The Mechanistic Insight Into ROS Production By Lactate Dehydrogenases In Cancer Cells

Increased hydrogen peroxide(H2O2)production is a characteristics of cancer cells,but the underlying mechanism is not completely understood.According to current understanding,H2O2production in cancer cells involves 2 steps:mitochondrial electron transporter chain(ETC)and enzymes likes NADPH oxidases generate superoxide radical,then mitochondrial superoxide dismutase(SOD)2 and cytosolic SOD 1 catalyzes the disproportionation of superoxide,converting two molecules of superoxide to one molecule of H2O2 and one molecule of molecular oxygen.Previously,it has been reported that rabbit lactate dehydrogenase(LDH)could catalyze one electron reduction of NADH to produce H2O2,with the superoxide as an initiator of the reaction.Although these studies built solid theoretical basis for LDH-involved H2O2 production,it has not attracted much attention since then.Up to date,it is unclear if LDH-catalyzed production of H2O2 is physiologically relevant.In theory,LDH-catalyzed H2O2 production should also present in cancer cells,on the following basis.First,superoxide radical,the initiator and amplifier,can be generated from various sources,such as mitochondria.Second,most cancer cells exhibit very high glycolysis rate,termed Warburg effect,which can efficiently cycle NAD~+to NADH,which is a substrate to maintain the free radical chain reactions catalyzed by LDH.Finally,increased level of LDHA is characteristic of many tumors.Based on the abundant sources of superoxide,adequate flux of NADH generation,and very high activity of LDH in cancer cells,we propose that LDH may contribute significantly to H2O2 production and oxidative stress in cancer cells in vitro and in vivo.To verify the hypothesis,we performed following experiments.1.H2O2-generation activity of LDH.The difference of the catalytic rate of LDHA and LDHB in H2O2 production is unknown.LDHB per unit wise displayed significantly higher activity than LDHA with respect to H2O2 generating.H2O2 generation rate was dependent on the concentration of LDH enzymes and the concentration of NADH.The H2O2 generation was inhibited by LDH inhibitor FX11(NAD binding site)and oxamate(pyruvate binding site),with the former being a significantly stronger inhibitor than the latter,suggesting that the enzyme catalytic core was necessary to H2O2 generation activity.Mitochondria isolated from cancer cells,normal tissues or solid tumors enhanced the hydrogen peroxide-producing activity of LDHA and LDHB.In human cervical cancer cell line He La and mouse breast cancer cell line 4T1,LDHA or LDHB knockout significantly reduced H2O2production.Re-expressing LDHA in LDHA-knocking out cell restored to a large extent H2O2production.Collectively,the data demonstrated that superoxide generated by mitochondria could initiate LDH to generate hydrogen peroxide in both the cell-free system and in cancer cells.2.The antioxidative and prooxidative activity of LDH in cancer cells.The effect of LDH knockout on cellular H2O2 production indicated that LDH functions as a pro-oxidant.On the other hand,the effect of perturbation of LDH on oxidative stress is a more complicated issue,when total ROS is concerned,as H2O2 only represents one species of numerous ROS.Previous reports demonstrated that LDH-inhibition enhances cellular ROS production,indicating that LDH functions as an antioxidant and it is interpreted that LDH inhibition redirects pyruvate to mitochondrial metabolism thereby enhancing ROS production.Since the antioxidative activity of LDH relies on mitochondria,this activity of LDH should be exhibited in mitochondria intact cells but not mitochondria defective cells(ρ0 cells).If this logic is correct,the antioxidative and prooxidative functions of LDH in living cells could be dissected.We demonstrated that inhibition of LDH activity by small molecule inhibitor FX11 or by si RNA knockdown induced a significant increase of total cellular ROS in He La or HCT116 cells,whereas the same treatments induced a significant decrease of ROS in He La/ρ0 or HCT116/ρ0 cell,hence the prooxidative and antioxidative activity of LDH in cancer cells was dissected.3.The quantitative relationship between mitochondrial ROS/superoxide and LDH-mediated cellular ROS.We sought to test if there is a quantitative relationship between mitochondrial ROS/superoxide and LDH-mediated cellular ROS.We treated He La cells with serial concentrations of antimycin,an inhibitor to mitochondrial complex III.Antimycin induced mitochondrial and cytosolic ROS in He La cells in a concentration-dependent manner.FX11 treatment enhanced cytosolic ROS in He La cells when antimycin concentration was low but inhibited cytosolic ROS when antimycin concentration was high.On the other hand,FX11 treatment inhibited cytosolic ROS in He La/ρ0cells regardless of with or without antimycin.The results suggested that LDH in mitochondria-intact cancer cells could switch between antioxidative and prooxidative activity,depending on the strength of oxidative stimuli.4.The relationship between mitochondrial ROS/superoxide and LDH-mediated cellular ROSWe treated cells with ETC modulators,such as rotenone,antimycin,FCCP,or oligomycin.These agents increased mitochondrial ROS/superoxide and total cellular ROS.In the presence of FX11,mitochondrial superoxide level with or without treatment of rotenone,antimycin,or oligomycin was comparable with each other.FX11 treatment increased total cellular ROS level in the control cells but reduced ROS in the treated cells to a similar level.The results suggest that without agents interfering with ETC,LDHs inhibit ROS production,with agents interfering with ETC,LDH enhances ROS production.5.The relevance of LDH with anticancer agent-induced ROS.It is recognized that Anti-tumor agents Piperlongumine(PL)and Phenethyl isothiocyanate(PEITC)kill cancer cells by increasing intracellular ROS,but the mechanism was unknown.We found that PL and PEITC could increase mitochondrial as well as cytosolic ROS.LDHA or LDHB knockout significantly reduced cytosolic ROS but not mitochondrial ROS.Hence,the results support the notion that PEITC-and PL-enhanced ROS production involves ROS initiation in mitochondria and LDH-mediated amplification of cytosolic ROS.6.The effect of LDH knockout on intra-tumor oxidative stress.Using mouse tumor xenograft models(He La and 4T1 cells),we found that LDHA or LDHB knockout decreased oxidative stress,assessed by the ROS indicators 4-Hydroxynonenal(4-HNE)and protein carbonylation level in tumor xenografts,indicating that LDHA or LDHB knockout decreases the oxidative stress in tumor.As ROS stabilizes hypoxia-inducible factor-1 alpha(HIF-1α)and promotes tumor progression,we determined if LDHA or LDHB knockout,which reduces oxidative stress,could also reduce HIF-1αprotein level in tumors.The result showed a significant decrease of HIF-1αin tumors formed by LDH-knockout cells.Together,the results support that LDHA or LDHB plays dominant prooxidative activity.Taken together,we conclude that LDHA and LDHB contribute significantly to H2O2 production in cancer cells.Moreover,LDHA and LDHB could switch between antioxidative and prooxidative activities in responding to different oxidative stimuli.In mouse tumor xenograft model,LDHA and LDHB demonstrate dominant prooxidative activity.The study provides a novel mechanism of hydrogen production and ROS regulation in cancer cells.