The Mechanism Of Ursolic Acid On Anti-tumor And Effects As Adjuvant For Murine H22Hepatocarcinoma Cells Vaccine

Malignant tumor is one of the main diseases that threaten human health. At present, themain treatment methods include operation, chemotherapy and radiotherapy but with sideeffects and poor efficacy. Therefore, search for side effects of small, safe and efficienttreatment is an important trend of antitumor research.Ursolic acid (UA) is one of the natural pentacyclic triterpene compounds and has manybiological functions. Recent studies have found that UA has antitumor and enhance theimmune function, however, the mechanisms of UA on anti-tumor still not clear.This paperstudies the UA on proliferation inhibition and induction of apoptosis mechanism of tumor cellin vitro, MAPK signal pathways and the changes of cytokine role in apoptosis induced by UA,UA tumor immune regulation in vivo and as immune adjuvant enhanced immunogenicity oftumor cell vaccine. This study provides reliable experimental evidence for further applicationdevelopment of UA in the field of antitumor.The main contents and conclusions of are asfollows:1. Antiproliferative and induces apoptosis mechanisms of UA in cervical carcinoma celllinesTo investigate the effect of UA on cell proliferation and apoptosis induction. MTT assaywas used to measure the proliferation of HeLa cells; flow cytometry (FACS) was used toanalysis the changes in cell cycle and apoptosis of HeLa cells; Western blot was detectedCaspase-3, Caspase-8, Caspase-9, Cytochrome C, Bcl-2, Bcl-xL, Bak, Bax protein expression;Real-time PCR detected the expression of HPV E6and E7gene on cervical cancer HeLa andSiHa cells. The results showed that UA inhibited the proliferation of HeLa cells in a dosedependent manner; the proliferation rate was91.80±3.26%after48h of40μM UA, IC50valuewas9.54±3.51μM. Measured results show that UA arrest HeLa cell cycle in G0/G1phase byflow cytometry, the result most significant of40μM ursolic effect48h. UA induced apoptosisof HeLa cells in a dose dependent manner, the early apoptosis rates were74.30±5.26%and83.87±2.27%after40μM and60μM UA after48h respectively and significantly higher thanthat in control group was2.20±1.15%(P<0.01). Western blot results showed that Caspase-3and Caspase-9activation of HeLa cells induced by UA and cleaved forms of Caspase-3andCaspase-9were observed; the expression of Cytochrome C protein increased in cytoplasm;the expression level of Bcl-2and Bcl-xL decreased, the expression level of Bax and Bakprotein expression increased and all in a certain time dependent manner. Procaspase-8proteinexpression was also reduced, but Caspase-8splicing tape was not detected. Real-time PCRshowed that the expression of HPV E6gene were significantly decreased in cervical cancerHeLa and SiHa cells (P<0.01), but the expression of E7gene did not show significant change.These suggested that UA has good proliferation inhibition activity for cervical cancer cells invitro. It can through induce apoptosis antitumor in vitro, research on the anti-apoptoticpathway indicated that could induce apoptosis of cervical cancer cells through mitochondrialpathway. Targets that UA anti cervical cancer may be that can inhibit HPV E6gene expression and promote apoptosis.2.The role of MAPK signaling pathway in UA induces HeLa cells apoptosis and thelevels of cytokinesMolecular mechanisms of UA induced apoptosis in tumor cells still not clear, we discussedthe effect of MAPK signaling pathway and the changes of cytokines in UA induced HeLa cellapoptosis. Western blot detected p38, p-p38, ERK, p-ERK, JNK, p-JNK protein expression inMAPK signaling pathway of different concentration of UA in Hela cells for48h; ERK1/2inhibitor U0126on the expression of ERK1/2and p-ERK1/2and p38inhibitor SB203580onthe expression of p38and p-p38; U0126and SB203580on the expression of apoptosisrelated proteins Bax,Bcl-2,Caspase-3and Cytochrome C. Real-time PCR tested the effects ofUA on the expression of DUSP genes; ELISA detected content of IL-2and IL-4cytokines incultured Hela medium. The results showed that UA decreased the expression of p-ERK1/2and p-p38,no change on the expression of p-JNK.; ERK1/2inhibitor U0126significantlyenhanced the UA induced Bax/Bcl-2ratio, enhance the expression of mitochondrialCytochrome C release and cleaved Caspase-3protein; p38inhibitor SB203580effect is notobvious. Real-time PCR results showed that UA enhanced DUSP1,2,4,5,6,7,9,10geneexpressions. ELISA results showed that the content of cytokines