Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

Abstract

Background: Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence.

Methods: Growth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant "bolus" administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models.

Results: MK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18-24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients' plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G₂ phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval.

Conclusions: There are two reasons why delayed addition of MK-8776 enhances sensitivity to gemcitabine: first, there is an increased number of cells arrested in S phase; and second, the arrested cells have adequate time to initiate recombination and thereby become Chk1 dependent. These results have important implications for the design of clinical trials using this drug combination.

Figure 1

Impact of gemcitabine and MK-8776 on cell cycle perturbation of MDA-MB-231 cells. A. Cells were incubated with 0 – 50 nmol/L gemcitabine for 24 h without (left) or with (right) 1 μmol/L MK-8776. The drugs were then removed and cells incubated for an additional 24 or 48 h. Cells were then analyzed for DNA content by flow cytometry. B. Similar to A except cells were incubated with gemcitabine for only the first 6 h, while MK-8776 was added only from 18–24 h.

Figure 2

Concentration and schedule dependence for the induction of DNA damage by gemcitabine plus MK-8776 in MDA-MB-231 cells. A. Cells were incubated with the indicated concentration of gemcitabine for 24 h without, or concurrently with 1 μmol/L MK-8776. Cell lysates were analyzed by western blotting. B. Cells were incubated with 1–10 nmol/L gemcitabine for 0 – 24 h, without or with 1 μmol/L MK-8776. The 24-h sample incubated with 1 nmol/L gemcitabine was run on the other western blots to compare band intensities. C. Cells were incubated with or without gemcitabine for 0–24 h, and MK-8776 was added for the last 2 h. D. Cells were incubated with or without gemcitabine for 24 h, and MK-8776 was added concurrently or for the final 6, 4 or 2 h. Parallel cultures were incubated with MK-8776 alone. Cell lysates were analyzed by western blotting.

Figure 3

Confocal imaging of RAD51 foci. A. MDA-MB-231 cells were cultured on coverslips with 10 nmol/L gemcitabine for 0 – 24 h then stained for RAD51 foci. 100 cells were scored for each condition. Values reflect the mean and range of 2 independent experiments. B. Cells were untreated or incubated with either 1 μmol/L MK-8776 for 6 h, 10 nmol/L gemcitabine for 24 h, or 10 nmol/L gemcitabine 0–24 h with 1 μmol/L MK-8776 added for the last 6 h. Cells were scored as in A. Significance was calculated using an unpaired t-test.

Figure 4

Identification of the optimum schedule for combining gemcitabine and MK-8776. The four indicated cell lines were incubated with gemcitabine for 6 h, and 2 μmol/L MK-8776 was added concurrently (column 2) or for a 6-h period at various times after removal of gemcitabine. After removal of drugs, cells were incubated for an additional 6–7 days and cell growth assayed based on DNA content. Experiments were performed in a 96 well format and results are expressed as 50% inhibition of growth of the culture. The values represent the mean and range for duplicate experiments. In addition, the mean and SEM of the values for additional experiments at 0 and 18–24 are presented in Table  1C.

Figure 5

Impact of gemcitabine and MK-8776 on two pancreas tumor xenografts. A. Mice were administered 150 mg/kg gemcitabine and tumors harvested at 18 h and 42 h. Serial sections from the tumors were stained for Ki67 and geminin and the ratio of geminin/Ki67 expressed as a percentage. Results represent the mean and SEM for at least 2 sections from 2–4 mice. B. Mice bearing AsPC-1 tumors were administered 150 mg/kg gemcitabine on days 1, 8, 15 (arrows); or 50 mg/kg MK-8776; or the combination of these two drugs with MK-8776 given either 30 min or 18 h after gemcitabine. Data are expressed as mean and SEM for each time point. “n” represents the number of mice in each group. After day 5, all gemcitabine-treated groups were significantly different from untreated mice (p < 0.05). Treatment with gemcitabine followed by MK-8776 after 30 min was not significantly different than gemcitabine alone. Treatment with gemcitabine followed by MK-8776 after 18 hours was significantly different from gemcitabine alone or when combined with MK-8776 after 30 minutes (p < 0.05). C. Mice bearing MiaPaCa-2 tumors were treated as in B. After 12 days, treatment with gemcitabine followed by MK-8776 after 18 hours was significantly different from either gemcitabine alone or when combined with MK-8776 after 30 minutes (p < 0.05).