P-glycoprotein Blockers Augment the Effect of Mitomycin C on Human Tenon's Fibroblasts

Abstract

Purpose: Mitomycin C (MMC), which induces apoptosis in human Tenon's fibroblasts (HTF), is frequently used to retard wound healing after glaucoma surgery. The aim of this in vitro study was to examine whether adjunctive Verapamil and Cyclosporine could augment the cytotoxic effect of MMC on HTF.

Methods: Fibroblast cell lines were established by explant culture from human tissue biopsy samples obtained during trabeculectomy procedures. Cells were exposed to MMC at varying concentrations (0.01-0.4 mg/ml) for 3 minutes, prior to washing in the presence or absence of the following drugs: Staurosporine (0.003mg/ml), Verapamil (2.5-0.25 mg/ml), or Cyclosporine (50-0.5 mg/ml). Following exposure, cells were cultured for 6 hours and surviving cells quantitated by haemocytometer counts.

Results: Both Verapamil and Staurosporine exhibited mild toxic effects on their own, but greatly enhanced the apoptotic effect of MMC. Staurosporine is too toxic to be considered clinically, so its augmentive effect on the activity of MMC was not studied further here. Doses as low as 0.25 mg/ml of Verapamil continued to show significant augmentation of the apoptotic effect of MMC Cyclosporine at a clinically used concentration (5 mg/ml) exhibited modest augmentation of the effect of MMC.

Conclusions: Verapamil and Cyclosporine in clinically acceptable concentrations potentiate the effect of MMC and may obviate the need for high dose antimetabolites in trabeculectomy; however, further preclinical study is required.

Translational relevance: Adjunctive Verapamil or Cyclosporine may allow lower dose MMC to be used in glaucoma filtration surgery while maintaining the same antifibrotic effects.

Keywords: Mitomycin C; P-glycoprotein; human Tenon's fibroblasts; wound healing.

Figure 1.

Photomicrographs comparing control HTF (A) with cells exposed to MMC at 0.4 mg/mL for 3 minutes followed by a further 24 hours incubation (B), as well as histograms showing the number of cells over time following such treatment (C), and the effect of varying concentrations of MMC (D). HTF had the characteristic fusiform appearance of fibroblasts in culture (A), and this was retained following treatment with MMC, although there did appear to be a reduction in cell number (B). Quantitation confirmed progressive reduction over 24 hours in HTF number following MMC treatment at 0.4 mg/mL (C), while lower concentrations of this agent had less effect on cell number (D). Error bars show SEM.

Figure 2.

Photomicrographs of HTF treated with Verapamil (V) (2.5 mg/mL) for 3 minutes and then further incubated for 24 hours without (A) or with (B) MMC treatment for a further 3 minutes at 0.4 mg/mL, as well as quantitation of surviving cell number following such V pretreatment with subsequent 3 minute exposure to MMC ranging from 0 to 0.4 mg/mL (C). V treatment alone had little effect on the appearance or number of HTF (A), but when combined with further MMC treatment did significantly reduce cell number and alter the morphology of surviving cells (B). (C) This effect of V at greatly potentiating the effect of MMC was seen at all concentrations of MMC studied (t-test P < 0.001), while varying the concentration of MMC did not have any clear effect (ANOVA, P > 0.05). Error bars show SEM. Asterisks denote statistical significant result.

Figure 3.

Photomicrographs of HTF treated Cyclosporine A (CA) (0.5 mg/mL) for 3 minutes and then further incubated for 24 hours without (A) or with (B) MMC treatment for a further 3 minutes at 0.4 mg/mL, as well as quantitation of surviving cell number following such CA pretreatment with subsequent 3 minute exposure to MMC ranging from 0 to 0.4 mg/mL (C). CA treatment alone had little effect on the appearance or number of HTF (A), but when combined with further MMC treatment did significantly reduce cell number and alter the morphology of surviving cells (B). (C) This effect of CA of greatly potentiating the effect of MMC was seen for MMC at 0.2 mg/mL and 0.4 mg/mL (t-test P < 0.001), but not at the lowest concentration tested. Error bars show SEM. Asterisks denote statistical significant result.

Figure 4.

Histograms showing the number of surviving HTF relative to control cells following initial pretreatment for 3 minutes with either Staurosporine (S) (0.3 μg/mL) (A), or Interferons α and γ combined (INF) at 500 and 100 U/mL respectively (B), followed in both cases by further incubation with MMC at concentrations ranging from 0 to 0.4 mg/mL. (A) Although S pretreatment did appear to have a modest effect in increasing sensitivity to MMC induced HTF death, this was only statistically significant when MMC was at a concentration of 0.2 mg/mL (P = 0.01, t-test). (B) Combined INF treatment significantly reduced HTF survival when cells were treated with MMC in a clearly dose dependent manner (P < 0.001). A three-way ANOVA test revealed a significant difference between treated groups (P < 0.001). Error bars show SEM. Asterisks denote statistical significant result.

Figure 5.

The effect of varying the pretreatment concentration of V, expressed as a percentage of 2.5 mg/mL, on the HTF cell counts of cells treated with 0.4 mg/mL MMC. Incubation time post treatment was 6 hours. At concentrations as low as 0.25 mg/mL V there was still marked augmentation of the effect of MMC. The effect was not as marked at the lowest dose of V tested (0.125 mg/mL). Error bars show SEM. The effect of V was statistically significant across all groups (P < 0.001).