The Mechanism Of Synergistic Effect Of Combination Treatment Of MK-2206 And Rapamycin In Neuroblastoma

Objective:Neuroblastoma(NB)is the most common pediatric extracranial solid malignant tumor.It derives from the neural crest and usually occurs primarily in the adrenal glands and it it contributes to approximately 15%of all pediatric cancer mortality.According to established prognostic factors(including the diagnostic age,the international NB stage system(INSS),DNA index,histological grade and MYCN gene amplification),NB patients were divided into low,moderate and high risk groups.It was commen that patients in the high-risk group had a poor prognosis and recurrences,and drug resistance was the biggest obstacle in the treatment.Therefore,it is particularly urgent to seek new drugs to improve the therapeutic effects and the prognosis of NB patientsAkt is a serine/threonine protein kinase that plays an important role in a variety of tumors.Enhanced Akt activity can be detected in NB tumor tissues,and NB patients with enhanced Akt activity tend to have a poor prognosis,indicating that the prognosis of NB patients is closely related to the activation of Akt.MK-2206 is an inhibitor of Akt,and our previous study found that MK-2206 could not only inhibit the NB cell growth in vivo and in vitro as a single drug,but also improve the sensitivity of NB cells to chemotherapy drugs.Some researchers have found that MK-2206 had synergistic effect with mTOR inhibitor RAD001 in hepatic carcinoma and cholangiocarcinoma cells.Our previous study found that MK-2206 and rapamycin obviously synergistically inhibited the cell growth in some NB cell lines,particularly those with MYCN amplification.The mechanisms of this synergistic effect had not been reported yet MYCN is an oncogene and encodes a transcription factor.Studies have shown that 20-30%of NB patients have MYCN amplification with poor prognosis,while about 40%of high-risk NB patients have MYCN amplification,so MYCN is closely related to drug resistance and prognosis of NB patients.MYCN amplification can be used as an independent factor to determine the poor prognosis of NB patients.Currently,the important role of MYCN in the synergistic effect of MK-2206 and rapamycin on NB has not been reported.This study can provide a new therapeutic option for MYCN-amplified NB patients with poor prognosis and drug resistance.This study will further explore the mechanisms of NB cell death induced by the combination treatment of MK-2206 and rapamycin and the important role of MYCN in this cell death which significancely guides the clinical treatment of NB patients with MYCN amplificationMethods:Four NB cell lines were used in this study which were MYCN-amplified cell lines NGP and SK-N-BE2(BE2)and MYCN-non amplified cell lines SK-N-AS(AS)and SH-SY5Y(SY5Y)1.CCK-8 assay was used to detect cell viability after the drug treatment2.The number of dead cells were observed by YOYO-1 staining3.IncuCyte Zoom was used to dynamically detect the changes of the cell conflunence,which indicated the changes of the cells proliferation or survival4.Gene microarray was used to screen out the differential genes of BE2 cells treated with combination of MK-2206 and rapamycin5.The ultrastructure changes of autophagy and necroptosis were observed by transmission electron microscopy6.Western Blot was used to detect the expression changes of autophagy related proteins(ATG5,ATG7,beclin-1 and LC3),necroptosis related proteins(RIPK1,RIPK3)and MYCN.7.Establish a nude mouse model of NGP cell transplantation tumor.Mice were divided into four groups:control group,rapamycin group,MK-2206 group and MK-2206+rapamycin group.Rapamycin was 5 mg/kg each day by intraperitoneal injection and MK-2206 was 200 mg/kg by oral 3 times a week.Tumor tissues were removed after 10 days and used for subsequent experiments8.HE staining was used to observe the changes of cell morphology of tumor tissues9.Immunohistochemical staining was used to detect the expression changes of necroptosis related proteins(RIPK1 and RIPK3)in tumor tissues10.siRNAs were transfected into NGP and BE2 cells to knock down MYCN and plasmid was transfection into AS and SY5Y cells to overexpress MYCN.Transfected cells were used for subsequent experiments11.The ECAR and OCR values were measured by the Seahorse Analyzer,indicating the changes of glycolysis and mitochondrial oxidative phosphorylation in NB cellsResults:1.The combination treatment of MK-2206 and rapamycin had a synergistic effect and was caspase-independent:in NGP and BE2 cells,the cell survival rate of MK-2206+rapamycin group was significantly lower than that of rapamycin group,the differences were statistically significant(P<0.01)and the number of dead cells was significantly increased.In addition,the caspase inhibitor z-VAD-fmk could not block NB cell death induced by the combination treatment of MK-2206 and rapamycin2.Microarray results were used to screen out differential expressed genes related to autophagy and necroptosis,such as ATG3,ATG5,ATG7,ATG9,RIPK3,TNFR,etc3.Autophagy mediated NB cell death induced by the combination treatment