A Proteomic Study On A Human Osteosarcoma Cell Line Saos-2 Treated With Diallyl Trisulfide

BackgroundOsteosarcoma is a primary malignant tumor of the skeleton, which frequently occurs in adolescent. Various therapeutic tools failed in radical cure, due to the character of infiltrating growth in osteosarcoma. Before 1970, all patients with osteosarcoma were treated by amputation but the 5-year survival rate was under 10% and almost all patients died within a year from diagnosis. Dramatic therapeutic improvement achieved in the last 30 years is a result of development of aggressive and efficient combination chemotherapy regimens. Thus, modern treatment programmes are typically multimodal, with surgery combined with both pre- and postoperative chemotherapy (neoadjuvant chemotherapy). Today, for localised osteosarcoma at onset treated with neoadjuvant chemotherapy associated with surgery, the percentage of patients cured varies between 60% and 70%. Several studies had indicated that the five-year survival rate of patients with osteosarcoma treated with neoadjuvant chemotherapy and limb salvage surgery can be more than 70%. However, in our clinical study, the disease free survival rate was 54.5%( the mean follow-up time was 47 months ). Although neoadjuvant chemotherapy is effective in improving patient survival, the frequent acquisition of drugresistant phenotypes and the occurrence of adverse reaction, such as myelosuppression, hepatotoxicity, toxicity of kidney, toxicity of heart, toxicity of nervous system, gastrointestinal reaction, are often associated with chemotherapy, which remain as serious problems. Therefore, there is a clear need for newer effective agents, which will be a therapeutic breakthrough for osteosarcoma in chemotherapeutics.Diallyl trisulfide(DATS), one of organosulfur compounds (OSCs) generated upon processing of fresh garlic, can inhibit proliferation of cultured cancer cells. Evidences have been accumulated to indicate that DATS can block cell mitosis and arrest cell cycle through regulating cyclin-dependent kinases (Cdks) and cyclins. Three biological processes, including mitochondrial signals, calcium homeostasis and oxidative stress, are typical events that result in or from apoptosis. Several components of apoptotic pathways have been widely investigated under DATS-induction, such as upregulation of Bax, Bad or Bak, downregulation of Bcl-2, activation of caspase-3 and -9. However, it seems that the previous studies limited on cancer cells from glandular organ or epithelial tissue. According to our available information, no related documents suggested that the anticarcinogenic effect of DATS has the selectivity in tissue origin. In the present study, we make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells, and the molecular mechanisms of DATS in suppressing cell proliferation. In this study, DATS was first applied to experimental study on drug treatment of osteosarcoma. Moreover, the combination of two-dimensional electrophoresis (2-DE) with mass spectrometry and database interrogations allowed us to identify the proteins differentially expressed in Saos-2 cells following DATS treatment. Therefore, our study can not only expand the anticarcinogenic extent of DATS, but also provide a novel insight into the drug treatment of osteosarcoma.PARTⅠEffects of diallyl trisulfide on cell proliferation in a human osteosarcoma cell line Saos-2 cellsObjectiveEvidences have been accumulated to indicate that DATS has the ability to suppress cell proliferation by blocking cell cycle progression and inducing apoptosis. However, it seems that the previous studies limited on cancer cells from glandular organ or epithelial tissue. According to our available information, no related documents suggested that the anticarcinogenic effect of DATS has the selectivity in tissue origin. In the present study, we make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells. After treated with DATS, cell morphologic change, cell cycle and apoptosis were used to analyze the effect on cell proliferation. Methods1 After treated with DATS at desired concentration for various time intervals, cell morphologic change was observed under inverted microscope.2 The effect of DATS on cell proliferation was detected by MTT [3-(4-5dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide] assay.3 After treated with DATS at desired concentration for various time intervals, cell cycle phase was analyzed by flow cytometry.4 After treated with DATS at desired concentration for various time intervals, the apoptotic rate was analyzed by flow cytometry.Results1 Cell morphologic changesAfter treated with 25μM, 50μM or 100μM DATS for various incubation time intervals, the shape deformations, such as shrinkage of cell bodies, the intercellular gaps becoming loose, cell lyses, condensation of nuclei, were observed under