Quercetin Inhibits Cell Migration And Invasion In Human Osteosarcoma Cells

?Background?Osteosarcoma is one of the most common malignant tumors of bone tissue in children and adolescents,with high malignancy and strong invasiveness.Surgical treatment combined with neoadjuvant chemotherapy greatly improved the survival rate of patients,but nearly half of the patients died from metastasis.Invasion and metastasis are the main biological characteristics of malignant tumors and the leading cause of death in patients with malignant tumors.Therefore,how to effectively prevent and treat metastasis is the key to improve the prognosis of patients with osteosarcoma.Quercetin is a natural flavonoid which has many biological functions,widely found in vegetables,fruits and plants.A large number of studies have reported that quercetin has anti-inflammatory,anti-allergic,dilated coronary arteries,anti-platelet aggregation,anti-oxidation,anti-cancer and regulates immune function.Quercetin has been recommended as a natural potential anti-cancer drug in Europe and America.Recent studies have found that quercetin can reduce the incidence of breast cancer,nasopharyngeal carcinoma,ovarian cancer and colon cancer.?Objective?The aims of the study are to investigate the effects of quercetin on invasion and metastasis of human osteosarcoma cells and molecular mechanisms in vivo and vitro,provide a theoretic basis for the potential anti-osteosarcoma effect of quercetin.?Methods??The effect of quercetin on osteosarcoma cell viability,cell cycle and cell apotosis?Human osteosarcoma cell line HOS and MG63 were resuscitation and subculture.Cells were seeded at a density of 5×10~4 cells/well in 24-well plates,and treated with quercetin(0?25?50?100?M)for 24 h.CCK-8 solution(40?l)was added to each well for 2 h at 37°C,and then the absorbance at 450 nm was measured by a microplate spectrophotometer.?5×10~4 cells were plated in 24-well plates,the cells were treated with quercetin(0?25?50?100?M)for 24 h,cells were harvested by trypsin-EDTA.The cell cycle of human osteosarcoma cells was determined according to the cell cycle kit.?Using FITC/PI cell apoptosis kit to evaluate the apoptotic cell.5×10~4 cells were plated in 24-well plates and treated with different concentrations of quercetin(0?25?50?100?M)for 24 h.Apoptotic cells were determined with an annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI)cell apoptosis kit(Invitrogen)according to the manufacturer's protocol.?The effect of quercetin on osteosarcoma cell invasion and cell migration?HOS and MG63 cells were seeded(5×10~4 cells/well)in 24-well plates in the appropriate culture medium,and grown to confluency.A sterile 10-?l pipette tip was subsequently used to create wounds,after which all the wells were washed with media PBS to remove cell debris.The cells were then treated with 0,25,50,and 100?M quercetin.Images were captured with an inverted microscope at different time points(0,12,and 24 h)post-quercetin administration.?The microporous membranes of each Transwell chamber were evenly coated with50?l liquefied Matrigel matrix glue and then incubated for 6 h at 37°C.The cells were incubated quercetin(0?25?50?100?M)for 24 h at 37°C.For cell invasion assay,HOS and MG63 cells(5×10~4)were seeded in the upper chamber in serum-free media,and medium supplemented with 10%FBS was added to the lower chamber,washed twice with PBS,fixed with methanol,and then stained with crystal violet.The cells that adhered to the upper surface of the chamber were carefully removed using cotton swabs,and those on the bottom surface of the membrane were imaged.The number of cells migrated to the lower side of the membrane was manually counted under microscope(×200)in five random fields.?The effect of quercetin on cell invasion and cell migration related genes mRNA and protein in osteosarcoma cells?Quantitative real-time PCR was used to detect the expression of HIF-1?,VEGF,MMP2 and MMP9 mRNA:2×10~5 cells/well were plated in 6-well plates,and treated with quercetin(0?25?50?100?M)for 24 h.Total RNA was extracted using Trizol according to the manufacturer's instructions.Reverse transcription was performed using TAKARA RT Kit.Quantitative real-time PCR was amplified by a standard PCR protocol using TAKARA PCR kit.The specificity of amplification was confirmed by running melting curve following each PCR run.?Western blotting to analyse the expression of HIF-1?,VEGF,MMP2 and MMP9:Cells