PFKFB3 Antagonist PFK15 Inhibits Autophagy And Proliferation In Rhabdomyosarcoma Cells

BackgroundRhabdomyosarcoma(RMS)is a kind of heterogeneous soft tissue malignancy characterized by skeletal muscle development,which originates from the mesenchymal tissue.Currently,the strategy of surgery combined with chemotherapy or radiotherapy remains the Standander treatment for rhabdomyosarcoma.However,in recent years,there has been no improvement in patients' 5-year survival rates.For the treatment on patients with rhabdomyosarcoma,drug resistance and metastasis are the two most common phenomena of treatment failure.Although some randomized chemotherapy trials have been introduced to explore new or more intensive treatment strategies,their efficacy has not been significantly improved.Therefore,more effective treatment strategies were urgently needed in clinic.Recent studies have investigated the pathogenesis of rhabdomyosarcoma from the viewpoint of molecular cell biology and molecular cytogenetics,and have further found the mechanism of the tumor.Autophagy is a complex self-digestion and balance of cell metabolism,which can be divided into micro-autophagy,macrophage and chaperone-mediated autophagy.Cells self-recognition,transport dysfunction and disuse organelles from small protein molecules to large mitochondria to the lysosome.Therefore,autophagy maintains the homeostasis of the intracellular environment based on constantly updated the function of elements in cells.This helps cells eliminate pathogens,accumulate proteins and damaged mitochondria.Autophagy also could provide amino acids for the body's metabolic needs.In recent years,autophagy has close relationship with other metabolic reactions in cells,such as cells mitosis,migration,glucose metabolism,inflammatory reaction and tumor biological behavior.Autophagy not only can promote the occurrence of tumors,but also inhibit tumorigenesis.So the relationship between autophagy and cancer is complex.Autophagy exerts its role as a tumor suppressor by clearing the damage and toxic components within the cell.However,once a tumor has formed,the survival characteristics of this cell's autophagy are transformed into unfavorable factors such as autophagy that allows tumor to adapt to energy compromise,emergency factors and the anti-chemotaxis effect.Therefore,the inhibition of autophagy may play a positive and effective role in the treatment of tumors.Tumor cells tend to convert most glucose to lactate even under aerobic conditions which were defined as aerobic glycolysis.This phenomenon was first found in tumor cells,called the Warburg effect.Although it is recognized as the need for a large number of biological raw materials for rapidly proliferating cells,the current mechanism remains unclear.Glycolysis could provide the basic ingredients needed for biosynthesis for the tumor cells.PFKFB3(6-phosphofructo-2-kinase)is a key regulator of glycolytic high-flux cancers through the catalytic synthesis of F26P2,which allosterically activates PFK-1,the rate-limiting enzyme for glycolysis.The PFKFB3 protein is overexpressed in a variety of cancers,including breast,prostate,colon,astrocytoma,ovarian,pancreatic and gastric cancers,whose expression or activity is associated with aggressiveness and poor prognosis of cancer.PFKFB3 could rapidly proliferat and express in tumor tissues by mitogenic stimuli activation.Studies confirm that PFKFB3 can be located in the nucleus,indicating the gene plays an important role in cell proliferation in a glycolysis-dependent manner.Inhibitors of PFKFB3 have also been developed,for example,3-(3-pyridinyl)-1-(4-pyridinyl)-2--2-propen-1-one,3-PO).However,their pharmacological activity was more than the inhibition activity on PFKFB3 protein,therefore,it is necessary to develop PFKFB3 inhibitors with low general toxicity and high selectivity for PFKFB3.As a novel PFKFB3 inhibitor,PFK15 has potent antitumor activity and can significantly reduce the uptake of 18FDG and the level of F2,6BP in grafted tumors.In addition,PFK15 plays important pro-apoptotic roles on cells transformation and tumors in vivo.In our study,we found that PFK15 could inhibit the basic autophagy of RD cells,accompanied by the inhibition of AMPK(AMP-activated protein kinase)signaling pathway.It has been reported that AMPK signal played a bidirectional role both on promoting and inhibiting autophagy.We often can observe the abnormality of the nucleus under fluoroscopy and electron microscopy when observing the autophagy induced by PFK15.Small nucleus,nucleoplasm and nuclear sprouting are common markers of nuclear damage,which also were phenotypes of cytogenetic damage.The study could contribute to a better understanding of the mechanism of cell autophagy regulation and on tumor survival based on the associations between aerobic glycolysis and autophagy,autophagy and AMPK signaling pathway.Moreover,the stability of the nucleus was also observed.PFK15,as the main research object,might provide a new target and theoretical basis for the treatment of rhabdomyosarcoma.Objective1.To study the significance of glycolysis in tumorigenesis and evaluate the efficacy of PFKFB3 inhibitor PFK15 on glycolysis of rhabdomyosarcoma cells.2.To evaluate the effect of PFK15 on rhabdomyosarcoma cell proliferation and cell viability.3.To investigate the effect of PFK15 on autophagy activity and signaling pathway in rhabdomyosarcoma cells and its mechanism.4.Explore the effect