Effect Of Cyclosporine A And Matrine On The Reversion Of Multidrug Resistance Of K562/ADM Line And Application Of Microfluidic Chip In The Study Of Drug Efflux Modulation

Objective:This paper was to study the reversal effect of cyclosporine A(CsA)and matrine(MAT)on multidrug resistance cell line K562/ADM and to investigate the reversal mechanism of this combination,and to provide theoretic evidence of the clinical application of them as resistance modifying agents,then to evaluate microfluidic chip in detecting the modulation of drug efflux of anticancer compounds by MDR modulators at single cell level.Method:The major group was divided as follows:(1)K562/S cell line; (2)K562/ADM cell line;(3)K562/ADM cell line with CsA;(4)K562/ADM cell line with MAT;(5)K562/ADM cell line with CsA and MAT.Cell toxicity and reversal time of CsA and/or MAT on tumor cells were determined by MTT assay.Cell apoptosis percentage was determined by flow cytometry.The reversal effect of CsA and MAT along with ADM on K562/ADM was discussed with the mentioned data above.Intracellular drug concentration was determined by spectrophotofluorometry,flow cytometry and microfluidic chip,respectively.In order to discuss the mechanism of reversal effect of CsA and MAT,expression of P-gp coded by MDR-1 gene was observed using immunohistochemical technique and changes of P-gp were determined by flow cytometry.Results:1.CsA or MAT could inhibit the growth of K562/S and K562/ADM,its IC₅₀ had no statistical difference(P＞0.05).Non-cytotoxic dosage and low-cytotoxic dosage of CsA and MAT were determined by MTT assay.Non-cytotoxic dosage was selected as reversal dosage;2.Used along with ADM,IC₅₀of both K562/ADM with CsA and K562/ADM with MAT were lower than that of K562/ADM(P＜0.01),but still higher than that of K562/ADM with CsA and MAT(P＜0.01).When two agents combined together, the reversal effect was better than that of the sum of CsA or MAT alone,coinciding with the increasing of cell apoptosis percentage determined by FCM;3.CsA and MAT (alone or combination)enhanced the intracellular ADM accumulation in K562/ADM, higher than that of K562/ADM(P＜0.01).The result of intracellular ADM accumulation detected by microfluidic chip coincided with the result detected by FCM; 4.lmmunohistochemical results showed the overexpression of P-gp in K562/ADM; 5.P-gp expression of K562/ADM with MAT was lower than that of K562/ADM(P＜0.01), but still higher than that of K562/ADM with CsA and MAT(P＜0.01).And no significant difference on P-gp expression was found between K562/ADM with CsA and K562/ADM(P＞0.05).Conclusions:1.CsA and MAT were effective antitumor drugs with inhibiting effect on tumor cells.K562/ADM had no drug resistance to CsA and MAT;2. Non-cytotoxic dosage of CsA and MAT could partly reverse the MDR of K562/ADM. The effect of combination of two agents was superior to that of single use due to their coordinate effect;3.A microfluidic chip that was capable of selecting and retaining single multidrug-resistant cancer cells can be used to investigate drug efflux inhibition in leukemia cell lines effectively.This novel method has an extensive application for drug selection and individual anticancer therapy in future;4.0ver expression of P-gp was related to mechanism of MDR;5.The reversing mechanism of CsA and MAT has relation to the decreasing of P-gp probably.