| Background and aim:Most of the pathological types of oesophageal cancer are squamous cell carcinoma.70%-80% of patients are already at a locally advanced stage at the time of diagnosis and are inoperable or cannot be cured by surgery alone.Radical radiotherapy is therefore the main treatment for patients with locally advanced inoperable oesophageal cancer.However,often due to reasons such as advanced age,poor health,difficulty in eating,or irregular treatment in the early stage,simultaneous radiotherapy is not an option at the time of consultation,and radiotherapy alone is the only option.Combined with highly effective and low-toxic drug sensitisation radiotherapy is currently the main clinical solution for patients with locally advanced squamous oesophageal cancer who are inoperable and intolerant to concurrent radiotherapy.In recent years,due to the discovery of the radiosensitising effect of molecularly targeted drugs and more targets for oesophageal cancer,more targeted drugs combined with radiotherapy to improve tumour sensitivity to radiation have been advocated,but the optimal time window for combined radiotherapy has been less studied.The multi-targeted antitumour drug anlotinib,a domestically produced small molecule tyrosine kinase inhibitor(TKI),has a dual inhibitory effect on tumour cell growth and tumour angiogenesis,and in the 2019 CSCO Guidelines for the Treatment of Esophageal Cancer and the Chinese Guidelines for the Radiotherapy of Esophageal Cancer(2019 edition),for non-surgically resectable locally advanced or advanced squamous carcinoma,anlotinib is also recommended as Class II.This experiment is intended to investigate the effect of anlotinib combined with radiotherapy on the radiosensitivity of esophageal cancer cells Eca-109 and Kyse-450 through in vitro studies,and then provide a basis for subsequent molecular mechanism studies and animal experiments.Methods:The six cell lines of human esophageal squamous carcinoma Kyse-410,TE-1,,Eca-109,Kyse-450,Kyse-150 and TE-12 were cultured in vitro,and the VEGFR2 protein expression levels in the six cell lines were firstly detected according to Western blotting,and Eca-109 and Kyse-450 were finally selected as the experimental cell lines.The CCK-8 method was used to verify the proliferation inhibition of Eca-109 and Kyse-450 cells by anlotinib,and the P value was used to determine the drug effect.The cell survival curves were calculated by software and radiological modeling,and the relevant parameters of radiosensitivity and radiosensitization ratio were calculated.Flow cytometry was used to detect the effect of each experimental group on the apoptosis and cycle of Eca-109 and Kyse-450 cell lines respectively.Results:1.Western blotting results of VEGFR2 protein expression in the six cell lines showed that the peak areas of VEGFR2 protein expression in Eca-109 and Kyse-450 were larger than those in the other four cell lines.The peak area of VEGF2 band was 16189.69±44.19.The Eca-109 and Kyse-450 cell lines were finally selected as the targets of the subsequent experiments.2.The results of the CCK-8 experiment showed that the effect of anlotinib(concentration: 5,10,20,40 and 80 μmol/L)on the two cell lines showed that all concentrations of anlotinib could inhibit the viability of Eca-109 and Kyse-450 cells,and the inhibitory effect showed an increasing trend with increasing concentration gradient and time,and showed concentration-dependent and time-dependent effects.The IC20 of Eca-109 and Kyse-450 cells were(1.30±0.19)μmol/L and(9.79±0.67)μmol/L,respectively,and the IC20 was used as the drug concentration for the subsequent experiments.3.The results of cell clone formation experiments showed that: the relevant indexes of cellular radiosensitivity were counted,and it was found that the Dq and D0 of the anlotinib combination irradiation group were smaller than those of the irradiation group alone,and the survival curves were fitted using a one-click multi-target model.The results showed that: Eca-109 cells: SER was 1.31 in the 24 h AR group,1.15 in the AR group and 1.23 in the 24 RA group;Kyse-450 cells: SER was 1.33 in the 24 AR group,1.08 in the AR group and 1.15 in the 24 RA group.the radiosensitizing effect of the two cells administered 24 hours before irradiation was higher than that of The radiosensitizing effect was more pronounced in the two cells administered 24 hours after irradiation and in the two cells irradiated with concomitant dosing.The survival curves were fitted to the L-Q model,and the α/β ratios of the two esophageal cancer cell lines were 4.11 Gy for Eca-109 and 3.88 Gy for Kyse-450,with lower α/β ratios,suggesting that the two esophageal squamous cancer cell lines have a strong ability to repair radiation damage.The α/β ratios were low,suggesting that the two esophageal squamous carcinoma cells have a strong ability to repair radiation damage.4.The results of flow cytometry showed that the combined irradiation group of anlotinib induced a significant increase in the apoptosis rate of both cell lines compared with irradiation alone(P<0.05).In both strains,the percentage of S phase was lower and the percentage of G2/M phase increased in the anlotinib combination irradiation group than in the irradiation alone group(P<0.05),indicating that the addition of the drug further aggravated the damage effect of radiation on esophageal squamous carcinoma Eca-109 and Kyse-450 cells.Moreover,irradiation-induced apoptosis was significantly increased by the addition of anlotinib,and the apoptosis rate was most pronounced in the pre-irradiation addition.Conclusion:Anlotinib inhibited both human esophageal cancer cell lines Eca-109 and Kyse-450 cells.The α/βratio measured when irradiated alone was low,and both cell lines had a high ability to repair radiation damage;moreover,the addition of anlotinib before irradiation was more likely to induce an increase in the apoptosis rate of both cell lines and to stall the cell cycle process,thereby increasing the radiosensitization of Eca-109 and Kyse-450 cells.Therefore,the optimal time window for combined irradiation with anlotinib is the addition of the drug before irradiation,which is a guideline for patients with advanced esophageal cancer to improve the therapeutic effect during clinical treatment. |
PDF Full Text Request