Critical role of reactive oxygen species (ROS) for synergistic enhancement of
apoptosis by vemurafenib and the potassium channel inhibitor TRAM-34 in
melanoma cells
Inhibition of MAP kinase pathways by selective BRAF inhibitors, such as
vemurafenib and dabrafenib, have evolved as key therapies of BRAF-mutated
melanoma. However, tumor relapse and therapy resistance have remained as major
problems, which may be addressed by combination with other pathway inhibitors.
Here we identified the potassium channel inhibitor TRAM-34 as highly effective
in combination with vemurafenib. Thus apoptosis was significantly enhanced and
cell viability was decreased. The combination vemurafenib/TRAM-34 was also
effective in vemurafenib-resistant cells, suggesting that acquired resistance
may be overcome. Vemurafenib decreased ERK phosphorylation, suppressed
antiapoptotic Mcl-1 and enhanced proapoptotic Puma and Bim. The combination
resulted in enhancement of proapoptotic pathways as caspase-3 and loss of
mitochondrial membrane potential. Indicating a special mechanism of
vemurafenib-induced apoptosis, we found strong enhancement of intracellular
ROS levels already at 1 h of treatment. The critical role of ROS was
demonstrated by the antioxidant vitamin E (α-tocopherol), which decreased
intracellular ROS as well as apoptosis. Also caspase activation and loss of
mitochondrial membrane potential were suppressed, proving ROS as an upstream
effect. Thus ROS represents an initial and independent apoptosis pathway in
melanoma cells that is of particular importance for vemurafenib and its
combination with TRAM-34