Cell cycle modulation by a multitargeted antifolate, LY231514, increases the cytotoxicity and antitumor activity of gemcitabine in HT29 colon carcinoma

The proliferation rate of HT29 colon carcinoma cells was decreased by the m ultitargeted antifolate (MTA), LY231514, This effect correlated with a buil dup of cells near the G(1)-S interface after 24 h of incubation, and a sync hronized progression of the population through 8 phase during the next 24 h , MTA treatment (0.03-3 mu M) was minimally cytotoxic (20-30%) to HT29 cell s after a 24-h exposure, and no dose response was observed, In contrast, th e nucleoside analogue gemcitabine (GEM) eras cytotoxic (IC50, 0.071 +/- 0.0 11 mu M; IC90, 0.648 +/- 0.229 mu M) after a 24-h exposure. We hypothesized that pretreatment of these cells with MTA would increase the potency of GE M by synchronizing the population for DNA synthesis. The cytotoxicity of GE M increased 27-fold when MTA was administered 24 h before GEM (IC50, 0.032 +/- 0.009 mu M; IC90, 0.094 +/- 0.019 mu M). In addition, an increase in ce ll kill for the combination compared with GEM alone (IC99, 12 mu M for GEM alone; IC99, 0.331 mu M for combination) was observed. No increase in poten cy or cell kill was observed when the two compounds were added simultaneous ly. 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 x enograft tumor model. At the doses tested, MTA alone (100 mg/kg) had a marg inal effect on tumor growth delay, whereas GEM (80 mg/kg) produced a statis tically significant tumor growth delay. In combination, the increase in tum or growth delay was greatest when MTA was administered before GEM, compared with simultaneous drug administration or the reverse sequence, e.g., GEM f ollowed 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, RITA induced cell cycle effects on HT29 cells that resulted in potentiation of the cytotoxicity of GEM. In vivo, combination o f these two drugs also demonstrated a schedule-dependent synergy that was o ptimal when MTA treatment preceded GEM.