| Energy metabolism reprogrammng(EMR),which represents rewiring their metabolism and energy production networks,favors cancer cells to adapt to the stressness in tumor microenvirment and to grow aggressively.Many studies have found that most of cancer cells are highly dependent on glucose for energy resource in the normal oxygen environment.The metabolic character of cancer cells is called “Warburg effect”.Aberrant cancer cells metabolism has been proven a promising anti-tumor agent in cancer therapy.However,there has been lack of an applicable therapy targeting cancer metabolism in clinic.The ketogenic diet(KD),comprised of high-fat,low-carbohydrate and appropriate protein,is studied extensively in the management of epilepsy for decades.Recently,many studies have shown that the KD exhibits anti-cancer effect on various tumors,including glioblastoma.However,the mechanisms of the KD in modulating GSCs and application of assessment have not been well understood.In this study,we investigated the therapeutic effects of the KD on recurrent GBM patients,and investigated the functions and regulatory mechanisms of ?HB in GSCs,the major component of ketone bodies in vitro.By evaluating inhibitory effects on GSCs,as well as glycolysis metabolism and the function of mitochondria,we elucidated the mechanism of the KD in GSCs.The experiment is divided into six parts:Part I: The therapeutic effect of the KD in three recurrence glioma patientsObjective: To evalutate the therapeutic efficacy,the clinical feasibility and the side effects of the KD in patients with recurrent glioma.To observe the differences in glycolytic metabolic pathways between malignant glioma tumor tissues and normal tissues.Methods: According to the KD clinical studies' enrollment and exclusion criteria,3 cases of relapsed gliomas in our hospital were treated with the KD,observing the reaction to the KD treatment and various physiological indicators;1.The expression of Glut1,HK1,HK2,PFK,PKM2,PKM1/2,LDHA and PDHA were detected bywestern blot asssyas in 3 cases of malignant glioma tumor tissues and normal brain tissues around the lesions.2.Graph Pad Prism 5.01 software was applied to count and analyze experimental dataResults: 1.After the KD therapy,2 of subjects suffered from hypoglycemia,elevated blood ketone bodies(2.0-3.8 mmol/L)and urinary ketone bodies(++-++++),but decreased blood glucose(5 ~ 6mmol/L).During the treatment of the KD,all patients victimed weakness and early gastrointestinal reactions;1 of these 3 patients developed joint pain which was induced by hyperkinesia;the symptom was relieved after treated with allopurinol.Two patients adhered to the treatment for 4 months.There was no evidence of recurrence during the treatment.One patient received the KD for 3 months.However,due to rapid tumor progression,the treatment was terminated.2.Western blot analysis showed that except from HK1,the expression of Glut1,HK2,PFK,PKM2,and LDHA,which were key enzymes in the glycolytic pathway,werehigher in glioma tissues than in normal tissues.However,PDHA,an enzyme that linked the mitochondrial metabolic pathway,was lowly expressed.These indicated that glioma tumor tissues were more dependent on the glycolytic pathway to maintain cell growth.Conclusion: 1.The KD are safe and feasible for the patients with recurrent glioma.They have a therapeutic effect on some gliomas,but can not be used as a single treatment program for malignant gliomas patients.2.Compared with normal brain tissues,glioma tumor tissues aberrantly increase the expression of glycolysis pathway.Part ?: The inhibitory effect of the KD on cell proliferation in GSCsObjective: To evaluate the influence of the ketogenic microenvironment on the proliferation of glioma stem cell line NCH421 k cells and two cases of primary isolated cultured glioma stem cells GSC#Y1,GSC#Y2.Methods: Different concentrations of ?