A triple combination of atorvastatin, celecoxib and tipifarnib strongly inhibits pancreatic cancer cells and xenograft pancreatic tumors

Abstract

Because K-Ras mutation and cyclooxygenase-2 (COX-2) overexpression are hallmarks of majority of pancreatic cancer patients, an approach to inhibit the progression and growth of pancreatic cancer using the simultaneous administration of agents that inhibit the function of both targets, should be considered. In the present study, we assessed the effects of atorvastatin (Lipitor), celecoxib (Celebrex) and tipifarnib (Zarnestra) on the growth of human pancreatic cancer. In the in vitro studies, we found that treatment of human pancreatic tumor cells with a combination of atorvastatin, celecoxib and tipifarnib had a stronger inhibitory effect on growth and a stronger stimulatory effect on apoptosis than each drug alone or for any combination of two drugs. We also found that treatment of Panc-1 cells with a combination of all three drugs strongly decreased the levels of phosphorylated Erk1/2 and Akt. In an animal model of xenograft tumors in severe combined immunodeficient (SCID) mice, we found that daily i.p. injections of a combination of atorvastatin, celecoxib and tipifarnib had a stronger inhibitory effect on the growth of the tumors in mice than each drug alone or for any combination of two drugs. The results of our study indicate that a combination of atorvastatin, celecoxib and tipifarnib may be an effective strategy for the treatment of pancreatic cancer.

Figure 1.

Proposed inhibitory effects of atorvastatin, celecoxib and tipifarnib.

Figure 2.

Effects of atorvastatin, celecoxib or tipifarnib on proliferation and apoptosis of Panc-1 cells. Panc-1 cells were seeded at a density of 0.2×10⁵ cells/ml in 35-mm tissue culture dishes (2 ml/dish) and incubated for 24 h. The cells were then treated with DMSO (2 μl/ml) or with various concentrations of atorvastatin (1–10 μM), celecoxib (1–10 μM) or tipifarnib (0.1–0.5 μM) in DMSO for 96 h. Each incubation mixture contained the same amount of solvent (DMSO; 2 μl/ml). (A) The number of viable cells was measured by a trypan blue exclusion assay and expressed as a percentage of solvent-treated control. (B) The number of apoptotic cells was determined by morphological assessment. Each value is the mean ± SE from three separate experiments.

Figure 3.

Effects of atorvastatin, celecoxib or tipifarnib alone or in combination on phosphorylation of Erk1/2 and Akt. Panc-1 cells were seeded at a density of 0.1×10⁶ cells/ml of medium in 100-mm tissue culture dishes and incubated for 24 h. The cells were then treated with atorvastatin (2 μM), celecoxib (2 μM) or tipifarnib (0.1 μM) alone or in combination for 24 h. Western blot analysis with anti-phosphorylated Erk1/2 (#4376, Cell Signaling Technology) and anti-phosphorylated Akt (#9275, Cell Signaling Technology) was used to determine the expression of phosphorylated-Erk and phosphorylated-Akt.

Figure 4.

Inhibitory effect of i.p. injection of atorvastatin, celecoxib or tipifarnib alone or in combination on the growth of subcutaneous Panc-1 tumors in SCID mice. Female SCID mice were injected subcutaneously with Panc-1 cells (2×10⁶ cells/0.1 ml) suspended in 50% Matrigel and RPMI medium. Injections of drug were started in mice after they had a tumor (0.6–1.0 cm wide and 0.6–1.0 cm long). The mice (6/group) received daily i.p. injections of vehicle (see below) or atorvastatin (ATOR; 2 μg/g body weight), celecoxib (CEL; 2 μg/g) or tipifarnib (TIP; 0.8 μg/g) alone or in combination. Each value is the mean ± SE. The linear trend in tumor growth for the atorvastatin + celecoxib + tipifarnib group was significantly different from that for any other group (p=0.0034).

Figure 5.

Inhibitory effect of i.p. injections of atorvastatin, celecoxib and tipifarnib in combination on the growth of orthotopic Panc-1 tumors in SCID mice. Female SCID mice were injected orthotopically with Panc-1 luc cells (1×10⁶ cells/mouse). After 2–3 weeks, mice with established Panc-1 tumors were randomized into two groups. One group of mice received daily i.p. injections with solvent and the other group of mice received daily i.p. injections with atorvastatin (2 μg/g) + celecoxib (2 μg/g) + tipifarnib (0.8 μg/g). Tumor size of each mouse was determined once a week using the IVIS system. Representative images are shown. (A and B) Mice before treatment. (C) An image taken from a mouse in the solvent control group 28 days after treatment. (D) An image taken from a mouse in the combination treatment group 28 days after treatment. (E) Each value for tumor mass is the mean ± SE from 4 mice.