| Osteosarcoma is the most common primary malignant bone tumor among children and adolescents.Currently,the traditional methods of osteosarcoma therapy include surgery and chemotherapy,but the therapeutic effect is not ideal.Over the past 30 years,the overall survival time of patients with metastatic osteosarcoma has not been improved significantly,and the 5-year survival rate of patients with distant metastasis is still below 20%.Therefore,it is urgent to find new therapeutic strategies against osteosarcoma."New use of old drugs" was proposed by James Black,a Scottish pharmacologist who won the 1988 Nobel Prize in Medicine for cimetidine,which is used to treat stomach ulcers.The pharmacokinetics,pharmacodynamics and toxicology of old drugs are clear,and the development of their new applications can greatly shorten the development cycle and save cost."New use of old drugs" is considered as one of the fastest and most effective strategies in the development of new drugs.Metformin is a US Food and Drug Administration-approved “star” drug used for diabetes mellitus type 2,and it was approved in the United State in 1995.Metformin was first reported to reduce the risk of cancer in patients with type 2 diabetes in 2005.Recent years,metformin has attracted much attention due to its anti-tumor effects.Iron,an important trace element,is necessary for life activities involved in DNA replication,energy metabolism,cell proliferation and cell respiration.Excessive iron can produce reactive oxygen species(ROS),which can lead to lipid peroxidation and DNA damage.The term "ferroptosis" was first proposed in 2012 by Stockwell.Ferroptosis,a novel form of regulated cell death,is characterized by the production of ROS from accumulated iron and lipid peroxidation.Ferroptosis may be a potential strategy for cancer therapy.Previous studies in this paper have shown that high-dose of metformin can induce ROS production in osteosarcoma cells,thereby promoting cell apoptosis in mitochondria dependent pathways.Studies have shown that combination of different drugs may produce synergistic effects.Then,whether metformin combined with iron preparations could synergistically induce apoptosis or death of osteosarcoma cells? Whether their combination therapy could reduce the dose of each drug? What are the mechanisms of action?The targeted drug delivery system can concentrate iron preparations in tumor tissues by static magnetic field,which can improve the efficacy of drugs and reduce the side effects.Therefore,whether static magnetic field could concentrate iron preparations in osteosarcoma tissues of mice? Whether their combination therapy could inhibit the growth of osteosarcoma?Accordingly,this study intends to investigate the research of metformin,iron preparations,static magnetic field and their combination treatment on osteosarcoma,and tries to elucidate their molecular mechanisms.Results showed that metformin dose-dependently inhibited the proliferation of osteosarcoma cells and osteosarcoma stem cells.Metformin(≥12.8 m M)significantly induced the apoptosis of osteosarcoma cells and osteosarcoma stem cells.At the same time,6.4 m M of metformin induced the autophagy of osteosarcoma stem cells by activating the AMPK/mTOR signaling pathway,and the disorder of autophagy further reduced the stemness features and the pluripotency of osteosarcoma stem cells.In addition,metformin inhibited the tumor growth of osteosarcoma by a mouse model of osteosarcoma.In the presence of the same concentration of iron,the utilization rates of iron preparations in osteosarcoma cells were ferrous sulfate(FAS),ammonium ferric citrate(FAC)and Iron dextran,respectively.The biological effects of osteosarcoma cells of FAS,FAC and Iron dextran are different.FAS and Iron dextran inhibited the cell viability of osteosarcoma cells and osteosarcoma stem cells only at high doses(iron content≥800 μM),but had no effect on cell apoptosis.However,FAC(iron content≥25 μM)significantly inhibited the cell viability of osteosarcoma cells,and FAC(≥100 μM of iron)induced apoptosis of osteosarcoma cells.Metformin(6 m M)combined with iron preparation FAC(50 μM of iron)significantly induced endoplasmic reticulum stress-mediated apoptosis in osteosarcoma cells in vitro.However,metformin(250 mg/kg/d,intraperitoneal injection)combined with FAC(100mg/kg/3d,intragastric administration)did not suppress the tumor growth of mice with osteosarcoma by a mouse model of osteosarcoma.We further evaluated the effect of metformin(250 mg/kg/d,intraperitoneal injection)combined with iron preparation FAC(100 mg/kg/3d,local injection in tumor)on tumor-bearing mice under 1.5 Tesla static magnetic field(60 min/d).The results showed that FAC could be concentrated in the tumor tissue of osteosarcoma mice.At the same time,metformin combined with FAC significantly inhibited the growth of osteosarcoma in tumor-bearing mice under 1.5 Tesla static magnetic field.We reported that exposure to 0.2 T-0.4 T moderate static magnetic field promoted the cell viability and the tumorspheres formation of osteosarcoma stem cells.Mechanistically,static magnetic field promoted the autophagic degradation of ferritin in osteosarcoma stem cells,which increased the concentration of intracellular available iron.By knocking down the cargo receptor for ferritinophagy-nuclear receptor coactivators 4(NCOA4)by siRNA,the self-renewal ability of osteosarcoma stem cells which promoted by static magnetic field was partially inhibited.In summary,the above results suggested that:(1)The iron content in clinical osteosarcoma tissues was significantly higher than those in adjacent tissues.(2)Metformin is expected to be a potential drug against osteosarcoma.(3)In vitro,metformin(6 m M)or FAC(50 μM iron)could not induce apoptosis of osteosarcoma cells,but the combination therapy could synergically induce apoptosis of osteosarcoma cells.In vivo,the combination therapy did not significantly inhibit tumor growth in osteosarcoma bearing mice.(4)Static magnetic field can concentrate FAC,metformin combined with FAC under static magnetic field significantly suppressed the tumor growth of osteosarcoma mice.(5)0.2 T-0.4 T static magnetic field promoted the self-renewal of osteosarcoma stem cells by NCOA4-mediated ferritinophagy. |
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