were seeded at a density of 2×10~5 cells/well in 6-well plates,cells were treated with quercetin(0?25?50?100?M)for 24 h.Cells were harvested in RIPA cell lysis buffer with proteinase inhibitor,and the protein concentration was determined using the BCA protein assay.40?g of proteins were subjected to electrophoresis in SDS-PAGE,transferred to PVDF,blocked in PBST containing 5%skimmed milk and then reacted with the primary antibody overnight at 4?,followed by incubation with second antibody for 1h at room temperature.The signals were detected using an ECL kit.ImageJ was used to analyse the gray value.?Animal studies?Groups of experiment:(1)the blank control group,normal saline;(2)positive control group,cisplatin group,2mg/kg;(3)quercetin groups:A.low concentration group,25mg/kg;B.middle concentration groups:50mg/kg C.high concentration group:100mg/kg.?Model establishment and the dosage project:A recombinant p Lenti-CMV-mCherry-linker-Luc-PGK puro plasmid was packaged into a mature lentivirus with 293T cells,after which the lentivirus was used to infect HOS cells to obtain stable transfectants.The cells in were centrifuged after trypsin digestion,and then suspended in normal saline to prepare 5x10~7/ml single cell suspension.?For our in vivo tumor experiments,3-week-old female BALB/c(nu/nu)nude mice were randomly assigned to five groups.After three days,we injected stably transfected HOS cells into the tail vein of each mouse.We then intraperitoneally injected 25,50 or 100 mg/kg quercetin into the mice in the corresponding groups twice daily for four weeks.Cisplatin was administered at a dose of 2 mg/kg and served as a positive control.The negative control mice were injected with vehicle(normal saline).Four weeks after treatment,the mice were sacrificed.Specifically,the mice were anesthetized with chloral hydrate and then abdominally injected with D-Luciferin before being killed by cervical dislocation.The lungs were inspected for metastases using bioluminescence imaging and Living Image Software.All of the above procedures and assays were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University.The inhibition rate was calculated as follows:[(C-T)/C]×100%,where T is the average fluorescence of the treated group,and C is the average fluorescence of the negative control group.?Results??The effect of quercetin on human osteosarcoma cell viability:We used a CCK-8assay to test the effects of various concentrations of quercetin(25,50,and 100?M)in HOS and MG63 cells 12h and 24h.The results showed that quercetin of different concentrations had no significant effect on the activity of HOS and MG63 in human osteosarcoma cells,and P>0.05 had no statistical significance.According to CCK-8,the effect of quercetin on the viability of human osteosarcoma cells was determined.25,50 and 100?M quercetin can be used as the stimulation concentration for subsequent experiments.?The effect of quercetin on the cell cycle of human osteosarcoma:We used the cell cycle detection kit to test the effects of various concentrations of quercetin(25,50,and 100?M)in HOS and MG63 cells 12h and 24h.The results showed that the different concentrations of quercetin(0,25,50,100?M)had no significant effect on the G0/G1,G2/M and S stages of human osteosarcoma cell lines.?The effect of quercetin on the apoptosis of human osteosarcoma cells:We used the Alexa Fluor 488 annexinV/Dead cell apoptosis kit to test the effects of various concentrations of quercetin(25,50,and 100?M)in HOS and MG63 cells 12h and 24h.The results showed that,compared with the control group of 0?M quercetin,quercetin did not cause obvious early or late apoptosis in human osteosarcoma cells.?Quercetin inhibits HOS and MG63 cell invasion:We examined cell invasion capacity with a three-dimensional Matrigel-coated filter after the indicated cell lines were treated with quercetin.The media containing different concentrations of quercetin in the lower chamber were incubated for 24h.The results show that treatment with quercetin significantly decreased cell invasion rates(treatment with25,50,and 100?M quercetin decreased HOS cell invasion rates in the corresponding cells to levels that were(83.98±2.76)%,(38.67±3.52)%,and(16.36±2.56)%of those in cells treated with 0?M quercetin,respectively.With the increase of quercetin concentration,the invasion ability of HOS cells was decreased.Treatment with 25,50,and 100?M quercetin decreased MG63 cell invasion in the