| Objective TPF(cisplatin + docetaxel +5-Fu)regimen is the most effective chemotherapy regimen for laryngeal cancer,but some patients show insensitivity to TPF regimen.The aim of this study was to investigate the mechanism of amygdalin on apoptosis in TPF-insensitive laryngeal cancer cells.Methods Human TPF-insensitive laryngeal cancer cells were cultured.(1)The proliferation of cells after amygdalin intervention at different time periods was detected by MTS method;Flow cytometry(Annexin V/PI double staining)was used to detect the changes of apoptosis rate after amygdalin intervention;Cell immunofluorescence was used to detect the changes of Bax protein co-localization with mitochondria after amygdalin intervention;RT-q PCR was used to detect the expression of apoptosis-related genes Bax,Bcl-2 and Caspase-3 m RNA levels after amygdalin intervention;Western blot to detect the expression of apoptosis-related proteins Bax,Bcl-2,cleaved Caspase-9 and active Caspase-3 after different concentrations of amygdalin intervention;(2)The proliferation of T,P,F and TPF mixture and amygdalin combined with TPF were detected by MTS method;Flow cytometry(Annexin V/PI double staining)to detect the changes of apoptosis rate of Control,TPF,amygdalin and amygdalin combined with TPF intervention;The m RNA expressions of apoptosis-related genes Bax,Bcl-2 and Caspase-3 were detected by RT-q PCR after the intervention of Control,TPF,amygdalin and amygdalin combined with TPF;(3)Establish TPF-insensitive laryngeal cancer cell transplantation tumor animal models,divided into saline treatment group,TPF treatment group(T: 20 mg/kg,P: 6 mg/kg,F: 17 mg/kg),amygdalin treatment group(80mg/kg)and amygdalin(80 mg/kg)combined with TPF(T: 20 mg/kg,P: 6 mg/kg,F: 17 mg/kg)treatment group,were administered by intraperitoneal injection,and samples were collected after 14 days of continuous intervention;TUNEL method to detect the changes of apoptosis in tumor cells of each treatment group;The expression of apoptosis-related protein active Caspase-3 in tumor tissue cells of each treatment group was observed by immunohistochemical staining;The expressions of apoptosis-related proteins Bax,Bcl-2,cleaved Caspase-9 and active Caspase-3 were detected by Western blot.Results(1)The results of MTS experiment showed that with the increase of amygdalin concentration and time,the proliferation of TPF insensitive laryngeal cancer cells could be significantly inhibited.The IC50 of 24 h,48 h,and 72 h were 14.21±1.98 mg/m L,3.44±0.62mg/m L and 2.69±0.21 mg/m L,respectively;Flow cytometry(Annexin V/PI double staining)was used to detect the apoptosis effect of TPF insensitive laryngeal cancer cells induced by amygdalin at different concentrations.It was found that the apoptosis rate increased with the increase of drug concentration,showing a dose-effect relationship;Cell immunofluorescence analysis showed that amygdalin at different concentrations induced the translocation of pro-apoptotic factor Bax protein from cytoplasm to mitochondrial membrane in TPF insensitive laryngeal cancer cells,suggesting that the translocation of Bax protein played an important role in amygdalin induced apoptosis of TPF insensitive laryngeal cancer cells;RT-q PCR and Western blot were used to detect the apoptosis-related genes and proteins of TPF-insensitive laryngeal carcinoma cells induced by amygdalin at different concentrations.The results showed that amygdalin significantly promoted the apoptosis of TPF-insensitive laryngeal carcinoma cells,and it was dose-dependent.(2)The results of the MTS experiment showed that the combination of amygdalin significantly improved the sensitivity of TPF,and amygdalin reduced the IC50 of TPF on TPF-insensitive laryngeal cancer cells from 1.28 μg/m L to 0.31 μg/m L.It also showed that the combination of amygdalin and TPF had a synergistic effect;Flow cytometry(Annexin V/PI double staining)showed that the percentage of apoptosis increased by 0.52% and 4.74%in TPF and amygdalin alone compared with the control group,and the group treated with amygdalin combination with TPF showed a 7.98% increase in apoptosis;RT-q PCR detection showed that the m RNA expression level of Bax in TPF group was increased compared with the control group(P < 0.05),and the m RNA expression levels of Bax and Caspase-3 in amygdalin and amygdalin combined with TPF group were significantly increased compared with the control group(P < 0.01).The m RNA expression level of Bcl-2 in TPF group was decreased compared with the control group(P < 0.05),and the m RNA expression level of Bcl-2 in amygdalin and amygdalin combined with TPF group was significantly decreased compared with the control group(P < 0.01).(3)The transplanted tumor volume was significantly smaller in the TPF group(P < 0.05)and in the amygdalin group(P < 0.01)compared with the control group,and the difference was extremely significant in the amygdalin combined with TPF group(P < 0.01).There was no significant difference in the body weight of nude mice compared with the control group in the amygdalin group and a significant decrease in body weight in the TPF group.The body weight of nude mice in amygdalin combined with TPF group was significantly higher than that in TPF group from day 8.The inhibitory rates of TPF group,amygdalin group and TPF combined amygdalin group were 21.4%,33.2% and 46.5%,respectively;TUNEL assay revealed that the green fluorescence area of transplanted tumor tissues was not significantly different in the TPF group compared with the control group,while the expression of green fluorescence area was significantly increased in the amygdalin group and the expression of green fluorescence area was significantly increased in the amygdalin combined with TPF group;Immunohistochemical results showed that compared with the control group,the positive rate of active Caspase-3 was highest in amygdalin combined with TPF group,followed by amygdalin group,and lowest in TPF group.Western blot analysis of apoptosis-related proteins showed that compared with the control group,the expressions of pro-apoptotic proteins Bax,cleaved Caspase-9 and active Caspase-3 were significantly increased in amygdalin group and amygdalin combined with TPF group.There was no significant difference in expression in TPF group.The expression of anti-apoptotic protein Bcl-2 was significantly decreased in amygdalin group and amygdalin combined with TPF group,but there was no significant difference in expression in TPF group.Conclusion1.Amygdalin can induce apoptosis of TPF-insensitive laryngeal cancer cells,and has a strong anti-TPF-insensitive laryngeal cancer effect in vitro.2.Amygdalin can effectively inhibit the tumor growth of TPF-insensitive laryngeal carcinoma cell transplantation model in nude mice,and has strong anti-TPF-insensitive laryngeal carcinoma effect in vivo.3.The combination of amygdalin and TPF regimen has a synergistic effect,suggesting that amygdalin can inhibit tumor cell proliferation in combination with other chemotherapeutic drugs.4.Activation of mitochondrial signaling pathway plays an important role in the anti-TPF-insensitive laryngeal cancer process of amygdalin. |
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