Exploration To The Mechanism Of Dual PI3K/mTOR Inhibitor NVP-BEZ235 Regulating Osteosarcoma Cancer Cells' Sensitivity To Cisplatin

IntroductionOsteosarcoma(OS)is a common malignant bone tumor predominantly seen in childhood and adolescence,accounts for nearly 2.4%of all malignancies in pediatric patients.Despite the combination of adjuvant chemotherapy and surgical removal of the tumor has improved the 5-year-survival rates dramatically,the 5-year-survival rates haven't improved in the past 30 years and the 5-year-survival rate of those with poor responses to chemotherapy is only 20%.What's more,patients with recurrent or metastatic diseases have a<20%chance of long term survival despite aggressive therapies.Although MAP(methotrexate,cisplatin,doxorubicin)regimen have been approved to be the gold standard in the treatment of OS,lots of patients are resistant to chemotherapeutic drugs.To some extent,the reason behind this may be due to the chemoresistance to anti-OS therapy.Although cisplatin belongs to the gold standard drugs in treatment of OS,high risk of cisplatin-associated nephrotoxcitiy,ototoxicity and gonadal dysfunction limit its individual doses.So,how to improve patient's sensitivity to chemotherapeutic drugs has become the focus of researchers.Although the exact mechanisms of OS chemoresistance have not been fully understood,several mechanisms were thought to playmportant roles in p chemoresistance of OS:1.decreased intracellular drug accumulation;2.drug inactivation;3.enhanced DNA repair;4.Perturbations in signal transduction pathways;5.Apoptosis and cell cycle-related gene expression turbulence;6.microRNA(miRNA)dysregulation;7.Cancer stem cells(CSCs)and drug resistance.Phosphoinositide 3-kinase(PI3K)-Akt-mammalian target of rapamycin inhibitor(mTOR)pathway takes part in lots of disparate cellular functions,including cell growth,proliferation,survival,apoptosis,chemo-resistance and is often constitutively activated in tumor cells.As PI3K-Akt-mTOR pathway has a vital function during chemoresitance and is always activated in human cancers,it is believed to be the target for inactivation in order to achieve better chemotherapy.Although the exact mechanisms of OS chemoresistance have not been fully explored,recently,several studies shows that PI3K-Akt-mTOR pathway is an important feature of OS and plays a vital role in progression and chemoresistance of OS.However,inactivation of PI3K-Akt-mTOR pathway has dual effect on chemosensitivity in cancer cells.Autophagy,an evolutionarily conserved process,mediates lysosomal degradation of cytoplasmic and cellular organelles,plays an important role in maintaining the homeostasis.It is generally thought that autophagy has four functions in cells confronted with different conditions:cytoprotective,cytotoxic,cytostatic and nonprotective.Researches have demonstrated that immunogenic signal,p53 status,apoptosis ability,HMGB1 redox status can determine the function of autophagy.Recently,autophagy targeting methods have been used to enhance chemosensitivity in tumor cells through switching function of autophagy,including OS.In 2015,President Obama put forward a project,named Precision Medicine Initiative(PMI).Precision Medicine,on the other hand,is an innovative approach which takes into account individual differences in people's genes,environments,and lifestyles.It gives medical professionals the resources they need to target the specific treatments of the illnesses we encounter,further develops our scientific and medical research,and keeps our families healthier.Although the exact mechanisms of OS chemoresistance have not been fully understood,several studies shows that PI3K-Akt-mTOR pathway is an important feature of OS and plays a vital role in progression and chemoresistance of OS.So,whether precisely inhibition of PI3K-Akt-mTOR pathway could enhance OS cancer cell's sensitivity to cisplatin is deserve our exploration.NVP-BEZ235,a dual PI3K/mTOR inhibitor,has been shown to inhibit the proliferation and induce apoptosis in OS cancer cells effectively.What's more,in some cisplatin-resistant tumor cell lines,NVP-BEZ235 can enhance their