Study On The Mechanism Of Tanshinone ⅡA Anti-osteosarcoma Mediated By Src Family Kinase

Osteosarcoma is the most common malignant tumor of the skeletal system. Due to the high rate of hematogenous metastasis, rapid progress, and the poor prognosis, osteosarcoma is one of the major death-causing diseases in teenagers. The occurrence and evolution of osteosarcoma is the result of multiple abnormal genes accumulation mainly about oncogene activation or tumor suppressor gene inactivation. Some studies have found that the abnormal expression and (or) activation of Src family kinases (SFKs) could promote the development of malignant tumor. Src can be activated by multiple signal pathways, Src protein is the center of the signal pathways. It has been confirmed that Src is associated with osteosarcoma closely.At present, besides the traditional targeting inhibitors will continue to be developed, looking for the natural, non-toxic side effects, efficient and new targeted drugs has become a research hotspot. Natural Chinese medicine have been widely payed attention at home and abroad for its extensive, exact curative effect and low toxicity. In many Chinese traditional medicine, Tanshinone II A (Tan IIA) is the one of the hotspot researchs. Tan IIA could inhibit osteoclast differentiation through Src. Therefore, Tan IIA may be a important auxiliary drug for anti-osteosarcoma.We have found that Tan IIA could inhibit cell proliferation, migration, invasion, and promote cell apoptosis in osteosarcoma MG-63 cell line, moreover, these effect were depended on concentration and time in vitro. While the mechanism of tanshinone anti-osteosarcoma is still unclear.Tan IIA could suppress osteosarcoma through multiple signaling pathways which are mediated by the Src, so we speculate the mechanism of tanshinone anti-osteosarcoma may be related to Src. In the signaling pathways which Src mediated, many proteins in MAPK/ERK, PI3K/Akt pathway are involved with the biological effect of osteosarcoma, therefore, it is necessary to focus on whether the effect of Tan IIA anti-osteosarcoma is mediated by MAPK/ERK, PI3K/Akt pathway. Although a Src inhibitors — dasatinib has been developed, and it has been confirmed that dasatinib could inhibit Src phosphorylation in osteosarcoma cell, prevent cell migration and invasion, induce cells apoptosis. In nude mice bearing osteosarcoma, dasatinib can inhibit Src been phosphorylating completely in primary tumor, but it can not prevent pulmonary. Therefore, it is necessary to compare the effect of Tan IIA and dasatinib to understand the difference between them.We propose to study the mechanism of Tan IIA anti-osteosarcoma with starting with Src, take MAPK/ERK, PI3K/Akt signaling pathway mediated bt Src as the main object, and use dasatinib as the reference, to provide the basis of theoretical study and clinical application. The research includes the following four parts.Part 1. The clinical and prognostic significance of Src and p-Src expression in patients with osteosarcomaObjective The expression of Src and phospho-Src (p-Src), is closely related to tumor invasion and metastasis. The aim of the present study was to investigate the expression of these proteins in osteosarcoma and their relationship with each other to provide a theoretical basis to understand the prognosis of osteosarcoma.Methods We selected surgically resected osteosarcoma specimens from 116 patients of Zhongnan Hospital of Wuhan University and Hubei Cancer Hospital, Hubei, China, between January 2000 and January 2010 with detailed follow-up data. Twenty osteochondroma specimens from the corresponding period were used as controls. Expression of Src and p-Src was detected in osteosarcoma and osteochondroma by immunohistochemistry. We analyzed the relationship of the two proteins and osteosarcoma patients’ prognosis.Results The expression of Src and p-Src in osteosarcoma was significantly higher than the expression level in osteochondroma (p<0.05). The expression levels of the two proteins, clinical stage, and tumor metastasis were significantly associated with survival time (p< 0.05), while there was no correlation between age or gender and survival time. The expression of Src and p-Src in osteosarcoma was positively correlated.Conclusion Src and p-Src can be used as an auxiliary indicator to determine a malignant phenotype of bone tumors, and that combined detection of Src and p-Src may indicate the prognosis of osteosarcoma.Part 2. The effect of Src on the biological characteristics of osteosarcoma cellObjective Src play the role of cancer gene in most tumors. We study the influence of Src on biological behavior of osteosarcoma cells in this part.Method Src-shRNA with the slow virus vector was stably transfected to MG-63 and U2OS cell lines. Then detected the expression of Src mRNA by RT-PCR and the expression Src, p-Src, p-ERK1/2, p-Akt proteins by western blot. Detected cell proliferation ability by CCK-8 and BRDU immunofluorescence, and cell invasion and migration ability by transwell and cell scratch test. The cell cycle was tested by flow cytometry instrument technology, and cell apoptosis was tested by AnnexinV/PI double staining.Results Immunofluorescence showed Src-shRNA was successfully stably transfected into osteosarcoma cells. The expression of p-Src, p-ERK1/2 and p-Akt were successfully suppressed after transfection, but the expression of Src protein and Src mRNA rises in MG- 63 cells, while, the expression of them had not obviously changed in U2OS cell. After transfection, cell