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

Abstract

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.

Figure 1. PAK Inhibitors decrease cell viability in a dose dependent manner in VS and meningioma cells

A. HEI193 and BenMen1 cell lines decreased 50% viability with PI-8 at 1.24 μM and 3.1 μM respectively. B. PI-15 decreased HEI193 and BenMen1 cell viability at IC50 1.8 μM and 0.6 μM respectively. C. Primary vestibular schwannoma and primary meningioma cells decreased 50% viability with PI-8 at 3.25 μM and 4.1 μM respectively. D. PI-15 IC50 at 2.1 and 2.2 μM decreased cell viability in primary VS and meningioma cells respectively. Error bars represent SEM.

Figure 2. PAK Inhibitors induce apoptosis in BenMen1 but not in HEI193 cells

A. TUNEL: PI-15 (2.5 μM) induces apoptosis in more BenMen1 cells than PI-8 (2.5 μM) compared to 0 μM (Negative Control). Hoechst dye shows nuclear staining of the cell sample in the positive control, negative control, and PI-8 treated cells while the PI-15 treated population of cells is very small. B. TUNEL: PI-8 or PI-15 at 2.5 μM did not induce apoptosis in HEI193 cells when compared to Negative control. In A and B Positive control conforms cells treated with DNase (Invitrogen, Carlsbad, CA) I. Representative pictures were taken with a 10X objective using the Delta Vision Deconvolution microscope. C. BenMen1 treated cells with PI-15 for 72 hours show increase in cleavage of the apoptosis initiator, caspase-9, into 39 and 37 kilodaltons (kDa), and of the executioner caspase-3 into 19 and 17 kDa at 2.5 μM when compared to the control. Total caspase-3 (35 kDa) decreases in treated cells at 2.5 μM. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) is the loading control.

Figure 2. PAK Inhibitors induce apoptosis in BenMen1 but not in HEI193 cells

A. TUNEL: PI-15 (2.5 μM) induces apoptosis in more BenMen1 cells than PI-8 (2.5 μM) compared to 0 μM (Negative Control). Hoechst dye shows nuclear staining of the cell sample in the positive control, negative control, and PI-8 treated cells while the PI-15 treated population of cells is very small. B. TUNEL: PI-8 or PI-15 at 2.5 μM did not induce apoptosis in HEI193 cells when compared to Negative control. In A and B Positive control conforms cells treated with DNase (Invitrogen, Carlsbad, CA) I. Representative pictures were taken with a 10X objective using the Delta Vision Deconvolution microscope. C. BenMen1 treated cells with PI-15 for 72 hours show increase in cleavage of the apoptosis initiator, caspase-9, into 39 and 37 kilodaltons (kDa), and of the executioner caspase-3 into 19 and 17 kDa at 2.5 μM when compared to the control. Total caspase-3 (35 kDa) decreases in treated cells at 2.5 μM. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) is the loading control.

Figure 3. PI-8 and PI-15 induces cell death by mitotic catastrophe in HEI193 cells

A. Control cells show phospho-merlin expression (red). Cell in the middles is bi-nucleated (DAPI: blue). B. HEI193 cells treated with 2.5-μM PI-8 show ATG5 expression (green and white arrows) in the nucleus of many cells. ATG5 expression is associated with autophagosomes in the cytoplasm (blue arrowheads). Phospho-merlin expression is visualized in centrosomes (yellow arrows) holding two nuclei together. C. HEI193 cells treated with 5-μM PI-8. Increased autophagosome formation associated with high levels of ATG5 and nuclear AGT5 is visualized. D. 2.5-μM PI-15 induced high ATG5 expression levels associated with autophagosomes in the cytoplasm and in the nucleus. Many centrosomes are visualized with phospho-merlin expression (yellow arrows). E. 5-μM PI-15 in HEI193 cells show ATG5 expression in the nucleus and associated with autophagosomes. Treatment was for 72 hours. Images were taken using the Delta Vision Deconvolution microscope. 60X.

Figure 4. PI-8 induces cell death by autophagy in BenMen1 cells

A. ATG5 (green) and LC3B (red) expression increases in BenMen1 cells treated with increasing doses of PI-8 at 48 hours. B. 24-hours BenMen1 treated cells show ATG5 and LC3B expression increase in a dose dependent manner. Merged: ATG5 and LC3B co-localization (yellow). C. Autophagy markers expression increased in BenMen1 cells treated for 72 hours with increasing doses of PI-8.

Figure 4. PI-8 induces cell death by autophagy in BenMen1 cells

A. ATG5 (green) and LC3B (red) expression increases in BenMen1 cells treated with increasing doses of PI-8 at 48 hours. B. 24-hours BenMen1 treated cells show ATG5 and LC3B expression increase in a dose dependent manner. Merged: ATG5 and LC3B co-localization (yellow). C. Autophagy markers expression increased in BenMen1 cells treated for 72 hours with increasing doses of PI-8.

Figure 4. PI-8 induces cell death by autophagy in BenMen1 cells

A. ATG5 (green) and LC3B (red) expression increases in BenMen1 cells treated with increasing doses of PI-8 at 48 hours. B. 24-hours BenMen1 treated cells show ATG5 and LC3B expression increase in a dose dependent manner. Merged: ATG5 and LC3B co-localization (yellow). C. Autophagy markers expression increased in BenMen1 cells treated for 72 hours with increasing doses of PI-8.

Figure 5. PI-8 and PI-15 inhibit PAK phosphorylation in vestibular schwannoma and meningioma cells

A. In HEI193 cells, progressive exposure to PI-8 decreased PAK phosphorylation at the threonine phosphorylation site. Progressive exposure to PI-15 decreased PAK phosphorylation at both threonine-423 and serine-144. Total PAK and AKT levels were stable in both treatment groups. At higher doses, PI-15 inhibits AKT phosphorylation at its threonine and serine phosphorylation sites. B. Progressive exposure to PI-15 decreases PAK phosphorylation at both phosphorylation sites. Total PAK and AKT levels were stable in both treatment groups. At higher doses, PI-15 inhibits AKT phosphorylation at its threonine and serine phosphorylation site.