Bupivacaine induces apoptosis through caspase-dependent and -independent pathways in canine mammary tumor cells

Local anesthetics have been reported to induce apoptosis in various cell lines. In this study, we showed that bupivacaine also induced apoptosis in DTK-SME cells, a vimentin⁺/AE1⁺/CK7⁺/HSP27⁺, tumorigenic, immortalized, canine mammary tumor cell line. Bupivacaine induced apoptosis in DTK-SME cells in a time- and concentration-dependent manner. Apoptosis-associated morphological changes, including cell shrinkage and rounding, chromatin condensation, and formation of apoptotic bodies, were observed in the bupivacaine-treated DTK-SME cells. Apoptosis was further confirmed with annexin V staining, TUNEL staining, and DNA laddering assays. At the molecular level, the activation of caspases-3, -8, and -9 corresponded well to the degree of DNA fragmentation triggered by bupivacaine. We also demonstrated that the pan-caspase inhibitor, z-VAD-fmk, only partially inhibited the apoptosis induced by bupivacaine. Moreover, treated cells increased expression of endonuclease G, a death effector that acts independently of caspases. Our data suggested that bupivacaine-induced apoptosis occurs through both caspase-dependent and caspase-independent apoptotic pathways.