| [Background]Osteosarcoma(Osteosarcoma,OS)is a primary malignant tumor derived directly from the skeletal system,characterized by tumor cells as immature or bone-like tissue.Typical osteosarcoma is a rare(0.2%of all malignant tumors)highly malignant tumors,with an estimated 3 cases per million per year.Osteosarcoma mainly occurs in the long bone,rarely appear in the soft tissue.Corresponding to the rapid development of skeletal system,the incidence reached its peak.Although modern multimodal therapy has significantly improved tumor resorption and long-term efficacy,25-50%of patients initially without metastasis,follow-up metastasis,which is still the main cause of death.Despite taking aggressive combined chemotherapy and surgery,more than 30%of patients develop lung metastases.The neoadjuvant chemotherapy drugs in the 1980s have greatly improved the 5-year survival rate of patients with osteosarcoma,but no further progress has been made since the treatment of osteosarcoma treatment or prognosis improvement.In patients with osteosarcoma in the lung and other parts of the distant metastasis,the long-term survival rate is still low,only 10-30%.At present,large doses of conventional chemotherapy drugs side effects,therefore,the new treatment goals should focus on exploring the search for safe and effective chemotherapy drugs.It is noteworthy that the number of cancer patients and cost of treatment are increasing,which has become a huge financial burden to the government.The cost of anticancer treatment in developed countries is spiraling,and its impact on the economy is increasingly becoming an event closely related to national health services.National public health systems strongly require effective and inexpensive anti-cancer strategies.In this regard,it is an efficient,realistic and inexpensive strategy to explore the "reuse" of drugs for cancer therapy,that is,the search for new therapeutic indications with existing drugs.Gene amplification,deletion,displacement and other mutations will cause tumorigenesis,bone tissue proliferation and differentiation abnormalities caused by genomic instability,will lead to the occurrence of osteosarcoma.Studies have confirmed that the occurrence of osteosarcoma and genome mutations are relevant.Changes in epigenetics such as DNA methylation,histone modification,chromosome recombination.have also been shown to play an important role in the development of tumors(including osteosarcoma).In Jaenisch et al,DNA methylation plays an important role in the development and progression of various cancers.Gonzalo and other studies have shown that the genetic changes that cause cancer are mainly methylation of tumor suppressor genes and low methylation of oncogenes.OS is characterized by frequent genomic instability,high heterogeneous karyotype,gene expression changes and recurrent epigenetic changes.Methylation of various genes has been demonstrated to be closely related to clinical indicators such as staging,grade,metastasis and prognosis of osteosarcoma.S-Adenosylmethionine(SAM)is an important molecule in biochemistry that donates their methyl group for many biochemical reactions and act as a precursor for forming higher polyamines.The polyamine helps to condense and stabilize DNA that controls the normal function of cells.It has been reported that SAM has an anti-cancer property and it is used as a therapeutic agent to treat cancer.Parashar found that SAM can inhibit the proliferation of osteosarcoma cells by slowing the cell cycle progression and by inducing apoptosis.However,the study of its molecular mechanism has just begun.SAM has the advantage of tumor therapy in that it is a naturally occurring molecule that is synthesized in the human body by methionine adenosine transferase isoenzyme and is FDA approved for nutritional supplements at low cost and very safe.Sox2 is also a key factor in maintaining the characteristics of osteosarcoma stem cells.Basu-Roy and other studies have found that in human and mouse osteosarcoma cell lines Sox2 showed high expression,through the endogenous Sox2 gene knockout or by RNA interference Sox2 gene expression,osteosarcoma cell invasion in vitro And transfer were significantly reduced.This means that Sox2 has the effect of promoting the occurrence and metastasis of osteosarcoma.The Wnt signaling pathway regulates gene transcription through the binding of ?-catenin and Tcf/Lef family,which is the cytosolic stability of ?-catenin.When the intracellular ?-catenin levels decreased,Wnt pathway closed;when ?-catenin levels increased,Wnt pathway open.Wnt/?-catenin signaling pathway plays an important role in the osteogenesis and osteogenesis of osteocytes,and its activation is one of the mechanisms that cause tumorigenesis and development.Studies have shown that activation of the Wnt/?-catenin signaling pathway promotes osteoblast hyperproliferation and inhibits the role of osteoclasts leading to osteosarcoma.Sox2 is a transcription factor belonging to the high mobility group HMG family,which plays a decisive role in the maintenance of pluripotency and self-renewal for embryonic stem cells.[Objectives]1?To observe the expression of Sox2 and ?-catenin in human osteosarcoma tissue,and to clarify the relationship between Sox2??-catenin protein and clinical features of osteosarcoma patients.2?To observe the effect of SAM on the activity of osteosarcoma cells.To investigate the regulatory effect of SAM on Sox2 and Wnt/?-catenin signaling pathway,and to explore the mechanism of SAM on osteosarcoma cell activity.3?To provide theoretical reference for the treatment of SAM to osteosarcoma.[Methods]According to the purpose of the study,this experiment is divided into the following three parts:1?Immunohistochemistry and Western blot were used to detect the expression of Sox2 and ?