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

•Bupivacaine induced apoptosis in DTK-SME, a canine mammary cancer cell line.•Bupivacaine induced apoptosis in DTK-SME in a time- and dose-dependent manner.•The apoptosis pathways included the extrinsic caspase-dependent pathway.•Mitochondria-regulated caspase-dependent and -independent pathways were activated.•Mitochondria play an important role in bupivacaine-induced apoptosis in DTK-SME.

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.