| BackgroundOut of the most common malignant bone tumor,osteosarcoma can be found in approximately 30%of the patients with malignant bone tumors.The ten-year survival rate of osteosarcoma patients is approximately 60%to 75%.At present,osteosarcoma is usually treated by preoperative chemotherapy,amputation or limb salvage tumor resection and postoperative chemotherapy.Chemotherapy drugs have obvious lethal effect on osteosarcoma cells and normal human cells,possibly further reducing the survival of osteosarcoma patients due to its side effect.Curcumin is used as a traditional medicine to treat a variety of diseases in China.Curcumin can exert anti-tumor effects by acting on the multiple mechanisms,such as transcription factors,apoptosis genes,and cell signaling molecules.Studies had shown that curcumin had obvious anti-cancer effect on liver cancer,stomach cancer,pancreatic cancer and other tumors.Curcumin has low toxicity and is well tolerated in humans.The p53 gene has been found to have the most closed relationship with malignant tumors up to date.It is also the most likely to have mutations in tumor cells.The normal function of p53 is to check the DNA damage point in the G1 phase and monitor the integrity of cell genes.The role of p53 in the apoptosis induced by curcumin is controversial.Some studies suggest that these apoptotic effects are p53-dependent,while other studies suggest that these apoptotic effects are p53-independent.The differences in these studies may be due to the different types of cancers and different cell lines they have studied.Because of the high incidence of p53 gene mutation in osteosarcoma cells,the clinical application of curcumin as an anti osteosarcoma drug will be really limited if curcumin inhibits osteosarcoma cells in a p53-dependent manner.ObjectiveThis article explores the inhibiting effect of different concentrations of curcumin on human osteosarcoma cells and investigate the potential molecular mechanisms of that.It is very important for the future clinical application of curcumin.Two different human osteosarcoma cell lines which have different p53 status were chosen in our study,p53-mutant MG-63 cells and p53 wild type U20S cells.We aimed to observe the inhibitory effect of different concentrations of curcumin on these two cell lines,study the effects of curcumin on cell proliferation,apoptosis and invasion of U20S and MG-63 cells,study the effect of curcumin against osteosarcoma cells with its concentration and time change,research the molecular mechanism of the anti-osteosarcorma effect of curcumin,analyze the differences of curcumin sensitivity between the two cell lines and discuss whether the effect of curcumin on osteosarcoma is p53-dependent.Materials and MethodsPart One:Effect of curcumin on the proliferation,apoptosis and invasion of human osteosarcoma p53-mutant MG-63 cell line1.Osteosarcoma p53-mutant MG-63 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences.2.MG-63 cells were divided into four groups,group A was the control group with PBS buffer solution;group B was added with curcumin at concentration of 5?mol/L;group C was added with curcumin at concentration of 10?mol/L;group D was added with curcumin at concentration of 20?mol/L.3.Proliferation assay.The proliferation of MG-63 cells was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT)assay.All cellular growth assays were performed in 96 well plates(1×104 cells/well).MG-63 cells were cultured at 370C in a 5%CO2 incubator with increasing concentrations of curcumin for 48 and 72 h,respectively.Cells were treated with 10 ?l MTT(5mg/ml)/well and incubated for 4 h at 37?.After dissolved in DMSO,the amount of formazan dye generated by cellular dehydrogenase activity was measured by absorbance at 490 nm with microplate autoreader.4.Flow cytometric analysis of apoptosis.MG-63 cells seeded in 12-well plates(1.5×105 per well)were treated with increasing concentrations of curcumin for 24 h before harvest.Apoptosis cellls were assessed by using Apoptosis detection kit as manufacturer's protocol.In brief,cells suspended in binding buffer incubated with AnnexinV-FITC and Propidium Iodide for 15 min at room temperature in the dark.The cells were then analyzed by flow cytometry performed on a FACScalibur cell analyser using CellQuest software.Cells undergoing apoptosis were defined as those positive for Annexin V.5.Invasion Assay.Matrigel was diluted 