P Cao1, X J Jiang2, Z J Xi1. 1. Department of Urology, Peking University First Hospital; Institute of Urology, Peking University, Beijing 100034, China. 2. Institute of Microbiology, Chinese Academy of Science, Beijing 100101, China.
Abstract
OBJECTIVE: To determine the mechanism of sunitinib-induced autophagy in renal cell carcinoma cells. METHODS: MTS assay was applied to detect the cell viability alteration under the treatment of sunitinib (2, 8 μmol/L). The sunitinib-induced autophagy as well as cell apoptosis was measured and compared after knocking down autophagy-related protein Beclin1 and microtubule associated protein 1 light chain 3 fusion protein (LC3) by RNA interference. The transmission electron microscope was used to observe the formation of autophagosomes in ACHN cells. The fluorescence microscope was used to monitor distribution and aggregation of endogenous LC3-II. The expressions of protein such as LC3-II, the autophagic regulation molecules protein kinase B/ mammalian target of rapamycin (Akt/mTOR) and the symbol of apoptosis poly ADP-ribose polymerase (PARP) were capable to be detected by immunoblotting assay. RESULTS: Sunitinib was able to significantly trigger cell viability loss in the renal carcinoma cell ACHN, which was both in a concentration-dependent and time-dependent manner (P<0.05). After reducing the autophagy by knocking down Beclin1 and LC3, the number of cleavage of PARP was increased remarkably, whereas there was nearly not any cleavage in the mock group. By the transmission electron microscope, there were more autophagic vacuoles in ACHN cells after being administrated with sunitininb compared with the control. And the nuclear-to-cytosol translocation as well as aggregation of LC3-II was presented after sunitinib treatment by the fluorescence microscope, which was the proof of the enhanced autophagy. According to the immunoblotting, sunitinib was able to increase the accumulation of LC3-II . At the same time, the result of sunitinib combined with chloroquine, a drug which blocked the fusion of autophagosomes and lysosomes, demonstrated that the increasing amount of LC3-II was due to the enhanced autophagy flux by sunitinib treatment in ACHN cells. However, phosphorylation of Akt as well as mTOR was decreased at the same time. The rapamycin (mTOR inhibitor) or knocking down Akt subunits could change the sunitinib-induced LC3 -II accumulation, whereas overexpression of Akt subunits decreased the autophagic flux, indicating that Akt/mTOR was the target of sunitinib in autophagy. CONCLUSION: Sunitinib induced autophagy via suppressing Akt/mTOR pathway, and the autophagy was involved in apopotosis.
OBJECTIVE: To determine the mechanism of sunitinib-induced autophagy in renal cell carcinoma cells. METHODS: MTS assay was applied to detect the cell viability alteration under the treatment of sunitinib (2, 8 μmol/L). The sunitinib-induced autophagy as well as cell apoptosis was measured and compared after knocking down autophagy-related protein Beclin1 and microtubule associated protein 1 light chain 3 fusion protein (LC3) by RNA interference. The transmission electron microscope was used to observe the formation of autophagosomes in ACHN cells. The fluorescence microscope was used to monitor distribution and aggregation of endogenous LC3-II. The expressions of protein such as LC3-II, the autophagic regulation molecules protein kinase B/ mammalian target of rapamycin (Akt/mTOR) and the symbol of apoptosis poly ADP-ribose polymerase (PARP) were capable to be detected by immunoblotting assay. RESULTS:Sunitinib was able to significantly trigger cell viability loss in the renal carcinoma cell ACHN, which was both in a concentration-dependent and time-dependent manner (P<0.05). After reducing the autophagy by knocking down Beclin1 and LC3, the number of cleavage of PARP was increased remarkably, whereas there was nearly not any cleavage in the mock group. By the transmission electron microscope, there were more autophagic vacuoles in ACHN cells after being administrated with sunitininb compared with the control. And the nuclear-to-cytosol translocation as well as aggregation of LC3-II was presented after sunitinib treatment by the fluorescence microscope, which was the proof of the enhanced autophagy. According to the immunoblotting, sunitinib was able to increase the accumulation of LC3-II . At the same time, the result of sunitinib combined with chloroquine, a drug which blocked the fusion of autophagosomes and lysosomes, demonstrated that the increasing amount of LC3-II was due to the enhanced autophagy flux by sunitinib treatment in ACHN cells. However, phosphorylation of Akt as well as mTOR was decreased at the same time. The rapamycin (mTOR inhibitor) or knocking down Akt subunits could change the sunitinib-induced LC3 -II accumulation, whereas overexpression of Akt subunits decreased the autophagic flux, indicating that Akt/mTOR was the target of sunitinib in autophagy. CONCLUSION:Sunitinib induced autophagy via suppressing Akt/mTOR pathway, and the autophagy was involved in apopotosis.