| Osteosarcoma(OS)is the most common primary malignant bone tumor,with an annual incidence of 0.2-3 per 100,000 children and 0.8-11 per 100,000 adolescents.Incidence peaks in the second decade of life.Osteosarcoma can attack on the surface of the bone,in or outside the bone,often with pulmonary metastases.Only 20% of patients had clinically detectable metastases,but the remaining 80% had undetectable micrometastases that were unknown at the time of diagnosis.Treatment of osteosarcoma includes surgical resection,preoperative and postoperative neoadjuvant chemotherapy(cisplatin,methotrexate,adriamycin),and intensive chemotherapy(ifosfamide and etoposide)in patients at high risk of metastasis.Neoadjuvant chemotherapy increased the 5-year survival rate of patients with osteosarcoma to about70 percent.However,the side effects of chemotherapy,resistance and tumor metastasis limit the clinical treatment of patients with osteosarcoma.Therefore,it is necessary to explore new bioactive molecules and therapeutic options to improve the therapeutic effect of osteosarcoma.Proscillaridin A is a kind of natural cardiac glycoside extracted from Urginea maritima and is often used to treat heart failure.Recent studies have found that Proscillaridin A is effective in many malignancies such as non-small cell lung cancer and glioblastoma,both in vivo and in vitro.Proscillaridin A has a variety of antitumor phenotypes,including anti-proliferation,inducing apoptosis,blocking cell cycle(such as G2/M block),promoting oxidative stress,and downregulation the expression of proto-oncogene MYC.However,the efficacy and mechanism of Proscillaridin A in the treatment of osteosarcoma remain unclear.In this study,we evaluated the antiosteosarcoma effect of Proscillaridin A in vivo and in vitro and its potential anti-tumor mechanism.In addition,we investigated whether Proscillaridin A could enhance the therapeutic effect of cisplatin on osteosarcoma by combining with the chemotherapy drug cisplatin and Proscillaridin A is a new therapeutic schedule for the treatment of osteosarcoma.Part-ⅠProscillaridin A inhibited the proliferation and invasion of osteosarcoma cells and promoted the apoptosis of osteosarcoma cells in vitro Objectives : Chemotherapeutic resistance and tumor metastasis impede the effectiveness of treatment for osteosarcoma,leading to poor prognosis.Proscillaridin A is a natural compound extracted from Urginea maritima,and is a kind of cardiac glycoside.It has been proved to have anti-proliferation activity in various malignant tumors,but the efficacy of this drug in the treatment of osteosarcoma remains unclear.In this section,we evaluated the effect of Proscillaridin A on osteosarcoma and explored its potential mechanism.Methods : The CCK-8 cytotoxicity assay was used to detect the effects of Proscillaridin A at different concentrations on the viability of human osteosarcoma cell lines 143 B,MG63 and human osteoblast cell line hFOB1.19.The effects of different concentrations of Proscillaridin A on the long-term proliferation of human osteosarcoma cells 143 B and MG63 were determined by plate cloning assay.Hoechst＿33258 staining and flow cytometry Annexin v-fitc /PI double staining were used to detect the effect of Proscillaridin A on the apoptosis morphology and apoptosis rate of osteosarcoma cells.Transwell invasion assay was performed to determine the effect of sarcomas A on the invasion ability of osteosarcoma cells.RT-PCR and Western blotting were used to determine the effects of Proscillaridin A on mRNA and protein expression levels of apoptosis-related protein Bcl-xl and matrix metalloproteinase MMP2.Results:1.The proliferation activity of human osteosarcoma cell lines 143 B and MG63 was reduced by Proscillaridin A,and the cell activity of human osteoblast cell line hFOB1.19 was less affected.In addition,Proscillaridin A decreased the number of plate clone spheres of human osteosarcoma cell lines 143 B and MG63.2.Proscillaridin A could make the nuclei of human osteosarcoma cell lines 143B and MG63 shrink and break,producing apoptotic bodies.Moreover,with the increase of concentration,the apoptosis rate of 143 B and MG63 cells increased.3.Transwell invasion experiment showed that the number of membrane penetrating cells in 143 B and MG63 cells decreased after treatment with Proscillaridin A, confirming that Proscillaridin A inhibited the invasion ability of osteosarcoma cells.4.RT-PCR and Western blot assay showed that Proscillaridin A inhibited the mRNA and protein expression levels of Bcl-xl and MMP2 in osteosarcoma cells.Conclusions: Proscillaridin A could inhibit the proliferation of osteosarcoma cells,but has little effect on normal osteoblast cells.Proscillaridin A could induce the apoptosis of osteosarcoma cells and inhibit their invasion ability.The molecular mechanism may be by down-regulating the expression of anti-apoptotic protein Bcl-xl and matrix metalloproteinase MMP2.These data confirmed the anti-osteosarcoma activity of Proscillaridin A.Part-Ⅱ Proscillaridin A inhibited the proliferation and invasion of osteosarcoma cells and promoted the apoptosis of osteosarcoma cells in vivo Objectives: The effect of natural products on the body and tumor is a complex biological process.In order to study the effects of Proscillaridin A on tumor and organism in vivo,this chapter of this project will