The Study On The Therapeutic Effects And Mechanisms Of A Novel Small Molecule Compound DMAMCL In Osteosarcoma

Objective: Osteosarcoma(OS)is the most common primary solid malignant bone tumor,which mainly originates from bone and in rare cases from soft tissue.Osteosarcoma is characterized by malignant mesenchymal cells that produce osteoid tissue or immature bone.It frequently occurs in children and adolescents.In addition,there is a second small peak in the age of 50.The incidence of male is 1.4 times that of female.The incidence rate is about 2-3/100 million per year.Although the 5-year survival rate of patients with osteosarcoma increased from less than 20% to 60-70% with the introduction of multimodal therapy,which combined with chemotherapy and surgery after 1980 s,it has not been significantly improved afterwards in the present decades.There are many factors that cause this situation,such as insensitivity to chemotherapy,drug resistance,recurrence and metastasis.Therefore,new treatment methods or new drugs are urgently needed to solve these problems,in order to improve the therapeutic effect and prognosis of osteosarcoma.A large number of studies have proved that in many kinds of tumors,including osteosarcoma,tumor stem cells exist and play an important role in drug resistance,recurrence and metastasis.DMAMCL is extracted from Compositae,a traditional Chinese medicine.Its precursor is stable sesquiterpene lactones synthesized by markel addition reaction.DMAMCL is a new antitumor drug independently developed in China.It has been proved that DMAMCL has a good antitumor effect in acute myeloid leukemia,glioma,rhabdomyosarcoma and other solid or non-solid tumors.Its active components have selective inhibition effect on tumor stem cells and drug-resistant tumor cells.The drug is in phase I clinical trials of adult brain gliomas in Australia and China(ANZCTRACTRN12618001934235,ChiCTR-OIC-17013604).To our knowledge,there is no study reports the effect of DMAMCL on osteosarcoma.This study aims to investigate the antitumor effect and mechanism of DMAMCL on osteosarcoma in vivo and in vitro,to find new drugs for osteosarcoma treatment,and provide theoretical basis for the clinical application of DMAMCL in osteosarcoma treatment.Methods: 1.Cell line and cell culture.In this study,five osteosarcoma cell lines(143B,MNNG,MG63,Saos-2,U-2OS),a mouse fibroblast cell line NIH3T3 and a human retinal epithelial cell line ARPE-19 were used.The cell lines were cultured in DMEM medium with 10% fetal bovine serum,2mM glutamine and antibiotics at 37°C in 5% CO2 incubator.The culture medium for 143 B was supplied with 0.015mg/ml 5-bromo-2'-deoxyuridine.2.Cell survival analysis.In vitro experiments,cell survival was detected by MTS assay,and the cell fusion rate was monitored by Incucyte Zoom live-cell imaging platform to reflect cell proliferation indirectly.3.Cell cycle and apoptosis analysis.The cell cycle was detected by PI(propidium iodide)and cell apoptosis was detected by AnnexinV and PI.Hoechst 33342 nuclear staining was used to detect cell apoptosis.Caspase-glo3/7 assay kit was used to detect the changes of Caspase3/7 activity before and after drug treatment.TUNEL cell apoptosis detection kit was used to detect the apoptosis of implanted tumor tissue in mice.4.Stemness analysis of tumor cells.The changes of osteosarcoma cell stemness were indirectly reflected by Colony formation assay and Sphere formation assay,and analyzed by Image J software.5.Analysis of protein expression.Western Blot was used to detect cell apoptosis,cell cycle and stemness related molecules expressions at protein level in cells and tumor tissues.6.Silencing the expression of target gene.The expression of Bax in osteosarcoma cells was silenced by siRNA transfection to study its function