The Heterocyclic Compound TEMPOL Inhibits Growth Of Cancer By Interfering With Metabolism

BackgroundChemotherapy is one of the main methods of cancer treatment,and drug side effects and drug resistance are the primary obstacles limiting its therapeutic effect.Tempol(4-hydroxy-2,2,6,6-Tetramethylpiperidine-1-oxyl,TPL),a stable nitroxide radical used as a contrast agent in magnetic resonance imaging,has been reported to induce cellular apoptosis in number of cancer cells.The toxicity effect of TPL is preferential to target cancer cells and its cytotoxicity depends on the elevation of cellular oxidative stress,TPL also induces the expression of the cycling dependent kinase inhibitor p21wafl/cipl and activates caspase-3 and Bax/Bcl-2 pathway.TPL is accumulated in mitochondrial and interacts with catalytic centers of respiratory chain complexes(complex Ⅰ,Ⅱ and Ⅳ),which impairs OXPHOS and reduces intracellular glutathione pool,and then induces oxidative stress and cellular apoptosis.Metabolic reprogramming is one of the features of cancer,Warburg effect and altered mitochondrial structure-function provide biosynthesis and NADPH production for building new biomass.And motochondrail plays an important role in energy production,redox regulation and apoptosis inhibition.In addition to glucose,glutamine is the main nutrient source.Many types of cancer cells exhibit glutamine addiction,and even some cancer cell lines couldn’t survive in the absence of glutamine,despite being in glucoserich media.Although TPL induces cellular apoptosis by elevating ROS level,the concentration of TPL in vivo is usually too low to induce oxidative stress.Therefore,this study detected whether can the lower-concentration TPL treatment also inhibits tumor growth.And we characterized the anti-proliferative effects of TPL in vitro and in vivo and elucidated the metabolic mechanisms involved in glucose and glutamine metabolism alteration in ovarian cancer cells by13C metabolic flux analysis.Result1.Cytotoxicity of TPL on cancer cellsFirst MTT assay showed TPL treatment reduced the viability of three types of cells in a concentration-dependent manner.Calcusyn software analysis indicated that TPL has a synergistic effect with DDP.2.Low concentration of TPL reduces ROS level and inhibits mitochondrial OXPHOSMTT assay showed that that low concentration(1mM)of TPL treatment for 48 hours significantly reduced cell proliferation and beyond 72hours induced cell apoptosis.To detect cellular ROS,emission fluorescence intensity in cells incubated in the presence of DCFH-DA was determined and found that cellular ROS level was increased in a concentration-dependent matter when the concentration of TPL was more than 2mM,while the ROS level was reduced by lower concentration TPL treatment(1mM),suggesting that dual effects of TPL on ROS production in cancer cells.To determine the effect of TPL on OXPHOS in cancer cells,we used extracellular flux analyzer Seahorse XF24e to measure the oxygen consumption rate(OCR).TPL treatment decreased basal and maximal mitochondrail OCR as well as ATP production,indicating inhibition of mitochondrial OXPHOS.The proton leak reduced by TPL treatment also indicated mitochondrial ROS production increasing.3.The effect of TPL on glycolysis in cancer cellsGC-MS-based13C metabolic flux analysis(MFA)and Seahorse extracellular flux analysis were used to analyze the effect of TPL on glycolysis of cancer cell.The result showed TPL treatment had less effect on glycolysis but reduced the production of pyruvate and inhibited serine pathway.4.TPL interferes with glutamine metabolism by inhibiting conversion between isocitrate and α-ketoglutarateGC-MS-based13C metabolic flux analysis(MFA)was used to explored the effect of TPL on cellular glutamine metabolism.The result showed TPL disturbed both glutamine anabolic and catabilic processes by inhibiting conversion between isocitrate and α-ketoglutarate.5.TPL inhibited growth of mouse colon cancer cells in xenograft animal modelWe next estabolished a xenograft mouse mode to examine whether TPL has an anti-tumor activity in immunocompetent mice by subcutaneous injection of mouse colon cancer cell line CT26 cells.High-dose TPL(1.16mM/kg)treatment inhibited the growth of tumor significantly.IHC assay showed decreases in cellular proferation and oxidative stress after TPL treatment.Besides,the low-dose of TPL counteracts the antitumor activity of DDP in mouse model.IHC assay and western blotting assay showed TPL treatment reduced cellular apoptosis induced by DDP.The lower ROS level was detected in TPL combined with DDP treatment than DDP treatment alone by flow cytometry assays.ConclusionWe confirmed that TPL inhibited the energy metabolism by interfering with glutamine metabolism,including glutaminolysis and reductive carboxylation,but not glycolysis.This mainly reaction inhibited by TPL was the conversion between citrate and a-ketoglutarat.Low-concentration TPL also inhibited OXPHOS of cancer cell and reduced ROS level.Hence,this study found that an anti-proliferation property of TPL on tumor in mouse model correlated with its inhibition of cellular metabolism.Furtherly,TPL treatment showed anti-tumor effect,which reduced cellular proferation but didn’t elevate the cellular ROS level in xenograft animal model.Our findings also hinted tant antioxidant may decreased the therapeutic effect of anticancer chemotherapeutic drugs,which play a cytotoxic role by elevating ROS level.In general,this study predicts that TPL is a potential anti-tumor agent that inhibits tumor growth via disturbing glutamine metabolism and provides a new possible mechanism for other antioxidants.