of MK-2206 and rapamycin:after NGP and BE2 cells were pretreated with autophagy inhibitor 3-MA,the cell viability of the 3-MA+MK-2206+rapamycin group were respectively2.1 times and 2.3 times higher than the MK-2206+rapamycin group,the differences were statistically significant(P<0.01).The number of autophagosomes in the MK-2206+rapamycin group increasing was observed by the transmission electron microscopy.The results of Western Blot showed that the expression levels of autophagy related proteins ATG5,ATG7,Beclin-1 and LC3 ? were increased in the MK-2206+rapamycin group4.Necroptosis mediated NB cell death induced by the combination treatment of MK-2206 and rapamycin:after NGP and BE2 cells were pretreated with necroptosis inhibitor Nec-1,the cell viability of the Nec-1+MK-2206+rapamycin group were respectively 2.4 times and 2.2 times higher than the MK-2206+rapamycin group,the differences were statistically significant(P<0.01).The typical characteristics of necroptosis,such as cytoplasm swelling and plasma membrane integrity deficiency,were only ovserved by the transmission electron microscopy in the MK-2206+rapamycin group.Western Blot results showed that the expression level of necroptosis related protein RIPK1 was decreased and RIPK3 was increased in the MK-2206+rapamycin group5.Combination treatment of MK-2206 and rapamycin induced cell death in NB tumor tissues of nude mice and autophagy and necroptosis related proteins changed:the HE staining results of NGP tumor cells in nude mice showed that the cells in the MK-2206+rapamycin group showed deeply eosinophilic dying and the nuclear shrinkage Immunohistochemistry staining showed that the expression level of RIPK1 was decreased and the expression level of RIPK3 was increased.Western Blot results showed the expression levels of autophagy related proteins ATG5,ATG7,Beclin-1 and LC3 II were increased and the expression level of necroptosis related protein RIPK1 was decreased and RIPK3 was increased6.Knocking down MYCN in NB cells attenuated the cell death induced by the combination treatment of MK-2206 and rapamycin:after knocking down MYCN in MYCN-amplified NGP and BE2 cells by transfecting siRNAs,NGP and BE2 cells were treated with MK-2206 and rapamycin in combination,CCK-8 assay results showed that cell viability increased by 35.2%(si MYCN#1)and 40.7%(si MYCN#2)in NGP cells and cell viability increased by 44.4%(si MYCN#1)and 40.7%(si MYCN#2)in BE2 cells,the differences were statistically significant(P<0.01).Western Blot results showed that after knocking down MYCN in NGP and BE2 cells,increased expression levels of ATG5,ATG7,Beclin 1,LC3 ?,RIPK3 protein decreased and decreased expression level of RIPK1 increased after the combination treatment of MK-2206 and rapamycin.7.Overexpressing MYCN in AS and SY5Y cells(MYCN-non amplified)could enhance the sensitivity to the combination treatment of MK-2206 and rapamycin overexpressing MYCN in AS and SY5Y cells by transfecting plasmid in AS and SY5Y cells,cells were treated with combinatioin of MK-2206 and rapamycin,CCK-8 assay results showed that the AS cell viability was 76.34%in MK-2206+rapamycin group and the cell viability decreased to 46.21%after overexpressing MYCN in MK-2206+rapamycin group;the SY5Y cell viability was 74.93%in MK-2206+rapamycin group and the cell viability decreased to 50.97%after overexpressing MYCN in MK-2206+rapamycin group,the differences were statistically significant(P<0.05)IncuCyte Zoom was used to observe the cell confluence of AS and SY5Y cells and the results showed that the cell confluence of the MK-2206+rapamycin group was significantly lower than the single drug group.Western Blot results showed that after overexpressing MYCN in AS and SY5Y cells,the expression levels of ATG5,LC3 ?,RIPK3 increased and the expression level of RIPK1 decreased8.Glycolysis level decreased after the combination treatment of MK-2206 and rapamycin in NGP cells:the ECAR value of NGP cells treated by MK-2206 and rapamycin in combination decreased which meant the the glycolysis level decreased 9.The expression level of MYCN was positively related to the glycolysis level and negatively related to the mitochondrial oxidative phosphorylation level:when knocking down MYCN in NGP cells,the ECAR value decreased and the OCR value increased,which indicated that the glycolysis level decreased and the mitochondrial oxidative phosphorylation level increased.When overexpressing MYCN in AS cells,the ECAR value increased and the OCR value decreased,which indicated that the glycolysis level increased and the mitochondrial oxidative phosphorylation level decreasedConclusion:1.NB cell death induced by the combination treatment of rapamycin and MK-2206 was not dependent on caspase2.Autophagy and necroptosis were involved in mediating NB cell death induced by the combination treatment of rapamycin and MK-22063.NB cell death induced by the combination of rapamycin and MK-2206 was dependent on the expression of MYCN4.The expression of MYCN was positively related to the glycolysis level and negatively related to the mitochondrial oxidative phosphorylation level.