Studies On Effects Of Ursolic Acid On Proliferation, Apoptosis And Associated Mechanism In Human Tongue Carcinoma Tca8113 Cells

Oral cancer is one of the most common malignant tumor of the top ten at present, while the incidence of tongue cancer ranks the first among the oral cancer in China. The properties of tongue cancer are growth-fast, highly malignant, highly invasive. Comprehensive treatment usually involves surgery, radiotherapy, chemotherapy etc, but the five-year survival of patients is still low. To explore positively effective preventive measures and drugs becomes very urgent and necessary.Ursolic acid is able to resistant to varieties of carcinogens, tumor promoters. In vitro experiments confirmed that it can inhibit a variety of tumor cell proliferation and induce their apoptosis. There are few studies about the effects of ursolic acid on the oral cancer, especially to tongue cancer research, and it is still rare at home and abroad.In vitro culturing human tongue Tca8113 cells were used to observe the role of ursolic acid. On this basis, MTT methods, flow cytometry, immunocytochemistry and RT-PCR, Western Blot techniques were used to observe molecular biology mechanism of proliferation, apoptosis in Tca8113 cells cultured in ursolic acid. Based on the experiments in vitro, human tongue carcinoma Tca8113 cells were seeded subcutaneously into the nude mice to establish tumor model. In order to explore the effects and mechanism on tumor and the toxicity to nude mice of metabolism ursolic acid, the appropriate concentration of ursolic acid was applied to therapy the tumor-bearing nude mice. Then explore new drugs, develop new ideas and methods for ursolic acid in treatment of tongue cancer. And provide experimental materials for development, application of ursolic acid in treatment of oral cancer. This study was divided into the following three parts:Part 1: Effects of ursolic acid on proliferation and apoptosis in human tongue carcinoma Tca8113 cellsMethods1. Morphology changes of Tca8113 cells without treated and treated by UA were observed with Optical microscope.2. The proliferation change of Tca8113 cells after treated with UA at 6.25,12.5,25, 50μmol/L for 12,24,36,48,60,72h was detected respectively by MTT assay to observe the toxic effects of UA to Tca8113 cells.3. After human tongue cancer Tca8113 cells were cultured 0,24,48,72h in concentration of 25μmol/L ursolic acid, the apoptotic rate and cell cycle distribution of Tca8113 cells in the functional level were detected by Flow CytoMeter (FCM).4. The changes of Ultramicrostructure in the cells treated with UA at 25μmol/L for 72h were observed by transmission electron microscope.5. Statistical analysis: The statistical package SPSS 13.0 was used for all analysis. Data was indicated with mean±standard deviation (x±s). Two-way ANOVA analysis was used in MTT assay results and one-way ANOVA analysis in FCM results. The significant level wasα= 0.05.Results1. The morphology changes of Tca8113 cells treated by UA were observed in Optical microscope.2. The proliferative activity of Tca8113 cells treated with UA was inhibited obviously, showing a dose-dependent and time-dependent apparently. There were significant difference between different concentrations groups and different time groups (P<0.01). The rate of growth inhibition of the Tca8113cells treated with 50μmol/L UA for 72h was 68.80%.3. The results of Flow cytometry showed: With the prolonging of UA role, the apoptosis rate of the cells increased from 2.57% to 24.51% gradually (There were significant differences, P<0.01). There was no significant change of G2/M stage cells proportion (P>0.05), but that S stage cells proportion decreased gradually (P<0.01) and that G0/G1 stage cells proportion increased obviously (P<0.01).4. The typical ultramicrostructure changes of the apoptotic cells, such as concentration and margination of nuclear chromatin and apoptotic body, appeared in the Tca8113 cells treated with UA at 25μmol/L for 72h under transmission electron microscope.Part 2: Effects of ursolic acid on human tongue carcinoma Tca8113 cell cycle and apoptotic mechanismsMethods1. The protein expression status of NF-κB,VEGF,Bc1-2 and Bax in the cells treated with UA at 25μmol/L for 0,24,48 or 72h was respectively detected by immunocytochemistry.2. The mRNA expression status of NF-κB,VEGF,Bc1-2 and Bax in the Tca8113 cells treated with UA at 25μmol/L for 0,24,48 or 72h was respectively detected by reverse transcription-polymerase chain reaction (RT-PCR) methods and the test results were investigated through semi-quantitative analysis. We hoped to know proliferation inhibition and promoting apoptosis mechanism of Tca8113 cells by UA.3. The protein expression status of NF-κB,VEGF,Bc1-2 and Bax in the Tca8113 cells treated with UA at 25μmol/L for 0,24,48 or 72h was respectively detected by Western Blot and the test results were investigated through semi-quantitative analysis. The proliferation inhibition and promoting apoptosis mechanism of Tca8113 cells were furtherly studied.4. Statistical analysis: The statistical package SPSS13.0 was used for all analysis. Data was indicated with mean±standard deviation (*±s). one-way ANOVA analysis was used in results. The significant level wasα＝0.05.Results1. Immunocytochemical staining results: Brown color of NF-κB was within the cytonucleus and cytoplasm. Brown color of VEGF was within the cytoplasm. Brown color of Bc1-2,Bax was within the cytoplasm.2. RT-PCR Results: After the action of ursolic acid, mRNA expression of NF-κB, VEGF, Bc1-2 in Tca8113 cells decreased gradually, while, mRNA expression of Bax increased gradually. Bcl-2/Bax ratio is lowered gradually. There was time-dependent. There was Statistical difference (P<0.05).3. Western Blot Results: After the action of ursolic acid, protein expression of NF-κB, VEGF, Bcl-2 in Tca8113 cells