Inhibiting p21-Activated Kinase Induces Cell Death in Vestibular Schwannoma and Meningioma via Mitotic Catastrophe.

HYPOTHESIS: p21-activated kinase (PAK) regulates signaling pathways that promote cell survival and proliferation; therefore, pharmacological inhibition of PAK will induce cell death in vestibular schwannomas (VS) and meningiomas. BACKGROUND: All VS and many meningiomas result from loss of the neurofibromatosis type 2 (NF2) gene product merlin, with ensuing PAK hyperactivation and increased cell proliferation/survival. METHODS: The novel small molecule PAK inhibitors PI-8 and PI-15-tested in schwannoma and meningioma cells-perturb molecular signaling and induce cell death. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay analyzed PAK inhibitors' effect on cell viability, cell cycle, and cell death, respectively. Western blots evaluated activation and expression of cell proliferation, apoptotic, and mitotic catastrophe markers. Light microscopy evaluated cell morphology, and immunocytochemistry analyzed cellular localization of phospho-Merlin and autophagy-related protein. RESULTS: Treatment with PI-8 and PI-15 decreased cell viability at 0.65 to 3.7 µM 50% inhibitory concentration (IC50) in schwannoma and meningioma cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling and immunocytochemistry studies show that PI-8 and PI-15 induce mitotic catastrophe but not apoptosis in HEI193 cells while in BenMen1 cells, PI-8 induces autophagy and mitotic catastrophe. PI-15 induces apoptosis in BenMen1 cells. PAK inhibitor treated cells show phospho-Merlin localized to over-duplicated centrosomes of dividing cells, multiple enlarged nuclei, and misaligned/missegregated chromosomes-markers for mitotic catastrophe. Increased autophagy-related protein levels in the nucleus confirmed this cell death type. PI-8 and PI-15 inhibits PAK in both cell lines. However, only PI-15 inhibits v-akt murine thymoma viral oncogene homolog in BenMen1 cells. CONCLUSION: PAK inhibitors induce cell death in schwannoma and meningioma cells, at least in part, by mitotic catastrophe.

Zebrafish swimming behavior as a biomarker for ototoxicity-induced hair cell damage: a high-throughput drug development platform targeting hearing loss.

Hearing loss is one of the most common human sensory disabilities, adversely affecting communication, socialization, mood, physical functioning, and quality of life. In addition to age and noise-induced damage, ototoxicity is a common cause of sensorineural hearing loss with chemotherapeutic agents, for example, cisplatin, being a major contributor. Zebrafish (Danio rerio) are an excellent model to study hearing loss as they have neurosensory hair cells on their body surface that are structurally similar to those within the human inner ear. Anatomic assays of toxin-mediated hair cell damage in zebrafish have been established; however, using fish swimming behavior--rheotaxis--as a biomarker for this anatomic damage was only recently described. We hypothesized that, in parallel, multilane measurements of rheotaxis could be used to create a high-throughput platform for drug development assessing both ototoxic and potentially otoprotective compounds in real time. Such a device was created, and results demonstrated a clear dose response between cisplatin exposure, progressive hair cell damage, and reduced rheotaxis in zebrafish. Furthermore, pre-exposure to the otoprotective medication dexamethasone, before cisplatin exposure, partially rescued rheotaxis swimming behavior and hair cell integrity. These results provide the first evidence that rescued swimming behavior can serve as a biomarker for rescued hair cell function. Developing a drug against hearing loss represents an unmet clinical need with global implications. Because hearing loss from diverse etiologies may result from common end-effects at the hair cell level, lessons learned from the present study may be broadly used.