Apoptosis Of Nasopharyngeal Carcinoma Cells Induced By Rhamnocitrin In Nervilia Fordii

Objective: The purpose of this research are to investigate the inhibitory effects of Nervilia Fordii methanol extracts(NFME)and its flavonoids rhamnocitrin on nasopharyngeal carcinoma cells CNE-2 and C666-1 in vitro,and determine the induction of NFME and rhamnocitrin apoptosis of nasopharyngeal carcinoma cells and explore the molecular mechanism of rhamnocitrin on nasopharyngeal carcinoma cells CNE-2 and C666-1 in vitro.Methods: Ultrasonic extraction method was used to extract the Nervilia Fordii methanol extracts,aluminum trichloride colorimetric method was used to detect the total flavonoid content in NFME.MTT was used to detect the effects of NFME and rhamnocitrin on the activity of CNE-2 and C666-1 cells;Clonogen formation experiment to verify the effect of NFME and rhamnocitrin on the colony forming ability of CNE-2 and C666-1 cells;Hoechst apoptosis staining test to detect the apoptosis of CNE-2 and C666-1 cells by NFME and rhamnocitrin effect;Flow cytometry confirmed that rhamnocitrin in Nervilia Fordii has the ability to induce apoptosis of CNE-2 and C666-1 cells;Western blot was used to detect apoptosis-related proteins(Caspase3,Caspase8,Caspase9,PARP,Survivin,Bax,Bcl-2,Mcl-1)in nasopharyngeal carcinoma cells;Western Blot detected the effects of rhamnocitrin on the expression of Akt,ERK and IGF-1R proteins in nasopharyngeal carcinoma cells at different concentrations and times;The effect of Akt pathway inhibitor LY294002 and ERK pathway inhibitor U0126 on the activity of CNE-2 and C666-1 cells was examined.Results:(1)The total flavonoid content was 4.72±0.13mg/g.(2)MTT results showed that compared with the blank control group,NFME had inhibitory effects on CNE-2 and C666-1 cells at 250?g/ml(P<0.05);rhamnocitrin was at 10,20,40,80 ?M had an inhibitory effect on C666-1 cells(P<0.05),and 40,80?M had an inhibitory effect on CNE-2 cells(P<0.01).(3)Compared with the control group,NFME inhibited the colony formation of CNE-2 cells at 0.2,0.4,and 0.8 mg/ml compared with the control group(P<0.05),and inhibited C666-1 at 0.4 and 0.8 mg/ml.Cell colony formation(P<0.01);compared with the blank control group,rhamnocitrin inhibited the formation of CNE-2 and C666-1 cell colonies at 20,40,80 ?M(P<0.01).(4)Hoechst apoptotic staining results showed that compared with the control group,CNE-2 and C666-1 cells showed stronger blue fluorescence and cell apoptosis at 0.2 and 0.4 mg/ml(P<0.01);Rhamnocitrin showed apoptosis at 40 and 80 ?M in CNE-2 cells(P<0.01),and C666-1 cells showed apoptosis at 80 ?M(P<0.01).(5)Flow cytometry results showed that compared with the control group,rhamnocitrin could induce apoptosis of CNE-2 and C666-1 cells(P<0.05).(6)Western Blot results showed that rhamnocitrin induced CNE-2 and C666-1 cells activate Caspase3,Caspase8,and Caspase9,thereby inducing apoptosis of nasopharyngeal carcinoma cells;inhibiting the expression of anti-apoptosis related proteins Survivin,Bcl-2,Mcl-1;activating Bax expression.Next,it was found that rhamnocitrin inhibited the phosphorylation of the protein IGF-1R on the cell membrane.At the same time,the downstream signaling pathways Akt and ERK of IGF-1R were detected,and it was found that rhamnocitrin significantly inhibited the phosphorylation of Akt and ERK1/2,and the inhibition was concentration-and time-dependent.(7)MTT results verified that the inhibitory effect of Akt pathway inhibitor LY294002 and ERK pathway inhibitor U0126 on nasopharyngeal carcinoma cells was similar to that of rhamnocitrin on nasopharyngeal carcinoma cells.Conclusion: Nervilia Fordii has an anti-nasopharyngeal carcinoma cell line in vitro,in which the flavonoid rhamnocitrin can induce the apoptosis of CNE-2 and C666-1 in nasopharyngeal carcinoma cells,and its mechanism of action may be through the IGF-1R pathway to inhibit its downstream Akt and ERK signaling pathways,and then induce apoptosis.