The Reversal Effect Of Afatinib On ABCB1-mediated Multidrug Resistance In Human Ovarian Cancer And Its Mechanisms

Background and ObjectiveMultidrug resistance (MDR) means that cancer cells produce resistance to the cytotoxic effects of numerous antineoplastic drugs that are structurally and mechanistically unrelated. The most common cause of MDR results from the overexpression of cell membrane-bounded ABCB1 transporter (ATP-binding cassette subfamily B member 1). Paclitaxel (Substrate drug of ABCB1 transporter) and platinum combination chemotherapy has remained the global first-line chemotherapy regimen for patients with advanced ovarian cancer. However, most of ovarian cancer cases will eventually develop drug-resistant disease and succumb. It has been widely reported that ABCB1 is also highly expressed in human ovarian cancer and its overexpression is correlated inversely with a benign response to chemotherapy and good clinical prognosis. Therefore, it is of vital medical significance to overcome or reverse ABCB1-mediated MDR in patients with ovarian cancer.To reverse ABCB1-mediated MDR, tremendous efforts have been made to discover or synthesize ABCB1 inhibitors which either inhibit the efflux function of ABCB1 transporter or attenuate the expression of ABCB1. Although lots of ABCB1 inhibitors were identified or even been tested in clinical trials, none of them have been approved, mainly because of several problems such as toxicity or pharmacokinetic uncertainty. One efficient solution is to rediscover new applications for existing drugs with known pharmacokinetics and safety profiles.The development of antitumor drugs has shifted from traditional cytotoxic drugs to molecular targeted drugs in recent years. Nowadays, tyrosine kinases have become the second most important group of drug targets after G-protein-coupled receptors. It is reported that several TKIs could inhibit the efflux function of ABCB1 and thus enhance the anticancer efficacy of chemotherapeutic drugs. However, the mechanisms are still not very clear. Furthermore, the binding mode between TKIs and ABCB1 was rarely investigated.Afatinib is the first irreversible multi-targeted TKI. An irreversible tyrosine kinase blockade may result in the longer suppression of correlated pathways than reversible TKIs, thus producing stronger therapeutic effects. In consideration of its dual receptor and irreversible inhibition, we wonder whether afatinib is also superior in reacting with ABCB1 and whether the irreversible inhibition will bring some unique effects. However, no related studies focusing on the reversal effect of afatinib on ABCB1-mediated MDR have been reported.In this study, in vitro, in vivo and in silico experiments were performed to determine whether afatinib could resensitize multidrug resistant cancer cells to conventional chemotherapy drugs by interacting with ABCB1 and to uncover its mechanisms. This study will further illuminate the interaction mechanisms between TKIs and ABCB1. This study will also investigate the differences and advantages of irreversible TKIs in reversing ABCB1-mediated MDR when compared with reversible TKIs.MethodsMTT assay was performed to investigate the cytotoxicity of afatinib as well as its reversal effect on MDR in multidrug-resistant ovarian cancer cells. siRNA assay was applied to knockdown the expression of ABCB1 to investigate the correlation between ABCB1 and the reversal effect of afatinib on MDR. AnnexinV-FITC/PI staining assay and TUNEL assay were performed to investigate the effect of afatinib on paclitaxel induced apoptosis in multidrug-resistant ovarian cancer cells in vitro and in vivo, respectively. Xenograft assay was applied to investigate the reversal effect of afatinib on MDR in vivo. Rhodamine 123 accumulation assay, in vitro transport assay, in vivo imaging assay and ATPase activity assay were performed to investigate the effect of afatinib on the function of ABCB1 and the underlying mechanisms. Western blot, RT-PCR, immunohistochemistry, tissue immunofluorescence and cell immunofluorescence assays were applied to investigate the effect of afatinib on the expression of ABCB1 and the underlying mechanisms. Pharmacophore model assay was performed to investigate the structural basis of afatinib as an ABCB1 inhibitor. Homology modeling assay and docking simulation assay were applied to investigate the binding mode between afatinib and ABCB1. DNA sequencing assay was performed to investigate the correlation between the reversal effect of afatinib on MDR and the related gene mutations.Results1. Afatinib reversed ABCB1-mediated MDR in ovarian cancer cells in vitroThe half maximal inhibitory concentration values (IC50) of paclitaxel in A2780 and A2780T cells were 0.039 ± 0.003μM and 198.92 ± 14.85μM, respectively. The IC50 values of adriamycin in A2780 and A2780T cells were 1.17 ± 0.06μM and 27.69 ± 2.11μM, respectively. The IC50 values of paclitaxel in SKOV3 and SKOV3-DDP cells were 0.033 ± 0.002μM and 41.79± 3.67μM, respectively. The IC50 values of adriamycin in SKOV3 and SKOV3-DDP