Meclofenamic Acid Restores Gefinitib Sensitivity by Downregulating Breast Cancer Resistance Protein and Multidrug Resistance Protein 7 via FTO/m6A-Demethylation/c-Myc in Non-Small Cell Lung Cancer

Abstract

Background and objective: Gefitinib (GE) is a first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) for patients with advanced non-small cell lung cancer (NSCLC) carrying EGFR activating mutations. However, drug resistance limits the clinical efficacy of gefitinib and ultimately leads to extremely poor clinical benefit. Meclofenamic acid (MA) is a non-steroidal anti-inflammatory drug (NSAID) that relieves moderate and severe pain. In the present study, we aim to determine the MA sensibilization of GE in NSCLC.

Methods: MTT assay was conducted to determine the synergistic effect of MA with GE in GE-sensitive and -resistant cell lines based on the Chou-Talalay method. The Annexin V-PI flow cytometry analysis was conducted to evaluate apoptosis. Western blot assay was used to detect alterations of EGFR downstream molecules. Tritium-labeled GE accumulation analysis was used to determine the efflux activity of GE. Dot blot assays were conducted to determine m6A levels after the MA and GE co-administration. Western blot evaluated the expression of FTO, c-Myc, MRP7, BCRP, and apoptotic proteins.

Results: MA showed a significant synergistic effect with GE in GE-resistant NSCLC cells; co-administration of MA with GE induced caspase-related apoptosis in resistant NSCLC cells. Moreover, EGFR downstream molecules, including Akt and MAPKs pathways, were significantly inhibited by the MA-GE combination. Short-term incubation of MA did not alter the efflux of GE; however, after incubation for 24 h, the accumulation of tritium-labeled GE significantly increased. A mechanism study showed that co-administration of MA and GE significantly downregulated BCRP and MRP7 expression in GE-resistant cells; increased N6-methylation was also observed after co-administration. The FTO/c-Myc was determined as target pathways on MA and GE co-administration mechanisms.

Conclusion: Our findings provide novel therapeutic approaches for GE-resistant NSCLC by combination use with MA through FTO-mediated N6-demethylation.

Keywords: M6A; drug resistance; gefitinib; meclofenamic acid; non-small cell lung cancer (NSCLC).

Figure 1

The antiproliferative effects of MA and/or GE on gefitinib-resistant NSCLC cells. (A–D) The parental PC9 and H292 as well as gefitinib-resistant PC9/GR and H292/GR cells were treated with various concentrations of GE or MA; the antiproliferative effects were determined by MTT assay following 48 h of incubation. Cells were treated with GE and MA as a single agent or in combination for 48 h, and MTT assays were used to analyze the cell viability. The Chou–Talalay method was conducted to calculate the combination index (CI); a CalcuSyn software were used to analyze the data. (E–H) illustrate the combination ratio/viability, while (I–L) show the fraction affected/combination index. Data are presented for at least three independent repetitions and were illustrated as mean ± SD.

Figure 2

MA and GE synergistically induced caspase-3-associated apoptosis in gefitinib-resistant NSCLC cells. Apoptotic cells in PC9/GR (A) and H292/GR (B) exposed to the single drugs or combination are presented in dot blot. Western blot results show dynamic caspase-3 face after PC9/GR (C) or H292/GR (D) exposed to single or combination drugs.

Figure 3

MA and GE synergistically inhibited the activity of the EGFR-related signaling pathway. Western blot results showed the expression of p-EGFR, p-Akt, and p-ERK1/2 in PC9/GR (A) and H292/GR (B) cells exposed to MA and GE for single or combination use. (C–H) showed the statistical analysis of the Western blot results (A, B). Data are presented for at least three independent repetitions and were illustrated as mean ± SD. ***p < 0.001, ****p < 0.0001.

Figure 4

MA significantly enhanced the accumulation of GE in gefitinib-resistant cells. (A) The collection of GE in parental PC9 and PC9/GR cells exposed to MA. (B) The accumulation of GE in parental H292 and H292/GR cells exposed to MA. Data are presented for at least three independent repetitions and were illustrated as mean ± SD. ***p < 0.001.

Figure 5

MA and GE synergistically inhibited the expression of BCRP and MRP7. BCRP and MRP7 in PC9/GR (A) and H292/GR (B) cells. (C, D) The membrane protein expression of BCRP and MRP7 in PC9/GR and H292/GR cells after MA and GE for single or in combination exposed. (E, F) The total cell lysate protein expression of BCRP and MRP7 in PC9/GR and H292/GR cells after MA and GE for single or in combination exposed. Data are presented for at least three independent repetitions and were illustrated as mean ± SD. *p < 0.05.

Figure 6

MA and GE in combination upregulated FTO-mediated MYC M6A modification. (A, B) Dot blot shows the M6A modification level in PC9/GR and H292/GR cells exposed to MA and GE for a single drug or combination. (C, D) Expression of FTO and MYC determined by Western blot assays in PC9/GR and H292/GR cells. (E, F) The m6A modification level of MYC cells was assessed by Me-RIP assay in PC9/GR and H292/GR cells exposed to MA and GE for a single drug or in combination. Data are presented for at least three independent repetitions and were illustrated as mean ± SD. *p < 0.05, ***p < 0.001.