changed but no regularity.These Suggested that ERK1/2signaling pathway involved in UA inducing cell apoptosis, UAmay by enhancing DUSPs gene expression to stimulate the MAPK signaling pathway toinduce cell apoptosis and can by changing cytokine secretion regulation of cell apoptosis.3. Antitumor effect and immune regulation activity of UA on Hepatoma H22in miceTo investigate the antitumor effect and immune regulation activity of UA on H22in mice.MTT assay was used to measure antiproliferative activity, and characterized apoptosis byflow cytometry (FCM) analysis in vitro. The models of mice bearing Hepatic cancer H22were established and randomized into5groups (10in each group),the model group (NS),CTX positive control group (25mg/kg.d), the UA high dose group (40mg/kg.d),middle dosegroup (20mg/kg.d) and low dose group (10mg/kg.d), each group given by ip for consecutive15days. The growth carves of xenografts in mice were drawed and the tumor inhibitory rate,the thymus index and spleen index were calculated, lymphocyte proliferation of spleenstimulated by ConA and LPS were detected by MTT, T cell subsets CD4+,CD8+were detectedby FCM and the levels of Th cytokine IL-2, TNF-α, IL-4in serum were detected by ELISA.The results showed that In vitro UA suppressed the growth of H22cells in a dose-dependent,the inhibitory rate was75.27±3.36%with40μmol/L for48h.The apoptotic rate in atime-dependent, with20μmol/L for48h was36.4%±0.2, there was significant difference withcontrol (P<0.01). In vivo, the inhibitory rate of UA high dose group and CTX group was39.14%and46.79%, respectively, which were significantly higher than that of the modelgroup (P<0.01).Compared with the model group, spleen index of high dose UA group weresignificantly lower (P<0.05), the T and B lymphocyte proliferation of spleen weresignificantly higher (P<0.01). The T cell subsets CD4+T expression and CD4+/CD8+T ratewere significantly increased with high dose UA compared with the model group (P<0.01), but CD8+T expression has no significantly change (P>0.05).The cytokine IL-2, TNF-α in serumexpression were promoted with high dose UA, which was difference with the model group(P<0.05), meanwhile the IL-4expression was reduced, which was significantly differencewith the model group (P<0.01). The results indicated that UA can inhibit tumor growth bothin vitro and in vivo. It could play a role of anti-tumor activity in vivo by regulating the body’sabnormal immune function in tumor-bearing state.4. Immunoadjuvant effect of UA for murine H22hepatocarcinoma cell vaccineTo study the immunoadjuvant effect of UA co-immunized with murine H22hepatocarcinoma cell vaccine. The mice were injected lysate of H22hepatocarcinoma cell andUA co-culture as vaccine three times. After one week of the last immunization the H22cellmodel were set up. The tumor growth curve and survival rate of the mice were observed. Theproliferations of T lymphocytes were measured by MTT and the levels of serum cytokinesIL-2and IL-4were determined by ELISA. CD4+, CD8+lymphocyte populations weredetermined by flow cytometry. The serum antitumor specific antibodies were determined byimmunofluorescence, ELISA and Western blot. It showed that after the mice were immunizedwith the tumor vaccines treated with UA as immunoadjuvant. Compared with model group,the tumor growth was inhibited obviously (P<0.05), the life span of vaccine group wassignificantly prolonged (P<0.01). The T and B lymphocyte proliferations wre significantlypromoted (P<0.01), the ratio of CD4+/CD8+was promoted obviously (P<0.01) and the levelsof serum cytokines IL-2and IL-4significantly higher (P<0.01).The specific binding ofantiserum and antigen was found and the higher levels of serum antibody was detected. It isconcluded that UA as adjuvant could remarkably improves the immunogenicity of H22tumorvaccines and the mice cellular immunity and humoral immunity could enhance on antitumor.In summary, UA induces apoptosis through mitochondrial intrinsic pathway in cervicalcancer cells and ERK1/2signaling pathway plays an important role in the process. UA canenhance the ability of immunoregulatory of H22mice and induced inhibition of tumor growthin mice. UA can promote immunogenicity of H22hepatoma cells and stimulate strong andlong-lasting antitumor immune protection of normal mice. UA may play a role ofimmunoadjuvant in the preparation of protective antigen. This study provides a goodtheoretical basis for the development of UA as antitumor drugs and as adjuvant of tumorvaccine.