| Objective: To investigate the effects of curcumol on IGF-1R/PI3K/Akt/GSK-3β pathway and its downstream molecules in human nasopharyngeal carcinoma CNE-2 cells in vitro;and explore cell proliferation inhibition,cell-cycle arrest and cell apoptosis phenomena induced by curcumol and the potential molecular mechanisms.Aims to lay a foundation for developing novel inhibitor of IGF-1R and identifing effective targets for molecular targeted therapy of nasopharyngeal carcinoma,and provide scientific basis for improving the efficacy of curcumol in the clinical treatment of cancer.Methods: CNE-2 cells in logarithmic growth phase were seeded in culture plates and intervented with increasing concentrations of curcumol(12.5,25,50 and 100μg/mL),in the meanwhile,a negative control group was set;Changes of cell proliferation viabilities of CNE-2 cells after 24,48,72 and 96 h treatment with different concentrations of curcumol were detected by MTT assay;A plate clone formation experiment was performed to assess differences in clonogenic and proliferation abilities of CNE-2 cells after treatment with different concentrations of curcumol;Flow cytometry was used to detect cell cycle progression and apoptosis of CNE-2 cells after curcumol treatment for 48h;The mRNA and protein expression of IGF-1R/PI3K/Akt/GSK-3β pathway and its downstream cycle-related and apoptosis-related molecules in CNE-2 cells were detected by Q-PCR and Western blotting.50μg/mL of curcumol and 50ng/mL of IGF-1 acted on CNE-2 cells respectively or cooperatively,and a negative control group was set;The reversal effects of IGF-1 on the anti-tumor effect of curcumol in CNE-2 cells were evaluated by assays of cell proliferation,cell cycle,cell apoptosis and detection of IGF-1R/PI3K/Akt/GSK-3β pathway as well as its downstream molecules expression.25μg/mL,50μg/mL and 100μg/mL of curcumol was combined with 50ng/mL IGF-1 respectively,and acted on CNE-2 cells,in the meanwhile,a negative control group was set;The inhibitory function of curcumol on the proliferation-promoting effect of IGF-1 was evaluated by cell proliferation assay,cycle-related proteins,apoptosis-related proteins and IGF-1R/PI3K/Akt/GSK-3β pathway detections.Results:(1)Curcumol significantly inhibited the proliferation of human nasopharyngeal carcinoma CNE-2 cells in a dose-and time-dependent manner;(2)Curcumol inhibited the formation and proliferation of CNE-2 cells clones,and the number of cell clones was negatively correlated with the concentration of curcumol to some extent;(3)Treatment with different concentrations of curcumol for 48 h caused CNE-2 cells arrest in G0/G1 phase,the number of cells in S phase and G2/M phase were significantly reduced.Down-regulation of CDK2,CDK4,Cyclin D1,Cyclin E and upregulation of p21 and p27 can accout for this phenomenon.(4)Curcumol treatment for 48 h induced CNE-2 cells apoptosis,which was related to the increase of the ratio of Bax/Bcl-2,the up-regulation of p53 and cleaved PARP as well as the down-regulation of MDM2;(5)Curcumol significantly down-regulated the mRNA and protein level of IGF-1R in CNE-2 cells;(6)Curcumol inhibited the activation of PI3K/Akt/GSK-3β signaling pathway in CNE-2 cells and significantly decreased the expression of p-p85,p-Akt and p-GSK-3β;(7)IGF-1 markedly reversed the proliferation inhibition,cell-cycle arrest,apoptosis induction and IGF-1R/PI3K/Akt/GSK-3β pathway inhibition induced by curcumol;(8)Curcumol effectively inhibited the growth stimulatory effect of IGF-I on CNE-2 cells.Conclusions: Curcumol treatment led to G0/G1-phase arrest through down-regulation of cyclins,CDKs expression and up-regulation of p21,p27 expression,mediating proliferation inhibition of nasopharyngeal carcinoma cells in a dose-dependent and time-dependent way;Curcumol treatment induced apoptosis of nasopharyngeal carcinoma cells by disrupting MDM2-p53 interaction,up-regulating Bax/Bcl-2 ratio,and initiating PARP apoptosis pathway;Curcumol treatment significantly inhibited IGF-1R/PI3K/Akt/GSK-3β pathway in nasopharyngeal carcinoma cells,which was characterized by the reduction of IGF-1R,p-p85,p-Akt,and p-GSK-3β;IGF-1 significantly reversed the proliferation inhibition,cell-cycle arrest,apoptosis induction and IGF-1R/PI3K/Akt/GSK-3β pathway inhibition induced by curcumol in nasopharyngeal carcinoma cells;Curcumol inhibited the growth stimulating effect of IGF-1 on nasopharyngeal carcinoma cells and supressed the activation of IGF-1R/PI3K/Akt/GSK-3β pathway,thus,triggered biological effects including proliferation inhibition,cell-cycle arrest and apoptosis induction. |
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