cells were 0.97 ± 0.07μM and 18.13± 1.54μM, respectively. These IC50 differences between drug-sensitive cells and drug-resistant cells were statistically significant (P<0.01). The drug-resistant A2780T and SKOV3-DDP cells showed significant resistant phenotype to ABCB1 substrate drugs (paclitaxel and adriamycin) than drug-sensitive A2780 and SKOV3 cells.The expression levels of ABCB1 protein and MDR1 mRNA in A2780T cells were 83.3 folds and 142.9 folds more than that in A2780 cells, respectively. The expression levels of ABCB1 protein and MDR1 mRNA in SKOV3-DDP cells were 37.0 folds and 23.3 folds more than that in SKOV3 cells, respectively.Afatinib strongly decreased the IC50 values of paclitaxel and adriamycin in ABCB1-overexpressing A2780T and SKOV3-DDP cells in a concentration-dependent manner. Afatinib at concentrations of 0.625μM,1.25μM and 2.5μM significantly decreased the IC50 values of paclitaxel in A2780T cells by 1.72 folds,10.08 folds and 22.45 folds, respectively (P<0.01). Afatinib at concentrations of 0.375μM,0.75μM and 1.5μM significantly decreased the IC50 values of paclitaxel in SKOV3-DDP cells by 1.78 folds,4.26 folds and 7.27 folds, respectively (P<0.01). Afatinib at concentrations of 0.625μM,1.25μM and 2.5μM significantly decreased the IC50 values of adriamycin in A2780T cells by 4.44 folds,7.05 folds and 17.64 folds, respectively (P<0.01). Afatinib at concentrations of 0.375uM,0.75μM and 1.5μM significantly decreased the IC50 values of adriamycin in SKOV3-DDP cells by 1.98 folds,5.06 folds and 14.50 folds, respectively (P<0.01). In contrast, afatinib didn’t alter the cytotoxicity of paclitaxel and adriamycin in A2780 and SKOV3 cells, which expressed low levels of ABCB1 protein. In addition, the reversal effects of afatinib on MDR to paclitaxel and adriamycin in A2780T and SKOV3-DDP cells were stronger than that of the same concentrations of lapatinib (reversible TKIs) or lOuM verapamil (a function inhibitor of ABCB1).Knockdown of ABCB1 in A2780T and SKOV3-DDP cells could significantly decrease the IC50 values of paclitaxel and adriamycin in them. The IC50 values of paclitaxel and adriamycin in A2780T/ABCB1-and SKOV3-DDP/ABCB1- cells further decreased when afatinib was combined.2. Afatinib reversed ABCBl-mediated MDR in vivoThe average weights of tumors were 0.707 ± 0.229 g,0.655± 0.289 g,0.247± 0.088 g and 0.113± 0.079 g for control group, paclitaxel group, afatinib group and the combination group, respectively. The combination of paclitaxel and afatinib not only significantly delayed the growth of A2780T xenografts with an inhibition rate of 84.02%, but also induced significant tumor necrosis and tumor regressions.3. Afatinib enhanced the paclitaxel-induced apoptosis in vitro and in vivoAfatinib significantly increased the percentages of apoptosis induced by paclitaxel in multidrug-resistant A2780T and SKOV3-DDP cells in vitro. Furthermore, TUNEL assay results showed that abundant apoptotic nuclei were clearly exhibited in tumor tissues of the combination group. Moreover, combining paclitaxel with afatinib induced remarkable nucleus rupture and nuclear mass leak in tumor tissues, indicating a high occurrence ratio of tumornecrosis.4. Afatinib inhibited the efflux function and stimulated the ATPase activity of ABCB1Afatinib remarkably increased the intracellular accumulation of rhodamine 123 in ABCB1-overexpressing A2780T cells (P<0.05), while having no effect on that in A2780 cells (P>0.05). More meaningfully, afatinib also significantly increased the accumulation of rhodamine 123 in A2780T xenografts by 2.28 folds when compared with rhodamine 123 group (P<0.01). Afatinib significantly decreased the efflux of rhodamine 123 in A2780T cells (P<0.05) while having no effect on that in A2780 cells (P>0.05). To sum up, afatinib significantly increased the accumulation of rhodamine 123 in A2780T cells by inhibiting the efflux function of ABCB1. In addition, both afatinib and paclitaxel stimulated the ATPase activity of ABCB1 during a short-time incubation with recombinant human ABCB1 membranes. The concentrations required for 50% stimulation of the ATPase activity of ABCB1 were about 2.5μM for afatinib and 70.1 μM for paclitaxel.5. Afatinib attenuated the expression of ABCB1 by inhibiting the activation of NF-κB pathwayAfatinib could dramatically attenuate the expression of ABCB1 protein and MDR1 mRNA in ABCB1-overexpressing A2780T and SKOV3-DDP cells in a concentration-dependent manner, whereas lapatinib having no effect on that. Furthermore, afatinib could also attenuate the expression of ABCB1 protein in A2780T xenografts in vivo. Moreover, both PDTC (a specific NF-κB inhibitor) and p65-specific siRNA could attenuate the expression of ABCB1 protein in A2780T cells. Afatinib also attenuated the expression of the NF-κB subunit p65 both in the nucleus and the cytoplasm in A2780T cells. Meanwhile, afatinib decreased the phosphorylation and degradation of IκBα in A2780T cells. Like PDTC, afatinib abrogated both the constitutive and LPS-induced nuclear translocation of p65 in