Effect of methotrexate/dexamethasone combination on epithelial-mesenchymal transition and inflammation gene expression of human RPE cells in-vitro

Abstract

Introduction: RPE cells serve as an experimental model for studying a retinal disease called proliferative vitreoretinopathy (PVR). The pathological background of PVR involves uncontrolled cell proliferation, increased inflammation, and enhanced epithelial-mesenchymal transition (EMT), which have been the focus of various research studies. The present study aimed to explore the effects of combination therapy using methotrexate (MTX) and dexamethasone (DEXA) on the expression of genes involved in apoptosis, inflammation and EMT in retinal pigment epithelial (RPE) cells.

Methods: Our study design comprised two sets of experiments. First, we assessed the effect of MTX serial dilutions (0.5x, x, 2x, and 4x, where x = 100 μg/mL) on RPE cells to determine the optimal concentration of MTX that promotes apoptosis-related gene expression without altering inflammatory-related gene expression. Second, we investigated the influence of MTX (at the selected dose) alone or in combination with DEXA (50 μg/mL) on apoptosis, inflammation, and EMT-related gene expression in RPE cells at the transcriptional level.

Results: Treatment with 100 μg/mL MTX demonstrated a pro-apoptotic effect according to the expression level of BAX and BCL-2 in RPE cells. The combination of MTX (100 μg/mL) and DEXA significantly reduced the expression of inflammation-related genes (IL-1b, IL-6), indicating a synergistic anti-inflammatory effect. However, there was no significant effect on the expression of genes related to EMT (TGF-β, CD90, β-Catenin, Snail), except for a partial neutralization of the reducing effect of MTX on ZEB1 and α-SMA genes.

Discussion: Our study highlighted the potential pro-apoptotic effect of MTX (at 100 μg/mL) on RPE cells and the synergistic anti-inflammatory impact of MTX/DEXA combination therapy. Nevertheless, this combination did not significantly affect genes associated with EMT. Further research is required to elucidate the clinical implications of these findings in the management of PVR.

Keywords: EMT; PVR; RPE; apoptosis; dexamethasone; inflammation; methotrexate.

FIGURE 1

Study design and workflow.

FIGURE 2

Morphological features of RPE cells. Panels (A,B) present phase-contrast images of RPE cells from passages 1 and 6, respectively (magnification ×20). In the first passage, RPE cells exhibited high pigmentation (indicated by arrows) and intercellular connections (depicted by dashed circles) (A). However, pigmentation and cell junctions diminished in subsequent passages (B). Agarose gel electrophoresis of RPE65 RT-PCR products for RPE cells from the first and sixth passages revealed a decrease in the level of RPE65 expression in the sixth passage compared to the first passage (confirming the typic characteristics of RPE cells). To assess this, 100 ng of total RNA was used from each passage of RPE cells. P1 denotes RPE65 mRNA expression in RPE cells from the first passage, and P6 represents RPE65 mRNA expression in RPE cells from the sixth passage (C).

FIGURE 3

The apoptosis-related mRNA levels in RPE cells subjected to (A) serial dilutions of MTX (0.5x, x, 2x, and 4x, where x = 100 μg/mL) and (C) with MTX (100 μg/mL), DEXA (50 μg/mL), and combination of MTX + DEXA (B). The dashed line represents the mRNA level in the controls (set as 1) following a 24-h treatment period. The graph presents the mean value of relative expression ± standard deviation of the mean. Statistical significance is denoted by * for P < 0.05 and # for P < 0.10, as determined by One-way ANOVA analysis (post hoc LSD, n = 3).

FIGURE 4

The inflammation-related mRNA levels in RPE cells exposed to (A) serial dilutions of MTX (0.5x, x, 2x, and 4x, where x = 100 μg/mL) and (B) MTX (100 μg/mL), DEXA (50 μg/mL), and combination of MTX + DEXA. The dashed line represents the mRNA level in the controls (set as 1) following a 24-h treatment period. The graph presents the mean value of relative expression ± standard deviation of the mean. Statistical significance is denoted by * for P < 0.05 and # for P < 0.10, as determined by One-way ANOVA analysis (post hoc LSD, n = 3).

FIGURE 5

The relative expression of EMT-related genes in RPE cells treated with MTX (100 μg/mL), DEXA (50 μg/mL), and combination of MTX + DEXA. The dashed line represents the mRNA level in the controls (set as 1) following a 24-h treatment period. The graph presents the mean value of relative expression ± standard deviation of the mean. Statistical significance is denoted by * for P < 0.05 and # for P < 0.10, as determined by One-way ANOVA analysis (post hoc LSD, n = 3).