Reversal Of P-gp Mediated Multidrug Resistance And Synergistic Enhancement Of Anticancer Activity Of Chemoagents By Honokiol

Background:Multidrug-resistance(MDR) and toxic side effects of chemoagents are the two major obstacles to the successful cancer chemotherapy.Multidrug-resistance(MDR) is a phenomenon refers resistance of cancer cells to many structurally and mechanically unrelated anticancer drugs. Over- expression of P-glycoprotein (P-gp) is the most frequent cause of MDR. P-gp is a transmembrane protein and functions as an ATP-dependent drug transporter which unilaterally transports the intracellular drugs out of the cells, and the intracellular drugs in the MDR cancer cells were kept at sub-lethal level, by which cancer cells circumvent the attack by the anti-cancer drugs. Prevention and reversal of MDR in cancer cells is an emergent problem to be solved.Another serious problem is the toxic side effect of chemoagents. During chemotherapy, the toxic side effects restrict the "tolerable maxium dose" of chemoagents used in cancer patients, sometimes even stop the chemotherapy.Honokiol, a naturally occurring compound present in Magnolia grandiflora, has several important medicinal use. In the previous research, we found that honokiol can induce apoptosis of cancer cells via p53-independent pathways, and is a potent inhibitor of the growth of human colon cancer cell RKO as ascietes as well as solid tumor in nude mice. Recently, we also reported that honokiol can induce a necrotic cell death through mitochondrial permeability transition pore in cancer cells.In the previous study, we found honokiol can reverse MDR in cancer cells and modulate the expression of HIF-1α, which is the upstream modulating target of P-gp. In this study we intend to confirm if the honokiol can downregulate P-gp expression in MDR cancer cells and if honokiol can enhance the anticancer activities of chemoagents.Objectives:To investigate the modulating effect of honokiol on P-gp expression in MDR cancer cells, and the reversal effect on sensitiveness of the latter toward chemoagents; To evaluate the preventive ability of honokiol against ADM induced drug-resistance in cancer cells; To analyze the combination effects of honokiol with chemotherapeutic drugs on cancer cells; To investigate the in vivo therapeutic effect of honokiol combined with ADM on nude mice bearing implanted MDR tumor.Results:1. Reversal and prevention of P-gp mediated multidrug resistance by honokiol1.1 The inhibitory effect of honokiol on the function of P-gpRh-123 is a substrate for P-gp and is widely used as an indicator for testing the activities of P-gp. We adopted a time dependent assay to investigated the inhibitoryeffect of honokiol on P-gp function. The results demonstrated that pre-incubation of MCF-7/ADR cells with honokiol within 24 hrs did not give rise to a significant increase of intracellular Rh-123 accumulation, while a significant increase of intracellular Rh-123 uptake was observed at the pre-incubation time of 48 hrs. These results clearly indicated that honokiol is not an inhibitor of P-gp, but a presumably down-regulator of P-gp.1.2 The modulating effect of honokiol on the expression of P-gp and its mRNATo prove if honokiol is a down-regulator of P-gp, we incubated MCF-7/ADR cells with honokiol for 12 to 72 hrs. FCM detection results indicated that honokiol was able to down-regulate P-gp expression in MDR cancer cells in a time-dependent manner and a dose-dependent manner.We then tested if down-regulation of P-gp was associated with the reduced expression of its mRNA. Real-time PCR detection results showed the well correlation of the reduction of P-gp with its mRNA, indicating, at least partially, the honokiol-induced decrease of P-gp is due to the down-regulation of its mRNA transcription.1.3 Modulation of intracellular Rh-123 accumulation and efflux by honokiolWe further tested if the reduced P-gp expression was also functionally associated with the recovery of drug accumulation in the drug resistant cells. The FCM detection for Rh-123 results indicated that inhibition of expression of P-gp in MCF-7/ADR cells by honokiol was functionally associated with the recovery of drug accumulation and decrease of drug efflux in the cells.1.4 Partial recovery the sensitivity of MCF-7/ADR cells to ADM by honokiolTo assess the effect of honokiol on ADM-induced toxicity, we performed a MTT colorimetric assay to determine the ADM-induced cytotoxicity toward MCF-7/ADR. MTT results showed pretreatment of MCF-7/ADR with honokiolsignificantly increased the sensitivity toward ADM. The IC₅₀ of Adr toward MCF-7/ADR with pretreatment of honokiol was 6.2 ±1.9 μg/ml, approximately 3-folds lower than that (17.7 ± 4.9 μg/ml) for MCF-7/ADR without pretreatment of honokiol.1.5 Prevention of ADM induced drug-resistance in cancer cells by honokiolThe real-time PCR detection results showed that honokiol did not inhibit the increase of mdr1 mRNA and P-gp expression level induced by ADM in sensitive K562 cells.2. Analysis of combination effects of honokiol with chemoagents2.1 Analyzing combination effects of honokiol with chemoagents on cancer cells using CLAccording to the calculated CI, we found that honokiol could synergistically enhance the anti-cancer activities of chemoagents except VP-16 towards MCF-7 cells. The synergism effect is a little more significant in drug-resistance cancer cells than in sensitive cells.2.2 Analysis of honokiol for the enhancing the anticancer activities of chemoagents using DRI.The Dose Reduction Index (DRI) can reflect the decreasing folds of chemoagents' concentration after addition of honokiol with a fixed concentration. According to the calculated DRI, we found that addition of honokiol with a fixed concentration can sensitized the cancer cells towards chemoagents except VP-16 towards MCF-7 cells.3. In vivo therapeutic effect of honokiol combined with ADM on nude mice bearing implanted MCF-7/ADR tumorThe results showed that honokiol can enhance the inhibitory effect of ADM onimplanted tumor growth. Eighteen days after drug administration, we found, in honokiol+ADM group, the mean volume of MCF-7/ADR implanted tumor decreased about 21.71% with statistical significance as compared with the ADM only group (P<0.05). The mean surviving time (MST) in combination group showed a prolongation compared with control group and ADM only group.Conclusions:1. Honokiol can reverse P-gp mediated multidrug resistance and partially recover the sensitivity of multidrug-resistance cancer cells towards chemoagents. This effect is, at least partially, due to the down-regulation of its mRNA transcription.2. Honokiol can synergistically enhance and sensitize the anti-cancer activities of chemoagents in drug-resistant and sensitive cancer cells.3. Honokiol can enhance the inhibitory effect of ADM on implanted tumor growth and prolong the MST of tumor-implanted nude mice.