The naturally occurring flavonoid nobiletin reverses methotrexate resistance via inhibition of P-glycoprotein synthesis

Abstract

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA). However, after long-term treatment, some patients develop resistance. P-glycoprotein (P-gp), as an indispensable drug transporter, is essential for mediating this MTX resistance. In addition, nobiletin (NOB), a naturally occurring polymethoxylated flavonoid, has also been shown to reverse P-gp-mediated MTX resistance in RA groups; however, the precise role of NOB in this process is still unclear. Here, we administered MTX and NOB alone or in combination to collagen II-induced arthritic (CIA) mice and evaluated disease severity using the arthritis index, synovial histopathological changes, immunohistochemistry, and P-gp expression. In addition, we used conventional RNA-seq to identify targets and possible pathways through which NOB reverses MTX-induced drug resistance. We found that NOB in combination with MTX could enhance its performance in synovial tissue and decrease P-gp expression in CIA mice compared to MTX treatment alone. In vitro, in MTX-resistant fibroblast-like synoviocytes from CIA cells (CIA-FLS/MTX), we show that NOB treatment downregulated the PI3K/AKT/HIF-1α pathway, thereby reducing the synthesis of the P-gp protein. In addition, NOB significantly inhibited glycolysis and metabolic activity of CIA-FLS/MTX cells, which could reduce the production of ATP and block P-gp, ultimately decreasing the efflux of MTX and maintaining its anti-RA effects. In conclusion, this study shows that NOB overcomes MTX resistance in CIA-FLS/MTX cells through the PI3K/AKT/HIF-1α pathway, simultaneously influencing metabolic processes and inhibiting P-gp-induced drug efflux.

Keywords: P-glycoprotein; PI3K/AKT/HIF-1α; glycolysis; methotrexate-induced drug resistance; nobiletin; rheumatoid arthritis.

Figure 1

Effect of NOB on the arthritis severity in CIA mice and P-gp in synovial tissue.A, effect of NOB on the arthritis index. B, a microcomputer tomography image of the hind paw of a CIA mouse. C, bone surface/volume ratio (BS/BV; mm⁻¹). D, H&E staining of ankle joint tissue in each group. (The scale bar represents 100 μm) Red arrow indicates inflammatory cell infiltration; green arrow indicates disordered collagen fiber arrangement; and black arrow indicates artery. E, histological score of each group. F–H, levels of IL-1β, IL-6, and TNF-α in mice ankle tissue. I, immunohistochemistry results about P-gp (the scale bar represents 100 μm). J, the area distribution of P-gp. K, Western blot results. CIA, collagen II-induced arthritis; IL, interleukin; NOB, nobiletin; P-gp, P-glycoprotein. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2

The chemical effect of NOB on CIA-FLS viability and cytotoxic effects, expression and activity of P-gp, and apoptosis of NOB and MTX in two cell lines.A, the chemical structure of NOB. The cells are cultivated with increasing concentrations of NOB (0, 10, 20, 40, 80, and 100 μM) for 24 h (B) and 48 h (C). Methotrexate (25, 50, 100, 250, and 500 μg/ml) efficiently decreased the viability of CIA-FLS cells (D) and CIA-FLS/MTX cells (E) at 24, 48, and 72 h. F, the combination of NOB and MTX synergistically decreased the viability of CIA-FLS/MTX cells at 72 h. The cell viability was measured by CCK-8 assay. G, MTX (100 μg/ml), NOB (80 μM), and the combination of NOB with MTX treatment (24 h) induced apoptosis in CIA-FLS/MTX cells. Apoptosis was measured by Annexin V/PI staining and flow cytometry (p < 0.05). H, the quantitative results of apoptosis rate. I, the expression of P-gp in CIA-FLS and CIA-FLS/MTX cells. J, Western blot results. Nobiletin (80 μM), MTX (100 μg/ml), or their combination affected P-gp expression in CIA-FLS/MTX cells. K, RT-qPCR results. L, the effect of NOB on P-gp substrate efflux activity was assessed. CIA, collagen II-induced arthritis; CCK-8, cell counting kit-8; FLS, fibroblast-like synovial cells; MTX, methotrexate; NOB, nobiletin; P-gp, P-glycoprotein; RT-qPCR, quantitative reverse-transcription PCR. ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

RNA-seq results.A and B, the DEGs consequence. C, cluster analysis of DEGs. D, gene ontology enrichment analysis. E, Kyoto Encyclopedia of Genes and Genomes analysis. DEGs, differentially expressed genes.

Figure 4

Effects of NOB on the AKT/HIF-1α signaling pathway in CIA-FLS/MTX cells.A, hypoxia-inducible factor-1 α expression in CIA-FLS and CIA-FLS/MTX cells. B-D, the relative expression of AKT, PI3K, and HIF-1α in different groups. E, quantitative reverse-transcription PCR results of HIF-1α. NOB, nobiletin; CIA, collagen II-induced arthritis; AKT, protein kinase B; FLS, fibroblast-like synovial cells; HIF1-α, hypoxia-inducible factor-1 α; MTX, methotrexate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

The effect of NOB on glycolysis status in CIA-FLS/MTX cells.A, ATP production. B, glucose consumption. C, lactate export. Western blot images of (D) PKM2, (E) GLUT1, (F) LDH, and (G) HK-II protein expression levels. H–K, quantitative reverse-transcription PCR results. CIA, collagen II-induced arthritis; FLS, fibroblast-like synovial cells; GLUT1, glucose transporter 1; HK-II, hexokinase-II; LDH, lactate dehydrogenase; MTX, methotrexate; NOB, nobiletin; PKM2, pyruvate kinase isozyme type M2.

Figure 6

Effects of siRNA transfection on the AKT/HIF-1α signaling pathway and the expression of P-gp. A–C, knockdown efficacy of AKT and HIF-1α. D–H, relative expression of AKT, p-AKT, HIF-1α, and P-gp in different groups. AKT, protein kinase B; HIF1-α, hypoxia-inducible factor-1 α; P-gp, P-glycoprotein.

Figure 7

The mechanism through which NOB reverses MTX-induced RA resistance. Nobiletin downregulated the AKT/HIF-1α signaling pathway, thereby maintaining the drug efflux caused by the activation of P-gp. In addition, NOB significantly inhibits glycolysis and metabolic activity of CIA-FLS/MTX cells for energy and nutrition supply, thereby increasing the efficacy in response to MTX in RA. AKT, protein kinase B; CIA, collagen II-induced arthritis; HIF1-α, hypoxia-inducible factor-1 α; MTX, methotrexate; NOB, nobiletin; P-gp, P-glycoprotein; RA, rheumatoid arthritis.