The Effects And Mechanisms Of Diallyl Trisulfide And Simvastatin On Osteosarcoma Cells

Part ⅠThe Effects and Mechanisms of Diallyl Trisulfide on Osteosarcoma CellsBackgroundOsteosarcoma is the most frequent form of non-hematopoietic, primary bone tumor occurring mostly in young adults and adolescents. The current favored treatment for osteosarcoma involves neoadjuvant chemotherapy, followed by surgery and chemotherapy again, which has led to a significant improvement of five-year survival rate (60-70%) in patients without metastases. However,35-55% of osteosarcoma patients with initially localized disease subsequently experience recurrence, and the inductions of drug-resistant and unwanted side effects are involved in chemotherapy which inadequately treat the disease. There is an urgent need to discover new natural or synthetic compounds with potential to prevent osteosarcoma progression and improve patient survival rates.Diallyl trisulfide (DATS) is one of the main activity compounds which are sulfide with allyl group. Considerable researches and epidemiology studies revealed that DATS has a broad-spectrum anti-neoplastic activity. It can induce apoptosis of multiple cancer cells, such as those of human gastric, colon, breast and prostate. In addition, several results suggested that the reactive oxygen species (ROS) played an important role in DATS-induced death of cancer cells. However, recently, some studies showed that DATS-induced apoptosis were involved with PI3K/Akt signaling pathways. Our previous studies demonstrated that DATS could suppress osteosarcoma cells proliferation and reverse drug resistance and lower the ratio of CD133+cells in conjunction with methotrexate. However, it is unclear if the apoptosis induced by DATS in osteosarcoma cells is related to PI3K/Akt signaling pathway. There is little research referred to the effect of DATS on human osteosarcoma cells as well as molecular mechanism.In the present study, the effect of DATS and possible molecular mechanism were further studied in human osteosarcoma cells. Our data built a foundation for DATS as a promising therapeutic agent for the treatment of osteosarcoma.Purposes1. To observe the effects of DATS on osteosarcoma cells.2. To investigate the possible molecular mechanisms and signaling pathway by which DATS exerted anti-osteosarcoma effects.Methods1. After the MG-63 and MNNG/HOS cells were treated with different concentrations of DATS for 24h,48h and 72 h, the cell viability was determined using a CCK-8 assay.2. After the MG-63 and MNNG/HOS cells were treated with different concentrations of DATS, the cells were examined under a phase-contrast microscope.3. After the MG-63 and MNNG/HOS cells were treated with different concentrations of DATS, cell cycle distribution were measured by flow cytometry. The expressions of cyclin D1, p21 and p27 were measured by Western blot analysis.4. After the MG-63 and MNNG/HOS cells were treated with different concentrations of DATS, cell apoptosis were measured by flow cytometry using Annexin V-FITC/PI staining.5. After the MG-63 and MNNG/HOS cells were treated with different concentrations of DATS or NAC, the intracellular ROS was examined by fluorescence microscope and flow cytometric analyses using the fluorescent probe DCFH-DA.6. After the MG-63 and MNNG/HOS cells were treated with different concentrations of DATS or NAC, the cells were incubated with JC-1. Mitochondrial Membrane Potential can be measured by the ratio of red/green fluorescence intensity of JC-1.7. After The MG-63 and MNNG/HOS cells were treated with DATS, total proteins were extracted and the interest proteins expressions were detected by Western blot. We determined the expressions of PI3Kp110β、PI3Kp85α、Akt、 phosoho-Akt、Bad、Bax、Bcl-2、Bcl-XL、cytochrome c、caspase-9、caspase-3 and cleaved PARP.Results1. DATS inhibited MG63 and MNNG/HOS cell viability in a dose-and time-dependent manner. DATS had less of an influence on MNNG/HOS cells than MG63 cells.2. For the morphological changes of MG63 and MNNG/HOS cells after incubation with DATS, the control group cells showed a typical polygonal and intact appearance, whereas the DATS-treated cells displayed dose-dependent