Experimental Study On The Effect Of Curcumin To Human Osteosarcoma Cell Line

Background:Osteosarcoma is the most common type of bone malignant tumor,which onset mostly between children and adolescent. Prognosis of osteosarcoma is very poor for recurrence and metastasis. Treatment of osteosarcoma includes surgery,chemotherapy,radiotherapy and Integrated treatment.The application of neoadjuvant chemotherapy has improved cure rates remarkably.Further studies have shown that multidrug resistance(MDR)to chemotherapeutic agents is a major barrier to the successful treatment of osteosarcoma.To explore new and effective chemotherapeutics will conduce to the therapy of osteosarcoma.Curcuma is a kind of Chinese traditional medicine in common use, it has been widely used as a spice,to color cheese and butter,as a cosmetic,and in some medicinal preparations.Curcumin is a kind of hydroxybenzene pigment picked-up from curcuma,it is the major effective component,and it has various medical functions,such as antiinflammatory,antioxidant,anticoagulation,anti-artherosclerosis and anticarcinogenic effects etc,especially as a kind of well-perspectived anti-tumor drug,more and more researches have been attached on it,and it has be,come a hotspot in recent years.The mechanism of curcumin is extensive,thereinto,people pay more attention to its inducing tumor cell apoptosis.Many studies have showed that,curcumin can prevent or reduce the growth and proliferation of many kinds of tumor cells cultured in vitro,and induce apoptosis of tumor cells,such as prostatic carcinoma, nasopharyngeal carcinoma,gastric carcinoma,colon carcinoma, hepatoma,etc.Simultaneouly,curcumin can reverse MDR mediated by p-glycoprotein.But few research about curcumin's function to osteosarcoma has been done.In the present study,the proliferation of U-2OS cells was measured by MTT assay;the morphological changes and percentage of apoptotic nuclei in U-2OS cells treated with curcumin were assayed by Hoechst 33258 staining,and apoptosis was determined by Annexin V/PI double staining technique and flow cytometry.The activities of caspase-9,8 and 3 were analyzed by Caspase Colorimetric Assay Kit.To explore the molecular mechanisms of curcumin to U-2OS or U-2OS/ADM. PartⅠThe effects of curcumin on human osteosarcoma U-2OS cell line in vitroObjectives:To observe the effects of curcumin on cell growth,and apoptosis in U-2OS cells,and explore the molecular mechanisms of curcumin on human osteosarcoma.Methods:The proliferation of U-2OS cells was measured by MTT assay.The morphological changes and percentage of apoptotic nuclei in U-2OS cells treated with curcumin were assayed by Hoechst 33258 staining,and apoptosis was determined by Annexin V/PI double staining technique and flow cytometry.The activities of caspase-9,8 and 3 were analyzed by Caspase Colorimetric Assay Kit.Results:(1)Curcumin significantly inhibited the proliferation of PC-3 cells in a concentration-and time-dependent manner.(2)A morphological change was observed in U-2OS cells.After 24 or 48h treatment with curcumin(20μmol/L),condensation of chromatin occurred,and blebbing nuclei and granular apoptotic bodies appeared. The results of FACS showed that the percentage of apoptotic cells in the PC-3 cells treated with curcumin(20μmol/L)for 12,24,or 48 h were (13.81±1.07)%,(27.03±2.32)%or(40.76±3.96)%,respectively.After exposure to different concentrations of curcumin(10,20,or 40μmol/L) for 48 hours,the U-2OS cells occured a lot of apoptosis cells,and percentage of apoptotic nuclei respectively were(11.26±3.03)%, (32.15±2.88)%or(55.24±4.40)%.Similarly,the results of FACS also indicated a dramatic increased in apoptosis cells,and the percentage of apoptotic cells were(16.26±1.44)%,(34.45±2.55)%or(60.46±4.01)%, respectively.The activation of Caspase-9,8 or 3 were markedly increased in U-2OS cells treated with curcumin(20 or 40μmol/L)at indicated periods.Conclusions:(1)Curcumin can significantly inhibit the proliferation of U-2OS cells.(2)Curcumin can induce apoptosis of U-2OS cells.(3)The effects of curcumin on induction of apoptosis may be related to the increase of Caspase-9,8 or 3' activity in U-2OS cells.(4)Curcumin provide a novel theory and method in the therapy for human osteosarcoma clinically. PartⅡThe reverse effects of curcumin on MDR of human osteosarcoma cell line in vitroObjectives:To investigate the reversion of P-gp mediated multidrug resistance of U-2OS/ADM cells with curcumin in vitro.Methods:The reversal efficacy of curcumin was measured by MTT assay.The effects of curcumin on Rh-123 uptake and efflux were analyzed by flow cytometer.RT-PCR technique was used to examine the MDR1 mRNA level,P-gp was detected by Western blot analysis.Results:(1)MTT demonstrates curcumin(20μmol/L)can increase the cytotoxicity of Adriamycin to U-2OS/ADM cells.(2)The result of FCM shows that Curcumin can increase the accumulation of Rh-123 and increase the cytotoxicity of Adriamycin to U-2OS/ADM cells in a dose-dependent manner.(3)The mRNA expression of MDR1 in U-2OS/ADM cells was more higher than that in U-2OS cells,and with few influence on it when treated with curcumin(10,20 or 30μmol/L)for 48 hours by RT-PCR.(4)The protein expression of P-gp in U-2OS/ADM cells was more higher than that in U-2OS cells,and with few influence on it when treated with curcumin(10,20 or 30μmol/L)for 48 hours by Western blot.Conclusions:(1)The reversal mechanism of curcumin was blocked the function of P-gp in U-2OS/ADM cellular membrane and have no effect on the expression of MDR1 and P-gp. (2)The expression of MDR1 and P-gp in U-2OS/ADM cells was increased significantly,which possibly mediated MDR in osteosarcoma.