| Objective:Osteosarcoma is a kind of malignant primary bone tumors,see more at 13-19 years old teenage children,have progressed fast,easy to lung metastasis and poor prognosis,men than women are more likely to come on,the current clinical mainly uses the neoadjuvant chemotherapy combined surgery,radiation therapy,targeted therapy,gene therapy,and immune therapy in the treatment of osteosarcoma,such methods as curative effect quickly,the effect is remarkable.However,while the above methods are effective and rapid in the treatment of osteosarcoma,their limitations are increasingly exposed,so it is necessary to find a more safe and effective treatment for osteosarcoma.Andrographolide(AG),the main component of andrographolide,has been shown to have antitumor effects in recent years.However,there are few reports that AG can affect osteosarcoma.Sphingolipid metabolism is an important lipid metabolism pathway in human body,which can affect a variety of biological activities in human body.Although osteosarcoma has been shown to be associated with sphingolipid metabolism,no effect of AG on sphingolipid metabolism has been reported.Our study was to explore the AG influence on bone sarcoma HOS and U2 OS cells,and further explore the specific effect of AG on the bone sarcoma cells and the mechanism of action,thus revealing the AG may affect bone sarcoma cell signaling pathways,and AG in osteosarcoma may target,osteosarcoma provide new options for clinical treatment and new ideas.Methods:Normal cultured human osteosarcoma cells were the control group(AG concentration was 0),and human osteosarcoma cells containing 5,10,20,50,and 100μM AG were the experimental group.Cell density was counted and survival rate of human osteosarcoma cells was calculated(cell density in the experimental group/cell density in the control group ×100%).The effect of AG on the proliferation ability of each group was detected by plate cloning experiment.Transwell chamber experiment examined the effect of AG on invasion and migration ability of each group.The effect of AG on cell cycle distribution in each group was detected by flow cytometry.Western blot assay was used to detect the effect of AG on the expression levels of sphingosinol kinase 1(SphK1)and adhesion spot kinase(FAK)in each group.Results:(1)On the premise of the same AG action time,compared with the control group,the survival rate of human osteosarcoma cells in each experimental group decreased significantly(P<0.01),and the results showed concentration dependence.At the same time,under the premise of the same AG concentration,the longer the AG action time,the lower the survival rate of human osteosarcoma cells,the results showed a time-dependent.(2)When the AG concentrations were 0,5,10,20,50,and 100,the population number of HOS cells in human osteosarcoma was 33.33±2.52,25.00±2.00,21.67±2.08,14.67±2.08,5.67±1.53,6.00±1.00,and the population number of human osteosarcoma U2 OS cells was 53.33±1.53,33.67±1.53,26.33±1.53,23.67± 2.53,14.67±2.52,13.00±2.65,respectively.It was suggested that the higher the AG concentration,the less the colony number of human osteosarcoma cells,the differences were statistically significant(P < 0.01),and the results showed a concentration dependence.(3)The relative invasion rates of HOS cells in the control group and the experimental group were 1.00±0.00 and 0.40±0.03,respectively;the relative invasion rates of U2 OS cells in the control group and the experimental group were 1.00±0.00 and 0.24±0.02,respectively.Compared with the control group,the invasion rates of human osteosarcoma cells in the experimental group were significantly reduced,and the differences were statistically significant(P<0.05).(4)The relative migration rates of HOS cells in the control group and the experimental group were 1.00±0.00 and 0.22±0.02,respectively;the relative migration rates of U2 OS cells in the control group and the experimental group were 1.00±0.00 and 0.14±0.01,respectively.Compared with the control group,the cell cycle distribution of human osteosarcoma cells in each experimental group showed significant changes.(5)In the HOS cell line,with the change of AG concentration,the proportion of G1 phase increased,the proportion of S phase did not change significantly,and the proportion of G2 phase decreased,with statistically significant differences(P<0.01).In U2 OS cell lines,with the change of AG concentration,the proportion of G1 phase increased,the proportion of S phase decreased,and the proportion of G2 phase decreased,the differences were statistically significant(P<0.01).(6)In HOS cells,the relative expression levels of SphK1 in the control group and the experimental group were 1.00±0.00 and 0.81±0.02,respectively.In U2 OS cells,the relative expression levels of SphK1 in the control group and the experimental group were 1.00±0.00 and 0.83±0.02,respectively.Compared with the control group,the expression levels of SphK1 in human osteosarcoma cells in the experimental group were significantly reduced,and the differences were statistically significant(P<0.05).(7)In HOS cells,the relative expression levels of FAK in the control group and the experimental group were 1.00±0.00 and 0.14±0.02,respectively.In U2 OS cells,the relative expression levels of FAK in the control group and the experimental group were 1.00±0.00 and 0.21±0.01,respectively.Compared with the control group,the expression levels of FAK in human osteosarcoma cells in the experimental group were significantly reduced,and the differences were statistically significant(P<0.05).Conclusion:1.AG has a killing effect on both HOS and U2 OS cells in human osteosarcoma,which is time-dependent and concentration-dependent.Specifically,AG can inhibit the proliferation,invasion and migration of HOS and U2 OS cells in human osteosarcoma.2.AG can change the cycle distribution of HOS and U2 OS cells in human osteosarcoma and increase the G1 phase ratio,which further confirms that AG can inhibit the proliferation of HOS and U2 OS cells in human osteosarcoma.3.AG can reduce the expression levels of SphK1 and FAK in HOS and U2 OS cells,affect spingolipid metabolism in HOS and U2 OS cells,and inhibit SphK1 pathway,which may be related to the killing effect of AG on HOS and U2 OS cells. |
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