in the mechanism of DMAMCL effect on osteosarcoma cells.7.In vivo experiments.Osteosarcoma cell lines(143B,MG63)were implanted in the subcutaneous of nude mice,and the antitumor effects of different doses of DMAMCL were studied when the implanted tumor grew to the specified range,the tumor growth and the survival of mice were analyzed.8.Statistical analysis.SPSS 22.0 was used to analyze the experimental data.The data of in vitro experiment was decribed as mean ± standard deviation(`X± SD);the data of in vivo experiment was described as mean ± standard error(`X± SE).T-test was performed to compare the experimental data between two groups.The experimental data was regarded as statistically significant if P<0.05.Log rank test was used to analyze the experimental data of mouse survival analysis among multiple groups,it was considered that the experimental data had statistical significance if P<0.05.The experimental data curve was drawn by GraphPad Prism 7 or Photoshop 6 software.Results: 1.DMAMCL inhibited the survival of osteosarcoma cells.Five osteosarcoma cell lines(143B,MNNG,MG63,Saos-2,U-2OS)and the control cell lines NIH3T3 and ARPE-19 were treated with DMAMCL at different concentrations for 72 hours,and then the cell survival was detected by MTS assay.The results showed that DMAMCL caused concentration dependent cell death in osteosarcoma cells.The IC50(50% inhibitory concentration)ranged from 11.73 to 13.16?M.The IC50 of NIH3T3 cells was 34.64 ?M;the IC50 of ARPE19 cells was 42.01 ?M,which was about 3 times and 4 times of OS cells.At the same time,Incucyte Zoom live-cell imaging platform was used to detect the fusion rate of osteosarcoma cells to further reflect the cell proliferation rate.The trend was consistent with the change of cell survival detected by MTS assay.2.DMAMCL inhibited the growth of osteosarcoma in vivo.For in vivo experiments,the nude mice inoculated with osteosarcoma cells(143B,MG63)were treated with DMAMCL(75mg/kg and 100mg/kg)for 21 days.The results showed that DMAMCL inhibited the growth of OS tumor.The osteosarcoma treated with DMAMCL showed a dose-dependent growth inhibition,the percentage of the tumor volume of treatment group(75mg/kg and 100mg/kg)comparing to control group was 70.69% and 48.01% in 143 B,83.26% and 64.29% in MG63 xenograft modle at the end point of the treatment.The difference of tumor growth between the 100mg/kg group and the control group was statistically significant.3.DMAMCL treatment prolonged the survival time of mice bearing osteosarcoma.In order to study the effect of DMAMCL on the survival rate of mice implanted with osteosarcoma cells,we treated nude mice,which were implanted osteosarcoma cells(143B,MG63)with DMAMCL and recorded the number of days from the start of treatment to the end of the study.Kaplan-Meier survival curves showed significant survival advantage in DMAMCL-treated xenografts compared to control group.The median survival time of the control group,DMAMCL 75mg/kg treatment group and 100mg/kg treatment group were 18 days,24 days(P = 0.0417)and 29 days(P = 0.0118)in 143 B tumor bearing mice;29 days,37 days(P = 0.0026)and 37 days(P = 0.0039)in MG63 tumor bearing mice.The difference between the control group and 75 mg/kg treatment group or 100 mg/kg treatment group was statistically significant.4.DMAMCL blocked the cell cycle of osteosarcoma cells in G2/M phase: We treated ostomeosarcoma cells(143B,MNNG,MG63,Saos-2,U-2OS)with DMAMCL and used flow cytometry to detect cell cycle.The analysis showed that with the increase of concentration,the proportion of osteosarcoma cells in G2/M phase increased significantly.The proportion of G2/M cells increased by 13.73% in 143 B cells,19.76% in MG63 cells,10.50% in MNNG cells,10.05% in Saos-2 cells and 22.49% in U-2OS cells.At the same