Cinobufagin Induces Apoptosis Of Osteosarcoma Cells Through Inactivation Of Notch Signaling

Osteosarcoma,also known as osteogenic sarcoma,is a primary malignant tumor of bone.The incidence of osteosarcoma in primary bone tumors is second only to plasma cell myeloma,and its histological features are in most cases Tumor cells produce bone-like or immature bones.In some cases these are very immature,so it is difficult to distinguish between bone-like tissue or tissue.It Often need other methods to clarify tumor classification,for example,the use of histochemical methods to determine the level of alkaline phosphatase in the tumor;tissue immunology and other methods.Osteosarcoma is highly malignant,prone to drug resistance and early distant metastasis,of which the most common lung metastasis,mortality and morbidity are higher.Induction of tumor cell apoptosis is an important goal of treatment,in recent years,many scholars look to find new drugs to seek better treatment.Cinobufacini is the skin of the Chinese Bufo gargarian made by hydration extraction and made of traditional Chinese medicine preparation,with detoxification,water swelling,blood stasis ulcer effect,its chemical composition is more complex,including indole alkaloids,reducing sugar,Amino acids and toad toxin,toad tryptamine,etc.,Cinobufacini as a traditional medicine of the motherland,which has a low toxicity,broad spectrum of anti-cancer and many other advantages,has become China’s clinical application of a wide range of tumor.The Cinobufagin(CB)is one of the major toxic ligands extracted from cinobufen and has a good antitumor effect.Previous studies have shown that cinobufagin can play an anti-tumor effect by inhibiting tumor cell proliferation and inducing apoptosis.The study found that its liver cancer,breast cancer,small cell lung cancer,colon cancer,prostate cancer and other malignant tumors have a good anti-cancer effect.However,the study of the application of toad venom to osteosarcoma is rare.In this study,we investigated the effects of the toxin on the proliferation and apoptosis of MG63,U20S,143B cells in human three kinds of osteosarcoma cell lines,and examined their effects on apoptosis-related mRNA and protein expression.The role of Notch in the apoptosis of osteosarcoma-induced osteosarcoma cells was studied by further studying the pathways of lentivirus transfection.The results were further studied in order to be more close to clinical practice,Persuasive results and conclusions,in order to clarify the anti-tumor mechanism of toad venom to provide a solid experimental basis and theoretical basis.Part1 Research of Cinobufagin inhibits proliferation and induces apoptosis in osteosarcoma cell lines and its possible mechanismObjeectives:To investigate the effects of Cinobufagin inhibits proliferation and induces apoptosis in osteosarcoma cell lines,MG63,U20S and 143B and the possible mechanism.Methods:Different concentrations of cinobufagin(0,20,50,100,150,200,250 nmol/L)were applied in osteosarcoma cell lines,MG63,U20S and 143B.CCK-8 assay was used to detect the expression of osteosarcoma cells in each group of osteosarcoma cells survival rate,Hoechst 33258 staining method was used to observe the apoptotic body from the morphology,Annexin V-FITC/PI double staining combined with flow cytometry to detect the apoptosis rate of osteosarcoma MG63,U20S and 143B cells,RT-PCR And Western Blot were used to detect apoptosis-related mRNA and proteins,and Notch signaling-related mRNA and protein were also observed.Results:The result of CCK-8 assay indicated that Cinobufagin significantly inhibited cell proliferation in a dose-and time-dependent manner.Marked changes in cell apoptosis was clearly observed after cinobufagin treatment.The results of flow cytometry showed that the apoptotic rate increased with the increase of the concentration of cinobufagin.The results of RT-PCR and Western Blot showed that the expression of bax(pro-apoptotic protein)increased gradually and the expression of bcl-2(anti-apoptotic protein)decreased gradually after 72 hours of cinobufacin treatment Bax/bcl-2 ratio was also significantly increased(P<0.05).The expression of Notchl,Hes-1,Hes-5 and Hey-L was negatively correlated with the time and concentration after Cinobufacini treatment.Conclusion:Cinobufagin can induce apoptosis of human osteosarcoma cells,and its effect is concentration and time-dependent and its mechanism may be related to inhibition of Notch signaling pathway.Part 2 Research on