In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients

Abstract

The present study was performed to investigate the capability of gemcitabine and pemetrexed to synergistically interact with respect to cytotoxicity and apoptosis in T24 and J82 bladder cancer cells, and to establish a correlation between drug activity and gene expression of selected genes in tumour samples. The interaction between gemcitabine and pemetrexed was synergistic; indeed, pemetrexed favoured gemcitabine cytotoxicity by increasing cellular population in S-phase, reducing Akt phosphorylation as well as by inducing the expression of a major gemcitabine uptake system, the human equilibrative nucleoside transporter-1 (hENT1), and the key activating enzyme deoxycytidine kinase (dCK) in both cell lines. Bladder tumour specimens showed an heterogeneous gene expression pattern and patients with higher levels of dCK and hENT1 had better response. Moreover, human nucleoside concentrative transporter-1 was detectable only in 3/12 patients, two of whom presented a complete response to gemcitabine. These data provide evidence that the chemotherapeutic activity of the combination of gemcitabine and pemetrexed is synergistic against bladder cancer cells in vitro and that the assessment of the expression of genes involved in gemcitabine uptake and activation might be a possible determinant of bladder cancer response and may represent a new tool for treatment optimization.

Figure 1

Concentration-dependent cytotoxicity of gemcitabine (dFdC), pemetrexed (MTA), and their combinations in T24 and J82 bladder cancer cells. Each data point represents the percentage of proliferating cells with respect to untreated control and is the average of three independent experiments. Bars, s.d.

Figure 2

Isobologram analysis of pharmacologic interaction of gemcitabine (dFdC) – pemetrexed (MTA) combinations in T24 and J82 cells.

Figure 3

Percentage of cells with damaged DNA after drug treatments with gemcitabine (dFdC), pemetrexed (MTA) and their combinations in both cell lines (A). Upper panels, morphological appearance of control and treated cells. Modulation of hENT1 and dCK expression by pemetrexed and modulation of TS and GARFT expression by gemcitabine in comparison with control in T24 and J82 cells (B). Right panel, representative plot of dCK and GAPDH expression in control and treated J82 bladder cancer cells. Columns, mean values obtained from three independent experiments; bars, s.d. ^*P<0.05 with respect to control, ^**P<0.05 with respect to gemcitabine.

Figure 4

Reduction of P-Ser473 Akt by gemcitabine (dFdC) and pemetrexed (MTA) in bladder cancer cell lines T24 and J82. ^*P<0.05 with respect to control.

Figure 5

Gene expression of gemcitabine-related genes in 12 bladder cancer patients. Values of gene expression were calculated by the GAPDH/target gene ratio in triplicate experiments.