Combinational regimen with celecoxib and atorvastatin causes cell cycle arrest and induces apoptosis in colon cancer cells

2361

Chemopreventive effects of non-steroidal anti-inflammatory drugs (NSAIDs) have been demonstrated in colorectal cancer. However, NSAIDs alone may not be strong candidates for colorectal cancer chemoprevention due to their potential side effects. Studies both in clinical trials and in animals have shown prominent chemopreventive efficacy of a combined treatment of NSAIDs and statins, a class of cholesterol-lowering drugs, in colorectal cancer. In this study, we investigated the effects of the celecoxib/atorvastatin combination treatment using two human colon cancer cell lines, HCT116 and HT29. Celecoxib moderately inhibited the growth of both cell lines with a similar IC₅₀ of 30-50 μM, while atorvastatin showed stronger growth inhibitory effect in HCT116 cells than in HT29 cells (IC₅₀: 7-8 μM vs. 20-30 μM) after treatment for 48-96 h. In combination, celecoxib and atorvastatin acted synergistically at various concentrations to suppress the growth of both cell lines. For example, at 48 h, treatment of HT29 cells with celecoxib (25 μM) and atorvastatin (10 μM) together resulted in 80% growth inhibition, whereas, treatment of the cells with celecoxib or atorvastatin only produced 17% or 5.7% growth inhibition, respectively. Cell cycle analysis indicated that, at 24 h, the celecoxib/atorvastatin combination treatment caused cell cycle arrest in G1 phase, whereas at 48 h or longer, significant apoptosis (indicated by sub-G1 population) was induced. Subsequent analysis with Annexin V/PI fluorescent staining showed that combination treatment with celecoxib (25 μM) and atorvastatin (1 μM) to HCT116 cells for 48 h induced early apoptosis by 27% and late apoptosis/necrosis by 12%. Treatments with celecoxib and atorvastatin alone did not increase apoptotic cell population compared to the control. Western immunoblots suggest that the combinational effects of celecoxib/atorvastatin on cell cycle arrest and apoptosis was associated with up-regulation of p21, p27 and pRB; down-regulation of phospho-ERK, phospho-p90rsk, phospho-GSK-3β and hyperphosphorylated pRB; and activation of caspase-3. It is noteworthy that our results also suggest the Cox-2-independent mechanisms are involved in the effects of the combined treatment with celecoxib/atorvastatin. Taken together, the results demonstrate a strong synergy between the actions of celecoxib and atorvastatin in colon cancer cells, and provide a system for further mechanistic investigation (supported by grant CA88961).