Autophagy, hypoxic stress and migration capability in gefitinib-sensitive and -resistant PC-9 cells

• Main Authors: Ya-Ting Chang, 張雅婷
• Other Authors: Anya Maan-Yuh Lin
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/34658600850961451211

碩士 === 國立陽明大學 === 藥理學研究所 === 99 === Autophagy, known as an intracellular self-eating mechanism, is suggested as a protective mechanism against unfavorable conditions, including starvation and hypoxia which reportedly involves in invasion and metastasis of tumor cells. Excess autophagy has been demonstrated in tumor cells under limited nutrition and oxygen supply. Up-regulation of autophagy machinery has been suggested in cells undergoing structural remodeling, such as metastasis. Cancer cells derive multiple aggressive functions concurrently with development of resistance to effective treatment. In my thesis, the involvement of autophagy in the migration and drug resistance was studied using PC-9 and PC-9/gef cells. PC-9/gef is a gefitinib-resistant cells that acquired 200-fold resistance after long-term exposure of PC-9 adenocarcinoma lung cancer cells in medium containing escalating concentrations of gefitinib. In normoxic condition, PC-9/gef cells had slightly increased migration compared with PC-9 parental cells using wound healing assay. At the same time, higher HIF-1? and LC3-II levels were demonstrated in PC-9/gef cells. Hypoxic treatment (1% O2 for 16 hours) further increased HIF-1?? LC3-II and migration in both PC-9 and PC-9/gef cells. Two autophagy inhibitors, including Bafilomycin A1 (which increased LC3-II levels by inhibiting fusion of lysosomes with autophagosomes) and 3-methyladenine (which inhibits autophagy, 3-MA) were used to delineate the role of autophagy in migration. Both Bafilomycin A1 and 3MA attenuated migration in PC-9 and PC-9/gef cells under both normoxic and hypoxic conditions. Furthermore, Atg7 siRNA not only inhibited Atg7 protein expression but also inhibited migration in PC-9 and PC-9/gef cells under both normoxic and hypoxic conditions. The involvement of autophagy in tumor cell growth was investigated using both in vitro and in vivo studies. Using MTT assay, 3MA had no effect on PC-9 cells growth but significantly inhibited the cell growth of PC-9/gef cells. While chloroquine alone (which increased LC3-II levels by inhibiting lysosomal fusion) is capable of inducing LC3-II levels in both PC-9 and PC-9/gef cells, combination of chloroquine and gefitinib did not augment gefitinib-induced cytotoxicity in PC-9 cells. However, combination of chloroquine and gefitinib significantly enhanced gefitinib-induced cytotoxicity in PC-9/gef cells. Using xenograft study, concomitant treatment of chloroquine (60mg/kg/day, i.p.) and gefitinib (50mg/kg, oral) was found to reverse gefitinib resistance in nude mice carrying PC-9/gef tumors. These data indicate that PC-9/gef cells have elevated LC3-II which may contribute to invasion, metastasis and malignancy of the drug resistance. Furthermore, combination of gefitinib and autophagy inhibitors may be therapeutically useful in reversing gefitinib resistance in treating non small cell lung cancers.