| BackgroundOsteosarcoma is the most common type of bone cancer and the second leading cause of cancer-related deaths in children and adolescents,which shows an incidence of 3.4 cases per million people every year worldwide.Osteosarcoma stem cell is a highly malignant tumor cell,it develops fast,and in the early stage shows pulmonary metastasis,in this way brings great pains to patients and families.Combination of surgery and adjacent chemotherapy is still the conventional therapeutic regimens for osteosarcoma patients.Despite of the significant improvements in diagnosis and therapy over the last decades,about 60–70% osteosarcoma patients exhibit no benefit from these treatment.The 5-year survival in patients with localized osteosarcoma is remained at 50% approximately,and only 15% for five-year survival estimation in the patients with lung metastasis.Therefore,novel and effective agents are urgent needs for improving osteosarcoma therapeutic efficiency.Cancer stem cells(CSCs)are a small number of tumor-forming andself-renewing cells within osteosarcoma tissues.Cancer stem cells are associated with the occurrence,recurrence,drug resistance,invasion and metastasis of malignant tumors.Therefore,the development of specific agents targeting osteosarcoma stem cells will provide a promising strategy for therapeutic improvement.Resveratrol(trans-3,4',5 trihydroxystilbene,Resveratrol)is a natural small polyphenolic compound which can be extracted from several plant species,such as mulberries,peanuts and grapes.Intensive studies have been performed in the fields of natural medicine or nutriology during the last decade.Resveratrol shows a beneficial role in inhibiting cancer progression,including leukemia,prostate cancer and gastric cancer.Moreover,resveratrol also induces CSCs apoptosis in pancreatic cancer.However,the function and mechanism of resveratrol on human osteosarcoma CSCs is rarely reported.STAT3 signaling pathway shows a pivotal role in cancer cell survival and disease progression.Activated STAT3 is observed in a variety of cancer cells,which is a promising therapeutic target to attenuate disease progression.Recent studies supported a critical role of STAT3 signaling activation in CSCs survival.Further analysis of STAT3 pathway in human osteosarcoma stem cells will provide critical proofs for optimized therapy.PART 1 RESEARCH OF THE INHIBITION BIOLOGICAL EFFECTS OF RESVERATROL ON OSTEOSARCOMAObjectiveThe effects of resveratrol on the growth of osteosarcoma cells were studied by in vitro and in vivo experiments.The effect of resveratrol on apoptosis of osteosarcoma cells was studied by in vitro experiments.Methods1.Determination of cell viability of human osteoblast cell line hFOB1.19 and osteosarcoma cell lines MG-63 and MNNG / HOS;Cells were treated with various concentrations of resveratrol for 48 hours(0,10,20 or 40?M).Cell proliferation was measured with a CCK-8 kit using a microplate reader.2.Colony formation ability of MG63 and MNNG / HOS cells was determined.Resveratrol(20?M)or equal vehicle treatment was administrated.Comparing the colony formation rate between the experimental group and the control group.3.MG63 and MNNG / HOS cells were treated with resveratrol(40?M)for 48 hours.Osteosarcoma cells were harvested after resveratrol treatment.Cell apoptosis was analyzed with Annexin V-FITC Apoptosis Detection Kit.Then measured with FACS Aria II flow cytometer.4.MG63 and MNNG / HOS cells were treated with resveratrol(40?M)for 48 hours.Caspase 3 activity was analyzed with Caspase 3Activity Assay Kit.Activity of caspase 3 was indicated by pNA concentrations.5.MG63 and MNNG / HOS cells were treated with resveratrol(40?M)for 48 hours.Apoptosis related proteins(cleaved PARP and caspase3,Bax,Bcl-2 and Bcl-xL)were measured with Western blot assays.6.Mice were randomly separated into resveratrol group and control group(n = 5 per group).mice were randomly separated into resveratrol group and control group(n = 5 per group).Tumor volume was measured every other day.Xenografts were harvested and imagined after 21 days.All animal-related procedures were approved by Animal Care and Use Committee of University.Results1.Resveratrol inhibits cell growth