sensitivity to cisplatin through inducing autophagy.Whether NVP-BEZ235 synergizes cisplatin sensitivity or not in OS cancer cells is unknown.ObjectiveExplore the mechanism of NVP-BEZ235 regulating osteosarcoma cancer cells'sensitivity to cisplatin in vitro and in vivo.Materials and MethodsIn vitro,CCK-8 assay and combination index were used to determine half maximal inhibitory concentration of NVP-BEZ235 or/and cisplatin and synergistic interaction between NVP-BEZ235 and cisplatin on U20S and Saos-2 cell lines.Western blot assay was used to determine activity of PI3K-Akt-mTOR pathway after treatment with cisplatin or/and NVP-BEZ235 on U20S cells.Function of autophagy induced by NVP-BEZ235 or/and cisplatin was determined by Annexin V-PI dual staining assays and Western blot with or without autophagy inhibition by 3-MA or ATG-5 siRNA.In vivo,effect on tumor growth,proliferation and apoptosis treated with NVP-BEZ235 or/and cisplatin were determined in mouse xenograft model.Results1.NVP-BEZ235 synergistically enhances the anti-proliferative effect of cisplatin on OS U20S cellsFirst,effect of NVP-BEZ235 or/and cisplatin on the proliferation of U20S cells was determined by CCK-8 assay.CCK-8 assay shows a differential response of U20S cells to different compounds.To further determine whether the inhibitory effects were synergistic,the CalcuSyn software was used to determine the CI values to ascertain synergism(CI<1),additive effect(CI=1)or antagonism(CI>1).The CI values are showed in Table 2 and CI values for combination of NVP-BEZ235 with cisplatin were all less than 1.0,indicating a synergistic anti-proliferative effect.2.Different regulation of PI3K-Akt-mTOR pathway activity in U20S cells by cisplatin or/and NVP-BEZ235.Western blot assays were used to determine PI3K-Akt-mTOR pathway regulated by cisplatin or/and NVP-BEZ235.Results showed that PI3K-Akt-mTOR pathway activity can be transiently activated in OS U20S and Saos-2 cells by cisplatin in a concentration dependent manner.Whereas,NVP-BEZ235 alone or plus cisplatin treatment decreased the PI3K-Akt-mTOR pathway activity at all times in U20S and Saos-2 cells.3.NVP-BEZ235 synergistically induces apoptosis in osteosarcoma cells treated with cisplatinTo better understand the mechanism underlying the combined anti-proliferative activity observed in the CCK-8 assays,cells were exposed to NVP-BEZ235 or/and cisplatin,then stained with Annexin V-FITC and PI and analyzed by flow cytometryor fluorescence microscopy examination in U20S and Saos-2 cells.Results show that cisplatin or NVP-BEZ235 alone could induce apoptosis,combined NVP-BEZ235 and cisplatin induced more apoptosis than cisplatin alone.In addition,cleaved PARP and cleaved caspase-3 were significantly elevated compared to the cisplatin or NVP-BEZ235 groups.All these suggest that NVP-BEZ235 synergistically induces apoptosis in U20S cells treated with cisplatin.4.The synergistic effects of combined NVP-BEZ235 and cisplatin weren't due to enhanced activation of the p53 or TAp73 pathwayWestern blot and qPCR assays were used to detect expressions of p53,TAp73 and their target genes(NOXA,PUMA,P21).Surprisingly,expression of p53 and TAp73 were down-regulated in combined NVP-BEZ235 and cisplatin treatment groups than cisplatin treatment groups.What's more,levels of downstream transcriptional targets of p53 and TAp73 including NOXA,p21 and PUMA were also decreased at both protein and mRNA level.So,the synergistic effects of the combined NVP-BEZ235 and cisplatin weren't due to enhanced activation of the p53 or TAp73 pathway.5.NVP-BEZ235 sensitizes OS cells to cisplatin through switching function of autophagy induced by cisplatinFirst,Western blot and fluorescent LC3 puncta analysis assays were used to detect whether NVP-BEZ235 or/and cisplatin induces autophagy and autophagy inhibition 3-MA(3-Methyladenine),CQ(Chloroquine Phosphate)inhibits autophagy in U20S and Saos-2 cells.Next,functions of autophagy induced by cisplatin or/and NVP-BEZ235 in U20S cells were analyzed by flow cytometry or Western blot assays after exposed to 3-MA,cisplatin,NVP-BEZ235,singly or in combination.Results show that