changes in cell shape, such as membrane blebbing, cell rounding and shrinkage, poor adherence and floating shapes.3. DATS treated groups significantly increased at the G0/G1 phase in a dose-dependent manner compared with the control groups. Co-treated with DATS and NAC, the DATS-induced G0/G1 phase arrest were completely reversed. There was a dose-dependent decrease in level of cyclin D1, whereas the protein levels of p21 and p27 were up-regulated.4. The apoptotic ratios of both cells treated with DATS increased in a dose-and time-dependent manner. Co-treatment with NAC completely blocked the DATS-induced apoptosis.5. The intracellular ROS generation was markedly elevated from 16 h treatment with DATS and increased in a dose-and time-dependent manner.6. DATS induced disruption of mitochondrial membrane potential (Δψm). NAC significantly restored the Δψm level compared with DATS treated cells.7. DATS induced apoptosis through ROS-dependent downregulation of PI3K/Akt pathway and mitochondrial apoptotic pathways. After the MG63 and MNNG/HOS cells were treated with DATS, the expressions of PI3Kp110β, PI3Kp85α, Akt and p-Akt were down-regulation. And the levels of Bcl-2 and Bcl-XL proteins decreased, whereas the expression of Bad and Bax increased. In addition, the expression levels of cytochrome c, caspase-9, and caspase-3 and cleaved PARP were up-regulated in a dose-dependent manner respectively. DATS served as an inhibitor of PI3K/Akt pathway in DATS-induced apoptosis, and the combined treatment groups showed a more powerful synergistic effect to trigger apoptosis. However, the presence of NAC almost completely blocked DATS-induced down-regulation of PI3K/Akt pathway.Conclusion1. DATS exerts cytotoxic and anti-proliferative effects on human osteosarcoma MG63 and MNNG/HOS cells.2. DATS induces MG63 and MNNG/HOS G0/G1 phase cell cycle arrest, which was involved with increase of intracellular ROS, down-regulation of cyclin D1 and up-regulation of p21 and p27 expressions.3. DATS induces increase of intracellular ROS and thus resulted to collapse of Δψm.4. DATS-induced apoptosis in MG63 and MNNG/HOS cells is mediated by inactivation of the PI3K/Akt signaling axis and through mitochondria apoptosis pathway, which are ROS-dependent.Part ⅡThe Effects and Mechanisms of Simvastatin on Osteosarcoma CellsBackgroundOsteosarcoma is the most common primary tumor of bone in children and young adults. Because these tumors have a high propensity to metastasis, most often to the lung, they are ranked among the most frequent causes of cancer-related death. The current favored treatment for osteosarcoma involves neoadjuvant chemotherapy, followed by surgery and chemotherapy again. Although surgical techniques and chemotherapeutic strategies have been improved during the past few years, the 5-year survival rate of osteosarcoma patients has not significantly increased. Thus, there is a great need to develop more effective treatments for this deadly disease.Statins, which are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), have been widely prescribed for the treatment of hypercholesterolemia and are among the most widely used pharmaceutical agents in the world. Recently, statins have received more attention for their anticancer effects. Evidence is accumulating that statins exert the tumor cytotoxicity through the inhibition of cell proliferation, induction of apoptosis, or inhibition of angiogenesis. Of all statins, Simvastatin (Sim), a lipophilic member, have showed more potent anti-tumor activity in vitro and in vivo in previous studies. Numerous experimental data confirmed that Sim inhibits a variety of cancer cells growth, including ovarian cancer, bile duct cancer, renal cancer, prostate cancer and hepatic cancer. Evidence is mounting to support claims that Sim exert the anti-tumor effects by inducing apoptosis and inhibiting cell cycle progression through a variety of cell signaling pathways. However, there is little research referred to the effects of Sim on human osteosarcoma cells as well as molecular mechanisms. Thus, in the present study, the effects of Sim on osteosarcoma and possible molecular mechanisms were further studied. Our findings