A2780T cells. In conclusion, afatinib could attenuate the expression of ABCB1 in drug-resistant ovarian cancer cells by inhibiting the activation of NF-κB pathway.Afatinib remarkably blocked the phosphorylation of Akt without affecting total Akt levels in A2780T cells. Meanwhile, LY294002, a specific inhibitor of PI3K/Akt pathway, also significantly suppressed the nuclear translocation of p65 in A2780T cells. Moreover, the combination of afatinib and LY294002 induced additive inhibitory effect on ABCB1 expression in A2780T cells, suggesting that afatinib might suppress NF-κB activity through inhibiting the PI3K/Akt pathway.6. Blockage of the MAPK/p38 pathway contributed to the reversal effect of afatinib on MDR in ovarian cancerInhibition of the MAPK pathway may partially restore the sensitivity to chemotherapeutic drugs in cancer cells. Afatinib significantly blocked the phosphorylation of EGFR, HER-2 and p38 in A2780T cells, whereas it didn’t alter the expression levels of total EGFR, HER-2 or p38. In addition, Afatinib didn’t alter the expression levels of total or phosphorylated ERK1/2 as well as JNK in A2780T cells. Since p38 and AKT are the upstream regulatory proteins of the NF-κB pathway, these results indicated that afatinib could attenuate the expression of ABCB1 via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB pathway.7. Mutations in the tyrosine kinase domain of EGFR were not correlated with the reversal effect of afatinib on MDR in ovarian cancer in this studyMore than 90% of EGFR mutations occur in the tyrosine kinase domain, which are encoded by exon 18, exon 19, exon 20 and exon 21 of the EGFR gene. Sequencing assay results showed that all the four exons were wide-type in both drug-sensitive cells (A2780 and SKOV3) and multidrug-resistant cells (A2780T and SKOV3-DDP). These results indicated that mutations in the tyrosine kinase domain of EGFR were not correlated with the reversal effect of afatinib on MDR in ovarian cancer.8. Structural basis of afatinib as an ABCBl inhibitorThe pharmacophore model of selective ABCB1 inhibitors had five features, including one aromatic center (AR), one hydrophobic center (HP) and three hydrogen bond acceptors (HBAs). Afatinib formed an impressive alignment with this model. To be specific:the aromatic ring was mapped onto the benzene ring of afatinib. The hydrophobic center was mapped onto the fluorine atoms and the connected benzene ring of afatinib. Three HBAs were mapped onto the three oxygen atoms of afatinib. These results indicated that afatinib had the pharmacophore features of selective ABCB1 inhibitors.9. Binding mode between afatinib and ABCBlDocking simulation assay results showed that afatinib could be successfully docked into both a substrate binding domain and an ATP binding domain of the generated ABCB1 model. The docking score for the ATP binding domain (-14.05 kcal/mol) was very close to that of the substrate binding domain (-14.09 kcal/mol). The interactions between afatinib and the ATP binding domain of ABCB1 included three hydrogen bonds and an arene-hydrogen interaction. The oxygen atom of oxolane in afatinib formed two hydrogen bonds with the backbone of Gly533 and Gly534. Besides, the tertiary amine of afatinib formed a hydrogen bond with the side chain of Lys536 while the N-containing heterocyclic of afatinib formed an arene hydrogen interaction with Ala560. DNA sequencing results showed that the normal human cells (liver LO2 cells) and four kinds of ovarian cells were all wide-type in these sites of MDR1 gene. In addition, afatinib treatment also didn’t induce gene mutations in these four sites in both drug-sensitive A2780 cells and multidrug resistant A2780T cells. These results indicated that mutations in these four sites might not be correlated with the reversal efficacy of afatinib on MDR in ovarian cancer in this study.ConclusionsTo sum up, afatinib could significantly reverse ABCB1-mediated MDR in ovarian cancer cells both in vitro and in vivo. Unlike reversible TKIs, afatinib had a distinctive dual-mode action in reversing ABCB1-mediated MDR. Afatinib not only inhibited the efflux function of ABCB1 and stimulated its ATPase activity, but also attenuated its expression transcriptionally via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB pathway. Furthermore, apart from a substrate binding domain, afatinib could also bind to an ATP binding domain of ABCBl through forming hydrogen bonds with Gly533, Gly534, Lys536 and Ala560 sites. Importantly, mutations in these four binding sites of ABCB1 and the tyrosine kinase domain of EGFR might not be correlated with the reversal effect of afatinib on ABCB1-mediated MDR in ovarian cancer in this study. This study strongly advocates for the combination treatment of afatinib and ABCB1 substrate drugs (paclitaxel and adriamycin) to obtain better chemotherapeutic effects and to reverse MDR. This study can facilitate the rediscovery of superior ABCB1 inhibitors from molecular targeted drugs to provide a more effective and safer way of resensitizing ABCB1-mediated MDR.