of PFK15 on nuclear damage and apoptosis of rhabdomyosarcoma.5.To study the effect of PFK15 on glucose metabolism and autophagy signaling pathways and their mechanisms.6.Explore the role of PFK15 in inhibiting rhabdomyosarcoma in vivo.Research methods1.Human rhabdomyosarcoma cell line cultured in vitro with different concentrations of PFK15 was used to determine the effect of PFK15 on glycolysis.The best concentration of PFK 15 was determined by measuring the amount of lactic acid released from the culture supernatant.2.The best concentration of PFK 15 was used to rhabdomyosarcoma cells.The MTS colorimetry,clone formation assay and immunoblotting were used to evaluate the effect of PFK15 on proliferation,viability and signal pathway of tumor cell lines.3.Use of PFK 15 with optimal concentration on rhabdomyosarcoma cells,the effect of PFK15 on autophagy detected by fluorescence microscopy,Western blot,and transmission electron microscopy.The autophagy inhibitors CQ and autophagic substrates SQSTM1/p62 were analyzed to detect the cell autophagy flow.4.The autophagy suppression model was established by silencing the Atg5 and Atg7 genes with siRNA.The effect of PFK15 on cleavage of the caspase-dependent apoptosis marker PARP-1 was evaluated by Western blotting.5.To evaluate the effect of PFK15 on the expression level of phosphorylated Akt(pAkt)in rhabdomyosarcoma cells and the effect on expression levels of phosphorylated AMPK(pAMPK)and its substrate phosphorylated ACC(pACC).The inhibition of pAMPK and its substrate pACC in rhabdomyosarcoma cells was compared with 3Po.6.Treatment of rhabdomyosarcoma cells with AMPK signaling agonist AICAR before PFK15 treatment.MTS colorimetric assay,clone formation assay and Western blotting,observed the effect of PFK15 on habdomyosarcoma cell activity,autophagy and signal Path effect.7.Based on the autophagy inhibition model of siRNA silencing Atg5 and Atg7,the expression levels of RAD51 and H2AX were evaluated by immunoblotting.8.A solid tumor model of nude mice was constructed to observe the effect of PFK15 on the proliferation in vivo.Results1.PFK15 significantly inhibited glycolysis in rhabdomyosarcoma cells in a dose-dependent manner.Compared with the control group,4 ?M and 6 ?M PFK15 significantly reduced lactate production by 20%and 27%.Therefore,the optimal concentration of PFK15 on rhabdomyosarcoma cells treatment was 6?M.2.PFK15 inhibits colony growth and causes abnormal nuclear morphology.The integrity of the nuclear membrane of PFK15-treated cells was disrupted,and the nuclear morphological changes including protrusions,nuclear condensation,chromosome aggregation into chromatin were detected in the nucleus.3.Cell viability was measured by MTS assay.PFK15 significantly reduced the survival rate of rhabdomyosarcoma cells compared with the control group.When treated with 3BP(25 ?M),cell viability was also significantly reduced.In addition,when cells were treated with PFK15 in combination with 3MA(1 mM),cell viability was further reduced,exhibiting a stronger synergistic effect.4.PFK15 significantly attenuated the accumulation of membrane vacuoles.In immunoblot experiments,PFK15 did not significantly increase LC3-? protein levels,or not promote the degradation of autophagic substrate p62.The level of LC3-? in PFK15 combined with autophagy inhibitor CQ was not increased further than PFK15 alone group,and there was no change or even decrease in autophagic substrate p62,indicating that PFK15 inhibited autophagic flow.5.Western blot analysis was performed to assess the effect of PFK15 on PARP-1 and PFK15 was found to induce cleavage of PARP-1 in a dose-dependent manner.Cleavage of PARP-1 is reduced when Atg5 and Atg7 were silenced.6.PFK15 inhibited the levels of phosphorylated AMPK and its substrate pACC in tumor cells by 44%and 20%,respectively.Similar results were observed with 3PO(another inhibitor of PFKFB3).PFK15 could significantly upregulatethe expression of p-Akt that inhibits autophagy by activating mTOR complex I.7.PFK15 also activates the negative regulator of autophagy mTOR complex I and pAkt signaling pathway,thus inhibiting the autophagy of RD cells.8.Compared with PFK15 alone,AMPK agonist AICAR attenuates PFK15 activity loss,autophagy inhibition and signal pathway regulation in RD cells.9.Based on the autophagic inhibition model of Atg5 and Atg7 knockdown,PFK15 affected the expression levels of RAD51 and H2AX,which may be the cause of nuclear damage.10.PFK15 injection significantly reduced the volume and weight of solid tumors in vivo.Conclusion1.PFK15 reduces glycolysis in rhabdomyosarcoma cells.2.PFK15 can reduce the survival rate of rhabdomyosarcoma cells and inhibit the formation of cell clones.3.PFK15 treatment increased the micronucleus phenomenon of RD cells,and inhibited the expression of RAD51 and increased the level of H2AX protein,which is the expression of DNA damage.4.PFK15 can significantly inhibit tumor growth in vivo.5.PFK15 inhibits the autophagic flow of RD cells.6.PFK15 can induce apoptosis of RD cells,which can be inhibited by the inhibition of early autophagy,which means that autophagy plays a protective role.7.PFK15 inhibits the AMPK signaling pathway in RD cells.8.PFK15 can significantly up-regulate p-Akt expression and p-mTOR levels.9.The application of AMPK agonists can attenuate the inhibitory effects of PFK15 on cell viability,autophagy,and AMPK.PFKFB3 may serve as an upstream functional regulator of AMPK.