demonstrated that Sim could inhibit the human osteosarcoma cells proliferation in vitro and in vivo through multimechanisms, implying that Sim may be a promising therapeutic drug for osteosarcoma patients.Purpopse1. To observe the effects of Sim on osteosarcoma cells.2. To investigate the possible molecular mechanisms by which Sim induced osteosarcoma cells apoptosis.Methods1. After the MNNG/HOS cells were subjected to different concentrations of Sim for 24,48 and 72 h, the cell viability was determined using CCK-8 assay.2. Cell migration (wound-healing) assay was conducted to determine the capacity of cell migration.3. The determinations of invasion assay was performed through 24-well Transwell Insert coated with Matrigel. Meanwhile, after the MNNG/HOS cells were treated with different concentrations of Sim, the expressions of MMP-2 and MMP-9 were determined by Western blot analysis.4. After the MNNG/HOS cells were treated with different concentrations of Sim, cell cycle distribution was analyzed using flow cytometer. In addition, the expressions of cyclin D1, CDK2, CDK4, p21 and p27 were determined by Western blot analysis.5. The apoptosis of MNNG/HOS cells was examined by flow cytometry using Annexin V-FITC/PI staining. In addition, the expressions of PI3K, Akt, p-Akt(Ser473), Bax, Bcl-2 and cleaved PARP were determined by Western blot analysis.6. We used insulin-like growth factor-1 (IGF-1), one of the most potent activators of the PI3K/Akt signaling pathway, and LY294002, a specific PI3K inhibitor, to further test the contribution of PI3K/Akt pathway in Sim-induced apoptosis. The expressions of PI3K, Akt, p-Akt(Ser473), Bax, Bcl-2 and cleaved PARP were determined by Western blot analysis.7. We established animal models of xenograft tumor and then investigate the anti-cancer effect of Sin in vivo/The tumor volume and body weight mice were measured every week.Results1. Sim significantly inhibited MNNG/HOS cell proliferation in a dose-and time-dependent manner.2. The wound healing assay showed that Sim significantly suppressed the migration ability of MNNG/HOS cells in a dose-dependent manner compared with the control groups.3. Transwell assay showed that Sim significantly reduced the invasive ability of MNNG/HOS cells in a dose-dependent manner compared with the control groups. The protein expressions of MMP-2 and MMP-9 were markedly inhibited in a dose-dependent manner in Sim-treated cells compared with the control groups.4. Sim caused significant accumulation of cells in G0/G1 phase in a dose-dependent manner in MNNG/HOS cells. MNNG/HOS cells exposed to Sim treatment for 48 h exhibited a dose-dependent decrease in cyclin D1, CDK2 and CDK4 expressions. Whereas the expressions of p21 and p27 were remarkably enhanced. These findings coincide with the Sim-induced cell cycle arrest in the G0/G1 phase.5. The apoptotic effects of Sim significantly increased in dose-dependent manner compared with control groups. Sim induced apoptosis via inactivation of PI3K/Akt pathway and enhancement of Bax/Bcl-2 expression.6. IGF-1 significantly activated PI3K/Akt signaling pathway. However, the Sim treatment had a reversal of roles, even simulated partly the effects of LY294002 on MNNG/HOS cells by inactivation of PI3K/Akt signaling pathway. Co-treated with Sim and IGF-1 almost completely reversed the IGF-1-induced up-regulation of PI3K and p-Akt, which suggested that blockage of PI3K/Akt pathway might account for Sim-induced apoptosis.7. During the treatment, the mice showed tolerance to Sim and maintained normal activities. Sim significantly reduced the tumor volume. Furthermore, there was no significant toxicity to mice treated with Sim.Conclusion1. Sim inhibited osteosarcoma cell proliferation in culture and tumor growth in vivo.2. Sim suppressed MNNG/HOS cells migration and invasion by down-regulation of expressions of MMP-2 and-9.3. Sim exerted the anti-tumor effects on MNNG/HOS cells by inducing G0/G1 phase arrest, which was involved with down-regulation of cyclin D1, CDK2 and CDK4 as well as up-regulation of p21 and p27 expressions.4. Sim induced MNNG/HOS cells apoptosis, the possible mechanism was involved in inactivation of PI3K/Akt signaling pathway.