Targeting Autophagy To Enhance Paclitaxel Sensitivity In Esophageal Carcinoma

Objective: In order to investigate the role of autophagy modulated by chloroquine(CQ) in the process of esophageal carcinoma treated by PTX, including explore the related molecular mechanisms to provide experimental evidence for the clinical application of the potential chemosensitizer.Methods: CQ was applied as an autophagy inhibitor to study the esophageal carcinoma cells EC109 co-treated with PTX in vitro, then observed the changes of the growth and proliferation, migration and invasion ability of EC109.1. The growth and proliferation of EC109 cells were detected by CCK8;2. The migration and invasion abilities were checked through scratch test and cell invasion Transwell chamber test;3. The colony formation ability were measured by soft agar;4. Detected the phosphorylation level of Akt,mTOR,p70S6 K,4EBP1 in the PI3K/Akt/mTOR cell signaling pathway, as well as the autophagy marker LC3 and p62 through Western Blot;5. Observed the autophagy changes by transfecting GFP-LC3 plasmid after the separate and co-treatment of PTX and CQ;6. Observed the changes of the growth and proliferation, migration and invasion ability of EC109 after knocking down the expression of mTOR by lentiviral shRNA embedding mTOR.Results:1. CQ and PTX could supress the growth and proliferation of EC109 cells in a time and dose-dependent manner and so does the migration and colony formation ability; the suppressive effect on EC109 cells will be augmented after the co-treatment of CQ and PTX;2. The Akt/mTOR pathway was supressed on CQ or PTX exposure, which resulted in the down-regulation of phosphorylated mTOR(p-mTOR) and phosphorylated Akt(p-Akt), the phosphorylated p70S6K(p-p70S6K) and phosphorylated p-4EBP1 were decreased after CQ treatment, in contrast with PTX; the co-treatment of CQ and PTX lead p-p70S6 K and p-4EBP1 expressed higher than CQ group, lower than the PTX group;3. The autophagy marker LC3 and p62 elevated after exposure to CQ while PTX didn’t influence the expression of them significantly and the expression of LC3 and p62 were lower than CQ group, higher than the PTX group after CQ used in combination with PTX;4. The number of autophagosome was increased after CQ treatment, but didn’t change significantly after PTX exposure by transfecting with GFP-LC3. And the autophagosome was fewer than CQ group, more than the PTX group after CQ used in combination with PTX;5. The growth and proliferation, migration and colony formation ability of EC109 were supressed, and the sensitivity of EC109 to CQ and PTX were elevated after knocking down the expression of mTOR by transfecting lentiviral shRNA embedding mTOR;6. The expression of p-Akt,p-p70S6 K and p-4EBP1 were decreased simultaneously, along with the increase of LC3 and p62 after the down-regulation of mTOR.Conclusion:1. CQ and PTX both could suppress the growth and proliferation, the migration and colony formation ability of EC109;2. CQ could enhance the suppressive effect of PTX on the growth and proliferation of EC109 cells by modulating autophagy;3. CQ and PTX could suppress the growth of tumor probably by activating the Akt/mTOR pathway;modulating the Akt/mTOR pathway could refrain the growth and proliferation, migration and colony formation ability of EC109, as well as elevate the sensitivity of EC109 to CQ and PTX;4. CQ could be used in clinical as an chemosensitizer and modulating autophagy may be an effective and potential strategy for the treatment of esophageal carcinoma.