Verapamil enhances the antiproliferative effect of chemotherapeutic reagents and inhibits tumor growth of chemoresistant side population in pancreatic cancer

Introduction: Verapamil is an L-type calcium channel blocker and has been used in the treatment of hypertension, angina pectoris, cardiac arrhythmia, and most recently, cluster headaches. Increasing evidence shows that Verapamil is able to enhance the cytotoxic effect of certain chemotherapies and reverse multidrug resistance by competitively inhibiting drug transport through P-glycoprotein. The side population (SP) is a small subtype of tumor cells with stem-like properties which could substantially be blocked by verapamil. In this study, we focus on the therapeutic potential of verapamil on stem-like SP cells and its positive effect as a chemotherapy promoter in pancreatic cancer.

Material and methods: From the highly metastatic pancreatic cancer cell line L3.6pl a gemcitabine resistant variant (L3.6pl_Gem) was developed and further validated by IC-50 determination. Hoechst33342 staining was used to detect the SP proportion in both sensitive and resistant L3.6pl cells_. Proliferation assays, clonogenic assays, migration assays and apoptosis assays were performed in the presence of verapamil alone and combined with gemcitabine treatment. SP cells sorted from L3.6pl_Gem were implanted orthotopically in nude mice and treated with verapamil by daily i.p injections.

Results: After continuous treatment with gemcitabine, the IC50 of L3.6pl_Gem significantly increased from 6.11ng/ml±0.93 to 119.77ng/ml±5.12 (p<1E-9). In parallel, the percentage of SP cells rose from 0.9%±0.22 to 5.38%±0.99 (p<5E-17). Verapamil treatment inhibited cell proliferation in both sensitive and resistant cells and increased chemosensitivity to gemcitabine. After 4 weeks daily therapy with verapamil, the in vivo data indicated a significant inhibition of primary tumor growth initiated by chemoresistant SP cells as compared to control groups.

Conclusion: Overall, our findings demonstrate that verapamil could reverse the sensitivity of gemcitabine in resistant pancreatic cancer cells and might inhibit tumorigenesis by targeting side population providing evidence for a new clinical feature of this 'old' reagent.