Function And Mechanism Analysis Of Autophagy In The Antitumor Effects Of Betulinic Acid In Colorectal Cancer Cells

Colorectal cancer is one of the common malignant tumor for people. In recent years, its incidence has a significantly increasing trend and the cure rate is low, which seriously threats human health. Advances in the prevention and treatment of cancer require the continued development of novel chemotherapeutic agents. One highly promising class of natural compounds are the triterpenoids with betulinic acid as the most prominent representative. Betulinic acid has been reported to produce toxic effects on tumor cells such as lung cancer, breast cancer, but the treatment effect has not yet been reported in colorectal cancer. Addtionally, Autophagy belongs to type II programmed cell death, and autophagy mechanism has become a new hot spot for tumor therapy in the current study. Therefore, this study mainly studied the effect of betulinic acid against colorectal cancer and the role of autophagy in the process of betulinic acid against colorectal cancer.(1) Betulinic acid induced apoptosis through the inhibition of cell proliferation in colorectal cancerColorectal cancer cells HCT116, SW480 and HT29 cells were treated with control and different concentrations of betulinic acid for 48 h.The results of cell prolifiation assay showed that betulinic acid dramaticlly restrained proliferation of colorectal cancer cells in a dose-dependent way.To evaluate apoptosis, Flow cytometry apoptosis detection by Annexin-V / PI double staining, observation of nuclear shape by DAPI and detection of apoptotic related proteins by western were carried out to evaluate apoptosis. Flow results showed apoptosis rates of betulinic acid treatment group significantly increased compared to control, and the rates of apoptosis increased in a dose-dependent manner. DAPI staining showed that the degree of condensation nuclei and the number of karyopycnosis cells were gradually increasing after treated with increasing betulinic acid. Western Blot revealed that pro-apoptosis factor Caspase 3 was much more activated and PARP was much more cleaved in betulinic acid treatment group than control.These results strongly suggested that betulinic acid suppressed the proliferation of colorectal cancer cells by inducing colorectal cancer cells apoptosis.(2) Betulinic acid promoted occurrence of autophagy and formation of autophagic flowTEM results found that intracellular autophagic vacuoles and the number of cells containing more than 5 autophagic vacuoles in betulinic acid treatment group significantly increased compared to the control group in HCT116 cells. Acidic autophagic vacuoles were stained by acridine orange and results showed that the number of acidic autophagic vacuoles increased significantly in three kinds of colorectal cancer cells with increasing of betulinic acid treatment concentration, strongly suggesting that betulinic acid induced autophagy occurence. In the same time, Laser scanning confocal microscope or fluorescence microscope results showed intracellular LC3 spots and the number of cells containing LC3 spots had an successively increase with the increase of betulinic acid concentrations. Indirect immunofluoresence results showed that LC3 spots had a notable increase in betulinic acid treatment compared to control. Western Blot showed LC3-I was significantly transformed into LC3-II in three kinds of colorectal cancer cells. RT-PCR showed that betulinic acid significantly activated the expression of autophagy key genes. These results strongly suggested betulinic acid promoted autophagy process of colorectal cancer cells.Addtionally, immune colocalization displayed that betulinic acid could promote the increase of autolysosomes. While, application of chloroquine or protease inhibitors, respectively, associated with betulinic acid further facilitated accumulation of LC3-II comparied to control, prompting that betulinic acid promoted the formation of autophagy flow in colorectal cancer cells.(3) Betulinic acid promoted autophagy by inhibiting Akt-mTOR signaling pathwayAs we all known, Akt-mTOR regulates autophagy classic signaling pathway, inhibiting autophagy process.Western Blot results showed that p-mTOR, p-Akt protein expression was significantly decreased in betulinic acid treatment group compared to control group, suggesting that betulinic acid activated autophagy by inhibiting Akt-mTOR signal pathway in colorectal cancer cells.(4) Inhibition of autophagy could enhance betulinic acid-induced proliferation inhibition effect of colorectal cancer cellsAdministration of autophagy inhibitor chloroquine or inhibition of expression of autophagy key gene ATG5 by siRNA interference, cell proliferation assay showed autophagy inhibition significantly enhanced betulinic acid-induced colorectal cancer cells growth inhibition rates. Simultaneously, flow cytometry apoptosis detection by Annexin-V/PI double staining method displayed autophagy inhibition significantly promoted betulinic acid-induced apoptosis. Western Blot results demonstrated that inhibition of autophagy promoted cleave of pro-apoptotic factor PARP and decline of anti-apoptotic factor bcl-xl, which conversely suggested that autophagy could inhibit the effect of pro-apoptosis and proliferation inhibition induced by betulinic acid in colorectal cancer cells.Above all, this study not only confirmed the betulinic acid inhibited the proliferation of colorectal cancer cells by promoting colorectal cancer cells apoptosis, but also confirmed betulinic acid activated autophagy by decreasing Akt-mTOR signaling pathway, while autophagy inhibiton enhanced betulinic acid-induced proliferation inhibition effect. This study preliminarily interpreted the effects and mechanism of betulinic acid against colorectal cancer and laid a solid foundation for further study of the mechanism and future rational use of betulinic acid as new anticancer drugs, which possesses a positive theoretical and practical significance.