inverted microscope. In addition, many cells became round, floated and necrotic. Moreover, these morphologic changes had concentration- and time-dependent phenomenon. However, as compared to the treated groups, the cell morphology in control group had no visible changes.2 DATS inhabits the proliferation of Saos-2 cellsThe antiproliferative effect of DATS Saos-2 cells, was examined through exposing them to different concentrations of DATS (25μM, 50μM or 100μM) for 24, 48, 72h and 96h, respectively. These results indicated that DATS can exert significant inhibition to Saos-2 cells proliferation in concentration- and time-dependent manner.3 Cell cycle analysisThe cell cycle of Saos-2 cells was arrested at G0/G1 phase when the cells were treated with DATS. The more concentration of DATS used, the more Saos-2 cells were blocked at G0/G1 phase. In addition, with the treated time prolonged, the percentage of arrest at G0/G1 phase was increased.4 Apoptosis analysis The results indicated that DATS can induce apoptosis in Saos-2 cells. Furthermore, the effect of DATS-induced apoptosis was increased in concentration- and time-dependent mannerPARTⅡExperimental study on the molecular mechanisms of diallyl trisulfideObjectiveIn PARTⅠ, we make attempt to investigate the effects of DATS on a human osteosarcoma cell line, Saos-2 cells. The results demonstrate that DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in a dose- and time-dependent manner.In an effort to approach the molecular mechanisms of DATS on Saos-2 cells, we used 2-DE and mass spectrometry to examine the protein profiles of Saos-2 cells treated with DATS.Methods1 After treated with or without 50uM DATS for 48h, total proteins in Saos-2 cells were extracted. The response of protein expression in Saos-2 cells induced by DATS was analyzed by 2-DE.2 The gel spots verified as the significant changes in spot volume were separated and transferred into matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to obtain newer or existing proteins.Results1 2-DE patterns of Saos-2 cells treated with or without DATSThe proteins extracted from Saos-2 cells were resolved on 2-DE. Upon 2-DE images, the proteins in Saoa-2 cells either with or without DATS treatment behaved electrophorectically in similar modes. After scanned by Amersham Imagescanner, the image analysis was conducted with ImageMaster 2D Platinum. The total spots were 316±6 (n=3) and 310±3 (n=3) in the control and DATS-treated groups, respectively. The threshold of the significant change in 2-DE spots was defined as 3-folds of change in spot volume upon comparison of average gels between the treated and control groups. A total of 36 spots were defined as DATS-sensitive spots in Saos-2 cells, including 22 downregulated spots and 14 upregulated spots. The results indicated that a total of 36 spots were defined as DATS-sensitive spots in Saos-2 cells, including 22 downregulated spots and 14 upregulated spots.2 Identification of differentially expressed proteins by MALDI-TOF MSThe spots from the 2-DE gel were subjected to trypsin digestion and MALDI-TOF MS analysis. On the basis of the data of mass spectrometry, 36 spots matched with the proteins, in which 27 spots were ascertained as unique proteins including 18 downregulated and 9 upregulated. These proteins are involved in many biological functions, such as cell growth and apoptosis, cell differentiation and mobility, cell structure and basic metabolism, protein biosynthesis and degradation.ConclusionIn the present study, DATS was first applied to experimental study on drug treatment of osteosarcoma. We make attempt to investigate the effect of DATS on human osteosarcoma cell line Saos-2 cells, and the molecular mechanisms of DATS in suppressing cell proliferation. Using inverted microscope and MTT assay, we found that DATS has the ability to suppress cell proliferation of Saos-2 cells in dose- and time-dependent manner. Moreover, cell cycle phase and apoptotic rate were analyzed by flow cytometry; and the results suggested that DATS can block Saos-2 cells at G0/G1 phase and induce apoptosis. The proteomic data provided novel insight into a massive response of protein expression in Saos-2 cells induced by DATS, and demonstrated that some of DATS-sensitive proteins were related with several biology pathways.In summary, the results of the present study indicate that DATS has the ability to suppress cell proliferation of Saos-2 cells by blocking cell cycle progression and inducing apoptosis in dose- and time-dependent manner. Therefore, our study can not only expand the anticarcinogenic extent of DATS, but also provide a novel insight into the drug treatment of osteosarcoma. In addition, the DATS-sensitive proteins in our study might elucidate the anticarcinogenic effects of DATS and be applied as therapeutic targets for gene therapy of osteosarcoma.