The Role Of Mitochondrial Pathway In Lidocaine Induced Apoptosis In Human Osteosarcoma Cell Lines And Its Underlying Mechanisms

Part ? Effect of Lidocaine on Cell Activity in Human Osteosarcoma Cell Lines and Its Underlying MechanismObjective:To evaluate the effect of local anesthetic lidocaine on osteosarcoma cell activity in vitro and its underlying pathway.Methods:Human osteosarcoma cells(HOS/MNNG,MG63,U2OS)in good condition were used.Before the drug treatment,seed the cell in 96-well culture plates in 1*104 per well.After 24 hours,different concentrations of lidocaine were added to the culture plates,DEPC water of the same volume was added in the control group and cultured for 24 hours,48 hours,and 72 hours respectively.The cell viability was measured using a Cell Counting Kit-8(CCK-8)assay kit,and the cells cycle were examined by flow cytometry.The activity of the mitochondrial membrane potential was detected by the JC-1 assay,and apoptosis-associated protein and mitochondrial pathw ay key proteins were detected by Western-blotting.Results:Local anesthetic lidocaine reduced the activity of osteosarcoma cells in a time-dependent and dose-dependent manner,and 4m M lidocaine was the appropriate concentration.After treated with lidocaine,the cell cycle was arrested and the mitochondrial membrane potential was decreased.The expression of BAX protein was increased,anti-apoptotic protein BCL-2 was attenuated,and expression of cytochrome C(Cyt-c),Cleaved-Caspase 3,and Caspase 9 were also increased.Conclusions:The toxic effects of lidocaine on osteosarcoma cells were dose-and time-dependent,and apoptosis was induced by mitochondrial apoptosis pathway.Part ? The Effect of Lidocaine on Cisplatin Chemosensitivity of Osteosarcoma Cells and Its MechanismObjective:To evaluate the effect of local anesthetic lidocaine on cisplatin,a chemotherapeutic agent,in the treatment of osteosarcoma and possible apoptosis pathway in vitro.Methods:Human osteosarcoma cells(HOS/MNNG,MG63,U2OS)with good growth status were used.Before the drug treatment,cells were seeded in a 96-well culture plate at in 1*104 per well.After 24 hours,different concentrations of cisplatin were used in the culture plates.In the culture medium,the control group was added with equal volume of DEPC water,and culture for 48 hours.The cell activity was assayed using the Cell Counting Kit-8(CCK-8)assay kit,the optimal cisplatin concentration was determined.Re-seed the plates,4m M lidocaine,10?M cisplatin,and 4m M lidocaine plus 10?M cisplatin contained in the medium were added to the culture plate.The cell cycle was detected by flow cytometry,and the change in mitochondrial membrane potential was detected by the JC-1 assay.Apoptosis-related and mitochondrial pathway key protein expression were detected by western blotting.Results:The chemotherapy drug cisplatin reduced the activity of osteosarcoma cells in a time-dependent and dose-dependent manner,and the concentration of 10?M cisplatin was the most suitable concentration.The combined treatment group was more cytotoxic than the local anesthetic or chemotherapy drug alone.After treatment with cells,the cell cycle was arrested,the mitochondrial membrane potential was decreased,the expression of pro-apoptotic protein BAX increased,the expression of anti-apoptotic protein BCL-2 decreased,and the mitochondrial pathway-associated proteins cytochrome C(Cyt-c)and Cleaved-Caspase.3,Caspase 9 expression increased either.Conclusions:The toxic effect of cisplatin on osteosarcoma cells is dose-and time-dependent.Lidocaine has a signif icant sensitizing effect on the chemotherapy effect of osteosarcoma cells and is mediated through mitochondrial apoptosis pathway.Part ? Therapeutic Effect of Lidocaine in The Antitumor Activity of Cisplatin Against Tumor Growth of Osetosarcoma Cell Xenografts in Vivo and Its Underlying MechanismObjective:Osteosarcoma model were establish successfully in vivo,lidocaine,cisplatin,cisplatin plus lidocaine were injected intraperitoneally to observe the effect of the drug on tumor size in vivo,and the expression of Cleaved Caspase3 and PCNA was detected by immunohistochemistry.Methods:Human osteosarcoma cells(HOS/MNNG)with good growth were harvested and mixed with medium.BD matrigel and culture medium with cells were mixed in 1:1 and planted in the right armpit of nude mice.When tumor growing to 20mm3,30mg/kg lidocaine(2times/week),3mg/kg cisplatin(1time/week),cisplatin plus lidocaine through intraperitoneal injection for 30 days,and the tumor volume of all group were calculated,then the tumor was embedded,and perform H&E,CleavedCaspase3,PCNA immunochemistry staining.Results:The HOS/MNNG cell line can establish osteosarcoma model in vivo successfully.Compared with the control group,lidocaine,cisplatin,and lidocaine plus cisplatin group can reduce the tumor volume,and the combination group is more effective than the single drug group.Conclusions:The treatment of Lidocaine plus cisplatin can inhibit the occurrence and develop-ment of tumors more effective than drugs alone,and its efficacy is better.