Objective To evaluate the synergistic effect of everolimus on cisplatin-mediated cytotoxicity against human cutaneous squamous cell carcinoma COLO-16 cells.Methods Cultured COLO-16 cells were divided into several groups to be treated with everolimus at different concentrations of 50,100 and 200 nmol/L or 25 μmol/L cisplatin for 12 and 24 hours.Acridine orange (AO)-labeled autophagic vesicles combined with lysomal enzyme inhibitors (E64d and pepstatin) were used to detect the levels of autophagy and autophagic flow.Western blot analysis was performed to track the conversion of the autophagosome marker microtubule-associated protein 1 light chain-3 (LC3)-Ⅰ to LC3-Ⅱ,as well as to detect cleavage levels of Caspase 3 and poly-ADP-ribose polymerase (PARP).Lactate dehydrogenase (LDH) assay was conducted to detect cell death,and Annexin V-EGFP staining to evaluate cell apoptosis.Results The LC3-Ⅱ / LC3-Ⅰ ratios (LC3-Ⅰ conversion to LC3-Ⅱ) after 12-and 24-hour treatment did not differ among the 50-,100-and 200-nmol/L everolimus groups (12 hours:3.52 ± 0.21 vs.4.03 ± 0.39 vs.5.05 ± 0.22,P ＞ 0.05;24 hours:3.38 ± 0.26 vs.3.29 ± 0.06 vs.6.57 ± 0.16,P ＞ 0.05),but were significantly higher in the three everolimus groups than in the control group receiving no treatment (12 hours:2.07 ± 0.05,P ＜ 0.05;24 hours:2.61 ± 0.16,P ＜ 0.05).After 12-hour treatment,no significant differences were observed in the ratio of LC3-Ⅱ to β-actin between the 50-nmol/L everolimus + E64d + pepstatin group (1.26 ± 0.40),100-nmol/L everolimus ± E64d + pepstatin group (1.16 ± 0.34),200-nmol/L everolimus + E64d + pepstatin group (1.21 ± 0.39) and E64d + pepstatin group (1.19 ± 0.27,P ＞ 0.05).Moreover,there was no significant difference in the percentages of autophagic vesicle-positive cells between the 100-nmol/L everolimus + E64d + pepstatin group and E64d + pepstatin group (2.06％ ± 0.61％ vs.1.68％ ± 0.62％,P ＞ 0.05).After 24-hour treatment,the everolimus + cisplatin group showed significantly increased rate of cell death compared with the cisplatin alone group (42.58％ ± 0.93％ vs.18.20％ ± 1.46％).However,no significant differences were observed in the cleavage levels of Caspase 3 and PARP,the number of annexin V-labelled cells and ratio of LC3-Ⅱ to β-actin between the everolimus + cisplatin group and the cisplatin-alone group (P ＞ 0.05).Conclusion Everolimus has a synergistic effect on the cisplatin-mediated COLO-16 cell death,and this effect does not depend on cell apoptosis or autophagy.

Objective To investigate molecular mechanisms underlying the synergistic damage to the human squamous cell carcinoma cell line COLO-16 by everolimus and cisplatin.Methods In the signaling pathway experiment,COLO-16 cells were divided into 4 groups:control group receiving no treatment,50,100 and 200 nmol/L everolimus groups treated with 50,100 and 200 nmol/L everolimus respectively.In the combined experiment,COLO-16 cells were divided into another 4 groups:control group,50 nmol/L everolimus group,25 mol/L cisplatin group,and 50 nmol/L everolimus + 25 mol/L cisplatin group.Western blot analysis was performed to analyze changes in mammalian target of rapamycin (mTOR) pathway,Akt pathway,DNA damage-related pathway and Csk homologous kinase (Chk) pathway.Results After the treatment with everolimus at different concentrations of 50,100 and 200 nmol/L for 12 and 24 hours,the phosphorylation levels of mTOR at ser2448 and ser2481 as well as Rictor at thr1 135 in COLO-16 cells were all decreased compared with the control group.However,there were no significant changes in the phosphorylation levels of downstream signals ULK1 at ser757,p70 S6 at thr389 and PKCα at thr638/64.The treatment with everolimus did not change the total protein level and phosphorylation of Akt.After the treatment with cisplatin for 12 and 24 hours,the phosphorylation levels of Rictor at thr1135 and Chk1 at ser345 were significantly increased,but the treatment with everolimus alone showed no such effects.After the combined treatment with everolimus and cisplatin for 12 and 24 hours,the upregulation of Chk1 and Rictor phosphorylation were significantly inhibited compared with the cisplatin alone group.Conclusions mTOR signaling is sensitive to everolimus in COLO-16 cells,but its targeted pathway is not regulated simultaneously to develop a cascade reaction.Everolimus may increase the cisplatin-induced death of COLO-16 cells by inhibiting the activation of Chk 1,but can not aggravate DNA damage induced by cisplatin.

Objective To evaluate the regulatory effects of lycopene on the key signaling receptors in human cutaneous squamous cell carcinoma cell line COLO16.Methods Cultured COLO16 cells were divided into 6 groups to be treated with lycopene at different concentrations of 0,5,10,15,20,and 25 μmol/L,respectively,for 24 hours (control group and 5,10,15,20,25 μmol/L lycopene groups),followed by estimation of the cell viability by lactate dehydrogenase (LDH) assay.Lycopene at a safe concentration was selected based on the LDH assay,and used for the determination of expression of signaling receptors,and Western blot analysis was performed to measure the expression of key signaling receptor proteins,including epidermal growth factor receptor (EGFR),glucocorticoid receptor (GR),retinoic acid receptor-alpha (RAR-α),retinoid X receptor-alpha (RXR-α),androgen receptor (AR) and progesterone receptor (PR).Statistical analysis was carried out by one-way analysis of variance (ANOVA),Tukey multiple comparison teat and Brown-Forsythe test with the SPSS software.Results After 24-hour treatment with lycopene at different concentrations,there were significant differences in the rate of cell death among these groups (F =13.116,P ＜ 0.05),and the rate of cell death in the 25 μmol/L lycopene group significantly differed from that in the control group (P ＜ 0.05).Therefor,lycopene at concentrations of 5,10 and 20 μmol/L were selected to treat COLO16 and HaCaT cells as well as human epidermal keratinocyte (HEK) for 24 hours in the following experiment.The treatment with lycopene significantly decreased the phosphorylation level of EGFR (P ＜ 0.05),but significantly increased the expression of GR protein (P ＜ 0.05),and showed no significant effects on the protein expression of RAR-α,RXR-α,AR,and PR in COLO16 cells.After 24-hour treatment with lycopene at concentrations of 5,10 and 20 μmol/L,there were no significant changes in the phosphorylation level of EGFR protein or the expression of GR protein in HaCaT cells and HEK (all P ＞ 0.05) compared with those without lycopene treatment.Conclusion Lycopene can decrease the viability of COLO16 cells,inhibit the activation of EGFR protein,and up-regulate the expression of GR,and these effects may be specific for tumor cells.