-catenin in human osteosarcoma tissue.The relationship between the expression of Sox2??-catenin and the clinicopathological parameters in osteosarcoma tissue was analyzed.To study the effect of Sox2 and ?-catenin on the occurrence and metastasis of osteosarcoma and its mechanism organizational evidence.2?The effect of SAM on the proliferation of human osteosarcoma U20S cells was detected by MTT assay.The apoptosis of U20S cells was detected by flow cytometry.The activity of Caspase-3,8,9 was detected by the Caspase Activity Assay Kit.Cell scratches were used to detect the migration of osteosarcoma cells.Western blot and real-time quantitative PCR were used to detect the expression of Sox2,?-catenin and C-myc.To observe the effect of SAM on the proliferation,apoptosis and migration of human osteosarcoma U20S cells,and to explore the mechanism of SAM in human osteosarcoma cells.3?The osteosarcoma model of nude mice was established with osteosarcoma K7M2 cell line.The histological features were observed by HE staining.The effect of SAM on the proliferation of tumor cells was observed and the safety was analyzed.The expression of apoptosis-related proteins Bax and Bcl-2 were detected by Western blot.Western blot and real-time quantitative PCR were used to detect the expression of Sox2,P-catenin and C-myc.The possible mechanism of SAM was investigated in vivo.[Results]1?The positive rate of Sox2 and ?-catenin in osteosarcoma tissues was 80.77%and 75.0%,respectively,which was significantly higher than that in osteochondroma tissues(P<0.05).The positive expression rates of Sox2 and ?-catenin were significantly different between different Enneking clinical stages(P<0.05).The positive rates of Sox2 and ?-catenin were significantly different between the group with metastasis(94.74%and 78.95%)and those without metastasis(72.73%and 51.51%)(P<0.05).The expression of Sox2 and ?-catenin was not correlated with age,sex,lesion and pathological type(P>0.05).2.The results of in vitro experiments showed that the OD value of the experimental group was significantly higher than that of the control group(P<0.05),with the increase of SAM dose(50,100,200,400?M)and the prolongation of time(24,48,72 hours),SAM can significantly inhibit the proliferation of U20S cells in a dose-dependent and time-dependent manner.Flow cytometry showed that 200?M SAM was used in U20S cells 24 and 48 of osteosarcoma,and the apoptotic rate increased gradually with the prolongation of time(P<0.05).U20S cells treated with 48 hours apoptosis rate was significantly higher than that of 24 hours,so SAM could induce apoptosis of human osteosarcoma U20S cells,and there was time-dependent.In the scoring experiment,SAM was treated for 48 hours.Compared with the control group,the distance between the experimental group was significantly increased(P<0.01)with the increase of SAM(50,100,200,400?M).SAM significantly reduced the ability of U20S osteosarcoma cells to migrate.50,100 ?M SAM was administered to U20S cells for 24 hours,48 hours,and the activity of Caspase-8,Caspase-9 and Caspase-3 was not significantly enhanced(P>0.05)compared with the control group,There was no significant increase in activity of the three enzymes with time and drug concentration.200?M SAM acted on human osteosarcoma U20S cells for 24 hours,compared with the control group,the activities of Caspase-8,Caspase-9 and Caspase-3 were significantly increased(P<0.05).The activity of the enzyme was not significantly increased at 48 hours compared with 24 hours(P>0.05).The activity of the enzyme was not significantly increased at 48 hours compared with 24 hours(P>0.05).400 ?M SAM was administered to U20S cells for 24 hours,the activity of Caspase-3,Caspase-8 and Caspase-9 was significantly enhanced(P<0.05)compared with the control group and 200?M group.The enzyme activity was not significantly increased(P>0.05)between the group of drug acted for 48 hours compared with 24 hours.The expression of protein and mRNA levels of Sox2,?-catenin and C-myc in the osteosarcoma U20S cells were detected by Western blot and real-time fluorescence quantitative PCR after 48 hours of SAM treatment.Compared with the control group,the expression level of the experimental group was significantly decreased(P<0.05).3.In vivo experiments,the appearance and histological examination confirmed that K7M2 cells successfully established nude mice osteosarcoma model.Compared with the control group,SAM can significantly inhibit tumor growth.At the same time,nude mice no significant decline in body weight,indicating that SAM has obvious anti-tumor effect and good safety.The expression of Bcl-2 protein was down-regulated in SAM group,and Bax showed high expression.Compared with the control group,the difference was statistically significant(P<0.05).The expression of Sox2,?-catenin and C-myc in the SAM treatment group were significantly lower than those in the control group(P<0.05),regardless of protein level or mRNA level.Indicating that SAM may down-regulate the expression of Sox2 and reduce the activity of Wnt/?-catenin signaling pathway in the treatment of osteosarcoma.[Results Conclusion]1?The expression of Sox2 and ?-catenin in human osteosarcoma increased,and the expression level was closely related to the clinical stage and distant metastasis of osteosarcoma.2?SAM has significant anti-proliferative and inhibition of migration effect on osteosa-rcoma U20S cells line.lt can induce apoptosis of osteosarcoma cell by enhancing the activity of Caspase-3,8,9.3?SAM may play a therapeutic role through the reducing of the expression of Sox2and the Wnt/?-catenin signaling pathway of osteosarcoma U20S cells line.5?SAM provides a novel theory and a new methodon human osteosarcoma treatment clinically. |
PDF Full Text Request