Treatment of pancreatic cancer that cannot be surgically resected currently relies on minimally beneficial cytotoxic chemotherapy with gemcitabine.As the 4th leading cause of cancer death in the United States with dismal survival statistics, pancreatic cancer demands new and more effective treatment approaches.Resistance to gemcitabine is nearly universal and appears to involve defects of the intrinsic/mitochondrial apoptotic pathway.The bioactive sphingolipid ceramide is a critical mediator of apoptosis initiated by a number of therapeutic modalities.Interestingly, insufficient ceramide accumulation has been linked to gemcitabine resistance in multiple cancer types, including pancreatic.Taking advantage of the fact that cancer cells frequently have more negatively charged mitochondria, we investigated a means to circumvent resistance to gemcitabine by targeting delivery of a pyridinium-conjugated ceramide (Pyr-L-Cer6) to cancer cell mitochondria.Pyr-L-Cer6 was effective in initiating apoptosis by causing mitochondrial depolarization in pancreatic cancer cells but demonstrated significantly less activity against nonmalignant pancreatic ductal epithelial cells.Furthermore, we demonstrate that the mitochondrial membrane potentials of the cancer cells were more negative than nonmalignant cells, and that dissipation of this potential abrogated cell killing by Pyr-L-Cer6, establishing that the effectiveness of this compound is potential-dependent.Pyr-L-Cer6 selectively accumulated in and inhibited the growth of xenografts in vivo, confirming the tumor selectivity and therapeutic potential of pyridinium-conjugated ceramides in pancreatic cancer.Interestingly, gemcitabine resistant pancreatic cancer cells became more sensitive to subsequent treatment with Pyr-L-Cer6, suggesting that this compound may be a uniquely suited to overcome gemcitabine resistance in pancreatic cancer.This study was supported in part by National Institute of Health National Cancer Institute P01 CA97132-06, P30 CA138313 and C06 RR015455.