| Esophageal cancer is one of the most common malignancies of the digestive tract,and its mortality rate ranks the sixth in the world.And the incidence rate of esophageal cancer is very high,which has posed a great threat to people’s health in China.Existing clinical data show that although there are many standardized treatments for esophageal cancer,most patients are diagnosed in the middle and late stages,leading to poor treatment effect and poor prognosis of esophageal cancer.Even if detected early,the highest survival rate after treatment is only up to 5 years.Nowadays,the research focus of tumor treatment is the introduction of autophagy,which is involved in physiological processes such as angiogenesis and apoptosis-related factors in the cell cycle.Its treatment methods include chemotherapy,radiotherapy and targeted therapy.Our lab has been devoted to studying the role and mechanism of macrolides antibiotics in anti-tumor.In previous studies,we have proved that Doramectin(DRM)could inhibit the proliferation of glioma cells and esophageal cancer cells,and the specific mechanism is to promote the apoptosis of esophageal cancer cells.This study further explored the role and mechanism of autophagy induced by DRM in esophageal cancer,as well as the effect of autophagy on apoptosis,so as to provide reliable theoretical research data for the possibility of DRM as a tumor suppressor drug.In this study,Eca109 and EC9706 cells were treated with different concentrations of DRM and treated with the same drug concentration for 24 h,48 h and 72 h,respectively.We could detect cell proliferation by MTT assay.With the increase of DRM concentration,the viability of esophageal cancer cells decreased significantly.Using the cell scratch assay,we know that with the increase of DRM concentration,the ability of cell migration is significantly reduced(P < 0.001).These results indicated that DRM significantly inhibited the growth and migration of esophageal cancer cells.In order to further clarify the DRM-induced autophagy of esophageal cancer and its main mechanism,we first observed the cell morphology through transmission electron microscopy.After DRM treatment,autophagosomes appeared in esophageal cancer cells.Then Western blot assay was used to determine the protein expression levels of autophagy related proteins and the Akt/m TOR signaling pathway.Compared with the control group,with the increase of DRM concentration,the protein expression levels of LC3Ⅱ/LC3Ⅰ and Beclin-1 were significantly up-regulated,on the contrary,the protein expression levels of p62 were significantly down-regulated(P < 0.05),p-Akt/Akt and p-m TOR/m TOR in the pathway were significantly down-regulated(P < 0.05).These results suggest that DRM could induce autophagy in esophageal cancer cells by inhibiting the Akt/m TOR signaling pathway.Apoptosis and autophagy are different from each other,but also related to each other.It is very important to understand the relationship between apoptosis and autophagy.In this experiment,chloroquine(CQ)was used as autophagy inhibitor.In this study,MTT assay was used to detect the proliferation of esophageal cancer cells after autophagy intervention.The results showed that the proliferation of esophageal cancer cells was significantly decreased by inhibiting autophagy induced by DRM(P < 0.001).At the same time,the cloning ability of esophageal cancer cells was detected by cell cloning assay,and the results showed that the cloning ability of esophageal cancer cells was significantly reduced by inhibiting autophagy induced by DRM(P < 0.05).In order to further clarify the reasons for the decreased cell proliferation and cloning ability after autophagy inhibition,we detected cell apoptosis by flow cytometry,and the number of apoptosis of esophageal cancer cells increased significantly after autophagy inhibition(P < 0.001).These results indicatethat DRM-induced autophagy could inhibit apoptosis.In order to further explore the DRM effect on esophageal cancer cell at the genetic levels,we detected the expression of Akt/m TOR signaling pathway genes and other autophagy-related genes by transcriptome sequencing.Compared with the control group,the expression levels of Akt and m TOR genes in LC3,p62,Beclin-1 and the Akt/m TOR signaling pathway were not significantly different,but the expression levels of autophagy-related genes such as ULK1,Atg13 and Atg16L2 were significantly up-regulated(P < 0.001).Combined with the previous Western blot results,the regulation effect of DRM on autophagy of esophageal cancer cells may be at the level of post-transcription,translation or post-translation regulation,but the specific mechanism is needed to further explore.In order to determine whether DRM could induce autophagy in esophageal cancer cells in vivo and inhibit the effect of autophagy on apoptosis,we established a mouse tumor-bearing model of Eca109 cells.During the treatment with DRM,the tumor volume was measured daily with a vernier caliper,while the body weight of the nude mice was measured with a balance.The results showed that the body weight of the nude mice did not change significantly after the intervention of DRM,but the tumor volume was significantly reduced.The expression of autophagy-related proteins were detected by Western blot,Compared with the control group,the expression levels of LC3 and Beclin-1 were significantly increased with the increasing of DRM concentration,and on the contrary,the expression of p62 was significantly decreased(P < 0.05).By HE staining,we found that DRM had no significant damage to tumor tissue and main organs of nude mice.The expressions of autophagy proteins were detected by immunohistochemical assay.The expression of LC3 protein was significantly up-regulated,whereas the expression of p62 protein was significantly down-regulated.By TUNEL assay,we were able to find that inhibition of DRM-induced autophagy significantly increased the number of esophageal cancer cell apoptosis.These results suggest that DRM could significantly inhibit the proliferation of esophageal cancer cells and induce autophagyin vivo.In addition,interfering autophagy could inhibit the apoptosis of esophageal cancer cells.These results suggest that DRM could significantly inhibit the proliferation of esophageal cancer cells in vitro and in vivo,and induce autophagy of esophageal cells through the Akt/m TOR signaling pathway.In addition,induced autophagy by DRM could inhibit apoptosis in esophageal cancer.Therefore,DRM is expected to be an effective drug for the treatment of esophageal cancer,and this study will provide an experimental basis for its clinical application. |
