Enhanced Malignant Phenotypes of Glioblastoma Cells Surviving NPe6-Mediated Photodynamic Therapy are Regulated via ERK1/2 Activation.

Tatsuya Kobayashi,Yoshitaka Narita,Takakazu Kawamata,Hideki Yamaguchi,Satoru Takeuchi,Koichi Ichimura,Daisuke Kawauchi,Kentaro Mori,Eita Uchida,Arata Tomiyama,Naoki Otani,Shun Yamamuro,Yohei Otsuka,Kosuke Kumagai,Makoto Miyazaki,Nobuyoshi Sasaki,Yoshihiro Muragakı ,Toshimasa Toyo′Oka ,Manabu Takahashi ,Koichiro Wada

doi:10.3390/cancers12123641

Abstract

Simple SummaryThe molecular machineries regulating resistance against photodynamic therapy (PDT) using talaporfin sodium (NPe6) (NPe6-PDT) in glioblastomas (GBM)s and mechanisms underlying the changes in GBM phenotypes following NPe6-PDT remain unknown. Herein, we established an in vitro NPe6-mediated PDT model using human GBM cell lines. NPe6-PDT induced both caspase-dependent and -independent GBM cell death in a NPe6 dose-dependent manner. Moreover, treatment with poly (ADP-ribose) polymerase inhibitor blocked NPe6-PDT-triggered caspase-independent GBM cell death. Next, it was revealed resistance to re-NPe6-PDT, migration, and invasion of GBM cells that survived following NPe6-PDT (NPe6-PDT-R cells) were enhanced. Immunoblotting of NPe6-PDT-R revealed that only ERK1/2 activation exhibited the same trend as migration. Importantly, treatment with the MEK1/2 inhibitor trametinib reversed resistance against re-NPe6-PDT and suppressed the enhanced migration and invasion of NPe6-PDT-R cells. Overall, enhanced ERK1/2 activation is suggested as a key regulator of elevated malignant phenotypes of GBM cells surviving NPe6-PDT.To manage refractory and invasive glioblastomas (GBM)s, photodynamic therapy (PDT) using talaporfin sodium (NPe6) (NPe6-PDT) was recently approved in clinical practice. However, the molecular machineries regulating resistance against NPe6-PDT in GBMs and mechanisms underlying the changes in GBM phenotypes following NPe6-PDT remain unknown. Herein, we established an in vitro NPe6-mediated PDT model using human GBM cell lines. NPe6-PDT induced GBM cell death in a NPe6 dose-dependent manner. However, this NPe6-PDT-induced GBM cell death was not completely blocked by the pan-caspase inhibitor, suggesting NPe6-PDT induces both caspase-dependent and -independent cell death. Moreover, treatment with poly (ADP-ribose) polymerase inhibitor blocked NPe6-PDT-triggered caspase-independent GBM cell death. Next, it was also revealed resistance to re-NPe6-PDT of GBM cells and GBM stem cells survived following NPe6-PDT (NPe6-PDT-R cells), as well as migration and invasion of NPe6-PDT-R cells were enhanced. Immunoblotting of NPe6-PDT-R cells to assess the behavior of the proteins that are known to be stress-induced revealed that only ERK1/2 activation exhibited the same trend as migration. Importantly, treatment with the MEK1/2 inhibitor trametinib reversed resistance against re-NPe6-PDT and suppressed the enhanced migration and invasion of NPe6-PDT-R cells. Overall, enhanced ERK1/2 activation is suggested as a key regulator of elevated malignant phenotypes of GBM cells surviving NPe6-PDT and is therefore considered as a potential therapeutic target against GBM.

Highlights

Glioblastoma (GBM) is the most common malignant intracranial tumor arising from the brain parenchyma and has the poorest prognosis, with median survival of only 12–15 months despite standard multidisciplinary treatment with maximal surgical resection, followed by chemoradiotherapy [1].This poor prognosis can be attributed to the limited surgical excision of GBM, due to its highly infiltrative growth into the normal brain tissue, need to minimize the damage to normal brain tissue due to surgery, and high resistance of this tumor to chemoradiotherapy
Moderate GBM cell death induced by photodynamic therapy (PDT) with 15 μg·mL−1 NPe6 in both lines was partially blocked by the pan-caspase inhibitor z-VAD, while moderate cell death induced by
By comparing the activation of molecules downstream of receptor tyrosine kinases (RTKs) [Akt, STAT3, ERK1/2, and Src family kinases (SFKs)] as well as JNK1/2 by immunoblotting, we demonstrated that only ERK1/2 activation was upregulated in both NPe6-PDT-R

Summary

Introduction

Glioblastoma (GBM) is the most common malignant intracranial tumor arising from the brain parenchyma and has the poorest prognosis, with median survival of only 12–15 months despite standard multidisciplinary treatment with maximal surgical resection, followed by chemoradiotherapy [1] This poor prognosis can be attributed to the limited surgical excision of GBM, due to its highly infiltrative growth into the normal brain tissue, need to minimize the damage to normal brain tissue due to surgery, and high resistance of this tumor to chemoradiotherapy. Following the uptake of a photosensitizer by specific target cells, highly toxic free radicals are produced in these cells through light-induced excitation of the photosensitizer; these radicals induce cell death Based on this principle, PDT-mediated specific killing of target tumor cells without damaging the surrounding normal tissue has been attempted and approved as a novel therapy against refractory tumors, including GBMs, in recent years [4,5]. In patients with GBM, intraoperative PDT using laser and talaporfin sodium (mono-l-aspartyl chlorin e6, NPe6)

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