Influence Of Naringenin On P-gp In K562/A02Cells And Its Relationship Between NF-κB

Background:Naringenin is a plentiful dihydroflavone compound with favorable biologicalactivity. Researches show that naringenin can obvious inhibit the activity of P-gp,then influence itself or other drug’s pharmacokinetics and even pharmacodynamics.Naringenin also can inhibit the expression and activity of NF-κB, then influence theexpression of target gene and protein which result in some pharmacological effectssuch as anti-inflammation, anti-oxidation, anti-tumour and so on. But the reportsabout the influence of naringenin on the expression of the MDR1mRNA and P-gp isfew, in addition, the relationship between naringenin, the expression of MDR1mRNAand P-gp, and NF-κB is unclear. In consideration of NF-κB is one of the importantnucleus transcription factor in many cell signaling pathways, studying the influenceof naringenin on the expression of the MDR1mRNA and P-gp, and its relationshipbetween NF-κB can provide the new eyes for the research of the molecular biologicalmechanism that the influence of naringenin on the P-gp.Objectives:The aim of this study was to discuss the molecular biology mechanism of theinfluence of naringenin on P-gp.Based on the successfully constructed K562/A02cellmodel with high expression of MDR1mRNA and P-gp,we explored the influence ofnaringenin on the transcription of MDR1gene and the expression of P-gp, and therelationship between NF-κB by RT-PCR and Western-blot.Methods:1. Cultivate K562/A02cells regularly.Maintain the drug resistance with1μg/mlADM.Drug free cultivate for2weeks before the experiment. Observe the status ofcells and draw the cell growth curve.2. Test the toxicity of PDTC, Adriamycin and naringenin on the K562/A02cellsby MTT. Choose the conditions under which the cell viability was over80%for thefollowing experiments.3. Incubate K562/A02cells with different concentrations of NF-κB specific inhibitor PDTC and NF-κB inducer Adriamycin alone or together for48h, then detectand estimate the expression of MDR1mRNA and P-gp of K562/A02cells by RT-PCR and Western-blot.4. Incubate K562/A02cells with different concentrations of naringenin alone ortogether with5μM Adriamycin for48h, then detect the expression level of MDR1mRNA and P-gp of K562/A02cells by RT-PCR and Western-blot, to estimate theinfluence of naringenin on the expression of MDR1mRNA and P-gp in K562/A02cells.5. Incubate K562/A02cells with500μM naringenin,25μM PDTC alone ortogether with5μM Adriamycin for48h, then detect the expression level of MDR1mRNA and P-gp of K562/A02cells by RT-PCR and Western-blot. Discuss therelationship among naringenin, MDR1mRNA and P-gp and NF-κB by comparing thedifferences among groups.Results：1. Compared with blank group,the expression of MDR1mRNA and P-gp inK562/A02cells could be down-regulated after incubation with5～100μM PDTC for48h(P＜0.05), and the inhibition was increased with the concentration.2. Compared with blank group, incubation with0.5～15M Adriamycin for48h,0.5～15μM Adriamycin could up-regulate the expression of MDR1mRNA inK562/A02cells(P＜0.05),and1～15μM Adriamycin could up-regulate the expressionof P-gp in K562/A02cells(P＜0.05).The induction was increased with theconcentration.3. Compared with positive control group(5μM Adriamycin),after incubation with5～100μM PDTC in combination with5μM Adriamycin for48h,both25μM and50μM PDTC could down-regulate the expression of MDR1mRNA and P-gp ofK562/A02cells(P＜0.05).Besides,5μM PDTC could also down-regulate theexpression of P-gp(P＜0.05).All the inhibition was increased with the concentrationof PDTC.4. Compared with blank group, incubation with25～500μM naringenin for48h,100～500μM naringenin could down-regulate the expression of MDR1mRNA inK562/A02cells(P＜0.05),and50～500μM naringenin could down-regulate the expression of P-gp in K562/A02cells(P＜0.05).The inhibition was increased with theconcentration of naringenin.5. Compared with positive control group(5μM Adriamycin), incubation with25～500μM naringenin in combination with5μM Adriamycin for48h,both250μMand500μM naringenin could down-regulate the expression of MDR1mRNA inK562/A02cells(P＜0.05).50～500μM naringenin could down-regulate theexpression of P-gp of K562/A02cells(P＜0.05).The inhibition was increased with theconcentration of naringenin.6. Compared with25μM PDTC,the expression of MDR1mRNA and P-gp inK562/A02cells could be inhibited to the same extent by incubation with500μMnaringenin for48h(P＞0.05). Compared with25μM PDTC in combination with5μM ADM，The expression of MDR1mRNA and P-gp of K562/A02cells induced byAdriamycin could also be inhibited to the same extent by incubation with500μMnaringenin in combination with5μM Adriamycin(P＞0.05).Conclusions:1. The K562/A02cell model with high expression of MDR1mRNA and P-gpconstructed by this study was suitable and reliable to explore the relationship amongdrugs, MDR1mRNA, P-gp and NF-κB.2. Naringenin could inhibit the expression of MDR1mRNA and P-gp ofK562/A02cells.The mechanism may be that naringenin may inhibit the activation ofNF-κB, thus inhibit the transcription of MDR1, which lead to the down-regulation ofthe expression of MDR1mRNA and P-gp.