1:19 with serum-free DMEM and 50?l was used to coat each invasion chamber.1×105 MG-63 cells,treated with increasing concentrations of curcumin for 48h,while the lower compartment filled with 10%fetal bovine serum in DMEM.After incubation for 48 h,the Matrigel on the upper side of the filter was removed.The cells on the bottom were fixed with methanol and stained with 1 mg/ml crystal violet dye.The picture of invaded cells was obtained under a microscope.6.Statistical analyses.All data are expressed as the mean ± s.d.of at least three independent experiments.Statistical analysis was performed using a paired Student's t-test in studies with two groups and one-way analysis of variance in the studies with more than two groups;significance was defined as*P<0.05 and**P<0.01.Part Two:Study on the molecular mechanism of curcumin-induced apoptosis of human osteosarcoma p53-mutant MG-63 cell line Western blotting assay.MG-63 cell lines were treated with curcumin of 0-20?mol/L for 12 h and then they were subjected to Western blotting.The antibodies which were used for Western blot include antibodies against Bcl-2,Bax,cleaved PARP,Caspase-3,and ?-actin.At the indicated time,the treated cells were harvested and they were washed for two times with the ice-cold PBS(pH 7.4),and then they were lysed with ice-cold buffer for half an hour on ice.Centrifugation of the lysates was carried out at 12,000g at 4 ? for 15 min.Protein concentration was determined,and sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to separate equal amounts of protein.The separated proteins were then transferred to polyvinylidene difluoride membrane.After that the indicated antibody was used to probe the proteins.And then appropriate secondary antibody was used.Detection was carried out with the odyssey infra-red imaging system.Equal loading was confirmed by using primary antibodies against ?-actin.Part Three:Comparison and analysis of the different effects of curcumin on the proliferation,apoptosis and invasion of human osteosarcoma p53-mutant MG-63 cell line and p53 wild type U20S cell line1.U20S cells were divided into four groups,group A was the control group with PBS buffer solution;group B was added with curcumin at concentration of 5 ?mol/L;group C was added with curcumin at concentration of 10 ?mol/L;group D was added with curcumin at concentration of 20 ?mol/L.MG-63 cell lines were grouped by the same method as previously described.2.Proliferation assay.The proliferation of MG-63 and U20S cells was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT)assay.All cellular growth assays were performed in 96 well plates(1 × 104 cells/well).U20S and MG-63 cells were cultured at 37? in a 5%CO2 incubator with increasing concentrations of curcumin for 48 and 72 h,respectively.Cells were treated with 10?1 MTT(5mg/ml)/well and incubated for 4 h at 37?= After dissolved in DMSO,the amount of formazan dye generated by cellular dehydrogenase activity was measured by absorbance at 490 nm with microplate autoreader.3.Flow cytometric analysis of apoptosis.MG-63 cells and U20S cells seeded in 12-well plates(1.5×105 per well)were treated with increasing concentrations of curcumin for 24 h before harvest.Apoptosis cells were assessed by using Apoptosis detection kit as manufacturer's protocol.In brief,cells suspended in binding buffer incubated with AnnexinV-FITC and Propidium Iodide for 15 min at room temperature in the dark.The cells were then analyzed by flow cytometry performed on a FACScalibur cell analyser using CellQuest software.Cells undergoing apoptosis were defined as those positive for Annexin V.4.Invasion Assay.Matrigel was diluted 1:19 with serum-free DMEM and 50?l was used to coat each invasion chamber.1×105 MG-63 cells and U20S cells,treated with increasing concentrations of curcumin for 48h,while the lower compartment filled with 10%fetal bovine serum in DMEM.After incubation for 48 h,the Matrigel on the upper side of the filter was removed.The cells on the bottom were fixed with methanol and stained with 1 mg/ml crystal violet dye.The picture of invaded cells was obtained under a microscope.5.Statistical analyses.All data are expressed as the mean ± s.d.of at least three independent experiments.Statistical analysis was performed using a paired Student's t-test in studies with two groups and one-way analysis of variance in the studies with more than two groups;significance was defined