of osteosarcoma1.1 The results demonstrated that the IC50 of hFOB 1.19,MG-63 and MNNG/HOS to resveratrol was 1254?M,28.56?M and 20.57?M,respectively,which indicated selective cytotoxicity of resveratrol to osteosarcoma cells.1.2 resveratrol significantly inhibited the proliferation of osteosarcoma MG-63 and MNNG/HOS cell lines in 20 or 40?M,whereas hFOB 1.19 showed no significantly cell viability inhibition effects(P<0.05).1.3 colony formation assays also confirmed the long-term cell growth inhibitory effects of resveratrol to osteosarcoma cells,MG-63 andMNNG/HOS cell lines in 40 ?M(P<0.05).1.4 we evaluated the therapeutic efficacy of resveratrol on tumor growth of osteosarcoma in vivo.We observed no evidence of noticeable side effects during the experimental period.Significant inhibitory potency was observed on tumor growth in resveratrol treatment group.The mean tumor volumes of resveratrol-treated group were significantly lower compared to control group.2.Resveratrol induces apoptotic cell death in osteosarcoma cells2.1 Flow cytometry analysis was performed to measure the percentage of apoptotic cells with 40?M resveratrol treatment.Compared to vehicle control group,resveratrol treatment significantly increased early and late apoptosis percentages of MG63 and MNNG/HOS cells(p < 0.01,respectively.).2.2 Increased activities of caspase 3 were observed in resveratrol treated MG63 and MNNG/HOS cells(p < 0.01,respectively.).2.3 the pro-apoptotic effects of resveratrol were also indicated by the induced cleavage of PARP,caspase3 and increased Bax,as well as downregulation of Bcl-2 and Bcl-xL.Conclusion1.These data suggest that resveratrol suppresses osteosarcoma cells growth in vitro and in vivo.2.These results indicate that resveratrol promotes apoptosis of osteosarcoma cells.PART 2 RESEARCH OF THE INHIBITION BIOLOGICAL EFFECTS OF RESVERATROL ON OSTEOSARCOMA STEM CELLSObjectiveThe effect of resveratrol on osteosarcoma stem cells was studied by in vitro experiments.Methods1.FACS sorted CD133+ cells were treated with different concentrations resveratrol(0,10,20 or 40?M)for 48 h.Cell proliferation was measured with a CCK-8 kit using a microplate reader.2.Suspended tumor spheres were established with no-adhesive suspension culture system.Osteosarcoma cells formed floating spherical colonies within 7 days,with different concentrations resveratrol treatment(0,10,20 or 40?M).Tumor spheres with a diameter > 75 um were counted.Secondary spheroids were cultured and calculated,which further confirmed osteosarcoma stem cells elimination with resveratrol treatment.3.MG-63 and MNNG/HOS cells were seeded in ultralow attachment plate with serum-free stem cell medium and treated with different concentrations of resveratrol(0,10,20 or 40?M)for 7 days.The tumor cell spheres were collected and the expression of CD133 on the surface ofosteosarcoma cells was detected by immunofluorescence technique.This experiment used confocal microscopy and Image analysis was performed using ZEN software.4.After 48 h culture in gradient concentrations of resveratrol(0,10,20 or 40?M),the percentage of CD133+ cells were analyzed with a flow cytometry.5.Xenograft specimens were subjected to IHC staining.The expression of CD133 was detected in the Xenograft tumor after resveratrol treatment.Results1.Osteosarcoma cells formed floating spherical colonies within 7 days,whereas resveratrol treatment significantly reduced the volume of tumor spheres.Significantly decreased number of tumor spheres were observed with resveratrol treatment(P<0.05).Secondary spheroids were cultured and calculated,which further confirmed osteosarcoma stem cells elimination with resveratrol treatment(P<0.05).2.Immunofluorescence(IF)staining also showed decreased expression of CD133,a CSCs surface marker,in tumor spherical colonies of MG-63 and MNNG/HOS cells with the treatment of 40 ?M resveratrol.3.After 48 h culture in gradient concentrations of resveratrol,the percentage of CD133+ cells were significantly decreased with a flow cytometry analysis(P<0.05).4,FACS sorted CD133+ cells were treated with different concentrations resveratrol for 48 h,which indicated resveratrol significantly inhibited proliferation of