-hydroxybutyric acid(?HB)(concentration: 0 m M,1 m M,5 m M,10 m M,25 m M)were added to low-glucose(2.5 m M glucose)stem cell culture medium(Glow),and normal stem cell culture medium(glucose concentration = 25 m M)was set as the control group.The colony formation assay,CCK8 cell viability assay,and Annexin V-FITC/PI staining were performed to observe effect of ?HB-Glow in cell proliferation after culturing for 7 days.NCH421 k cells at different time points(5d,6d,7d)were detected by Edu assays.At the same time,normal mouse neural stem cells(NSCs)were taken to observe the effect of different concentrations of ?HB on the neurosphere formation ability.Results: 1.Compared with the control group,both the number and diameter of tumorspheres in ?HB-Glow were decreased dramatically.2.Edu assays found that the percentage of Edu-positive cells in the ?HB-Glow groups decreased in a time-depended manner in the NHB421 k cells.Edu positive cells weredetected in the 25 m M ?HB-Glow treatment group at 5,6,and 7 days.Edu positive percentages were: 24.01±6.17%,21.61±5.73%,and 12.48±0.65%,seperately.3.CCK8 assays showed that in NCH421 k cells,the OD values in the ?HB-Glow treated group were lower,comparing with the control group and the Glow group.4.Annexin V-FITC/PI double staining flow cytometry assays revealed that the percentage of apoptosis in NCH421 k cells were increased in the ?HB-Glow treated groups.The percentage of apoptosis in the ?HB-Glow treated groups(including early and late apoptosis)): 0 m M group 51.87±14.39%,1 m M group 60.40±11.30%,5 m M group 51.30±7.11%,10 m M group 69.23±10.30%,25 m M group 62.47±9.35%,and most of apoptosis were appeared in the period of early apoptosis.5.In NSCs,after cultured with different concentrations of ?HB(?HB concentrations: 0 m M,1 m M,5 m M,10 m M,25 m M,50 m M)for 7 days,the number of neurospheres did't decrease.On the contrary,?HB-Glow increased the number of neurospheres along with increasing concentrations of ?HB.Conclusion: The ketogenic microenvironment can inhibit cell proliferation and induce cell apoptosis in GSCs without affecting the proliferation in NSCs.Part ?: The inhibitory effect of the KD on stemness phenotype in GSCsObjective: To evaluate inhibitory effect of the ketogenic microenvironment on self-renewal ability in GSCs.Methods: NCH421 k cells treated with different concentrations of ?HB-Glow(?HB concentration: 0 m M,1 m M,5 m M,10 m M,25 m M)were replanted in the same ?HB-Glow.The colony formation assays were performed to evaluate the self-renewal ability in NCH421 k cells.In addition,CD133 staining and Sox2 expression were detected to observe the expression of stemness markers in ?HB-Glow.In vivo,NCH421 k cells in both the control group and ?HB-Glow groups were injected intracranially in situ to observe the tumorigenicity and tumor volume.Results:1.Compared with the culture group,both the number and diameter of secondary tumorspheres in the ?HB-Glow group were decreased.2.CD133 flow cytometry results showed that the percentage of CD133 positive cells in NCH421 k cells in 10 m M ?HB-Glow group were decreased,comparing with these in the control group and the Glow group.The real-time PCR and western blot assays indicated that the expression of the transcription factor Sox2 in NCH421 k cells was inhibited in ?HB-Glow.3.In vivo,NCH421 k cells in the ?HB-Glow group exhibits a poor tumorigenic ability(tumor/mice=1/4).Conclusion: The ketogenic microenvironment can inhibit the maintenance of stemness phenotype in GSCs.Part ?: The inhibitory effect of the KD on glycolysis metabolism in GSCsObjective: To evaluate inhibitory effect of the ketogenic microenvironment on glycolysis metabolism in GSCs.Methods: NCH421 k cells were divided into three groups: the control group,the Glow group and the ?HB-Glow group cultured for 7 days.Cell metabolism was detected by ATP assays,quantitative real-time PCR and western blot assays were used to detect the expression of metabolic enzymes.The activity of glycolytic enzymes(PFK,PK,and LDH)and lactic acid production were measured.The function of glucose transporter was analyzed by 2-NBDG staining.Results: 1.ATP assays showed that the intracellular ATP content in NCH421 k cells was decreased in ?HB-Glow,and the relative value of ATP content in NCH421 k cells was 0.068±0.11.2.Quantitative real-time PCR showed that the expression levels of Glut1,PFKM,and PDHA were inhibited in ?HB-Glow.Further,western blot assays found that in addition to PFK,the relative protein expression levels of the metabolic enzymes Glut1,HK1,HK2,PFK,PKM2,LDHA,and PDHA in the ?HB-Glow group were decreased,comparing