percentage of apoptotic cells in combined cisplatin and 3-MA groups were higer than cisplatin groups in both U20S and Saos-2 cells.However,apoptosis rates in combined 3-MA,cisplatin and NVP-BEZ235 groups were lower than combined cisplatin and NVP-BEZ235 groups in U20S cells,whereas,differences of apoptosis rates among combined cisplatin and NVP-BEZ235 groups and combined 3-MA,cisplatin and NVP-BEZ235 groups weren't significant.Similar results were obtained through detecting expressions of cleaved PARP by Western blot.Together,all these results show that NVP-BEZ235 sensitizes U20S cells to cisplatin through switching cisplatin induced cytoprotective autophagy to nonprotective.6.NVP-BEZ235 switches function of autophagy induced by cisplatin through downregulating HMGB1 expression.Western blot assays were used to detcet HMGB1 expression after U20S and Saos-2 cells treated by cisplatin or/and NVP-BEZ235.Results show that cisplatin upregulates HMGB1 expression.However,HMGB1 expressions were lower in combined NVP-BEZ235 and cisplatin treatment groups than cisplatin treatment groups.So,it seems that NVP-BEZ235 switches function of autophagy induced by cisplatin through downregulating HMGB1 expression.7.NVP-BEZ235 sensitizes OS cancer cells to cisplatin in vivo.To detect the in vivo efficacy of combined treatment of NVP-BEZ235 plus cisplatin,U20S xenograft mice models were established and divided into 4 groups:(1)mice treated with vehicle only,(2)mice treated with cisplatin only,(3)mice treated with NVP-BEZ235 only,(4)mice treated with combined cisplatin and NVP-BEZ235.Tumors were allowed to develop for 21 days after injection,and therapy was started on day 3 after tumor implantation.The tumor growth curve showed that volume of tumor in combined NVP-BEZ235 and cisplatin groups exhibited a decrease in contrast with the control,cisplatin and NVP-BEZ235 groups.On day 21,all mice were sacrificed and tumor volumes were measured.Consistent with above results,tumor volume in combined NVP-BEZ235 and cisplatin groups were lesser than mice treated with vehicle,cisplatin or NVP-BEZ235 alone.This indicates that NVP-BEZ235 could synergistically enhance growth inhibition effect of cisplatin in vivo.Then,a pronounced decrease in tumor cell proliferation(Ki67)and increase in apoptosis(TUNEL-positive tumor cells)were also noted in combination-treated xenografts based on immunostaining.Taken together,these data recapitulate the observations made in vitro and demonstrate that NVP-BEZ235 displays synergistic activity with cisplatin in vivo xenograft models.Then,a pronounced decrease in tumor cell proliferation(Ki67)and increase in apoptosis(TUNEL-positive tumor cells)were also noted in combination-treated xenografts based on immunostaining(Fig 6B).Taken together,these data recapitulate the observations made in vitro and demonstrate that NVP-BEZ235 displays synergistic activity with cisplatin in vivo xenograft models.Conclusions1.NVP-BEZ235 alone could inhibit the proliferation and induce apoptosis in OS U20S and Saos-2 cells;2.NVP-BEZ235 alone or combined with cisplatin could inhibit PI3K-Akt-mTOR pathway activity effectively,whereas,cisplatin could increase activity of PI3K-Akt-mTOR pathway in early times;3.NVP-BEZ235 synergistically enhances the anti-proliferative effect of cisplatin on OS U20S and Saos-2 cells;4.NVP-BEZ235 synergistically induces apoptosis in osteosarcoma cells treated with cisplatin;5.The synergistic effects of combined NVP-BEZ235 and cisplatin weren't due to enhanced activation of the p53 or TAp73 pathway;6.NVP-BEZ235 can synergize cisplatin sensitivity in OS cells through switching function of autophagy induced by cisplatin;7.NVP-BEZ235 switches function of autophagy induced by cisplatin through downregulating HMGB1 expression;8.NVP-BEZ235 can synergize cisplatin sensitivity in U20S xenograft SCID mice models;9.There is value in utilizing NVP-BEZ235 to sensitize OS cells to cisplatin.To sum up,the results of our work showed that NVP-BEZ235 could synergize cisplatin sensitivity in OS cells through switching function of autophagy induced by cisplatin and may be a promising adjuvant drug for OS.