decreased gradually, while, protein expression of Bax increased gradually. There was time-dependent. There was Statistical difference (P<0.05).Part 3:Study on inhibitory effect of UA on human tongue carcinoma xenograft tumors in nude miceMethods1. Tca8113 cells cultured in vitro were injected subcutaneously into the armpits of the 12 BALB/C-nu/nu female nude mice (4-6 weeks old), in order to establish human tongue cancer xenograft models. After the tumor formed, the mice were divided randomly into control group and experimental group, and 6 mice in each group. The mice in experimental group were injected into tumor tissue with UA at a dose of 0.05mg/g body mass each day, while those in control group were injected with PBS at the same volume(0.01M, pH7.4). The treatment was lasted 4 weeks.2. The mental status, diet situation and act of the mice were observed each day. The size of the tumors and the body weight of the mice were measured every week. And then evaluate the toxicity of UA.3. At the end of treatment, the mice were killed and the tumors were removed out from the killed mice and weighted to calculate inhibition rate of tumors growth.4. The histologic conditions in the tumors and main organs of the mice, such as encephalon, heart, liver, spleen and kidney, were observed under light microscope.5. The mRNA expression status of NF-KB,VEGF,Bc1-2 and Bax in the xenograft neoplasm were respectively detected by RT-PCR methods. The test results were investigated through semi-quantitative analysis. We hoped to know the mechanism of xenograft neoplasm inhibition by UA. 6. The protein expression status of NF-κB,VEGF,Bc1-2 and Bax in the xenograft neoplasm were respectively detected by Western Blot. The test results were investigated through semi-quantitative analysis. The proliferation inhibition and promoting apoptosis mechanism of xenograft neoplasm by UA were fuetherly studied.7. The AI of the xenograft tumors was detected by TUNEL methods.8. Statistical analysis: The statistical package SPSS13.0 was used for all analysis. Data was indicated with mean±standard deviation (x±s). Independent samples t-test and paired t test were respectively used about Changes of body mass and tumor tissue mass. One-way ANOVA analysis was used in RT-PCR and Western Blot results. The significant level was a= 0.05.Results1. Compared with the control group, the growth of the tumors in experimental group was slower, and tumor inhibitory effect was more evident with the prolonging treatment of UA. At the end of treatment, the differences of tumor mass and tumor volume between two groups of xenograft neoplasm in nude mice got respectively to 55.65% and 61.72% and there were significantly difference (P <0.05).2. In the course of treatment, no bad effects of UA were observed in the mice. The values of final body mass/initial body mass in two groups were both more than 0.8. This indicated that there were no side effects to the mice about UA. No organic lesion and tumor metastasis appeared in the main organs (encephalon, heart, liver, spleen and kidney etc) of the mice in two groups.3. Optical microscope detecting: Tissue necrosis size within the tumor in the experimental group was significantly larger than of the control group. And nuclear atypia of cells in the experimental group with phenomenon of apoptosis such as nuclear karyopycnosis was lower than of control group.4. RT-PCR Results: In experimental group, mRNA expression level of NF-κB, VEGF, Bcl-2 in tissue of xenograft neoplasm decreased, while, mRNA expression level of Bax increased. There was Statistical difference (P<0.05).5. Western Blot Results: In experimental group, protein expression level of NF-κB, VEGF, Bcl-2 in tissue of xenograft neoplasm decreased, while, protein expression level of Bax increased. There was Statistical difference (P<0.05).6. The number of apoptotic cells in the experimental group was much more than that in the control group. The AI in two groups was 1.63±0.30,17.39±2.78 respectively and there was significant difference between two groups(P<0.01).ConclusionIn the first two parts of this study, the proliferation, apoptosis and cell cycle of human tongue cancer Tca8113 cells were detected by MTT method, flow cytometry. The mRNA expression and protein expression of NF-κB, VEGF, Bc1-2 and Bax were detected by immunocytochemistry, RT-PCR technology and Western Blot. In the third part, the properties of Tca8113 cells xenograft neoplasm treated with UA in nude mice were detected by some methods mentioned above. Draw the following conclusion.1. UA was able to inhibit proliferation of human tongue cancer Tca8113 cells, and these effects showed time and dose dependence.2. UA was able to induce apoptosis of Tca8113 cells, and there was time dependence.3. UA was able to decrease NF-κB, VEGF, Bc1-2 mRNA and protein expression and increased Bax mRNA and protein expression of Tca8113 cells. The ratio of Bc1-2/Bax was altered and these effects had a time-dependence. This might be one of mechanisms of cells proliferation inhibition. 4. UA took part in regulating cell cycle in Tca8113 cells and stoped cell cycle at G0/G1 phase. The proliferation inhibition, apoptosis induction and inhibition of correlation genes of Tca8113 cells by UA might all be related to the cell cycle arrest at G0/G1 phase.5. The xenograft neoplasm of human tongue carcinoma Tca8113 cells was established successfully in nude mice. UA was confirmed to inhibit the growth of tongue xenograft tumors and had no significant toxicity and side effects.6. Resembling to the results of experiments in vitro, UA was able to regulate down the mRNA and protein expression of NF-κB,VEGF,Bc1-2 and regulate up mRNA and protein expression of Bax and induce cell apoptosis and inhibit growth of xenograft tumors in nude mice.This study provided some reference materials for the clinical application of ursolic acid.