The Mechanism Of FC101-induced Toxicity On Kidney Cells And The Protecive Role Of Coenzyme Q10

Fsarochromanone?FC101?,a mycotoxin produced by the fungus Fusarium equiseti,is frequently observed in the contaminated grains and feedstuffs,which is toxic to animals and humans.After consumption of contaminated food,FC101 might cause acute symptom or severe chronic degenerative and lead to cancer,which cause significant economic losses to the livestock industry.However,the underlying molecular mechanism remains to be defined.In this study,we found that FC101 executes its toxicity at least by inducing G₁ cell cycle arrest and inhibiting cell proliferation in COS7 and HEK293 cells.Furthermore,FC101 may induced cell death through caspase-dependent and-independent mechanisms.To further explore the mechanism of cell death,we found FC101 induced ROS generation and our finding supporting a role of ROS production in inducing apoptosis and autophagy.Furthermore,Our finding suggested that FC101 induced cytotoxicity through induction of ROS,at least by downregulation PP2A and PP5,leading to activation of JNK.The coenzyme Q₁₀(CoQ₁₀)takes part in the mitochondrial respiratory chain,accepts and transports electrons to oxygen,and at the same time the proton gradient promotes ATP synthesis.CoQ₁₀ is a well known natural antioxidant and cell metabolism activator.Clinical applications of CoQ₁₀ mostly focused on adjuvant treatment of congestive heart failure,viral hepatitis and cancer.Report on home and abroad showed CoQ₁₀ protected and strengthened the function of the cells from ROS mediated damage.However,the role of CoQ₁₀ in the cell death that induced by FC101 in kidney cells is not well characterized.In this experiment,we will study the mechanism of FC101-induced toxicity on COS7 and HEK 293 cells and the protective role of CoQ₁₀.Our experiment are divided into four parts as follows:1 Effect of FC101 on cell cycle progression in Kidney Cells COS7 and HEK 293cell were chosen and treated cell with FC101?0?5?M?for 6 days or 24?72 hrs.Cell morphology was analyzed with an inverted phase-contrast microscope.Cell survival rate was evaluated by one solution assay.Cell cycle was determined using a FACSCalibur flow cytometer.The expression of proteins were analyzed by western blot assay.In this study,we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner.Flow cytometric analysis showed that FC101 induced G₁ cell cycle arrest.Concurrently,FC101 downregulated protein expression of cyclin D1,cyclin-dependent kinases?CDKf4 and CDK6?,and Cdc25 A,and upregulated expression of the CDK inhibitors(p21^Cip1 and p27^Kip1),resulting in hypophosphorylation of Rb.Our results support the notion that FC101 executes its toxicity at least by inducing G₁cell cycle arrest and inhibiting cell proliferation.2 Effect of FC101 on Apoptosis and Autophagy in Kidney Cells COS7 and HEK293 cell were chosen and treated cell with FC101?0?5?M?or pretreated with pan-caspase inhibitor?Z-VAD-FMK?,autophagy inhibitor?CQ?,ROS scavenger?NAC?lhr and then treated with FC101?0?5?M?for 0?72h or infected with Ad-GFP-LC3 for 24h then treated with FC101 for 24h.Trypan blue exclusion assay was detected cell death.Annexin V-FITC/PI staining and DAPI staining were detected cell apoptosis.Immune fluorescence assay was detected autophagy.ROS generation was measured by CM-H2DCFDA fluorescence dye.Caspase-3/7 activity was analyzed using the Caspase-3/7 assay kit.The expression or activity of BAD?BAX?BAK?Bcl-2?Bcl-xL?Mcl-1?survivin?DR5?cleaved caspase 3?cleaved caspase 8?cleaved PARP?AIF?LC3A/B?H2A??