time,Western Blot showed that the expression of cyclinB1 increased,while the expression of CDC2 decreased after DMAMCL treatment.5.DMAMCL affected the stemness of osteosarcoma cells.After DMAMCL treatment for 24 hours,the expressions of CD133 and Nanog decreased significantly in osteosarcoma cells(143B,MG63,MNNG cells).In Colony formation assay,the number of clones in DMAMCL group was significantly less than that in control group(143B,MG63 cells).Statistical analysis showed that the clone number of DMAMCL treatment group decreased to 30.58% of the control group(P < 0.05)in 143 B cells and 32.11% in MG63 cells(P < 0.01).Consistent results were observed in the Sphere formation assay.Statistical analysis showed that in 143 B cells,the sphere formation rate of DMAMCL treatment group decreased to 29.62%(P < 0.01)of the control group,while in MNNG cells,the sphere formation rate of DMAMCL treatment group decreased to 12.05%(P < 0.01).6.DMAMCL induced apoptosis of osteosarcoma in vitro and in vivo.In cell cycle detection,it was found that with the increase of DMAMCL treatment concentration,the number of osteosarcoma cells in Sub G1 phase increased gradually.In order to further verify whether DMAMCL caused cell death by inducing apoptosis of osteosarcoma,the osteosarcoma cells(143B,MG63,Saos-2)were treated with DMAMCL(10?M,20?M,30?M)for 24 hours and then stained with AnnexinV/PI,Apoptotic cells were detected by flow cytometry.The results showed that the number of both the early and late apoptotic cells were increased in a dose dependent manner after DMAMCL treatment.The percentage of apoptosis cells increased from 1.45%(0?M)to 21.25%(30?M)in 143 B cells,from 3.03%(0?M)to 50.75%(30?M)in MG63 cells,and from 8.37%(0?M)to 16.85%(30?M)in Saos-2 cells.After DMAMCL treatment for 24 hours,we observed the increased karyopyknosis and karyorrhexis which were shown by Hoechst 33342 staining,significantly increased activity of Caspase3/7 by Gaspase-Glo 3/7 Assay,increased expression of cleaved-PARP,increased expressions of Bcl-2 family members Bax and Bak and decreased expression of Bcl-2 and Mcl-1.In vivo,compared with the control group,the number of TUNEL positive cells and the protein expression level of cleaved-PARP were significantly increased in osteosarcoma treated with DMAMCL.7.Bax mediated the osteosarcoma cell death induced by DMAMCL.As a key effector of Bcl-2 family members in the process of cell apoptosis,Bax expression significantly increased in DMAMCL treated OS cells.In order to further determine whether Bax is the target gene of DMAMCL,Bax si RNAs were transfected into osteosarcoma cells(143B and MG63)to down regulate Bax expression.The results showed that Bax siRNAs(#1,#2)transfected in 143 B cells and MG63 cells blocked the increase expression of Bax induced by DMAMCL.The MTS assay showed that compared to the DMAMCL-treated group,cell survival of DMAMCL+Bax-siRNA-treated groups significantly increased: 143 B cells(DMAMCL-treated group: 19.85%,DMAMCL+Bax-siRNA#1-treated group: 46.45%,DMAMCL+Bax-siRNA#2-treated group: 28.76%)and MG63 cells(DMAMCL-treated group: 37.12%,DMAMCL+Bax-siRNA#1-treated group: 50.44%,DMAMCL+Bax-siRNA#2-treated group: 53.04%).These data indicated that down-regulation of Bax attenuated the DMAMCL-induced cell death in OS cells.Conclusions: 1.DMAMCL induces dose dependent cell death of osteosarcoma which was more sensitive than non-tumor cells,inhibits cell cycle of osteosarcoma leading to G2/M arrest and inhibits stemness of osteosarcoma cells.2.DMAMCL,as a single drug,can inhibit the tumor growth of osteosarcoma xenograft model on mice and prolong the survival time of tumor bearing mice.3.DMAMCL induces osteosarcoma cell death by inducing apoptosis in vitro and in vivo.4.Bax mediates the osteosarcoma cell death induced by DMAMCL.