Gemcitabine kills proliferating endothelial cells exclusively via acid sphingomyelinase activation

Abstract

Gemcitabine is a widely-used anti-cancer drug with a well-defined mechanism of action in normal and transformed epithelial cells. However, its effect on endothelial cells is largely unknown. Acid sphingomyelinase (ASMase) is highly expressed in endothelial cells, converting plasma membrane sphingomyelin to pro-apoptotic ceramide upon activation by diverse stresses. In the current study, we investigated gemcitabine impact in primary cultures of endothelial cells. We find baseline ASMase increases markedly in bovine aortic endothelial cells (BAEC) as they transit from a proliferative to a confluent growth-arrested state. Further, gemcitabine activates ASMase and induces release of a secretory ASMase form into the media only in proliferating endothelial cells. Additionally, proliferative, but not growth-arrested BAEC, are sensitive to gemcitabine-induced apoptotic death, an effect blocked by inhibiting ASMase with imipramine or by binding ceramide on the cell surface with an anti-ceramide Ab. Confluent growth-arrested BAEC can be re-sensitized to gemcitabine-induced apoptosis by provision of exogenous sphingomyelinase. A highly similar phenotype was observed in primary cultures of human coronary artery endothelial cells. These findings reveal a previously-unrecognized mechanism of gemcitabine cytotoxicity in endothelium that may well contribute to its clinical benefit, and suggest the potential for further improvement of its clinical efficacy via pharmacologic modulation of ASMase/ceramide signaling in proliferative tumor endothelium.

Keywords: Apoptosis; Ceramide; Gemcitabine; Vascular endothelium.

Figure 1. ASMase activity elevates over time in cultured endothelial cells

A: BAEC were harvested at different days after seeding as indicated and ASMase activity was determined in cell lysates using bovine [¹⁴C-methylcholine]sphingomyelin. Confluence was judged visually using a phase-contrast microscope (day 0 was defined as the day the monolayer covered 100% of the surface of the well). B: ASMase activity was determined in absence of exogenous zinc, quantifying the lysosomal fraction of cellular ASMase. C: BAEC culture medium (serum-free) was collected after 24 hours and concentrated using 30 kDa molecular weight cut-off filter units. ASMase activity elevates over time in concordance with total cellular levels. Data (mean ± SD) are collated from 9 separate experiments.

Figure 2. Gemcitabine activates ASMase exclusively in proliferating BAEC

A: Time-course of ASMase activity in proliferating BAEC upon addition of 100 nM gemcitabine. B: Dose-response curve of ASMase activation at 5 minutes. C: Gemcitabine does not activate ASMase in growth-arrested BAEC. Data (mean ± SEM) are collated from 3 separate experiments.

Figure 3. Gemcitabine induces ASMase secretion

A: 15 minutes after adding gemcitabine to proliferating BAEC, medium was harvested and concentrated using a 30 kDa molecular weight cutoff spin column. ASMase activity (sphingomyelin conversion, nmol/mg/h) in concentrated medium samples was determined in the absence (open bars) or presence (grey bars) of exogenous zinc. Activity of the secretory ASMase is dependent on exogenous zinc. B: The rate of ASMase activity secreted per minute is linearly related to gemcitabine concentration. Baseline ASMase secretion rate (0 μM) was subtracted from the gemcitabine-stimulated samples. Data (mean ± SEM) are collated from 5 experiments.

Figure 4. Gemcitabine activates and induces secretion of ASMase exclusively in proliferating HCAEC

A: Total cellular ASMase activity in proliferating HCAEC upon addition of gemcitabine (10 μM). B: Stable ASMase activity profile upon gemcitabine addition to HCAEC, growth-arrested by 3 days confluence. C: Measurement of ASMase activity secreted into the medium. Gemcitabine induces time-dependent secretion of ASMase in proliferating HCAEC. D: ASMase secretion from growth-arrested HCAEC. Medium was collected and cells were lysed at the indicated time points after gemcitabine (10 μM) addition. Data (mean ± SEM) are collated from 4 separate experiments.

Figure 5. Proliferating endothelial cells are gemcitabine sensitive, in contrast to growth-arrested cells

Endothelial cells were either proliferating (closed circles), or confluent at growth-arrest (open circles). A: BAEC were exposed to gemcitabine and MTT conversion was measured as a readout for cell viability. Cells treated with vehicle as a control were set at 100%. B: After 16 hours gemcitabine exposure, BAEC cells were harvested and apoptosis was quantified by bis-benzimide nuclear staining. C: Pre-treatment (45 min) of BAEC with the pan-caspase inhibitor z-VAD-fmk blocks apoptosis induced by 10 μM gemcitabine. D: HCAEC were exposed to gemcitabine for 16 hours and apoptosis was quantified. Data (mean ± SD) are collated from 3 separate experiments each.

Figure 6. Pharmacologic ASMase inhibition and anti-ceramide antibody prevent gemcitabine-induced apoptosis

A: Pre-incubation with up to 150 μM imipramine inhibits gemcitabine-induced endothelial apoptosis. Imipramine was added to proliferating BAEC 30 minutes before 1.0 μM gemcitabine. B: Anti-ceramide monoclonal antibody blocks gemcitabine-induced apoptosis in proliferating endothelial cells. BAEC were pre-incubated with anti-ceramide antibody for 15 min prior to 1.0 μM gemcitabine. Cells were harvested after 16 hours and apoptosis was quantified by bis-benzimide staining. Data (mean ± SD) are collated from 3 separate experiments each.

Figure 7. Exogenous sphingomyelinase restores gemcitabine-induced apoptosis in confluent BAEC

A: Bacterial sphingomyelinase (Bacillus cereus; bSMase) was applied with or without 1 μM gemcitabine to a confluent monolayer of BAEC in a 2 cm² well. Apoptosis was quantified after 16 hours. Growth-arrested BAEC otherwise resist gemcitabine (leftmost bar). B: Anti-ceramide antibody blocks the apoptosis, induced by bSMase plus gemcitabine on confluent BAEC. Anti-ceramide antibody (10 μg/ml) was added prior to bSMase (5 units) and gemcitabine (1 μM) treatment. Asterisk denotes a p value < 0.05. The activity of a bSMase unit is defined by the hydrolysis of 1.0 μmol sphingomyelin per minute at 37°C. Data (mean ± CV) are collated from 3 separate experiments each.