| BackgroundOsteosarcoma has long been the third tumor threaten in both children and adults and shown an undesirably yearly increasing tendency.Even pulmonary metastasis is the chief factor to tumor associated mortality,however,promotion by routine scheme exhibits little efficacy.Moreover,mechanisms underlying tumorigenesis needs to be deeply expounded,further promoting clinical diagnosis as well as innovative,personalized therapy.ObjectivesTo explore the variation of profile between primary and metastatic diseases on the basis of clinical derived osteosarcoma tissue and further identify collapsed signaling pathways linked to clinical phenotype.To screen structure-based ginsenoside-Rh2 pharmacological targets as well as mechanisms involved with proliferation,apoptosis and migration.Methods1.Bioinformatics tools(Pharm Mapper,STRING)were applied to discover ginsenoside-Rh2 targeting biomolecules and extract interactive network along with osteosarcoma tumorigenesis.2.Crystal violet staining and MTT assay,with an arrangement of concentration gradient of Rh2,were designed to measure viabilities of cell lines 143 B and MG63.3.JC-1 staining assay was employed to detect transform of mitochondrial membrane potential stimulated by Rh2 at indicated time point.4.Wound healing and Transwell assay were utilized to monitor relative rate of cells migration with a lower dosage of Rh2.5.Western blot was used to measure expression levels of biomarkers associated with proliferation,apoptosis and P38/p-P38 signaling pathway.6.Limma was engaged with differentiation of expression profiles between primary and metastatic subgroups and GSEA,PCA analysis were designed to molecular classification.Results1.Summarized from reverse matching model of Pharm Mapper,there are total 152 targets,within which KIF11 and dihydroorotate dehydrogenase(DHODH)showed significance with respect of both of z' test and normalized fit score higher than 0.9.In addition,when cut off value of confidence equals 0.04,almost all of nodes contributed to an interactive network with P< 1.0e-16.2.Crystal violet staining showed that pigment intensity of both 143 B and MG63 declined via the increase of Rh2 concentration.Within 143 B,suppression of cell proliferation only depended on drug concentration but had no dependency with time course.Among subgroups of MG63,inhibitory effect of Rh2 exhibited a time and concentration related transformation(P<0.05).Besides,IC50 of both cells were approximately 53.05 and 54.16?M,respectively.3.Relative proportion of green fluorescence augmented after treatment with Rh2 gradient whereas intensity of red fluorescence showed a highes t degree in control.Wound healing and Transwell assays showed an inhibitory behavior on both 143 B and MG63 cells comparing to control group(P<0.05).4.Results of Western blot indicated that expression of apoptotic markers,such as cleaved-capase-3/8/9/PARP,increased whereas proliferative molecule PCNA declined.Moreover,level of p-P38 also upregulated with stimulation of Rh2.5.Differential profile according to Limma depicted 913 proceeding involved genes(log|FC|>1,P<0.05).Enrichment with GSEA showed abnormal activation of Chemokine pathway(ES=0.42,P<0.05)among metastatic tissue whereas in primary group,Alzheimer's disease and Huntington disease(FDR<0.25,P<0.01)were significantly enriched.PCA analysis displayed a variation of 35.6% on PC1,relative 12.5% on PC2.Conclusions1.Ginsenoside-Rh2 could affect cellular molecules on the basis of a structural model as well as intervene processing of osteosarcoma.2.Ginsenoside-Rh2 acts as a suppressor of osteosarcoma proliferation and migration as well as a promoter of early apoptosis.3.Blocking of malignancy duration by Ginsenoside-Rh2 might be achieved by stimulating of P38/p-P38 signaling.4.Variation of signaling transduction following differential expression enrichment concurrent with advancing of disease could facilitate clinic al phenotyping and personalized therapy. |
PDF Full Text Request