Abstract
BackgroundAutophagy is an evolutionarily conserved process through which cells degrade and recycle cytoplasm. The relation among autophagy, apoptosis and tumor is highly controversial until now and the molecular mechanism is poorly understood.MethodsCell viability and apoptosis were detected by CCK8, crystal violet staining, Hoechst333342 staining and flow cytometry. The expression of AMPK and ULK1 was analyzed by western blotting. Colon cancer growth suppression by NVP-BEZ235 or CQ in vivo was studied in a tumor xenograft mouse model.ResultsOur previous study revealed that NVP-BEZ235 suppressed colorectal cancer growth via inducing apoptosis, however later, we found it also initiated autophagy simultaneously. In this present study, our results show that NVP-BEZ235 induced autophagy through AMPK/ULK1 pathway in colon cancer cells. Blocking autophagy by knocking down AMPK or ULK1 inhibited cell proliferation and further promoted NVP-BEZ235 induced apoptosis. Meantime, the autophagy inhibitor chloroquine (CQ) shows obvious effect on inhibiting cell proliferation but not on inducing apoptosis, while it significantly increased NVP-BEZ235 induced apoptosis. Furthermore, the combinational therapy of NVP-BEZ235 and CQ shows synergistic antitumor effects in colon cancer in vivo.ConclusionNVP-BEZ235 induced AMPK/ULK1-dependent autophagy. Targeting this autophagy suppressed colon cancer growth through further promoting apoptosis, which is a potential therapeutic option for clinical patients.
Highlights
Autophagy is an evolutionarily conserved process through which cells degrade and recycle cytoplasm
Antibodies and reagents Primary antibodies against Unc-51-like kinase 1 (ULK1), p-ULK1, AMP-activated protein kinase (AMPK), p-AMPK, LC3-II, cleaved-caspase3 and Actin were purchased from Cell Signaling Technology (CST)
In order to examine whether NVP-BEZ235 would regulate autophagy in colon cancer cells, we evaluated the puncta of GFPLC3 in differently colon cancer cell lines by fluorescence microscope
Summary
Autophagy is an evolutionarily conserved process through which cells degrade and recycle cytoplasm. The relation among autophagy, apoptosis and tumor is highly controversial until now and the molecular mechanism is poorly understood. Autophagy is a process of self-destruction, cellular constituents including proteins and cytoplasmic organelles were orderly degraded and reusing [8, 9]. LC3 (microtubule-associated protein light chain 3) is widely used to test autophagic activity, including LC3-I (cytosolic) and LC3-II (membrane bound). PI3K/AKT/mTOR signal pathway plays an important role in cell proliferation, survival and metabolism [11]. PI3K/Akt, and mTOR have been found to be over-activated in colorectal adenocarcinoma and have become potential targets for treatment [12, 13]. NVP-BEZ235, a dual PI3K/mTOR inhibitor, showing great therapeutic potential in colorectal adenocarcinoma
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