-H2AX?ser139??p53?ATM?phospho-ATM?ser1981??ATR?phospho-ATR?ser428??NF?B?p65?,phospho-NF?B?p65??Ser139?were analyzed using the Western blot assay.Our results showed that FC101 induced apoptosis and autophagy in COS7 and HEK 293 cells.Moreover,FC101 upregulated expression of pro-apoptoic protein BAD,death receptor signaling protein DR5 and downregulated expression of anti-apoptoic proteins Mcl-1?Bcl-2?Bcl-xL and survivin.FC101 also induced expression of cleaved caspase8 and activating of caspase 3 and cleavage of PARP in a concentration-dependent manner.But no change in the expression of pro-apoptoic protein BAK and BAX was seen.After treatment with FC101,the cells displaying characteristic features of apoptosis with fragmented and condensed nuclei when used with DAPI staining.We also found FC101 induced autophagy,as indicated by the increased number of cells with LC3-punctate;the increased LC3?expression.Furthermore,FC101 induced ROS generation and our finding supporting a role of ROS production in inducing apoptosis and autophagy.FC101 activated NF?B and P53in a concentration-dependent manner and induced phosphorylation of DNA damage response factor,such as H2AX and ATM.These finding suggested FC101 maybe reduced DNA damage.3 The Mechanism of FC101-Induced Toxicity via Oxidative Stress and JNK Signaling Pathway in Kidney Cells COS7 and HEK 293 cell were chosen and treated cell with FC101?0?5?M?or pretreated with ROS scavenger?NAC?or JNK inhibitor?SP600125?for 1hr and then treated with FC101?0?5?M?for 24hrs;or overexpression of c-Jun,PP2A,PP5 by adenovirus interference then treated with FC101?0.5,1?M?for24h.ROS generation was measured by CM-H2DCFDA fluorescence dye.Cell morphology was analyzed with an inverted phase-contrast microscope.Cell survival rate was evaluated by one solution assay.The expression of Erk 1/2?JNK and p38 MAPK signaling pathway,protein phosphatases 2A?PP2A?and 5?PP5?were analyzed using the Western blot assay.Our data suggested that FC101-induced ROS activates JNK.Inhibition of JNK with kinase inhibitor?SP600125?,or overexpression of dominant negative c-Jun,prevented FC101-induced cytotoxity.Pretreatment with N-acetyl-L-cysteine?NAC?scavenged FC101-induced ROS,hindering activation of JNK and cytotoxity.We have further found that FC101-induced ROS inhibited serine/threonine protein phosphatases 2A?PP2A?and 5?PP5?,which was abrogated by NAC.Overexpression of PP2A and PP5 partially prevented JNK.Our finding suggested that FC101induced cytotoxicity through induction of ROS,at least by downregulation PP2A and PP5,leading to activation of JNK.4 CoQ₁₀ Protects COS7 cells from FC101-Induced Cytotoxicity through Inhibition of Oxidative Stress and JNK Pathway We used COS7 cells as a model of oxidative damage induced by FC101 and study the molecular mechanism that CoQ₁₀protected cells against FC101-indced damage.COS7 and HEK 293 cell were chosen and treated cell with FC101?0?5?M?or pretreated with CoQ₁₀ or JNK inhibitor?SP600125?for lhr or 30 min and then treated with FC101?0?5?M?for 6 days or 24hrs or overexpression of c-Jun by adenovirus interference then pretreated with CoQ₁₀ for lhr and then treated with FC101?1?M?for 24h.ROS generation was measured by CM-H2DCFDA fluorescence dye.Cell morphology was analyzed with an inverted phase-contrast microscope.Cell survival rate was evaluated by one solution assay.The expression phosphorylation of c-Jun and apoptotic-related proteins such as Bad,Bcl-xL,cleaved-caspase3 were analyzed using the Western blot assay.Our data suggested that FC101induced cytotoxicity,leading to cell death through induction of ROS.CoQ₁₀ acts by scavenging ROS production,inhibiting cell apoptosis to reduce cytotoxicity.CoQ₁₀potently reduced the FC101-induced decreased rate of cell survival,morphologic changes and fragmented and condensed nuclei.CoQ₁₀ could also downregulate expression of Bad,upregulation expression of Bcl-xL and decrease the activity of caspase-3.Moreover,CoQ₁₀could inhibit JNK activation.Furthermore,using JNK inhibitor?SP600125?or overexpression of c-Jun potentiated CQ₁₀ protection against FC101-induced cytotoxicity.Our finding indicated that FC101 induced cytotoxicity through induction of oxidative stress which was prevents by CQ₁₀ via targeting JNK.