Clostridium botulinum neurotoxin A induces apoptosis and mitochondrial oxidative stress via activation of TRPM2 channel signaling pathway in neuroblastoma and glioblastoma tumor cells

Abstract

Background

The Clostridium botulinum neurotoxin A (BTX) is a polypeptide produced by the bacterium Clostridium botulinum. In addition to the therapeutic actions of BTX against pain and neuromuscular disorders, it is acted as anticancerogenic effect through excessive mitochondria reactive oxygen species (ROS) production, apoptosis, and caspase activations. The TRPM2 cation channel is activated by ROS and ADP-ribose and it is inhibited by 2-aminoethyl diphenylborinate (2-APB) and N-(p-amylcinnamoyl) anthranilic acid (ACA). The aim of this study was an investigation of involvement BTX-induced TRPM2 activation on the mitochondria ROS production and apoptosis levels in the DBTRG glioblastoma and SH-SY5Y neuroblastoma tumor cells.

Material and methods

The DBTRG and SH-SY5Y cells were divided into four groups as control, BTX (5 IU for 24 h), BTX + ACA (25 µM for 30 min), and BTX + 2-APB (100 µM for 30 min).

Results

BTX treatment increased mitochondrial membrane depolarization (JC-1), mitochondrial (MitROS), and cytosolic (DHR123 and DCFH-DA) ROS levels, neuronal death (propidium iodide/Hoechst) rate, caspase −3, and −9 levels in the BTX group, although their levels were diminished in the BTX + ACA and BTX + 2-APB groups. The ACA and 2-APB treatments also decreased BTX-induced increase of TRPM2 cytosolic free Ca²⁺ concentration in the glioblastoma and neuroblastoma cell death.

Conclusions

BTX caused neuroblastoma and glioblastoma tumor cell death by activating the mitochondria ROS production via stimulating TRPM2 signaling pathways. BTX may serve as a potential therapeutic target via activation of TRPM2 for treating glioblastoma and neuroblastoma cells.

Keywords:

• Apoptosis
• Clostridium botulinum neurotoxin A
• glioblastoma death
• mitochondria oxidative stress
• TRPM2 channel

Author contributions

The cell culture, plate reader, laser confocal microscope and cell viability analyses in the current study were performed in 3^rd International Brain Research School, 25 June-1 July October 2018, Isparta, Turkey by O.A., A.Ö, M.G and M.N (http://2018.brs.org.tr/). M.N. and O.A formulated the present hypothesis. M.N. wrote the report. A.Ö. and M.G. were responsible for preparation of cell culture. O.A. was also responsible for the plate reader caspase analyses. M.N. was responsible for analyzing the laser confocal microscope. A.Ö. and M.G. made critical revisions to the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The study was supported by BSN Health, Analyses, Innovation, Consultancy, Organization, Agriculture, Industry and Trade Limited Company, Göller Bölgesi Teknokenti, Isparta, Turkey (Project No: 2018-21).