Study On Ursolic Acid Inducing Autophagic Cell Death In TC-1 Cancer Cell

BackgroundCell death is divided into apoptosis and non-apoptosis,non-apoptosis including necrosis, autophagy,etc. Apoptosis has come to be used synonymously with the phrase'programmed cell death'. It accommodates the body to grow normally by many regulators.It is important that by these regulators the body can keep steady. Also by these regulators we can control the tumor and cure the tumor. Apoptotic cell death,which proceeds by cell shringkage,chromatin condensation, nucleosomal DNA degradation and fragmentation of the cell into so-called'apoptotic bodies'. Activation of the caspase family of cysteine proteases gives rise to these characteristic morphological features.The teatment of cancer include surgery,radiotherapy,chemotherapy and so on. Chemotherapy is still one of the key therapeutic strategies against cancer. traditional cancer drug therapy evokes cell death by inducing apoptosis. One of the most important advances has been the recognition that resistance to apoptotic cell death is an important aspect of both tumorigensis and the development of resistance to anticnacer drugs.Therefore,other mechanisms of cell death have emerged as potential novel mechanisms for cancer therapies to induce cell death。Autophagy is a degradation mechanism involved in quality and quantity control of cytoplasmic proteins and organelles,regarded as a programmed cell death juxtaposed with apoptosis and necrosis. Autophagic cell death is characterized by the appearance of double-or-multiple–membrane cytoplasmic vesicles engulfing bulk cytoplasm and/or cytoplsmic organelles such as mitochondria and endoplasmic reticulum. Increasing number of studies propose autophagic cell death as the mechanism of action of some anticancer agents.It was suggested that autophagic cell death induction in cancers have a therapeutic value.Ursolic acid(UA) is a pentacyclic triterpenoid derived from berries leaves, flowers, and fruits of medicinal plants.UA shows several bioactivities,such as anti-tumor,anti-diabetic,lowering hyperlipemia,anti-inflammation,and antioxidant activities,et al.ObjectiveTo investigate whether ursolic acid can inhibit proliferation of TC-1 cancer cells which are positive for the expression of HPV-16 E6/E7 and induce autophagic cell death.MethodsBased on the culture of TC-1 cells in vitro,the concentrations of UA used in cell culture system were 0,1μmol/L,5μmol/L,10μmol/L,20μmol/L and 1,2,3,4,5days were selected as the termination point of cell culture.Inhibition of cell proliferation was determined by MTT assay;The cell morphologic changes respectively under the optical microscope and fluorescent microscope after the coloration of PI;The C57BL/6 mice bearing TC-1 cancer cells were established in vivo and inhibition ratios were calculated according to tumor weight; The effects of UA on apoptosis were observed by DNA ladder; The rate of early apotosis by Annexin V-FITC/PI;Expression of caspase-3 was detected by Western bloting; Autophagy observed by transmission electron microscope; Expression of LC-3 was detected by Western bloting.ResultsOptical microscope and MTT assay showed UA had a significant antiproliferative effect on TC-1 cells in a dose-and time-dependent manner significantly; The transplanted tumor of the treated group grew slowly. The weight of tumors in the high-dose group were smaller than those in the control group. The tumor inhibitive rate was 69 %;DNA"ladder"was absent in 0, 10μmol/L,20μmol/L, 30μmol/L treatment groups for 48h through agarose electrophoresis; No typical features of apoptosis can be found by flow cytometry;No change in the expression of caspase-3;Autophagosomes were found under transmission electron microscope;UA could induce autophagy in TC-1 cells,which was characterized by increasing the conversion form LC3-Ⅰto LC3-Ⅱ.Conclusion1.Ursolic acid inhibit proliferation and induce death in TC-1 cancer cells .2. UA has promising anticancer activity in vivo and in vitro.3. UA induce cell death through non-apoptotic pathway.4. UA can induce autophagic cell death in TC-1 cancer cells.5.UA may be used to treat cancer through a new mechanism through inducing autophagic cell death ,which provides new targets for the therapy for cancer.