corresponding cells to levels that were(87.55±4.98)%,(69.34±5)%,and(41.98±4.4)%of those in cells treated with 0?M quercetin,respectively).With the increase of quercetin concentration,the invasion ability of MG63 cells was decreased.These results showed that the ability of quercetin-treated HOS and MG63 cells to traverse the Matrigel-coated layer was decreased compared with that of untreated control cells,suggesting that quercetin inhibits HOS and MG63 cell invasion in osteosarcoma in vitro.?The effect of quercetin on the migration ability of human osteosarcoma cells:We performed wound healing assay to observe the effect of quercetin on the migration ability of human osteosarcoma cells.The results show that HOS and MG63 cells were cultured and then coincubated with different doses(0,25,50,and 100?M)of quercetin for various time intervals(0,12,and 24 h).Treatment with 25,50,and 100?M quercetin for 12 and 24 h decreased HOS cell migration rates in the corresponding cells to levels that were 72.28±22.34%,61.49±19.61%,and 49.66±9.33%and 59.40%±20.13%,42.78±11.34%,and 46.06±11.5%of those in cells treated with 0?M quercetin,respectively.Treatment with 25,50,and 100?M quercetin for 12 and24 h decreased MG63 cell migration rates in the corresponding cells to levels that were 39.49±5.9%,31.15±12.27%,and 20.34±4.25%and 36.39±3.09%,28.43±0.7%,and 21.26±7.47%of those in cells treated with 0?M quercetin,respectively).Moreover,the rate of proliferation inhibition increased as the concentration of quercetin increased.These data showed that quercetin inhibited cell migration in the indicated osteosarcoma cell lines in a dose-and time-dependent manner.?The effect of quercetin on human osteosarcoma cell related genes mRNA and protein:HOS was treated with quercetin(0?25?50?100?M)for 24h.Total RNA was extracted,reverse transcription,and then quantitative real-time PCR was performed.The results showed that compared with the 0?M quercetin control group,with the increase of quercetin concentration,the mRNA level of HIF-1?,VEGF,MMP2 and MMP9 gradually decreased,HIF-1?(0.65±0.05,0.08±0.02,0.04±0.01)?VEGF(0.25±0.09,0.27±0.01,0.01±0.01)?MMP2(0.48±0.06,0.15±0.05,0.14±0.07)?MMP9(0.28±0.08,0.12±0.02,0.08±0.02),P<0.05.The results demonstrated that treatment with 0,25,50 and 100?M quercetin significantly decreased HIF-1?(0.57±0.08,0.45±0.07,0.42±0.08,0.42±0.01),VEGF(0.51±0.04,0.48±0.09,0.37±0.10,0.25±0.03),MMP2(0.59±0.01,0.54±0.13,0.46±0.07,0.34±0.01)and MMP9(1.07±0.06,0.89±0.19,0.75±0.07,0.60±0.16),and protein expression in a dose-dependent manner in HOS cells compared with treatment with controls.With the increase of quercetin concentration,the protein levels of HIF-1?,VEGF,MMP-2 and MMP-9 decreased gradually.Quercetin inhibited mRNA and protein expression of HIF-1?,VEGF,MMP2 and MMP9 in human osteosarcoma cells in a dose-dependent manner.?Animal studies:To determine whether treatment with quercetin affects osteosarcoma in vivo,we injected stably transfected HOS cells into the tail veins of nude mice.Three days after implantation,the animals were treated with quercetin or an equivalent volume of normal saline.The results showed that quercetin significantly inhibited tumor growth in the lung in a dose-dependent manner(treatment with 25,50,and 100 mg/kg quercetin decreased tumor growth in the corresponding group of mice to levels that were 0.89±0.10,0.71±0.06,and0.63±0.05 of that in mice treated with saline,respectively;treatment with cisplatin decreased tumor growth in the corresponding group of mice to a level that was0.63±0.04 of that in mice treated with saline).The tumor inhibition rate was calculated according to Eq.1.Treatment with 100 mg/kg quercetin decreased tumor growth by 37.41%,and treatment with cisplatin decreased tumor growth by 36.46%compared with treatment with saline.These results indicated that quercetin could ameliorate tumor metastasis in vivo.?Conclusion??Various concentrations of quercetin(0,25,50,100?M)did not affect the cell viability of human osteosarcoma cells,and failed to induce cell cycle arrest and cell apoptosis in HOS and MG63 cells.?Quercetin inhibited cell invasion and cell migration in osteosarcoma cell lines in a dose-dependent manner.?Quercetin downregulates mRNA and protein expression of HIF-1?,VEGF,MMP2,and MMP9 in a dose-dependent manner in osteosarcoma HOS cells.?Animal experiment further confirmed that quercetin can inhibit the metastasis of lung metastasis in nude mice.