Anti-cancer Effect Of Nano Graphene Oxide On Osteosarcoma And Its Underlying Mechanism

Currently,osteosarcoma is one of the most harmful diseases in child and adolescent.Recently,with the rapid development of surgical techniques and orthopaedic implants,emerging therapies such as gene therapy and immune therapy appear constantly.So far,there is still no effective method for clinical osteosarcoma therapy.Although surgery,radiotherapy and chemotherapy are the traditional methods for the treatment of osteosarcoma,the trauma and low immunity affected the quality of life of young patients.Therefore,search for low toxicity and high tumor selectivity mehod is an urgent task,which supplied a novel study direction for clinical treatment of osteosarcoma.Graphene oxide?GO?has the unique physical and chemical properties,which is a kind of a single atomic layer of two-dimension carbon nano materials.It shows great potential in energy storage,optical,and especially in the field of biomedical.The existing research results show that GO has obvious inhibitory effect of lung cancer,breast cancer,prostate cancer in human,which reveals its great application potential in the field of anti-cancer.However,through the literature retrieval,the study of GO on osteosarcoma is rarely reported,and its underlying mechanism is not clear.Therefore,our research aims to study the GO anti-cancer effect and mechanism,which will provide new methods and theoretical basis for the treatment of osteosarcoma.The research content is divided into three parts as following shows.Part ? Study the inhibition and migration effects of GO on osteosarcoma cellsObjectives: GO was investigated for growth and migration ability of MG-63 and K₇M₂ osteosarcoma cells.We also established osteosarcoma heterotopic transplantation tumor models in mice,and observed the growth inhibition of osteosarcoma in vivo.Methods: For in vitro studies,Different concentration and time of GO was applied to MG-63 and K₇M₂ osteosarcoma cells,to observe morphological changes of cancer cells with microscope.The Live/Dead method was used to detect cell death;MTT was used to test cell viability;the ability of cells migration and invasion was studied by scratch healing assay.For in vivo studies,we established the mice buttock model with trasplanting human K₇M₂ osteosarcoma cells,grouping according to processing factors,The 1st group was given saline solution as negative control;The 2nd group was given GO as positive control?1mg/ml,1 times / 50 ?L,intratumor injection?.Drug was administreted at the begining five days,observing the change of mice generally vital signs and tumor size till 20 days.Results?1?GO could cause morphology change of cell apoptosis in K₇M₂ cells,but GO did not cause similar change in the MG-63 cells;?2?GO could obviously inhibit the growth and invasion ability of MG-63 and K₇M₂ cells,dependence on time and concentration,K₇M₂ cells are more sensitive than MG-63 cells;?3?GO could significantly inhibit inhibit the growth and transplantation of K₇M₂ cells.Conclusions:GO can change the morphology of MG-63 and K₇M₂ osteosarcoma cells,and inhibit the growth and invasion of osteosarcoma cells.Part ? GO induced apoptosis of K₇M₂ osteosarcoma cells through oxidative stressObjectives: To investigate GO induced oxidative stress level and apoptosis of MG-63 and K₇M₂ osteosarcoma cells.Methods: We measured the ROS?Reactive oxygen species?level changes of MG-63 and K₇M₂ cells processing with GO by DHE staining and flow cytometry;We detected GO inducing apoptosis of K₇M₂ through Annexin V and PI double staining test;Immunofluorescence method and Western Blot analysis distribution of Nrf-2 MG-63 and K₇M₂ after processing with GO;MTT method detect the affect of proliferation in K₇M₂ cells which was processed by GO combined with ROS inhibitors?NAC?;Flow cytometry detect ROS level of MG-63 cells which was processed by GO combined with Nrf-2 inhibitor?ML-385?;Western Blot method measure apoptosis protein expression of K₇M₂ cells after processing with GO.Results:?1?GO can increase levels of ROS in K₇M₂ cells,and induce cell apoptosis,but NAC is obviously improved the cell inhibition effect induced by the GO;?2?GO ddi not affect levels of ROS in MG-63 cells,but promote the Nrf-2 transposition from the cytoplasm to the nucleus,and the Nrf-2 inhibitor?ML-385?has significantly promote the GO inducing ROS;?3?the GO can obviously increase apoptosis related proteins expression?caspase-3,etc?of K₇M₂ cells,and decrease expression of apoptosis inhibiting protein?Bcl-2?.Conclusions:?1?GO can promote K₇M₂ apoptosis by oxidative stress,activation and apoptosis signaling pathways;?2?GO do not affect oxidative stress level of MG-63 cells,and it is closely related to the Nrf-2 transposition activation.Part ? Investigation of GO induced autophagy in MG-63 osteosarcoma cells through autophagosome formationObjectives: To study GO induced autophagosome formation and the impact of autophagy related protein expression in MG-63 and K₇M₂ osteosarcoma cells..Methods MDC staining test was used by observing autophagy body formation of MG-63 and K₇M₂ osteosarcoma cells after processing with GO;Western Blot method measure autophagy related protein expression of MG-63 and K₇M₂ osteosarcoma cells after processing by GO combined with autophagy inhibitor?Baf A1?.Results:?1?GO induced the occurrence of autophagy and the increased autophagy related protein expression of MG-63 cells,GO combined with autophagy inhibitor?Baf A1?increased expression level of LC3 II protein;?2?GO didnot induce autophagy of K₇M₂ cells.Conclusions: GO induced the occurrence of autophagy in MG-63 cells.