with the control group.In addition,2-NBDG staining showed that the percentage of 2-NBDG positive cells in the Glow group were decreased from 70.28±15.45% to 47.35±18.56%.Also,the activity of PFK,PK and LDH in the ?HBGlow group were inhibited,comparing with the control group.3.We detected an increase of the intracellular production of lactate in the ?HB-Glow group.Conclusion: The ketogenic microenvironment can inhibit glycolysis metabolism and increase the intracellular production of lactate in GSCs.Part?: The effect of the KD on cell mitochondria in GSCsObjective: To evaluate the effect of the ketogenic microenvironment on cell mitochondria in GSCs.Methods: NCH421 k cells were divided into three groups: the control group,the Glow group and the ?HB-Glow group cultured for 7 days.Transmission electron microscopy(TEM),JC-1 staining,TMRM staining and ROS probe labeling combined with flow cytometric analysis techniques were introduced to observe changes in cell mitochondrial function.Further,Mitro Tacer/ROS probe was combined with flow cytometry to observe changes in mitochondrial ROS production.Also,CD133/ROS/PI staining were used to observe changes in ROS generation in CD133-positive cells.In addition,western blot assays were introduced to detect the expression of related signaling pathway protein p-m TOR/m TOR,Hif-1a and Bcl2.Results: 1.TEM and JC-1 staining showed that the mitochondrial structure and function of NCH421 k cells in ?HB-Glow were damaged after ?HB-Glow treatment.2.We observed the culture environment of ?HB-Glow increased the intracellular ROS production from 16.18±9.85% to 23.89±9.58%,which were generated from cell mitochondria by the double staining Mitro Tracer/ROS.Further,TMRM staining and triple-labeled CD133/ROS/PI staining assays showed that the culture environment of ?HB-Glow inhibited the population of stemness-like cells in NCH421 k cells.3.Western blot analysis revealed that the protein expression of p-m TOR,Hif-1?,and mitochondrial anti-apoptotic protein Bcl2 in the ?HB-Glow group were decreased,comparing with the control group.Conclusion: The ketogenic microenvironment can damage cell mitochondria and increase the ROS production,which in turns,affecting cell proliferation in GSCs by regulating the expression of transcriptional regulatory molecules in related signaling pathway.Part?: The mechanism of the inhibitory effect of the KD in GSCsObjective: To elucidate the mechanism of the ketogenic microenvironment in GSCs.Methods: N-acetylserine(NAC)was used to scavenge ROS in NCH421 k cells and primary glioma stem cells GSC#Y1,GSC#Y2.After ROS scavenged,cell proliferation was detected by colony formation assays,CCK8 cell viability assay,and Edu staining.Also,ATP detection and 2NBDG staining were used to observe the changes in cellular metabolic function after ROS scavenged.TEM was introduced to observe the mitochondrial structural changes after ROS scavenged.In addition,western blot assays were used to detect the protein expression of cell stemness marker Sox2,key enzymes in cell metabolism,including Glut1,HK1,HK2,PFK,PKM2,LDHA,PDHA,and the signaling pathway protein molecules p-m TOR/m TOR,Hif-1a.Results: 1.After abrogating ROS in cells,the proliferation of GSCs in the ?HB-Glow group was restored,as both the number and diameter of the tumorspheres were increased compared with non-NAC treated group.CCK8 assays showed an increase in the OD value in the ?HB-Glow group,which were in accordance with Edu assays.2.Western blot assays were detected that the expression of cell stemness marker Sox2 were recovered after abrogating ROS.3.TEM revealed that the mitochondrial morphological structure was recovered after the removal of ROS.4.The ATP production and the protein expression of glycolysis key enzymes weren't increased in the ?HB-Glow group,which were accordance with the protein expression of signaling pathway molecules p-m TOR/m TOR,Hif-1a after ROS scavenged.Conclusion: The ketogenic microenvironment can inhibit cell proliferation,self-renewal ability and glucose transporter function by increasing ROS production in GSCs through mitochondrial damage.In addition,the ketone bodies may have an inhibitory activity in GSCs. |
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