Role Of The Abnormal HIF-Glycolysis-Aerobic Oxidation Pathway In Non-hodgkin Lymphoma And The Intervention Study

Background:The rapidly dividing cells can be targeted by the traditional anti-cancer medicines.In fact,despite those quickly dividing tumor cells,there also exist many malignant cells with slow proliferation.On the contrary,plenty of rapidly deviding cells can also be found among normal tissues.Therefore,using traditional medicine merely is not powerful enough to cure cancer or what’s the worest is that it may also have inevitable side effects on normal cells.How to improve the target activity and the efficacy of anticancer medicines and how to enhance the security are becoming more and more important.In 1920s,Warburg effect pointed out the special metabolic character of cancer cells:even under aerobic surroundings,tumor cells still depend on glycolysis to satisfy its energenic and biosynthetic demand rather than aerobic oxidation.The casual relationship between the aerobic glycolysis and the tumorigenesis remains controversial.The research of exploring the role of the obsessively activated glycolysis in lymphoma has been progressing.In addition,some studies have verified that the special metabolic phenotypes of cancer cells are regulated by PI3K/Akt/mTOR pathway,HIF-1αand the oncogene C-myc.It’s an active regulation rather than a passive course forced by the injury of the mitochondria.Interestingly,this course is reversible.Purpose:We imply that we can reduce the anti-cancer drug dose to minimize side effects and enhance the efficacy through inhibiting glycolysis by using glycolysis inhibitor or inhibiting PI3K/Akt/mTOR pathway.By virtue of the reversibility of the metabolism phenotype of cancer cells,both the inhibitors of the aerobic oxidation and glycolysis can be tested on malignant cells in order to attenuate the dual metabolitic pathway of cancer cells.Therefore,the resources of energy and substance for high biosynthesis can be completely blocked in consequence.Three sections are included in this thesis:in the frist place,to explore the clinical prognostic significance of HIF-1αand the critical proteins referring to glycolysis and aerobic oxidation among DLBCL patients;secondly,to study the mechanisms of the two inhibitors,glycolysis inhibitor 2DG and mTOR inhibitor RAPA,in sensitizing the SUDHL-4（DLBCL cell line）and Raji（BL cell line）to DOX,and to explore the efficacy when combining RAPA,OM and DOX on Raji and SUDHL-4 cells.At last,to look inward the way of BTZ,one kind of protesome inhibitor,working alone or in combination with OM on SUDHL-4 and Raji cells.Methods:To study the prognostic significance of the abnormal HIF-1α-Glycolysis-Aerobic Oxidation pathway in DLBCL,the immunohistochemical results,the serum biochemical parameters and PTE/CT values of all 83 patients were retrospectively analysed,and the evaluated HIF-1αand HKⅡ,which are critical to glycolysis,and SDHA,an important enzyme among TCA,were being under prognostic analysed as well.In order to verify whether 2DG and RAPA could sensitize the DLBCL and Burkitt’s lymphoma cell lines to DOX,to study how glycolysis inhibition plays its role in the efficacy of RAPA and to explore whether inbibiting both the glycolysis and aerobic oxidation by using RAPA and OM could extremely sensitize NHL cell lines to DOX,SUDHL-4 and Raji cells were treated with different concentrations of OM and RAPA.In addition,the normal human peripheral blood lymphocytes were also treated in the same way as a control.Next,the NHL cells were then treated with the combination of DOX and 2DG or RAPA,respectively,and even the combination of DOX,RAPA and OM.Cell proliferation and cytotoxicity were detected by CCK-8.Glucose consumption,lactic acid and succinate generation were measured by corresponding metabolic kits.Effects on gene transcription and protein expression related to metabolic pathway were determined by RT-qPCR and Western blot,respectively.Apoptosis and cell cycle distribution were analysed by flow cytometry（FCM）.To study the effects of the combination of BTZ and OM on NHL cell lines,different concentrations of BTZ alone or combination with OM were applied on both SUDHL-4 and Raji cells.Cell proliferation and cytotoxicity were detected by CCK-8.Concentrations of glucose,lactic acid and succinate were measured by corresponding metabolic kits.Effects on gene transcription and protein expression related to metabolic pathway were determined by RT-qPCR and Western blot,respectively.Apoptosis was analysed by flow cytometry（FCM）.Results:The retrospective analysis of the 83 newly diagnosed DLBCL patients showed that:Ki67,LDH,SUVmax,IPI,Ki67^hiBCL-2⁺,HIF-1α,HKⅡand SDHA were significantly correlated with both PFS and OS under the single factor analysis;when divided into two groups,the H-H-S group(HIF-1α⁺HKⅡ^hi SDHA^lo),meaning all HIF-1α,HKⅡand SDHA are abnormaly expressed,and the non H-H-S group,the prognostic risk factors such as LDH,Ki67,SUV_max and IPI differed significantly between the two groups（P<0.05）.The three-year progression-free survival rates among H-H-S and non H-H-S groups were 14.3%and 78.3%（P=0.001）and the three-year overall survival rates in H-H-S and non H-H-S groups were 14.3%and 89.9%（P=0.000）,respectively.The IC₅₀ valuesof RAPA were（148.198±5.004）nM and（139.866±6.812）nM,and OM were（0.306±0.3005）μg/ml and（0.331±0.2040）μg/ml for Raji and SUDHL-4 cells,respectively.However,the IC₅₀ values of RAPA and OM were（608.698±20.324）nM and（2.058±0.9320）μg/ml for the normal lymphocytes,respectively.RAPA,similar to 2DG,when combined with DOX could synergistically hamper the proliferation,the glucose consumption and the generation of lactic acid in lymphoma cells.Given the fact that RAPA owned a stronger inhibitive effect on glycolysis compared with 2DG when combined with DOX,we applied RAPA as glycolysis inhibitor in the subsequent experiments.Through combining RAPA and OM with DOX to realize the ambition of inhibiting the dual metabolic pathway simultaneously,more severe inhibitory effects above mentioned were observed.BTZ,when used individually,could effectively inhibit the cell proliferation of Raji and SUDHL-4 cells in a does-dependent way and suppress gene transcription and protein expression of c-MYC,HIF-1α,VEGF and GLUT1 at various degrees.In addition,key enzymes and proteins related to glycolysis,such as HKⅡand LDHA,and SDHA referring to aerobic oxidation were suppressed by BTZ.The consumption of glucose and the generation of lactic acid and succinate were also inhibited at the same time.FCM showed that BTZ could also induce apoptosis in both of the two cell lines.Furthermore,when BTZ was used in combination with OM,significantly synergistic effects were observed.Conclusion:It was the abnormal HIF-1α-Glycolysis-Aerobic Oxidation pathway featuring HIF-1α,HKⅡand SDHA that could tell the different prognosis among the newly diagnosed DLBCL patients.2DG,the glycolysis inhibitor,RAPA referring to inhibiting mTOR and BTZ could inhibit glycolysis successfully.When 2DG or RAPA was applied in combination with DOX,each inhibitor could sentisize the NHL cells to the anti-cancer drug and achieve a prominent glycolysisDinhition effect.Furthermore,the involvement of OM enhanced the extent of inhibiton mentioned above.The mechanism of this synergy phenomenon can be partially explained by attenuating both the glycolysis and the aerobic oxidation pathway.All in all,inhibiting the dual metabolic pathway provide a new strategy to cure the NHL.The abnormal HIF-1α-Glycolysis-Aerobic Oxidation pathway might act as a vital feature in NHL.RAPA in combination with OM may serve as a new adjunct therapeutic strategy for NHL.