The Combined Effect Of Dichloroacetate And Bortezomib On Human Multiple Myeloma

Section One The Combined Effect of Dichloroacetate and Bortezomib on Human Multiple Myeloma Cell Line RPMI8226Background:Multiple myeloma(MM)is a hematological malignancy,which is characterized by cloned proliferation of plasma cell population in the bone marrow.The key clinical features of MM include the production of a monoclonal Para protein,amyloidosis,renal insufficiency,anemia,increased BM angiogenesis,osteolytic bone lesions,severe bone pain,spontaneous fractures and hypercalcemia.In the past decade,there have been major advances in the treatment of MM.The introduction of novel agents such as Bortezomib(proteasome inhibitor)and thalidomide(immunomodulatory drug)has dramatically improved the outcome of MM patients.Bortezomib(PS-341),the first therapeutic proteasome inhibitor,was approved for refractory,relapsed,and newly diagnosed MM.Treatment of MM with Bortezomib has dramatically improved survival for patients with MM;however,some patients do not benefit from this treatment,and those responding ultimately acquire resistance.Combination therapy using lower doses of Bortezomib is an approach to increase efficacy and reduce side effects,thereby enhancing the likelihood of survival for MM patients.Recently,significant advances in both basic and translational research have enhanced understanding of disease pathogenesis and guided the development of new and more effective therapies.It has been proven that cancer cells including MM cells incline to obtain energy by exerting high glycolysis when compared with normal cells,even in the presence of oxygen.This phenomenon is known as Warburg effect.However,resistance to apoptosis is a survival advantage by aerobic glycolysis.Indeed,ongoing studies are investigating possible ways to exploit or interrupt tumor glycolytic metabolism in cancer cells.Dichloroacetate(DCA)is a synthesized glycolytic inhibitor,which selectively activates pyruvate dehydrogenase(PDH)by inhibiting pyruvate dehydrogenase kinase(PDK),thus converts cytosolic pyruvate to mitochondrial Acetyl-Co A,the substrate for the Krebs cycle.In this way,DCA reduces the resistance to apoptosis by inducing the conversion from aerobic glycolysis to oxidative phosphorylation.In conclusion,DCA could serve as a new therapy with Bortezomib in multiple myeloma.Objective:To determine the effect of glycolytic inhibitor DCA on the proliferation and apoptosis of human MM RPMI 8226 Cell Line,to evaluate the anti-tumor efficacy of the combination of DCA and proteasome inhibitor Bortezomib and to explore the potential mechanisms involved in these processes.Methods:1.Cell culture: MM RPMI 8226 Cell Line were cultured in vitro,and were used in the experiment in logarithmic phase.The effect of DCA and Bortezomib on the proliferation of MM RPMI 8226 Cell Line was evaluated by CCK-8 assay to determine the concentration when used in combination.2.The effect of drugs on the proliferation of Multiple Myeloma RPMI 8226 Cell Line was determined by Brd U staining: MM RPMI 8226 Cell Line were cultured and then treated by DCA or Bortezomib in single or in combination.The effect of drugs on the proliferation of MM RPMI 8226 Cell Line was determined by Brd U staining and Flow Cytometry.3.The effect of drugs on the apoptosis of MM RPMI 8226 Cell Line was determined by Annexin V-FITC/PI staining: MM RPMI 8226 Cell Line were cultured and then treated by DCA or Bortezomib in single or combination.The effect of drugs on the apoptosis of MM RPMI 8226 Cell Line was determined by Annexin V-FITC/PI staining and Flow Cytometry.4.The effect of drugs on the production level of lactate was determined by Elisa: MM RPMI 8226 Cell Line were cultured and then treated by DCA or Bortezomib in single or in combination.The effect of drugs on the production level of lactate was determined using lactate test kit.5.The effect of drugs on the expression level of Reactive oxygen species(ROS)was determined by CM-H2 DCFDA probe: MM RPMI 8226 Cell Line was cultured and then treated by DCA or Bortezomib in single or in combination.The effect of drugs on the expression level of ROS was determined using CM-H2 DCFDA probe.6.The expression levels of HIF-1α,p PDH-E1α(Ser-293),PDH-E1α(Ser-293)protein were analyzed by Western blot assay: MM RPMI 8226 Cell Line was cultured and then treated by DCA or Bortezomib in single or in combination.Protein was extracted and the expression levels of Hypoxia inducible factor-1α(HIF-1 α),Pyruvate dehydrogenase-E1αphospho S293(p PDH-E1α),Pyruvate