Effect Of Ganoderma Neutral Triterpene On Proliferation And Apoptosis In Human Nasopharyngeal Carcinoma CNE2 Cells

OBJECTIVE:To screen the active neutral triterpenes of Ganoderma lucidum against nasopharyngeal carcinoma,and to investigate the effect of active ingredients on proliferation and apoptosis in human nasopharyngeal carcinoma CNE2 cells.METHODS:（1）The neutral triterpene components of Ganoderma lucidum were separated and purified by silica gel column chromatography and preparative high performance liquid chromatography.The structure of the neutral triterpenes of Ganoderma lucidum was identified by spectral analysis and chemical methods.（2）For screening of active ingredients Methyl thiazolyl tetrazolium（MTT）method was used to detect the inhibitory effect of neutral triterpenes of Ganoderma lucidum on CNE2cells to screen the active ingredients.（3）The inhibition of active triterpenes on CNE2cell proliferation was detected by MTT,CFSE single parameter and colony formation assay.（4）PI staining was used to detect the effect of active triterpenes on CNE2 cell cycle.（5）Annexin V-FITC/PI double staining was used to detect the effect of active triterpene-induced CNE2 cell apoptosis.（6）Western blot was used to detect the effect of active triterpenes on the expression of proteins related to the Ras/ERK signaling pathway and mitochondrial pathway of apoptosis in CNE2 cells.（7）The effect of active triterpenes on mitochondrial membrane potential of CNE2 cells was detected by JC-1staining.（8）The effects of active triterpenes on the mitochondrial MPTP openness,intracellular reactive oxygen species（ROS）and Ca²⁺content of CNE2 cells were analyzed by fluorescence probe staining.RESULTS:（1）Six neutral triterpenoids and one sterol were isolated from Ganoderma lucidum,which were ganodermanontriol（1）,ergosterol peroxide（2）,ganoderol B（3）,ganodermanondiol（4）,ganoderone A（5）,lucialdehyde C（6）and lucialdehyde B（7）.（2）In the concentration range of 5-80μg/m L,compounds 1 and 3 had a weak inhibitory effect on the growth of CNE2 in human nasopharyngeal carcinoma cells,while the other compounds all showed certain cytotoxic activity in a concentration-dependent manner.The IC₅₀values of compounds 2,4,5,6 and 7 on human nasopharyngeal carcinoma CNE2 cells for 48h were 42.80±1.16、64.35±1.20、55.30±1.32、15.33±1.21和14.17±1.10μg/m L,respectively.（3）MTT assay showed that lucialdehyde B inhibited the growth of nasopharyngeal carcinoma cells CNE2 in a dose-and time-dependent,and the IC₅₀values of 24,48 and 72h were 25.42±0.87,14.23±0.93 and 11.60±0.77μg/m L,respectively.CFSE fluorescence labeling test showed that the proliferation of CNE2cells was significantly inhibited with a proliferation index of 12.70±0.46 after being treated with lucialdehyde B for 72h,while that of the control group was 31.44±1.73.The colony formation experiment showed that lucialdehyde B had an inhibitory effect on CNE2 cells colony formation,and the colony number of CNE2 cells was 72.33±2.52,63.00±4.36,24.67±2.08 and 9.33±2.08,respectively,after 12h of 0,10,20 and 40 l/m L lucialdehyde B.After 12h treatment with 0,10,20 and 40μg/m L lucialdehyde B,the number of CNE2 cells forming colonies was 72.33±2.52,63.00±4.36,24.67±2.08 and9.33±2.08,respectively.（4）Compared with the control group,lucialdehyde B arrested CNE2 cell cycle progression at the G2/M phase after 48h treatment.After 0,10and 20μg/m L lucialdehyde B treatment for 48h,the G2/M phase proportions of CNE2cells were 6.39,10.00 and 15.54%,respectively.（5）Lucialdehyde B could induce the apoptosis of CNE2 cells.Compared with the control group,the apoptosis rate of 40μg/m L lucialdehyde B for 48h significantly increased to 90.93±0.56%.（6）Lucialdehyde B dose-dependent down-regulated the protein expression levels of Ras,c-Raf,Erk1/2and their phosphorylation products p-c-Raf and p-Erk1/2,down-regulated the protein expression levels of caspase-9,caspase-3,PARP,bcl-2,and up-regulated the protein expression levels of Cleaved caspase-9,Cleaved caspase-3,Cleaved-PARP,and Bax.（7）Compared with the control group,the mitochondrial membrane potential of CNE2 cells decreased to a certain extent under the action of lucialdehyde B at different concentrations for 24h.（8）After treatment for 24h,lucialdehyde B can enhance the MPTP openness of CNE2 cells and increase the intracellular ROS and Ca²⁺levels in CNE2 cells.CONCLUSION:Lucialdehyde C and lucialdehyde B isolated from Ganoderma lucidum have a strong inhibitory effect on the growth of human nasopharyngeal carcinoma CNE2 cells,which may be the active components of Ganoderma lucidum against nasopharyngeal carcinoma.Lucialdehyde B inhibits the proliferation of human nasopharyngeal carcinoma cell CNE2,and its mechanism of action may be related to blocking cell cycle and inhibiting the proliferation signaling pathway of Ras/ERK.Lucialdehyde B induces CNE2 cells apoptosis,and its mechanism may be related to ROS accumulation and calcium overload in cells,down-regulation of Bcl-2,up-regulation of Bax,and initiation of mitochondrial apoptosis pathway.