osteosarcoma stem cells(CD133+cells subpopulation)(P<0.05).5.To further investigate the effects in vivo,IHC staining was performedwith the xenografts,which showed resveratrol treatment increased decreased CD133 expression in xenografts.Conclusion1.Resveratrol reduces cancer stem cell subpopulation in osteosarcoma.2.Resveratrol significantly inhibited proliferation of osteosarcoma stem cells.PART 3 RESEARCH OF THE INHIBITION MECHANISM OF RESVERATROL ON OSTEOSARCOMA STEM CELLSObjectiveTo investigate the effect of resveratrol on STAT3 signaling pathway in osteosarcoma stem cells.Methods1.MG63 and MNNG / HOS cells were treated with resveratrol(40?M)for 48 hours.JAK2/STAT3 pathway related proteins(Oncostatin M?p-STAT3?p-JAK2?STAT3 and JAK2)were measured with Western blot assays.2.MG63 and MNNG / HOS cells were treated with gradient concentrations of resveratrol(0,10,20 or 40?M)for 48 hours.AKT/NF-kB pathway proteins(p-PI3K(Tyr199),p-AKT(Ser473)? NF-kB(p65))and CD133 were measured with Western blot assays.3.Xenograft specimens were subjected to IHC staining.The expression of p-STAT3 was detected in the Xenograft tumor after resveratrol treatment.4.MG-63 cells were infected with a constitutively activated STAT3(STAT3-C).STAT3-C-overexpressing cells treated with resveratrol.Proteins(CD133?STAT3 and STAT3-Flag)were measured with Western blot assays.5.MG-6 and MG-63/STAT3-C were treated with different concentrations resveratrol(0,10,20 or 40?M)for 48 h.Cell proliferation was measured with a CCK-8 kit using a microplate reader.6.Suspended tumor spheres were established with no-adhesive suspension culture system.MG-63 and MG-63/STAT3-C formed floating spherical colonies within 7 days,with different concentrations resveratrol treatment(0,10,20 or 40?M).Tumor spheres with a diameter > 75 um were counted.7.NOD/SCID Mice were randomly separated into resveratrol group and control group(n = 5 per group).mice were randomly separated into MG-63 group and MG-63/STAT3-C group(n = 5 per group).Tumor volume was measured every other day.Xenografts were harvested and imagined after 21 days.All animal-related procedures were approved by Animal Care and Use Committee of University.Results1.Resveratrol inhibits JAK2/STAT3 pathway in osteosarcoma cells1.1 Western blot assays showed that decreased Oncostatin M,STAT3 and JAK2 phosphorylation was observed in MG-63 and MNNG/HOS cells with 40 ?M resveratrol treatment for 48 h.1.2 Decreased expression of osteosarcoma stem cell marker,CD133 was also observed with a gradient of resveratrol treatment.Meanwhile,PI3K/AKT/NF-kB signaling,a downstream pathway of JAK2/STAT3,wasalso examined in the gradient of resveratrol treated osteosarcoma cells.Significantly decreased p-PI3K(Tyr199),p-AKT(Ser473)and NF-kB(p65)protein expressions were showed in cells treated with resveratrol than vehicle controls.1.3 To further investigate the effects in vivo,IHC staining was performedwith the xenografts,which showed resveratrol treatment increased decreased p-STAT3 expression in xenografts.2.STAT3 activation attenuates cancer stem cells elimination effects of resveratrol2.1 To further confirmed the role of STAT3 activation in resveratrol treatment,MG-63 cells were infected with a constitutively activated STAT3(STAT3-C).Western blot assays showed that STAT3-C effectively increased CD133 expression in MG63 cells.More importantly,elevated CD133 expression was not attenuated by resveratrol treatment.2.2 CCK-8 assays showed that resveratrol failed to inhibit proliferation of STAT3-C-overexpressing cells.STAT3-C-overexpressing cells also significantly abrogated cell proliferation inhibition effects of resveratrol treatment.2.3 Tumor sphere formation assays also indicated that resveratrol failed to decrease the sphere number of STAT3-C-overexpressing cells.2.4 Further studies were also performed with planted xenografts invivo.Resveratrol effectively inhibited the growth of control xenografts but not in STAT3-C-overexpressing tumors.Conclusion1.Resveratrol eliminates cancer stem cells of osteosarcoma by STAT3 pathway inhibition2.STAT3 activation attenuates cancer stem cells elimination effects of resveratrol... |
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