as*P<0.05 and**P<0.01.ResultsPart One:Effect of curcumin on the proliferation,apoptosis and invasion of human osteosarcoma p53-mutant MG-63 cell line1.The effect of different concentrations of curcumin on the proliferation of MG-63 cellsIn order to study the effect of curcumin on the proliferation of MG-63 cell line,MG-63 cells were cultured under different concentrations of curcumin.The culture time was 48 hours and 72 hours respectively.The concentrations of curcumin were 5?M,10?M,20?M respectively.The results showed that the proliferation of MG-63 cells was significantly inhibited with the increase of the curcumin concentration.The degree of inhibition was in a concentration dependent manner.The relationship between the prolliferation of osteosarcoma cells and the length of the action time of curcumin was also studied in this experiment.The results showed that the degree of inhibition of osteosarcoma cells was significantly correlated with the length of the action time of curcumin.The inhibitory effect of curcumin in the 72 hours group was significantly higher than that in the 48 hours group.The difference was statistically significant.Based on the experiment,when the concentration of curcumin was between 0 and 20?M,the degree of the inhibition of the osteosarcoma cells proliferation increased with the increase of the action time and the concentration of curcumin.2.Curcumin could enhance the apoptosis level of MG-63 cells.The flow cytometry was used to evaluate apoptosis of MG-63 cells that were respectively treated with 5?M,10?M,20?M curcumin for 24h.The results showed that the apoptosis rate of MG-63 cells increased significantly with the increase of the concentration of curcumin,and the increase of apoptosis was positively correlated with the rise of the curcumin concentration.Based on the experiment,when the concentration of curcumin was between 0 and 20?M,the apoptosis rate of the osteosarcoma cells increased with the increase of the curcumin concentration.3.Effect of different concentrations of curcumin on the invasion ability of MG-63 cellsIn order to further verify the effect of curcumin on the invasion ability of osteosarcoma cells,we studied the inhibitory effect of curcumin on MG-63 cells by using the Transwell tumor invasion assay.The results showed that with the increase of the curcumin concentration,the invasion ability of MG-63 cells were significantly decreased.Part Two:Study on the molecular mechanism of curcumin-induced apoptosis of human osteosarcoma p53-mutant MG-63 cell line The expression level of apoptosis related proteins in MG-63 cells was affected by curcumin.In order to further investigate the effect of curcumin on the apoptosis of osteosarcoma cells,Western Blot was used to detect the expression level of Bcl-2,Bax,Caspase-3 and cleaved PARP with different concentrations of curcumin.It was found that the expression of Bcl-2 decreased significantly with the increase of the curcumin concentration,while the expression of Bax,Caspase-3 and cleaved PARP increased with the increase of the curcumin concentration.At the same time,the proportion of Bax/Bcl-2 increased with the increase of the curcumin concentration.Based on the experiment,it was found that the expression of several pro-apoptotic proteins of osteosarcoma cells was up regulated,and the expression of several anti-apoptotic proteins was inhibited.It is possible that curcumin promotes osteosarcoma cell apoptosis through the mitochondrial pathway by raising Bax/Bcl-2 ratio and activating Caspase-3.Part Three:Comparison and analysis of the different effects of curcumin on the proliferation,apoptosis and invasion of human osteosarcoma p53-mutant MG-63 cell line and p53 wild type U20S cell line1.The different effect of curcumin on the proliferation of p53-mutant MG-63 cells and p53 wild type U2OS cellsIn order to compare the different effect of curcumin on the proliferation of p53-mutant MG-63 cells and p53 wild type U20S cells,U20S and MG-63 cells were cultured under different concentrations of curcumin.The culture time was 48 hours and 72 hours respectively.The concentrations of curcumin were 5?M,10?M,20?M respectively.The results showed that the proliferation of U20S and MG-63 cells was both significantly inhibited with the increase of the curcumin concentration.The degrees of inhibition were both in a concentration dependent manner.MG-63 cells are more sensitive to