Experimental Study On The Effect Of Icaritin To Human Osteosarcoma Cells

Background Osteosarcoma is a kind of primary malignant tumor of bone, whose rate of incidence stands the second place of that of primary tumor of bone, following the plasma cell myeloma. It is usually seen on children and teenagers, which has high degree of grade malignancy, incidental recurrence and metastasis, and pretty bad prognosis. The primary treatments now include operation, chemo-treatment, radiation therapy and combined treatment. During recent years, amputation rate of osteosarcoma reduces apparently, which is owing to the progress of combined treatment giving priority to chemo-treatment in a large part. So to explore new and effective chemo-medicine continuously is significant to the treatment of osteosarcoma.Herba Epimedii is one of the Chinese traditional medicines, warm natured, and has effects of reinforcing the kidney yang and expelling wind and removing damness. The modern pharmacology research shows that the primary effective elements is flavonoid, as well as polysaccharide compound, alkaloid, lignanoid, linolenic acid, vitamin E, naphtha, trace element and so on.Herba Epimedii can improve the condition of myocardial ischemia, reduce the blood pressure, inhibit the thrombosis, promote hemopoietic function, regulate the immunity, resist the bone loss, aging and tumor and so on. The flavone monomeric ingredient extracted from Herba Epimedii is icariin, which has diverse biological activity, including improving cardiovascular efficiency, anti-tumor, influencing incretion, inhibiting platelet count, anti-ageing, regulating immunologic function and so on.As the ramification of icariin, icaritin comes from Herba Epimedii after multiple steps of extraction, silica gel chromatography and elution, and has functions including resisting oxidation, preventing and curing bone loss and breast cancer, promoting the growth and multiplication of osteoblast, curing angiocardiopathy, and keeping nerves from degenerative damage. The icaritin has the molecular formula of C21H20O6and the molecular weight368, which has similar molecular structure with its pro-material, icariin. The different part is that the glycosidic linkage in the place of A-ring,7rank is replaced by hydroxyl group. The oxidation resistant function will be strengthened concluding from the drug structure-activity relationship.The relative research of icaritin shows that the anti-tumor function is attracting more and more attention and being the hot focus of research in recent years. The icaritin has function of inhibiting multiplication and inducting apoptosis to multiple tumor cell strain (breast cancer,prostatic cancer,liver cancer,endometrial carcinoma,chronic granulocytic leukemia etc.) But until now, the above mentioned research about icaritin's function to osteosarcoma has no relevant report.Object Researching the effect of icaritin to human osteosarcoma cell in vitro culture on multiplication and apoptosis. Providing new theory and method to the osteosarcoma clinical treatment.Method1. Icaritin of different concentration is used to MG-63cell and in different acting time, observe the shape change of osteosarcoma cell MG-63by inverted microscope;2. Detect the multiplication state of cell by MTT colorimetry;3. Detect the cell apoptosis by Annexin V/PI double method and flow cytometry;4. Detect the activity of Caspase-9,8,3by Caspase active and quantitative determination kit.Results1. Observe the MG-63cell treated by icaritin by inverted microscope and find that the cell multiplication is inhibited along with the increase of drug concentration and acting time, and the cell gradually reduces and floats off, having the reduced diopter and dispersing in the field of view.2. Detect the MG-63cell treated by icaritin of different concentration of4μmol/L,8μmol/L,16μmol/L and32μmol/L by MTT colorimetry and find that higher the medicine concentration is, better the inhibition ratio is of icaritin to cell (P<0.05); longer the acting time is, more remarkable the inhibition function is to cell (P<0.05). When the icaritin concentration is64μmol/L, the inhibition ratio isn't higher than the concentration of32μmol/L and the multiplication inhibition effect to cell isn't apparently strengthened (P>0.05)3. The flow cytometry analysis:the cell apoptosis ratio is (14.17±1.16)%,(22.01±3.21)%,(28.71±2.17)%and (45.75±3.24)%respectively after24h's effect of icaritin of4μmol/L,8μmol/L,16μmol/L and32μmol/L; the cell apoptosis ratio is (24.21±2.17)%,(43.52±4.15)%,(71.57±3.26)%and (79.19±4.54)%respectively after48h's effect. The analysis is that along with the continuous increase of medicine concentration, the early and terminal apoptosis ratio of human osteosarcoma MG-63cell increase gradually after24h and48h's effects of icaritin of4μmol/L,8μmol/L,16μmol/L and32μmol/L, which has statistical significance (P<0.05). The apoptosis ratio of human osteosarcoma MG-63cell effected by medicine for48h is apparently higher than that of cell effected for24h under the same icaritin concentration, the4μmol/L,8μmol/L and16μmol/L groups are pretty apparent and the16μmol/L group is most obvious. So the icaritin function of inducting the apoptosis of human osteosarcoma MG-63cell has dependency on time and drug concentration, and its drug function trend of inducting the apoptosis of human osteosarcoma MG-63cell approximately agrees with MTT result.4. The Caspase-9,8,3activity of human osteosarcoma MG-63cell effected by icaritin of4μmol/L and8μmol/L for24h and48h has no apparent increase than control group and the activity increase trend of three kinds of enzymes is not apparent along with the change of function time and drug concentration (P>0.05). The Caspase-9,8,3activity of human osteosarcoma MG-63cell affected by icaritin of16μmol/L for24h increases apparently to the control group (P<0.05). The enzyme activity has no obvious increase after being effected by this medicine for48h to24h(P>0.05). The Caspase-9,8,3activity of human osteosarcoma MG-63cell affected by icaritin of32μmol/L for24h increases apparently to the16μmol/L group (P<0.05). The enzyme activity has no noticeable increase after being effected by this medicine for48h to24h (P>0.05)Conclusion1. The icaritin can inhibit the external multiplication of human osteosarcoma MG-63cells, presenting the dependency on time and concentration.2. The icaritin can induct the apoptosis of human osteosarcoma MG-63cells, presenting the dependency on time and concentration.3. The icaritin function of inducting apoptosis of human osteosarcoma MG-63cells may has something to do with the increase of Caspase-9,8,3activity.