Early effects of the antineoplastic agent salinomycin on mitochondrial function.

Salinomycin, isolated from Streptomyces albus, displays antimicrobial activity. Recently, a large-scale screening approach identified salinomycin and nigericin as selective apoptosis inducers of cancer stem cells. Growing evidence suggests that salinomycin is able to kill different types of non-stem tumor cells that usually display resistance to common therapeutic approaches, but the mechanism of action of this molecule is still poorly understood. Since salinomycin has been suggested to act as a K(+) ionophore, we explored its impact on mitochondrial bioenergetic performance at an early time point following drug application. In contrast to the K(+) ionophore valinomycin, salinomycin induced a rapid hyperpolarization. In addition, mitochondrial matrix acidification and a significant decrease of respiration were observed in intact mouse embryonic fibroblasts (MEFs) and in cancer stem cell-like HMLE cells within tens of minutes, while increased production of reactive oxygen species was not detected. By comparing the chemical structures and cellular effects of this drug with those of valinomycin (K(+) ionophore) and nigericin (K(+)/H(+) exchanger), we conclude that salinomycin mediates K(+)/H(+) exchange across the inner mitochondrial membrane. Compatible with its direct modulation of mitochondrial function, salinomycin was able to induce cell death also in Bax/Bak-less double-knockout MEF cells. Since at the concentration range used in most studies (around 10 µM) salinomycin exerts its effect at the level of mitochondria and alters bioenergetic performance, the specificity of its action on pathologic B cells isolated from patients with chronic lymphocytic leukemia (CLL) versus B cells from healthy subjects was investigated. Mesenchymal stromal cells (MSCs), proposed to mimic the tumor environment, attenuated the apoptotic effect of salinomycin on B-CLL cells. Apoptosis occurred to a significant extent in healthy B cells as well as in MSCs and human primary fibroblasts. The results indicate that salinomycin, when used above µM concentrations, exerts direct, mitochondrial effects, thus compromising cell survival.

Mitochondrial ion channels as oncological targets.

Mitochondria, the key bioenergetic intracellular organelles, harbor a number of proteins with proven or hypothetical ion channel functions. Growing evidence points to the important contribution of these channels to the regulation of mitochondrial function, such as ion homeostasis imbalances profoundly affecting energy transducing processes, reactive oxygen species production and mitochondrial integrity. Given the central role of mitochondria in apoptosis, their ion channels with the potential to compromise mitochondrial function have become promising targets for the treatment of malignancies. Importantly, in vivo evidence demonstrates the involvement of the proton-transporting uncoupling protein, a mitochondrial potassium channel, the outer membrane located porin and the permeability transition pore in tumor progression/control. In this review, we focus on mitochondrial channels that have been assigned a definite role in cell death regulation and possess clear oncological relevance. Overall, based on in vivo and in vitro genetic and pharmacological evidence, mitochondrial ion channels are emerging as promising targets for cancer treatment.

Induction of apoptosis in macrophages via Kv1.3 and Kv1.5 potassium channels.

We have previously shown that the mitochondrial potassium channel Kv1.3 (mtKv1.3) in T lymphocytes is a novel target of Bax. Mutation of Bax at lysine 128 (BaxK128E) abrogates its inhibitory effects on mtKv1.3 and prevents apoptosis. The importance of mtKv1.3 inhibition was underscored by the finding that membrane-permeant Kv1.3 inhibitors induced Bax/Bak-independent cell death and reduced the volume of an mtKv1.3-expressing tumor by 90% in a mouse model. However, the possible involvement of other Kv channels in apoptosis has not been clarified. Here we report that, like Kv1.3, Kv1.1 and Kv1.5 also interact with Bax. Transfection of Kvdeficient lymphocytes with Kv1.1 restores sensitivity to cell death in apoptosis-resistant CTLL-2 lymphocytes. SiRNA down-regulation of Kv1.3 and Kv1.5 expression in macrophages confers resistance to apoptosis. We further report that J774 macrophages express Kv1.3 and Kv1.5 in their mitochondria and that inhibition of both channels with specific membrane-permeant drugs can efficiently induce apoptosis in a macrophage cell line. Thus, our results indicate that the mechanism proposed for Kv1.3 can be extended to other Kv channels and suggest that membrane-permeant drugs may be a novel pharmacological tool for inducing apoptosis in macrophages, important players in the immune system. This result could be exploited for the depletion of tumor-associated macrophages, which have been shown to foster tumor growth.

Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis.

Members of the Bcl-2 family play key roles as proapoptotic (e.g., Bax) and antiapoptotic (e.g., Bcl-x(L)) regulators of programmed cell death. We previously identified the mitochondrial potassium channel Kv1.3 as a novel target of Bax. Incubating Kv1.3-positive isolated mitochondria with Bax triggered apoptotic events, whereas Kv1.3-deficient mitochondria were resistant to this stimulus. Mutation of Bax at lysine 128 (BaxK128E) abrogated its effects on Kv1.3 and the induction of apoptotic changes in mitochondria. These data indicate a toxin-like action of Bax on Kv1.3 to trigger at least some of the mitochondrial changes typical for apoptosis. To gain insight into the mechanism of Bax-Kv1.3 interaction, we mutated Glu158 of Bcl-x(L) (corresponding to K128 in Bax) to lysine. This substitution turned Bcl-x(L) proapoptotic. Transfection of double knockout (Bax(-/-)/Bak(-/-)) mouse embryonic fibroblasts (DKO MEFs) with either wild-type Bax, BaxK128E, or Bcl-x(L)E158K showed that apoptosis induced by various stimuli was defective in DKO MEFs and BaxK128E-transfected cells, but was recovered upon transfection with Bcl-xLE158K or wild-type Bax. Both wild-type Bax and BaxK128E can form similar ion-conducting pores upon incorporation into planar lipid bilayers. Our results point to a physiologically relevant interaction of Bax with Kv1.3 and further indicate a crucial role of a distinct lysine in determining the proapoptotic character of Bcl2-family proteins.

Combined radius and ulna reconstruction with a free fibula transfer.

A 20-year-old man sustained a high energy injury to his right forearm resulting in comminuted open fractures of the forearm with 6 cm bone loss of the radius and 7 cm bone loss of the ulna. A bone segment was removed from the middle of a free fibula transfer to produce a single flap with two vascularised bone segments to reconstruct both the radius and ulna defects. Step osteotomies were stabilised with a single lag screw for each fracture line and, with a rigid external fixation device on the ulna, two vein grafts were used to anastomose the flap pedicle to the recipient vessels in the forearm. The postoperative period was uneventful. X-rays and scintigrams confirmed good healing of the fractures and normal perfusion of the flap so that the external fixation device could be removed 3 months after surgery. One year after the injury, the patient underwent a functional evaluation showing excellent results with very good preservation of rotation of the forearm.