Cell Cycle Modulation by a Multitargeted Antifolate, LY231514, Increases the Cytotoxicity and Antitumor Activity of Gemcitabine in HT29 Colon Carcinoma

Abstract

The proliferation rate of HT29 colon carcinoma cells was decreased by the multitargeted antifolate (MTA), LY231514. This effect correlated with a buildup of cells near the G₁-S interface after 24 h of incubation, and a synchronized progression of the population through S phase during the next 24 h. MTA treatment (0.03–3 μm) was minimally cytotoxic (20–30%) to HT29 cells after a 24-h exposure, and no dose response was observed. In contrast, the nucleoside analogue gemcitabine (GEM) was cytotoxic (IC₅₀, 0.071 ± 0.011 μm; IC₉₀, 0.648 ± 0.229 μm) after a 24-h exposure. We hypothesized that pretreatment of these cells with MTA would increase the potency of GEM by synchronizing the population for DNA synthesis. The cytotoxicity of GEM increased 2–7-fold when MTA was administered 24 h before GEM (IC₅₀, 0.032 ± 0.009 μm; IC₉₀, 0.094 ± 0.019 μm). In addition, an increase in cell kill for the combination compared with GEM alone (IC₉₉, 12 μm for GEM alone; IC₉₉, 0.331 μm for combination) was observed. No increase in potency or cell kill was observed when the two compounds were added simultaneously. MTA pretreatment also potentiated the cytotoxicity of a 1-h exposure to GEM. These cell-based observations were extended to evaluate the schedule-dependent interaction of these two agents in vivo using a nude mouse HT29 xenograft tumor model. At the doses tested, MTA alone (100 mg/kg) had a marginal effect on tumor growth delay, whereas GEM (80 mg/kg) produced a statistically significant tumor growth delay. In combination, the increase in tumor growth delay was greatest when MTA was administered before GEM, compared with simultaneous drug administration or the reverse sequence, e.g., GEM followed by MTA. The effect of sequential administration of MTA followed by GEM was greater than additive, indicating synergistic interaction of these agents. Thus, in vitro, MTA induced cell cycle effects on HT29 cells that resulted in potentiation of the cytotoxicity of GEM. In vivo, combination of these two drugs also demonstrated a schedule-dependent synergy that was optimal when MTA treatment preceded GEM.