A Study Of The Inhibitory Effect Of Ginsenoside Rg3 On Human Osteosarcoma Cells And Its Mechanism

Background:Osteosarcoma is a malignant tumor originated from mesenchymal stem cells.The onset age of osteosarcoma presents a bimodal distribution,often occurring in adolescents or the elderly people.Due to most patients with osteosarcoma have metastatic or micrometastatic disease at the time of diagnosis,almost all patients receive multiagent chemotherapy before or after surgical resection.Patients tend to have poor tolerance and a high rate of recurrence.Ginseng is a herb native to northeast Asia and widely used in traditional medicine in east Asia.As one of the main active substances in ginseng,ginsenoside Rg3 has attracted more and more attention in recent years for its anti-tumor effect.The steroidal structure of ginsenoside Rg3 allows it to interact with cell membranes,membrane-bound ion channels,and intracellular and extracellular receptors to alter the transcriptional level of cells and thus plays an anti-tumor role.Methods:The effects of ginsenoside Rg3 on proliferation,apoptosis,migration,invasion and cell cycle of osteosarcoma cell lines MG-63,HOS and U2OS were studied by MTT,flow cytometry,cell scratch test and Transwell cell invasion test.We further selected MG-63 cells as the object,and explored the specific mechanism of ginsenoside Rg3 inducing autophagy,apoptosis and cell cycle arrest by Western blot,quantitative PCR,flow cytometry and small interfering RNA.Results:MTT assay showed that ginsenoside Rg3 inhibited the proliferation of three osteosarcoma cell lines MG-63,HOS and U2OS in a dose-dependent manner.Flow cytometry confirmed that ginsenoside Rg3 promoted apoptosis of the three osteosarcoma cell lines.Scratch assay test and Transwell cell migration test showed that ginsenoside Rg3 could inhibit the migration and invasion of three osteosarcoma cell lines.Western blot showed that the level of LC3-II/LC3-I in osteosarcoma MG-63 treated with ginsenoside Rg3 was significantly higher than that in control group.Western blot and quantitative PCR confirmed the increase of Caspase-3 gene and protein expression in ginsenoside Rg3 group,and flow cytometry confirmed the increase of apoptotic level in ginsenoside Rg3 group.When we used the representative autophagy pathway inhibitor 3-MA and ginsenoside Rg3 to treat MG-63 cells at the same time,western blot and quantitative PCR showed that 3-MA could significantly reverse the apoptosis and the changes of expression of autophagy-related proteins induced by ginsenoside Rg3.Silencing LC3 gene with small interfering RNA could significantly reverse the apoptosis and limitation of migration and invasion of MG-63 cells induced by ginsenoside Rg3.Western blot showed that ginsenoside Rg3 could decrease the expression levels of p-PI3K/PI3K,p-Akt/Akt and p-mTORC1/mTORC1,but increase the expression levels of LC3/β-actin.Adding PI3K agonist IGF-1 could reverse the change of expression levels of p-PI3K/PI3K,p-Akt/Akt,p-mTORC1/mTORC1and LC3/β-actin induced by ginsenoside Rg3.Flow cytometry showed that the proportion of osteosarcoma cells MG-63,HOS and U2OS in G₀/G₁ increased and the proportion of cells in S phase decreased after treatment with ginsenoside Rg3.Western blot showed that the expression of cyclin A and cyclin B in MG-63 cells treated with ginsenoside Rg3 did not change significantly,while the expression of cyclinD1,CDK-4 and CDK-6 decreased significantly,and the expression of upstream regulators p53 and p21 increased significantly.Silencing p53 gene by small interfering RNA technology can significantly reverse the cycle arrest induced by ginsenoside Rg3 and the changes of expression levels of p21,cyclinD1,CDK4 and CDK6.Conclusion:Ginsenoside Rg3 can reduce the expression of autophagy inhibitor mTORC1,induce autophagy of cells,increase the expression level of LC3-II/LC3I,and then induce the increase of Caspase-3 transcription and expression level and ultimately lead to apoptosis of MG-63 cells by inhibiting PI3K/Akt/mTORC1 pathway.Meanwhile,ginsenoside Rg3 can activate p53/p21 to inhibit the expression and activity of cyclinD1,CDK-4 and CDK-6,which are the key proteins in the transformation from G1 to S phase,so that osteosarcoma cells can be blocked at G0/G1 phase.