Genistein Potentiates Anti-cancer Effects Of Gemcitabine And Induces Apoptosis In Human Osteosarcoma Via Akt/NF-?B

Osteosarcoma(OS)is the most common primary bone tumor in children and young adults.With current therapies of OS,such as extensive surgical excision,radiotherapy and neoadjuvant chemotherapy,five-year survival rate has increased up to 65%?75%.But five-year survival rate is still less than 20%if patients have recurrence or metastasis of OS.Meanwhile,the effectiveness of cytotoxic drugs often declines due to acquired chemoresistance.Combination treatment could potentially result in better antitumor effectiveness at much lower drug doses,thus reducing the extent and severity of treatment-related toxicity.Gemcitabine(2',2'-difluorodeoxycytidine,dFdCyd)is an analog of cytosine arabinoside,following influx through the cell membrane via nucleoside transporters,gemcitabine undergoes intracellular conversion to the nucleotides gemcitabine diphosphate(dFdCDP)and triphosphate(dFdCTP)responsible for its cytotoxic actions.The cytotoxic activity of gemcitabine may be the result of several actions on DNA synthesis.dFdCTP competes with deoxycytidine triphosphate(dCTP)as an inhibitor of DNA polymerase.dFdCDP is a potent inhibitor of ribonucleoside reductase,resulting in depletion of deoxyribonucleotide pools necessary for DNA synthesis and,thereby potentiating the effects of dFdCTP.dFdCTP is incorporated into DNA and after the incorporation of one more nucleotide leads to DNA strand termination.This extra nucleotide may be important in hiding the dFdCTP from DNA repair enzymes,as incorporation of dFdCTP into DNA appears to be resistant to the normal mechanisms of DNA repair.Gemcitabine can be effectively inactivated mainly by the action of deoxycytidine deaminase to 2,2'-difluorodeoxyuridine.Also,5'-nucleotidase opposes the action of nucleoside kinases by catalysing the conversion of nucleotides back to nucleosides.Additional sites of action and self-potentiating effects have been described.It is reported that gemcitabine mono-chemotherapy is not effective,because of NF-?B activation.Many anticancer agents induce NF-?B nuclear translocation and activation of its target genes,which impinge on cellular resistance to anticancer agents.Flavonoids are polyphenolic compounds that are ubiquitously in plants.The flavonoids are phenyl substituted chromones(benzopyran derivatives)consisting of a 15-carbon basic skeleton(C6-C3-C6).Epidemiologic studies suggest that a diet that includes regular consumption of fruits and vegetables(rich in flavonoids)significantly reduces the risk of many cancers.Moreover,the flavonoids can induce the apoptosis of cancers.At the molecular level,flavonoids can trigger apoptosis through the modulation of Caspase,Bcl-2,PI3K/Akt in cellular signal transduction pathways to anticancer.In addition,the flavonoids play an important role in cell growth and kinase activity inhibition,suppression of the secretion of matrix metalloproteinases and of tumor invasive behavior,inhibition of angiogenesis,antioxidation and reversal of multidrug resistance.In view of the drug resistance of gemcitabine in the treatment of osteosarcoma and drug sensitization effect of genistein,we design the drug combination research.Our study contains the following two parts:(1)Osteosarcoma cell lines as the research object,we test the growth inhibition of human osteosarcoma cells by genistein,gemcitabine,and the combination,analysis apoptosis related gene or protein to clarify the main signal transduction pathways.(2)In the MNNG/HOS subcutaneous xenograft model,further histological evaluation of tumor tissues from mouse treated with gemcitabine and/or genistein,analysis related protein to clarify the main signal transduction pathways,verify the experimental results in vitro.Our study confirm that genistein potentiates anti-tumor effects of gemcitabine and induces apoptosis via the inactivation of Akt/NF-?B.Part one:Genistein potentiates anti-tumor effects of gemcitabine and induces apoptosis in vitroObjective:We investigated whether genistein could be employed as a novel strategy to enhance the anti-tumor activity of gemcitabine in human osteosarcoma cell lines to clarify its molecular mechanism.Methods:To investigate the cytotoxicity of gemcitabine or genistein on osteosarcoma,we treated human osteosarcoma cell lines with various doses of gemcitabine or genistein for 72 h.Then we assessed the effect of a combination of genistein and gemcitabine on cell viability by MTT assay.To examine whether the reduced viability of osteosarcoma cells was caused by apoptosis,we carried out TEM analysis,DNA Ladder,annexin V-FITC/PI double staining and TUNEL assay.Non-radioactive EMSA for detecting NF-?B-DNA binding activity in human osteosarcoma cell lines after treatment with 0.5?M gemcitabine,20?M genistein or the combination for 72 h.Western blot analysis of P-Akt,Bcl-xL,Bcl-2,and COX-2 in whole cell lysates of human osteosarcoma cell lines after treatment with 0.5?M gemcitabine,20?M genistein or the combination for 72 h.Results:MTT assay showed that cell growth was inhibited by gemcitabine or genistein in a dose-dependent manner.Analysis of osteosarcoma tumor cells in vitro by TEM revealed typical apoptotic morphological features induced by the combination of gemcitabine and genistein:denser cytoplasm,concentrated and aggregated karyotin,and the formation of dense,round apoptotic bodies.These observations were consistent with the morphological alterations detected by light microscopy.Cells treated by both agents displayed much higher rates of apoptosis(up to 44.15%)than cells treated by single agent(31.20%and 34.75%)or control cells(0.70%).Most apoptotic cells were in the early stages.In addition,to distinguish the apoptotic and necrotic cells,we performed the