dehydrogenase-E1α(PDH-E1α)protein were analyzed by Western blot assay.Results:1.DCA and Bortezomib could both inhibit the proliferation of MM RPMI 8226 Cell Line.The effect of inhibition on cell proliferation was obvious and equal when the concentration of DCA was 20 m M or Bortezomib was 5n M(p<0.05).This concentration level is suitable for the use in combination.2.DCA apparently inhibited the proliferation of MM RPMI 8226 Cell Line,induced cell cycle arrest at G0/G1 phase and induced the apoptosis of MM RPMI 8226 Cell Line.DCA obviously increased the sensitivity of MM RPMI 8226 Cell Line to Bortezomib.3.DCA apparently changed the metabolism of MM RPMI 8226 Cell Line by inducing the conversion from aerobic glycolysis to oxidative phosphorylation.This conversion is correlated with the degradation of HIF-1α protein,p PDH-E1α protein,lactate and the increase of ROS production.Conclusion:Glycolytic inhibitor DCA could effectively inhibit the proliferation and induce the apoptosis of MM RPMI 8226 Cell Line.Also,DCA obviously increased the sensitivity of MM RPMI 8226 Cell Line to Bortezomib.Its molecular mechanisms are associated with the degradation of HIF-1α protein,p PDH-E1α protein,lactate and the increase of ROS production.Section Two The Combined Effect of Dichloroacetate and Bortezomib on Multiple Myeloma Mouse XenograftBackground:Multiple myeloma(MM)is a malignant plasma-cell disorder that accounts for 1% of all cancers and 10%-15% of all hematologic malignancies.The use of novel therapeutic agents such as immunomodulators and proteasome inhibitors as well as hemapoietic stem cell transplantation in eligible patients has led to significant improvements in the survival of MM patients.Bortezomib is a proteasome inhibitor which has been proven to be cytotoxic to many kinds of tumor cells in vitro.Preclinical tumor xenograft in vivo has proven that Bortezomib could inhibit proliferation of tumor cells including multiple myeloma and now has been in clinical use.However,MM is still an incurable disease,with patients experiencing subsequent phases of remission and relapse,eventually leading to disease resistance and patient death when using Bortezomib in single.Dichloroacetate(DCA)is a glycolytic inhibitor,which selectively activates the oxidation of pyruvate.In Section One,we have demonstrated that using DCA and Bortezomib in combination could significantly improved the effect of Bortezomib on multiple myeloma,but its effect of combination therapy in vivo needs further investigation.Objective:To investigate the effect of glycolytic inhibitor DCA in combination with Bortezomib on multiple myeloma bearing mice in vivo and to explore the potential mechanisms involved in these processes.Provide theoretical and preclinical rationale for the combination therapy in clinical use.Methods:1.Tumor-bearing mouse model building: MM RPMI8226 Cell Line was cultured in vitro,and were injected into Non-Obese Diabetes/Severe Combined Immunodefic iency(NOD/SCID)mice subcutaneously to build multiple myeloma-bearing mouse model.2.Observe the general condition of mice,including mental status,activity,skin condition dietary and water intake.Continuously monitor the growth of tumor body growth in mice and measure its diameter regularly.3.Grouping and drug treatment: Mice were divided into 3 groups randomly and treat them by DCA,Bortezomib,DCA+Bortezomib respectively.Continue to observe the general condition of mice and measure the diameter of tumor body regularly.4.Immunohistochemical staining: Execute mice after a certain period.Separate and weigh the tumor body and then perform immunohistochemical staining to detect the expression and location of HIF-1 α and p PDH-E1 α protein.Results:1.MM mouse model was successfully built.After 7-14 days,palpable tumor was established in the left inguen of mouse.2.DCA in combination with Bortezomib could significantly inhibit the growth of tumor body and improve multiple disease parameters.3.The result of immunohistochemical staining suggests that the molecular mechanisms of combination therapy are associated with the degradation of HIF-1 α protein and p PDH-E1α protein.Conclusion:The combination therapy of glycolytic inhibitor DCA and Bortezomib could effectively inhibit the growth of tumor body in multiple myeloma bearing mice improve multiple disease parameters.Taken together,our data provide a novel insight into the potential application of DCA and Bortezomib for MM,and provides support for further clinical evaluation of the combination of DCA and Bortezomib for MM patients.