Many solid cancer patients are treated with platinum-based chemotherapies (e.g. cisplatin), but its accepted dose is limited due to ototoxic side effects of reduced cochlear and vestibular function. Previous studies provide evidence that these side effects may result from induction of the mitochondrial permeability transition pore (mPTP), which compromises mitochondrial function. The long-term aim of our project is to explore whether cyclosporine A (a known blocker of mPTP induction) and other pharmacotherapies that mimic its actions can be otoprotective against these effects of cisplatin. Our lab has previously shown that widespread vestibulo-toxicity can be induced without inducing hair cell death (Sultemeier and Hoffman 2017). Thus, sensory dysfunction is not wholly explained by hair cell or afferent neuron apoptosis; in fact, functional compromise due to non-apoptotic pathology is the more likely scenario is patients undergoing platinum-based chemotherapies.
The goal of the present study was to develop an in vitro model through which we can investigate cisplatin-induced mitochondrial compromise without inducing cell death. We used SH-SY5Y cells, a human neuroblastoma cell line, for our model. We used immunolabeling of TOM20 (a translocase of the outer mitochondrial membrane) to identify populations of mitochondria in our cells and as a proxy for mitochondrial compromise. A viability assay was conducted using trypan blue staining in order to optimize a cisplatin dose for our purposes. Lastly, we imaged our cells as Z stacks using confocal microscopy and image analysis.
The viability assay that was conducted for cells of the following conditions: untreated, 0.25, 0.5, 2.5, and 5 μM, induced 3%, 8%, 8%, 19%, and 9% reduction in cell viability, respectively. The viability assay also showed that most cells that were trypan blue positive (i.e. no longer viable) were beginning to detach from the coverslip. Thus, even at highest doses treated of cisplatin, at least 80% of the cells remained viable. TOM20 immunolabeling was diminished in cells receiving 5 μM cisplatin dose when directly compared with cells receiving doses of 0.25, 0.5, and 2.5 μM. The decrement of the TOM20 immunolabeling is consistent with compromise in mitochondrial populations. This finding will be further explored with in vivo or live cell imaging of mitochondrial membrane potential and/or extracellular flux analysis.
It is very likely that the cells treated at 5 μM of cisplatin are viable and have been compromised in mitochondrial function. Future directions will involve extracellular flux analysis to measure oxygen consumption as a more direct measure of mitochondrial function. These data establish an in vitro model for studying cisplatin-induced mitochondrial dysfunction. This will segue into using cyclosporine A and other pharmacotherapies mimicking its effects.