The Anti-tumor Effect (Including Cell Proliferation Apoptosis And Metastasis) Of Dihydroarteminin On Human-osteasarcoma And The Mechanism

PurposesArtemisinin is an extremely effective anti-malarial drug. Efficiencyand low toxicity anti-malarial effect, in addition to widely used to treatmalaria, more studies have shown that artemisinin also had significantanti-tumor effects. In this study, the active form of artemisinin: thedihydrotestosterone of artemisinin (Dihydroarteminin, DHA) as theresearch object, the effects on human osteosarcoma, and to explore thepossible molecular mechanism.Experimental methods and proceduresIn vitro, cultured human osteosarcoma cell line (MG63, U2OS,143B,and Saos2), the intervention of different concentrations of DHA onosteosarcoma were24and48h respectively, and then select the morecancerous and the more obvious characteristics of lung metastasis andappropriate range of DHA for follow-up experiments. Experimental groups:Control group, solvent group (0.1%DMSO), the experimental group (high,medium, low concentrations of DHA sequentially at15μM, were treatedwith25μM and35μM). The main experimental steps are as follows:1. In vitro, cultured human osteosarcoma cell line (MG63, U2OS, 143B, and Saos2), crystal violet staining and MTT (MTT) colorimetric todetect the discrepancy in cell proliferation, before and after the interventionDHA, qualitative and quantitative; colony formation-assay to detect theinfluence of DHA on human osteosarcoma in the ability of cellcolony-forming, crystal violet staining indicates the number ofcolony-forming, and calculate the colony forming efficiency. Screeningmore sensitive cell lines and the sensitive drug concentration range.Western blotting to detect differential expression of protein markers (suchas PCNA, cyclin D1, c-Myc) that are closely related to tumor cellproliferation.2. In vitro, Hoechst33258apoptosis staining and flow cytometry(FCM), before and after the treatment of DHA, observe the differentialexpression of human osteosarcoma cell cycles, apoptotic cellmorphological change. Western blotting analyzed these tumor markers ofapoptosis-related proteins (such as Bad, Bcl-2, cleaved-Caspase-3)differentially expressed.3. In Vitro, cultured human osteosarcoma cells143B, wound healingassay, Transwell chamber shuttle the experimental and Transwellartificial-matrigel shuttle the analog assay DHA treated humanosteosarcoma cell invasion and metastasis ability to change. Westernblotting was used to detect tumor metastasis invasion-related proteinmarkers (VEGF and MMP-9) expression differences.4. Wnt/β-catenin signaling pathway plays a non-negligible effect intumorigenesis and development. Therefore, by constructing β-cateninluciferase reporter gene (β-catenin-Luc, p-Top), before and after DHAtreatment, β-catenin activity intracellular detected. Differential expressionof critical regulator (such as GSK-3β, p-GSK-3β, β-catenin, Axin, the Dvl and E-Cadherin, and target genes COX-2, Cyclin D1and c-Myc) inβ-catenin signaling pathway by Western blotting.5. Construction of a mouse model of human osteosarcoma, oraladministration, observation of DHA in the body of the impact on humanosteosarcoma.Results1. The four strains of human osteosarcoma cells (MG63, U2OS,143B,and Saos2) performance is sensitive to DHA, the range of concentration forDHA at10~40μM treatment, proliferative activity decreased significantlyand cloning capabilities inhibited, and drug intervention effect increaseswith the concentration and the extension of time trend.143B cells moresensitive to performance, compared with the others, and the highest degreeof malignancy, prone to lung metastasis. Therefore, the143B cells choicedand the concentration range of10-40μM for subsequent experimental study.Inhibition of tumor proliferation-associated protein markers expression wasobvious, and a significant difference (P <0.05) can be seen in theexperimental group and the control.2. In accordance with the experimental groups, treatment of DHA,Hoechst33258staining and flow cytometry (FCM). The FCM results showthat DHA treatment compared with the control group, the percentage ofapoptotic cells was significantly increased up to67%; Hoechst33258fluorescence staining showed chromatin condensation, nucleusfragmentation showed typical apoptotic body, reducing the cell body.Biochemical Characteristics: chromatin degradation, nucleosomal DNAconnection between parts is degraded, to produce oligomer nucleosomalDNA fragment, i.e.,180-200bp lengths, integer multiple DNA fragments.Markers of tumor apoptosis-related protein expression increased up to56%, compared with the control group.3. Wound healing assay and artificial matrigel experimental resultsshow that DHA significantly reduced tumor cell metastasis and invasion.Secreted by the tumor cells themselves destroy tissue basement membraneand matrix metalloproteinases (MMP) was significantly inhibited, to adjustthe tumor metastasis ectopic generated vascular endothelial growth factor(VEGF) expression was significantly reduced. Thus, DHA is effective toinhibit the metastasis of tumor cells and ectopic angiogenesis.4. Of all these pathways, with a variety of tumor occurrence anddevelopment of related signaling pathways doctrine, more thorough studyof the mechanism, the Wnt/β-catenin signaling pathway, a key regulatoryfactor signaling pathways by comparing DHA before and after theintervention differentially expressed, the results showed that theWnt/β-catenin signaling pathway of tumor positive regulation of proteinfactors have varying degrees of suppression of expression; tumorprogression and negative regulation of protein factors, to varying degrees,increased expression. It is speculated that Wnt/β-catenin signaling pathwayplay an important regulatory role cannot be ignored in the development ofDHA interfering with human osteosarcoma.5. In vitro, study showed that DHA showed a significant and effectiveinterference for osteosarcoma. Therefore, the subject of build humanosteosarcoma xenograft models in mice by oral administration to observethe impact of solid tumors. Histopathological analysis showed that thehuman osteosarcoma mouse xenograft model, tumor tissue is typical bonetumor morphology performance. Lung tissue section pathological analysisshowed that the experimental group of mice did not occur visible lungmetastases, while the control group of mice model visible metastases confirmed osteosarcoma. The curve of tumor volume, tumor volumegrowth rate of the experimental group than in the control group wassignificantly inhibited.ConclusionsDihydroartemisinin, except as an effective anti-malarial drug, it alsohas significant anti-tumor effect. Significantly inhibited cervical cancer,colorectal cancer, this research project, it has significantly inhibit theproliferation and metastasis of human osteosarcoma cell invasion andpromote the biological effects of tumor cell apoptosis. Wnt/β-cateninsignaling pathway plays an important regulator in DHA treatment withhuman osteosarcoma.