the inhibitory effect of Cinobufagin on the growth of osteosarcoma in vivo and its mechanismObjectives:The aim of this study was to investigate the inhibitory effect of Cinobufagin on the proliferation of osteosarcoma and the ability to induce apoptosis in nude mice,and to explore the clinical application of Cinobufagin.Methods:In this study,we used the 143B osteosarcoma cell line to construct the osteosarcoma xenograft model of mice by subcutaneous inoculation and to detect the effect of the Cinobufagin on the growth of osteosarcoma.Negative control group was treated with the same amount of PBS,the blank control group did not make any treatment(healthy mice).The experimental group was given intraperitoneal injection of 4 mg/kg of Cinobufagin for 1 week,twice daily.The experimental procedure was carried out in the SPF animal experiment,and then placed in the SPF animal experimental center feeding.To observe the general condition of nude mice,including the amount of activity,feeding,urine,color,until the tumor appears,every 5 days with a vernier caliper to measure the smallest diameter of the tumor block(Width)and the largest diameter(Length),1umor volume(mm3)is estimated as:(minimum short diameter)2×(maximum long diameter)/2.The tumor volume was calculated according to the formula and the weight was recorded.After the success of tumor formation,8 weeks after continuous observation,the nude mice were sacrificed and the tumor tissue was removed from the skin.The size of the tumor was measured with a vernier caliper.The tumor tissue was divided into three parts,one for RT-PCR,one for Western Blot and the other in 4%paraformaldehyde for 24 hours for the production of paraffin sections.Sections were used for subsequent HE staining and immunohistochemical staining(IHC).Results:1.In vivo experiments showed that the experimental group of osteosarcoma model mice treated with 4 mg/kg of Cinobufagin for 7 days(twice daily),compared with the control group(the same amount of PBS treatment),the tumor’s volume of experimental group of mice was significantly smaller than the control group.2.There was no significant difference between the experimental group and the healthy mice which without tumor burden.3.The expression of bax mRNA(pro-apoptotic gene)in the experimental group was significantly higher than that in the control group,and the expression of bcl-2 mRNA was significantly lower than that of the control group,and the expression of bax/bcl-2 The ratio was also significantly increased(P<0.05).4.The expression of Notch signaling,mRNA,NICD1,Hes-1,Hes-5 and Hey-L in the experimental group were significantly lower than those in the control group(P<0.05).5.The survival rate of the experimental group was higher than that of the control group.Conclusions:Studies have shown that the Cinobufagin can inhibit the growth of human osteosarcoma model mice,induce tumor cell apoptosis and improve the survival rate of tumor-bearing mice in vivo.Part 3 Effects of Notch Signaling Pathway on the Proliferation and Apoptosis of Osteosarcoma CellsObjectives:To explore the effect of Notch signaling pathway on the proliferation and apoptosis of osteosarcoma cells,the role of Notch signaling pathway and its regulatory mechanism were discussed.Methods:Construction of Notch signaling reporter vector lentivirus,screening stable transfected osteosarcoma cell lines,by RT-PCR and Western Blot to verify the effect of induced expression.CCK-8 assay was used to detect the cell viability of the osteosarcoma of different Notch expression levels.Flow cytometry was used to detect the apoptotic rate of the osteosarcoma of the different Notch expression levels.Results:1.Stably transfected osteosarcoma cell lines can achieve the desired effect,resulting in Notch activation and Notch closure of the subtype.2.The cell viability of the activated cell line was the highest,and the survival rate of Notch-closed cell line was the lowest,and the survival rate of routine Notch-expressing cell line was in the mediately.3.The apoptosis rate of Notch-activation cell lines was the highest,and the apoptotic rate of routine Notch expression was in the mediately.Conclusions:Notch signaling pathway is resistant to the toxic effects of the Cinobufagin,which is resistant to the apoptosis induced by the Cinobufagin,which shows more resistant properties.Notch-closed to promote the toxic effects of the Cinobufagin and to promote the induction of apoptosis of Cinobufagin,which represent a sensitizing effect.