Research On The Nrf2-Keap1 Signaling Pathway And The Effect Of Doxorubicin In Leukemia K562 Cells

Background:Chemotherapy plays an important role in the comprehensive treatment of cancer.However,the side effects of chemotherapy drugs and drug resistance problems seriously affect the treatment effect.In particular,multidrug resistance(MDR)has reduced the efficacy of combined chemotherapy,leading to the failure of treatment.Doxorubicin(DOX)is one of the most widely used Anthracyclines(ANTs).Anthracyclines are periodic nonspecific antibiotics that kill cancer cells in each growth cycle.As the most effective broad-spectrum anticancer drugs,there are two problems(side effects and drug resistance)in the application of Anthracyclines.Anthracyclines produce reactive oxygen species(ROS)by metabolism,improve the oxidative level in cancer cells and induce apoptosis.A large number of reactive oxygen species can also cause cancer cell necrosis and produce anticancer effect.On the other hand,the increase of oxidation level induces the activation of oxidative stress defense pathway in cancer cells,and the expression increases of drug metabolism enzyme,leading to the increase of toxic and side reaction,inducing drug resistance.The mechanism of oxidative stress has been paid more and more attention in the effect of Anthracyclines and induced drug resistance.Nuclear factor E2 correlation factor 2/Kelch-like epichlorohydrin-related protein 1 signaling pathway(Nrf2-Keap1)is an important regulatory pathway of anti-oxidative stress in cells.Oxidative stress activates the pathway.Nrf2 was separated from the inhibitory protein Keap1.Under a variety of protein kinase phosphorylation,Nrf2 transfer into the nucleus.Nrf2 combined with the antioxidant response element(ARE),started a series of antioxidant genes and downstream ? detoxifying enzymes transcription,give play to the role of powerful antioxidant,restore oxidation-antioxidant balance in the cells.Activation of Nrf2-Keap1 signaling pathway activates downstream antioxidant enzymes that produce anti-oxidative stress.Activation of Nrf2-Keap1 signaling pathway raise drug metabolic enzyme gene transcriptional expression promote drug metabolism,also control other signaling pathways such as cell autophagy,apoptosis pathway,reducing chemotherapy drug curative effect,inducing side effects and the generation of induced resistance.Inhibition of Nrf2-Keap1 signaling pathway has become one of the important targets to reverse drug resistance in cancer cells.The combination of Nrf2-keap1 signaling pathway inhibitors and anticancer drugs can enhance the sensitivity of cancer cells to chemotherapy drugs,improve the therapeutic effect of chemotherapy drugs,and provide a new way for cancer treatment.The First Part Nrf2-Keap1 signaling pathway and the effect of Doxorubicin in leukemia K562 cellsObjective: To detect the protein level,messenger RNA(m RNA)expression of Nrf2-Keap1 signal pathway and autophagy in K562 cells by DOX treatment;to detect the Nrf2 protein level in K562 cells by DOX combined with Actinomycin.To investigate the correlation between Nrf2-Keap1 signaling pathway and the effect of DOX treatment in the leukemia cell K562.Methods: 1.CCK-8 method: The effect of DOX on the proliferation of K562 cells was detected;50% inhibition concentration(IC50)was calculated and the experimental concentration of DOX was determined.2.Western Blot: The proteins expression of Nrf2-Keap1 signaling pathway(Nrf2,Keap1,GCS,AKR1C1 and CBR1)and autophagy pathway(Beclin-1,LC3 and P62)in K562 cells by different concentrations of DOX was detected at 4h and 16 h.3.Real-time quantitative PCR: Relatively expressed m RNA levels of Nrf2-Keap1 signaling pathway genes(Nrf2,GCLM,AKR1C1 and CBR1)and autophagy pathway genes(Beclin-1,LC3 and P62)in K562 cells by different concentrations of DOX were detected at 4h and 16 h.4.Western Blot: to determine the half-life of Nrf2 protein after the combination of DOX and Actinomycin was applied to K562 cells.Results: 1.The activity of K562 cells showed concentration dependence when different concentrations of DOX acted on K562 cells at 48 h.In small dose,DOX had a stimulatingeffect on K562 cell growth.DOX significantly inhibited the growth of K562 cells with higher concentration.IC50:1.13?g/ml.2.The expression of Nrf2-Keap1 signaling pathway protein Nrf2 and AKR1C1 was increased when DOX was applied to K562 cells at 4h.There was no significant change in Keap1,GCS protein expression.In Autophagy pathway proteins,Beclin-1 and LC3 expression increased.P62 expression had no significant change.3.In Nrf2-Keap1 signaling pathway proteins,the expression of Nrf2,GCS and CBR1 was increased when DOX was applied to K562 cell at 16 h.There was no significant change in the expression of Keap1 and AKR1C1.In Autophagy pathway proteins,Beclin-1 and LC3 expression increased,P62 expression decreased.4.There was no significantly different in Nrf2 m RNA expression of K562 cells by different concentrations of DOX treatment at 4h(P > 0.05).The expression of Keap1,GCLM,AKR1C1 and CBR1 m RNA did not change significantly after DOX treatment at different concentrations in K562 cells at 4h.The expression of Beclin-1 m RNA was significantly increased,while the expression of LC3 and P62 did not change significantly.5.The difference of Nrf2 m RNA expression in the experimental group was not statistically significant between the negative control group and DOX at 16 h of K562 cells.GCLM,AKR1C1 and CBR1 m RNA expression increase.The expression of Beclin-1 m RNA was slightly increased,and the expression of LC3 and P62 m RNA decreased.6.Under the condition of inhibition protein synthesis by actinomycetin,DOX prolonged the half-life of Nrf2 protein.Conclusion: DOX can increase Nrf2 protein accumulation,activate Nrf2-Keap1 signaling pathway,and promote the transcription expression of downstream target gene GCLM,AKR1C1 and CBR1,then increase proteins accumulation.DOX can activate cellular autophagy.By improving autophagy level,DOX can also increase Nrf2 protein accumulation and activate Nrf2-Keap1 signaling pathway.The Second Part Study of Brusatol inhibition of Nrf2-Keap1 signaling with Doxorubicin effectObjective: To investigate the Brusatol inhibitory effects of Nrf2-Keap1 signal pathway on DOX treatment in K562 cells;and to explore the mechanism of brusatolimprove DOX inhibit the proliferation of K562 cells.To provide preliminary experimental basis for clinical application of brusatol.Method: 1.Western blot:To detect the expression of Nrf2-Keap1 signaling pathway proteins(Nrf2,Keap1 and AKR1C1)in K562 cells at 16 h with different concentrations of brusatol,and the appropriate concentration was chosen.2.Western blot:To detect the expression of Nrf2-Keap1 signaling pathway proteins(Nrf2 and CBR1)and apoptosis-associated protein(cleaved-caspase 3/caspase 3)after different concentrations of DOX treated K562 cells.3.CCK-8 method: To detect the activity of K562 cells by the combination of 100 n M brusatol and DOX treatment at 48 h.Results: 1.Brusatol can inhibit the expression of Nrf2 protein and its downstream AKR1C1 protein.There was no significant effect on the expression of Keap1 protein.100 n M is the appropriate dose.2.In DOX treatment,the joint brusatol decreased Nrf2 and CBR1 expression,and improve the expression of cleaved caspasase 3/ caspasase in k562 cells.3.Brusatol promotes K562 cells apoptosis induced by DOX.Conclusion: Brusatol can inhibit the accumulation of Nrf2 protein in K562 cells induced by DOX;inhibit the expression of nrf2-keap1 signaling pathway and downstream target gene;then reduce the anti-oxidative stress ability of K562 cells.The treatment of K562 cells with brusatol was enhanced to induce the apoptosis of K562 cells induced by DOX.