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.

Keywords: combination therapy; drug disposition; drug transporters; kinase signaling; renal proximal tubule.

Figure 1

Methotrexate uptake in ciPTEC-OAT1 and ciPTEC-OAT3. Methotrexate (MTX) significantly reduced the uptake of fluorescein (A) and inhibited the efflux of Hoechst33342, in ciPTEC-OAT1 (B) and GS-MF (C), in both ciPTEC-OAT1 and OAT3. Calcein efflux was not blocked by methotrexate in both cell lines (D), and Hoechst33342 activity in ciPTEC-OAT3 could not be determined. Model inhibitors Probenecid, KO143, MK571, and PSC833 were tested as positive controls. Data are presented as mean values ± SEM. Statistical analysis was performed via unpaired Student’s t test. ∗, p < 0.05 and ∗∗∗, p < 0.01 compared to control (CTRL). A schematic depiction of the potential interactions (E).

Figure 2

Expression and function of renal drug transporters upon cetuximab pretreatment. Cells were pretreated with cetuximab (CTX) before gene and protein expression was determined. The activity of renal drug transporter was evaluated using fluorescent substrates (OAT1, fluorescein; BCRP, Hoechst33342; P-gp, calcein and MRP4; GS-MF, respectively). Changes in transport activity are depicted in fluorescent intensity expressed in arbitrary units (a.u). CTX effectively reduced the expression and activity of OAT1 (A, B) and BCRP (C, D), while increasing the expression and activity of P-gp (E, F) and MRP4 (G, H). Protein expression was determined for OAT1 (60 kDa) and BCRP (72 kDa) (A, C lower panel), Na,K-ATPase was used as loading control (112 kDa). Data are presented as mean values ± SEM, n = 3. Statistical analysis was performed via unpaired Student’s t test. ∗, p < 0.05 and ∗∗∗, p < 0.01 compared to control (+EGF condition).

Figure 3

Cetuximab-mediated regulation of EGFR downstream signaling. Cetuximab (CTX) promoted the activation of STK and phospholipases leading to PI3K-AKT and MAPK-ERK regulation of renal drug transporters. Data in panel B is expressed as mean values ± SEM of a minimum of two independent assays performed in triplicate.

Figure 4

Cetuximab reduced methotrexate-induced cytotoxicity in ciPTEC-OAT1. Exposure to cetuximab (CTX) for 24 h reduced the uptake of methotrexate (MTX) in both CM (A) and SFM (B). CTX pretreatment effectively rests cell cycle progression (C). For cell viability assessments, cells were exposed to methotrexate (0–100 μM) for 24 h in SFM (D), MTX (100 μM) for 24 h in SFM, followed by 24 h of treatment with CM (E) or CTX. Cells were preconditioned for 24 h in SFM with CTX or U-0126 (2 μM), followed by 48 h of exposure to MTX (100 μM) (F). MTT assay was then performed. CTX preconditioning did not rescue MTX cytotoxicity in ciPTEC-OAT3 (G). Results are expressed in viability (%) compared to control (i.e., SFM-treated cells). CTX pretreatment effectively reduced MTX uptake and toxicity. Values are shown as mean ± SEM of minimally two independent experiments performed in triplicates. ∗, Significantly different from control (p < 0.05); #, significantly different from MTX (100 μM; p < 0.05).

Figure 5

Cetuximab reduced cisplatin-induced cytotoxicity. Cell viability in the presence of cisplatin (CDDP) was determined after cetuximab (CTX) pretreatment for 24 h in SFM, followed by exposure to CDDP (25 μM) for 24 h. MTT assay was then performed (A). Preconditioning of cells with CTX decreased CDDP cytotoxicity. Results are expressed in viability (%) compared to control (i.e., SFM-treated cells). CDDP toxicity after 72 h recovery in CM and CTX was determined after 48 h CTX pretreatment followed by 6 h of increasing CDDP concentrations (0–100 μM) (B). CDDP recovery was performed in CM to minimize cell stress. The absence of EGF ameliorated CDDP toxicity. The accumulation of ASP⁺ (OCT2 substrate) was reduced upon CTX pretreatment (C). Values are shown as mean ± SEM of minimally two independent experiments performed in triplicates. ∗, Significantly different from control (p < 0.05).