Poster V5 - Chemical Activation of Piezo1 and Combination Therapies Enhance TRAIL-induced Apoptosis in Glioblastoma

Introduction: Glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, has a mean survival of less than 15 months after standard treatment, including the administration of temozolomide (TMZ) [1]. Treatments may be ineffective if damaged tumor cells undergo DNA repair or acquire mutations that inactivate transcription factor p53 [2]. Tumor Necrosis Factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in multiple tumor types through a transcription-independent mechanism [3]. GBM is particularly resistant to TRAIL, but studies have found that mechanosensitive ion channel Piezo1 can be activated even under static conditions via Yoda1 agonist to induce TRAIL sensitization in other cancer cell lines [3]. This study examines the effects of Yoda1 on TRAIL-mediated apoptosis in two GBM cell lines: U87 cells (TMZ-sensitive) and LN18 cells (TMZ-resistant) [4].

Materials and

Methods: GBM cells were treated with DMSO (10μM), Yoda1 (10μM), DMSO + TRAIL (25 ng/mL), Yoda1 + TRAIL, and TMZ (50μM or 200μM), in which DMSO served as a vehicle control for all experiments. For TRAIL + TMZ combination treatments, U87 cells were treated with TRAIL (100 ng/mL) and TMZ concentrations including 200, 1000 and 2000 µM. An Annexin-V and Propidium Iodide (AV-PI) flow cytometry assay was used to measure cell viability 24 h (U87) or 4 h (LN18) after treatment. For this, cells were stained with 3% AV and 5% PI. A JC-1 flow cytometry assay was used to quantify mitochondrial depolarization of U87 and LN18 cells, using a JC-1 antibody at 40μM. A calcium flux assay was also performed via flow cytometry using 1μM Fluo-4 and 2μM Fura Red fluorescent dyes to quantify the calcium influx into GBM cells upon treatment. Cells were treated with 1μM, 5μM, and 10μM of DMSO and Yoda1. FlowJo software was used for gating and analysis. Additionally, U87 and LN18 cells were seeded onto glass coverslips and fixed after 48 h, followed by staining for Piezo1, DAPI, and actin. Images were acquired at 20x using a Zeiss LSM 710 confocal microscope, and analysis was performed using FIJI.

Results, Conclusions, and Discussions: Yoda1 + TRAIL treatments significantly decreased cell viability (Fig. 1A-C) and significantly increased the mitochondrial depolarization (Fig. 1D-E) for the Yoda1 + TRAIL treatment compared to control conditions in both cell lines. The LN18 cells were more sensitive to this treatment than the U87 cells, although the treatment duration was shorter. Yoda1 + TRAIL combination treatment more effectively at induced apoptosis in LN18 cells compared TMZ treatments alone (Fig. 1A-C). LN18 cells also showed a significant increase in calcium influx for the Yoda1 treatment at each concentration measured (Fig. 1F). No significant difference was observed in the calcium influx in U87 cells for these same Yoda1 concentrations, suggesting saturation of Piezo1 channels in these cells. This was further supported by Figure 1G, which shows that LN18 cells express significantly more Piezo1 than U87 cells. Treatment of U87 cells with TRAIL + TMZ increased the apoptosis compared to TRAIL alone (Fig. 1H). The percentage of depolarized mitochondria significantly increased in U87 cells treated with both drugs (Fig. 1I).

Yoda1 + TRAIL treatments increase apoptosis in both GBM cell lines and are more effective at inducing apoptosis in LN18 cells compared to TMZ, the current standard of care. The increased Piezo1 expression in LN18 cells correlates with increased sensitivity to the Yoda1 + TRAIL treatment compared to U87 cells. These results are supported by an increased mitochondrial depolarization upon Yoda1 + TRAIL treatment and suggest that the intrinsic TRAIL apoptotic pathway is executed, although other downstream pathways may also be activated. Currently, it is not known why the TRAIL + TMZ combination therapy increased the apoptosis and mitochondrial depolarization in the U87 cells, but future experiments will further explore this mechanism and test this treatment regimen with Yoda1 to further understand the effects of Piezo1 activation in GBM. This study demonstrates that mechanotransduction enhances the therapeutic effects of TRAIL, and introduces this combination therapy as an alternative, transcription-independent GBM treatment. This therapy may be useful for treating TMZ-resistant tumors or could be used in combination with TMZ to target two different apoptotic pathways in GBM cells, which may improve patient outcomes.

Acknowledgements (Optional): This work was supported by NIH Grant R01 CA256054.

References: [1] LE Schnöller, Radiat Oncol, 2022, 17, 79; [2] FA Lincoln, Cell Death Dis, 2018, 9, 1-14; [3] JM Hope, Cell Death Dis, 2019, 10, 1-15; [4] SV Knoblauch, ACS Omega, 2023, 8,16975−16986.