curcumin than U20S cells.With the same action time and under the same concentration of curcumin,the proliferation of both of the MG-63 cells and U20S cells was obviously inhibited,however,MG-63 cells were more inhibited than U20S cells,and the difference was statistically significant(P<0.05).Treated with 20?M curcumin for 72h,the inhibitory rate of MG-63 cells could reach to 80%.Based on the experiment,when the concentration of curcumin was between 0 and 20?M,the degree of the inhibition of the osteosarcoma cells proliferationincreased with the increase of the action time and the concentration of curcumin.MG-63 cell line was more sensitive to curcumin than U20S cell line.These results showed that curcumin inhibited the proliferation of osteosarcoma cells in a p53-independent manner.2.Curcumin could enhance the apoptosis level of p53-mutant MG-63 cells and p53 wild type U20S cells.In order to compare the different effect of curcumin on apoptosis of p53-mutant MG-63 cells and p53 wild type U20S cells,the flow cytometry was used to evaluate apoptosis of MG-63 and U20S cells that were respectively treated with 5?M?10?M,20?M curcumin for 24h.The results showed that the apoptosis rate of MG-63 cells and U20S cells increased significantly with the increase of the concentration of curcumin.The sensitivity of MG-63 cells to curcumin was much higher than that of U20S cells.The apoptosis rate of MG-63 cells was higher than that of U20S cells with the same concentration of curcumin.Especially when the concentration of curcumin was 20?M,the apoptosis rate of MG-63 cell line was about 84.21%,compared to 42.81%of U20S cell line.Under the same concentration of curcumin,the proliferation of MG-63 cells was more inhibited than that of U20S cells,and the apoptosis rate of MG-63 cells was also higher than that of U20S cells.Based on the experiment,when the concentration of curcumin was between 0 and 20?M,the apoptosis rate of the osteosarcoma cells increased with the increase of the curcumin concentration.P53-mutant MG-63 cells was also more sensitive to curcumin than p53 wild type U20S cells.These results showed that curcumin induced the apoptosis of osteosarcoma cells in a p5 3-independent manner.3.Different effect of curcumin on the invasion ability of p53-mutant MG-63 cells and p53 wild type U20S cellsIn order to compare the different effect of curcumin on the invasion ability of p53-mutant MG-63 cells and p53 wild type U20S cells,we studied the inhibitory effect of curcumin on U20S and MG-63 cells by using the Transwell tumor invasion assay.The results showed that with the increase of the curcumin concentration,the invasion ability of MG-63 cells and U20S cells were significantly decreased,while the inhibition of MG-63 cells by curcumin was more significant than that of U20S cells.These results showed that curcumin inhibited the invasion of osteosarcoma cells in a p53-independent manner.ConclusionBased on the experiment,the proliferation of osteosarcoma cell lines are significantly inhibited by curcumin,while the apoptosis of them are promoted and the invasion ability are significantly inhibited.The inhibition effect of curcumin on osteosarcoma is in a concentration dependent manner and time dependent manner.According to our experimental results,p53-mutant MG-63 cells are more sensitive to curcumin than p53 wild type U20S cells,and that suggests curcumin inhibits osteosarcoma cells in a p53-independent manner.Western Blot detection showed that with the increase of curcumin concentration,the expression of Bax,cleaved PARP and Caspase-3 obviously increased,but Bcl-2 expression gradually decreased,and Bax/Bcl-2 ratio increased with the rise of curcumin concentration.Generally speaking,curcumin inhibits osteosarcoma cells through the mitochondrial pathway by raising Bax/Bcl-2 ratio and activating Caspase-3 in a p53-independent manner.Curcumin is an important supplement to the traditional chemotherapy regimen,so a lot of research is being carried out about it around the world.A large number of p53 mutations exist in malignant tumors,and that results in widespread resistance to chemotherapy.Curcumin inhibits osteosarcoma cells in a p53-independent manner,and that suggests curcumin may have a good therapeutic effect on those malignant tumors which are resistance to traditional chemotherapy because of p53 dysfunction. |
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