| BackgroundMultiple myeloma(MM)is a malignant hematologic disease featured by abnormal proliferation of monoclonal plasma cells.MM patients have several major clinical symptoms,including increased blood calcium levels,renal insufficiency,anemia and bone lesions.In hematological tumors,the incidence of MM ranks second,only to lymphoma.In recent years,the incidence rate has increased.It has seriously affected people’s survival and quality of life.MM nowadays remains incurable,although novel treatment options(bortezomib,lenalidomide,daratumumab and others)have contributed to a doubling in the average life expectancy of MM patients.Therefore,a new pathophysiological mechanism and therapeutic target of MM hope to provide a potential strategy for finally overcoming the disease.In recent years,the abnormally high metabolic characteristic of tumor cells is different from normal cells.Glucose metabolism provides the necessary energy for tumor cells.Exploring glycolytic targets may be the new direction for treating MM.Studies have shown phosphofructokinase-1(PFK-1)is one of the rate-limiting glycolytic enzyme which turns fructose 6-bisphosphate(Fru-6-P)into fructose 1,6-bisphosphate(Fru-1,6-P2).The subtype of phosphofructokinase-2/fructose-2,6-bisphosphatas PFKFB3,which promotes the synthesis of Fru-2,6-P2 accelerates the glycolysis and tumor cell proliferation.PFKFB3 is highly expressed in many tumors,such as gastric cancer,breast cancer,head and neck cancer and so on.The increased protein is closely related to the poor prognosis.Our previous study found that PFKFB3 was also highly expressed in MM cell lines,and inhibition of PFKFB3 affected cell proliferation and promoted cell apoptosis.However,the influence on MM glycolysis and the signal network interacting with it are not clear.Therefore,this study further explores the influence of PFKFB3 on the survial of MM and its mechanism to provide a new glycolysis-targeted theory for MM treatment.Objective1.To clarify the expression of PFKFB3 in primary MM cells(patient bone marrow CD 13 8+cells)and MM cell lines;2.Inhibition of PFKFB3 enzyme activity to clarify its effects on glycolysis,growth and proliferation of MM cell lines;3.The lentiviral vector overexpressing PFKFB3 was transfected into MM cells to investigate the specific mechanism of PFKFB3 on MM cells.4.In vitro and in vivo experiments further explore the mechanism of PFKFB3 on MM growth and proliferation.Methods1.Western blot and qRT-PCR were used to detect the expression of PFKFB3 in primary MM cells and MM cell lines(RPMI8226,OPM2,ARP-1,MM.IS)2.The effects of PFKFB3 inhibitor PFK15 on the growth,proliferation and apoptosis of MM cell line were detected by CCK-8 method and trypan blue exclusion test.The inhibition of glycolysis level was detected by the related kits.3.Using the lentiviral overexpressing PFKFB3 to transfect MM cell lines(RPMI8226,OPM2),combined with inhibitor PFK15 to explore the signal network pathway involved in PFKFB3;4.Western blot and qRT-PCR were used to detect the expression of PFKFB3 in MM cells immersed in high glucose medium.The effect of metformin on PFKFB3 was detected by gene chip and Western blot.The combined effect of PFKFB3 inhibitor PFK15 and metformin was detected by CCK-8 method.The compusyn software was used to verify the combined effect:combination index(CI)<1 for synergy,=1 for additive effect,and>1 for antagonism.Flow cytometry was used to verify MM cell apoptosis with the two drugs combination.5.A NOD-SCID mouse model of myeloma was established with subcutaneous injection of MM cell line RPMI8226(1×107).PFK15 and metformin were used to inhibit PFKFB3 to test changes of tumor volume.The tumor marker proteins and related signaling pathway proteins were confirmed by immunohistochemical staining.Results1.The PFKFB3 expression was high in primary MM cells and MM cell lines(RPMI8226,OPM2,ARP-1,MM.1 S).2.PFKFB3 inhibitor PFK15 significantly inhibited the activity and growth rate of MM cells.The ability of glucose uptake and Fru-2,6-p2 production decreased,indicating that the glycolysis ability was inhibited.3.Flow cytometry AnnexinV/PI results showed that apoptosis increased with raised PFK15 concentration(P<0.05).Cell cycle was arrested in S phase.Tunel assay also confirmed the apoptosis effect of PFK15.After PFKFB3 was inhibited,Western blot results showed that the expression of Caspase3,Bak,parp-1 and P21 increased,CyclinDl and Mcl-1 decreased.4.Using pfkfb3-overexpressing MM cell line binding inhibitor PFK15 showed that PFKFB3 was positively correlated with theMAPKs/STAT1 signaling family.P38 expression was changed by the P38 inhibitor LY2228820,but PFKFB3 was not affected.The results showed that MAPKs signaling pathway was located in the downstream of PFKFB3.In addition,PFKFB3 was inhibited,DNA damage proteinγH2AX was increased5.The expression of PFKFB3 in MM cells immersed in high glucose medium was high.metformin and PFK15 inhibitor was synergistic(CI<1)analyzed by CCk-8 method,flow cytometry and compusyn software.6.The results of the in vivo experiment showed that PFKFB3 inhibitor PFK15 inhibited tumor growth,and the inhibitory effect was more significant when combining with metformin.The results of immunohistochemical staining were also consistent.Conclusions1.PFKFB3 is expressed in both myeloma cell lines and primary MM cells.2.PFKFB3 is inhibited to promote MM cells apoptosis and cell cycle is arrested in S phase.Glycolysis ability decreases,and glycolytic protein GLUT1 and HK2 are upregulated by a negative feedback action.3.PFKFB3 is related to MAPKs/STAT1 signal pathway and DNA damage mechanism when works on MM cells.4.PFKFB3 is highly expressed in MM cells especially immersed in high glucose environment.Metformin inhibits the PFKFB3 expression.It is further found that PFKFB3 inhibitor PFK15 has a combined effect with metformin.5.PFKFB3 inhibition,PFK15 combined with metformin could lower tumor burden significantly than single drug in xenograft model... |
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