 50?100?200nmol/L CPT as well as(25.81±5.99)%,(44.35±5.32)%,(65.81±8.28)%,(73.23±9.59)% respectively after 48 h treatment with 25?50?100?200nmol/L CPT,with a significant difference compared with the control groups at the corresponding time point(p all<0.001).After 24 h treatment with 50?100?200nmol/L CPT,the apotosis rates were(14.46±2.38)%,(19.15±1.59)%,(29.88±1.37)% respectively,showing a significant difference in comparison with the control group(p<0.001).The expressions of LC3? were significantly increased and p62 were decresed in the 5?10 nmol/L CPT groups.There were more bright green fluorescent dots in the cytoplasm of the experimental group(10 nmol/L CPT for 24h)than the control group,the proportion of autophagosome-positive cells were(36.67±4.55)% and(6.23±0.92)%respectively(t=6.546,p=0.0028).The transmission electron microscope showed the ultrastmctural morphology of autophagsome and autolysosome in Ha Ca T cells incubated for 24 h with 10nmol/L CPT.(2)The cell proliferation rates were inhibited by(33.90±3.65)% ?(51.72±5.06)% respectively in Ha Ca T cells after 24 h treatment with 2 ?mol/L?6 ?mol/L CPT as well as(25.81±5.99)%,(19.21±5.74)%?(52.09±3.01)%?(63.37±5.45)% respectively after 48 h treatment with 200nmol/L?2?mol/L?6?mol/L CPT,with a significant difference compared with the control groups at the corresponding time point(p all<0.001),the cell proliferation rate was inhibited by(7.59±1.82)% in primary keratinocytes after 24 h treatment with 200nmol/L CPT.After 24 h treatment with 200nmol/L?2?mol/L?6?mol/L CPT,the apotosis rates were(15.90±2.14)%,(29.33± 3.51)%,(35.28 ±3.05)% respectively,showing a significant difference in comparison with the control group(p<0.001).The expressions of LC3? were significantly increased and p62 were decresed in the 2?mol/L ? 6?mol/L CPT groups.There were more bright green fluorescent dots in the cytoplasm of the experimental group(2 ?mol/L CPT for 24h)than the control group,the proportion of autophagosome-positive cells were(60.16±8.78)% ?(38.96±13.12)% respectively(t=3.003,p=0.017).The transmission electron microscope showed the ultrastmctural morphology of autophagsome and autolysosome in primary keratinocytes incubated for 24 h with 2 ?mol/L CPT;the expression of LC3 in psoriatic lesions was significantly lower than that in normal epidermis detected by immunohistochemistry.Conclusions The low concentrations of CPT(5?10?25 nmol/L)can induce autophagy in Ha Ca T cells,but have no significant effect on the proliferation and apoptosis of cells.The high concentrations of CPT(50 ? 100 ? 200 nmol/L)can inhibite cell proliferation,induce cell apotosis and decrese the level of autophagy.2?mol/L?6?mol/L CPT can increase autophagy level in primary keratinocytes and inhibite cell proliferation and induce cell apoptosis at the same time.