invasion and migration ability had been significantly inhibited. Cells apoptosis had been induced, cell proliferation had been inhibited and cell cycle had been arrested in U2OS cell line after transfection, while these effect were not discovered in MG-63 cell line.Conclusion The carcinogenic effect of Src was mainly presented on promoting cell proliferation, invasion and migration, and inhibiting apoptosis, regulating cell cycle, but the regulating effect of Src on proliferation, apoptosis and cell cycle exhibited diversity in different cell lines.Part 3. The research of Tanshinone IIA anti-osteosarcoma mediated by Src in vitroObjective It has been reported that Tan IIA could supress the expression of Src protein, and Src could inhibit osteosarcoma cell invasion, migration, proliferation, and promote cell apoptosis. This section is to study the mechanism of Tan IIA anti-osteosarcoma mediated by Src.Methods Tan IIA, Src inhibitor dasatinib, and Tan IIA combined with dasatinib were added to osteosarcoma cell with stable transfection Src-shRNA and normal osteosarcoma cells, respectively. Then detected the expression of Src, p-Src, p-MAPK,2, p-ERK1/2, p-PI3K, p-Akt proteins and Src mRNA in each groups, and tested the changing of cell cycle, apoptosis, proliferation and invasive ability. Treated the above two cell lines with ERK inhibitor PD98059 and Akt inhibitors LY294002 respectively, then detected the expression of p-Src, p ERK 1/2, p-Akt, and the changing of cell cycle, apoptosis, proliferation and invasive ability.Results After the treatment of osteosarcoma cell with Tan IIA and (or) dasatinib, it was found that in MG-63 cell line, dasatinib could increase the expression of Src protein and Src mRNA, and inhibit p-Src, p-MAPK, p-ERK1/2, p-PI3K and p-Akt expression; in U2OS cell line, dasatinib could not effect on the expression of Src protein and Src mRNA, but it could inhibit p-Src, p-MAPK, p-ERK1/2, p-PI3K and p-Akt expression. Tan IIA could decrease the expression of Src, p-Src, p-MAPK, p-ERK1/2, p-PI3K, p-Akt protein and Src mRNA. Tan IIA combined with dasatinib have a synergistic effect on Src, p-Src, p-MAPK, p-ERK1/2, p-PI3K, p-Akt (p<0.05). The ability of Tan IIA inhibiting p-Src, p-PI3K, p-Akt was not significant difference between dasatinib, but Tan IIA could inhibit the expression of Src, p-MAPK, p-ERK more effectively than dasatinib (p<0.05). The effect of Tan IIA anti-osteosarcoma cell in in stable transfection of Src-shRNA group was more obviously than normal osteosarcoma cell group. Comparing with dasatinib, the ability of Tan IIA anti-osteosarcoma cell proliferation, apoptosis was not significant difference, but the ability of inhibiting cell invasion and migration was more obviously in U2OS cell line. In MG-63 cell line, the ability of Tan IIA inhibiting cell invasion and migration was more obviously than dasatinib, and Tan IIA could inhibiting cell proliferation and promote apoptosis, while the effect was not found in dasatinib. After treating osteosarcoma cell with ERK inhibitor and Akt inhibitor, it was found that PD98059 had a more obviously effect on inhibiting cell invasion and migration, while LY294002 was more effect on inhibiting cell proliferation and promoting cell apoptosis. PD98059 could significantly inhibit the expression of p-ERK1/2 but have no effect on p-Akt and p-Src; LY294002 could significantly inhibit p-Akt and partially inhibit p-ERK1/2 whlie have no effect on p-Src.Conclusion The impact of Tan IIA anti-osteosarcoma is mediated by MAPK/ERK and PI3K/Akt signaling pathways downstream of Src at cellular level. Compared to dasatinib, Tan IIA could more comprehensively suppresse biological behavior of osteosarcoma cell.Part 4. The research of Tanshinone IIA anti-osteosarcoma mediated by Src in vivoObjective We had confirmed in the above part that the impact of Tan IIA anti-osteosarcoma was mediated by MAPK/ERK and PI3K/Akt signaling pathways downstream of Src at cellular level. In this part, we propose to confirm that the effect of Tan IIA anti-osteosarcoma is also mediated by Src in vivo.Methods Cell suspension injection method was used for nude mice tumorigenicity assay on BLAB/C-nu/nu female nude mices. After the tumor successfully established, HE staining and X ray were used to confirm for osteosarcoma growth in-situ, and then divided the 20 tumor-burdened nude mice into two groups randomly. The experimental group was treated with Tan IIA, while control group was given saline lavage. After the treatment for two weeks, tested the effect of anti-osteosarcoma between the two groups by HE staining, tumor inhibition rate, pulmonary metastasis rate and X ray.Results Tibial osteosarcoma nude mice model had been successfully established by Cell suspension injection method, the success rates was 100%. The tumor inhibition rate was 64.09%. Compared to control group, the tumors were shrinked, tumor weight and pulmonary metastasis rate were decreased, cell nucleus were thin and part of the cells were high differentiation tested by HE staining in experimental group. The expression of Src, p-Src, p-MAPK, p-ERK1/2, p-PI3K, p-Akt proteins were reduced in experimental group, while the expression of the proteins were slightly higher in control group. The survival curve analysis showed a longer survive time in experimental group.Conclusion The impact of Tan IIA anti-osteosarcoma is mediated by MAPK/ERK and PI3K/Akt signaling pathway downstream of Src at animal level. Tan IIA could play a role of anti-osteosarcoma and inhibiting pulmonary.