Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters

Abstract

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Hepatocyte-Specific Deletion of EGFR in Mice Reduces Hepatic Abcg2 Transport Activity Measured by [11C]erlotinib and Positron Emission Tomography

“…Interestingly, there was an increase in hepatic Abcb1a/b expression levels (Fig. 7C), which is in line with another study that assessed changes in transporter expression levels in human conditionally immortalized proximal tubule epithelial cells after treatment with the EGFR recombinant antibody cetuximab; significant decreases occurred in ABCG2 mRNA and increases were seen in ABCB1 mRNA (Caetano-Pinto et al, 2017). Our data thus support the premise that hepatobiliary excretion of [ 11 C]erlotinib is, to a larger extent, dependent on Abcg2 than on Abcb1a/b, as k bile was decreased in EGFR Δhep mice despite an apparent upregulation of canalicular Abcb1a/b.…”

A Pharmacokinetic and Pharmacogenetic Analysis of Osteosarcoma Patients Treated With High‐Dose Methotrexate: Data From the OS2006/Sarcoma‐09 Trial

“…21 Studies in mice and humans have suggested that ABCG2, coding for the breast cancer resistance protein (BCRP) transporter has a major role in transporting MTX out of the liver and kidney. 39,40 BCRP is a multidrug transporter expressed in many tissues, 41 including the human kidney proximal tubule apical membrane. 42 In our cohort, GG genotype carriers for rs13120400 appear to have a moderate decrease on MTX exposure compared to AA or GA carriers under a recessive genetic model.…”

Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip

“…Human-derived immortalized renal cell models such as human embryonic kidney 293 (HEK 293) and human kidney 2 (HK 2) cells have shown to be a valuable tool in screenings in drug development, but have low or no endogenous expression of relevant drug transporters (48,49). To overcome these limitations, we have developed ciPTEC-OAT1 with proven suitability for nephrotoxicity and drug-transporter interactions studies (10)(11)(12)(13)(14). The broad range of drug transporter expression in ciPTEC-OAT1 provides a strong basis for highly predictive renal drug interaction studies.…”

“…Further transduction resulted in stable expression and functionality of OAT1 or OAT3 (10). Multiple studies have shown the potential of ciPTEC for studying drug transport and drug-induced toxicity cultured under conventional static conditions, often referred to as two-dimensional (2D) cell culture (10)(11)(12)(13). In addition, transepithelial barrier function, polarization, and transport of cations and anions have been demonstrated in ciPTEC cultured in a threedimensional (3D) model using a tailor-made hollow fiber membrane system exposed to flow (13,14).…”