TUNEL assay.The results showed that genistein could potentiate apoptosis of the osteosarcoma cells induced by gemcitabine.Constitutively active NF-?B-DNA binding activity was observed in nuclear extracts from cells.Compared with untreated control,gemcitabine treatment induced an obvious shift of NF-?B-DNA binding activity,while genistein treatment blocked the shift.These data demonstrated that genistein abrogated gemcitabine-induced activation of NF-?B-DNA binding activity.Akt phosphorylation,an indicator of Akt activation,was not up-regulated by gemcitabine.However,genistein significantly down-regulated Akt phosphorylation,and the combination treatment also down-regulated Akt phosphorylation significantly.In addition,we detected the levels of apoptosis related proteins which are regulated by NF-?B.Western blot analysis showed that the expression of NF-?B,Bcl-xL and COX-2 was significantly reduced in the combination treatment group compared to single-agent treatment group and control group.Moreover,caspase-3 activity and PARP cleavage were significantly induced by genistein and gemcitabine combination treatment.A significant decrease in the mitochondrial cytochrome c level was observed in the combination treatment group compared with single agent treatment group.Moreover,a PI3K/Akt-pathway inhibitor(LY-294002)or specific NF-?B inhibitor(BAY 11-7082)alone did not significantly affect cell viability in MNNG/HOS cells;however,combination of gemcitabine and LY294002 or BAY11-7082 strongly decreased the cell viability.There were significant differences between the combination group and gemcitabine group.Conclusions:These data suggest that gemcitabine treatment induced an obvious shift of NF-?B-DNA binding activity;genistein may potentiate anti-tumor e? ects of gemcitabine and induce apoptosis via the inactivation of Akt/NF-KB.Part two:Genistein potentiates anti-tumor effects of gemcitabine and induces apoptosis in vivoObjective:We investigated whether genistein could be enhance the anti-tumor activity of gemcitabine in xenograft model of osteosarcoma,and to clarify its molecular mechanism.Methods:Female BALB/c mice(weight 16-18 g)were maintained under specific conditions and supplied with sterile food and water ad libitum.Approximately 5×106 MNNG/HOS cells(suspended in 0.2 mL PBS)were injected subcutaneously into the right axillary fossa of each nude mouse under aseptic conditions.At the 5th day after the injection(day 1),mice were randomly assigned to one of four groups(n=6):(a)vehicle alone(control);(b)gemcitabine(80 mg/kg body weight),once every other day(i.v.injection);(c)genistein,everyday orally for 10 days;and(d)genistein and gemcitabine,following the schedule as for individual treatments.Genistein was dissolved in 0.1 mol/L Na2C03 and mixed with sesame seed oil at 2:1 ratio prior to gavage and delivered p.o.in 0.3 ml at a dose of 1 mg/d per mouse.All mice were killed on day 3 following last dose of treatment,and tumors were dissected and weighed.For routine HE staining,one part of the tissue was fixed in formalin and embedded in paraffin.Another part was rapidly frozen in liquid nitrogen and stored at-70?.HE staining confirmed the presence of tumors in each mouse.Tumor size was measured every three days with calipers and the volume was calculated by the following formula 0.5×a×b2,where "a" was the largest dimension and "b" the perpendicular diameter.Diet and water consumption as well as animal body weight were monitored regularly throughout the study.In addition,the mortality was monitored daily.Digital images of positively-stained fields were assessed by measuring the optical density(OD)of stained regions of tumor tissue.Brown granules in the cytoplasm or/and on the cell membrane represented positive staining,and staining intensity indicated the expression level of NF-?B and Bcl-xL.The expression of NF-?B and Bcl-xL was analyzed using the Image Pro-Plus software.TUNEL kit(Roche)was used to detect apoptosis in the xenograft tissue.We performed EMSA with nuclear proteins extracted from tumor tissues.Results:In the MNNG/HOS subcutaneous xenograft model,the combination treatment of gemcitabine with genistein showed significant inhibition of tumor growth,resulting in larger decreases in tumor volume and weight in xenograft model.Further histological evaluation of tumor tissues from mouse treated with gemcitabine and/or genistein showed that the ratio of nucleus to cytoplasm was reduced and the nuclei were polygonal and lightly stained.The sarcoma cells were loosely arranged and there were marked signs of widespread tumor destruction;coagulation and necrotic and apoptotic cells were observed.Tumors in the combination treatment group displayed stronger TUNEL staining than control group or single treatment group.In addition,to complement our in vitro results we first performed immunohistochemistry on the tumor tissues and found that the expression of Bcl-xL and NF-?B,two important molecules of tumor cell survival and metastasis,was significantly decreased in the combination treatment group compared to the control,although there was a mild reduction in the expression of these two proteins in the groups treated with genistein or cisplatin alone compared to the control.Next,we performed EMSA with nuclear proteins extracted from tumor tissues and the results showed that genistein treatment blocked the shift.These data demonstrated that genistein abrogated gemcitabine-induced activation of NF-?B-DNA binding activity in the xenografts.Finally,we detected the activation of Akt in the tumor tissues and immunoblot results clearly showed that Akt phosphorylation was induced by gemcitabine and down-regulated by genistein.Conclusion:Genistein potentiates anti-tumor effects of gemcitabine and induces apoptosis via inactivation of NF-?B/Akt in vivo.