establish a mouse tumor transplant model to detect the effect and mechanism of Proscillaridin A on tumor growth in vivo,and to detect the effect of various organs and functions.These results provide experimental basis for the treatment of osteosarcoma with Proscillaridin A.Methods: To establish the mouse xenograft model,osteosarcoma cells 143 B were transplanted subcutaneously.After culturing one week,1 mg/kg,2 mg/kg,4 mg/kg of Proscillaridin A or normal saline was intraperitoneally injected into the mice,once a day,and the weight and tumor size of the mice were recorded weekly.Mice were sacrificed after 4 weeks of treatment with Proscillaridin A and tumor tissues were taken.The expression levels of anti-apoptosis-related protein bcl-xl and matrix metalloproteinase MMP2 in osteosarcoma tissues were detected by immunohistochemistry.HE staining was used to detect the effects of Proscillaridin A on heart,liver,lung and kidney of mice.The effect of Proscillaridin A on the blood system was detected by serum test and blood count.Results:1.The growth of osteosarcoma could be inhibited by different concentrations of Proscillaridin A,and the degree of proliferation inhibition of osteosarcoma increased with the increase of drug concentration.2.HE staining showed that none of the Proscillaridin A treated mice had lung metastasis of osteosarcoma,and 2 of the 8 mice in the saline group had lung metastasis.3.Immunohistochemistry showed that Proscillaridin A inhibited the mRNA and protein expression levels of Bcl-xl and MMP2 in osteosarcoma cells.4.There was no significant difference in body weight of mice at different concentrations of Proscillaridin A.HE staining showed no significant damage to heart,liver and kidney.The serological index showed that the Proscillaridin A had no effect on the liver and kidney function of mice.The blood count showed that protothalonin A was not toxic to the blood system.Conclusions: Proscillaridin A can inhibit the growth and metastasis of osteosarcoma in vivo,and its mechanism may be mediated by down-regulating the expression of antiapoptosis-related protein Bcl-xl and matrix metalloproteinase MMP2.4 mg/kg/day of Proscillaridin A had no significant effect on body weight,heart,liver,kidney and blood system in mice,which provided experimental basis for the treatment of osteosarcoma with Proscillaridin A.Part-Ⅲ Experimental study on the inhibition of proliferation and stem cell characteristics of osteosarcoma cells and the promotion of apoptosis of osteosarcoma cells by combination of Proscillaridin A and cisplatin Objectives: Cisplatin(CDDP)has been shown to be an effective antitumor agent and is a first-line drug in the treatment of osteosarcoma.However,the side effects of cisplatin and the resistance of osteosarcoma to cisplatin limit its therapeutic application.It is necessary to study how to reduce the toxicity of cisplatin and increase the killing ability of tumor to improve the quality of life and therapeutic effect of patients with osteosarcoma.In this study,we elucidated the effect of Proscillaridin A combined with cisplatin on the treatment of osteosarcoma and explored its potential mechanism.Methods: Osteosarcoma cell line 143 B was treated alone or in combination with Proscillaridin A and cisplatin,and CCK-8 cytotoxicity assay was used to detect the effects of different treatments on the viability of human osteosarcoma cell line 143 B.Hoechst＿33258 staining and flow cytometry Annexin V-FITC /PI double staining were used to detect the effects of different treatments on the apoptosis morphology and apoptosis rate of osteosarcoma cells.The effect of different treatments on the stem cell potential of osteosarcoma cells was detected by the suspension clone ball test.RT-PCR and Western blot were used to detect the effects of different treatments on mRNA and protein expression levels of drug-resistant proteins T-gp and telomere TERT..Results:1.Compared with the single dose,the combination of drugs inhibited the proliferation activity of human osteosarcoma cell line 143 B more obviously and had a synergistic effect,with less plate clone spheres formation.2.Compared with single dose,the apoptosis rate of 143 B cells was increased in combination group.3.The suspension clone ball experiment showed that 143 B formed fewer suspension clone balls after treatment with the combination drug,confirming that the combination drug could inhibit the stem cell characteristics of osteosarcoma cells.4.RT-PCR and Western blot assay showed that the expression levels of stem cell related gene TERT and drug resistance related gene P-gp mRNA and protein in osteosarcoma cells increased after low concentration and short time of cisplatin treatment.Compared with the cisplatin group,the expression levels of P-gp andTERT in the combination group were significantly reduced.Conclusions: The combination of Proscillaridin A and cisplatin has a synergistic effect,effectively reducing the dose of cisplatin,inhibiting the proliferation and stem cell characteristics of osteosarcoma cells,and inducing their apoptosis.The molecular mechanism may be mediated by down-regulating the expression of drug-resistant related proteins P-gp and telomerase TERT.The combination of Proscillaridin A and cisplatin may be an effective treatment for osteosarcoma. |
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