Gamabufotalin induces a negative feedback loop connecting ATP1A3 expression and the AQP4 pathway to promote temozolomide sensitivity in glioblastoma cells by targeting the amino acid Thr794.

Cheng Chen,Jia‐Cheng Lou,Yu‐Long Lan,Xun Wang,Bo Zhang,Wen Lyu,Jin‐Shan Xing,Ji‐Liang Hu,Shuang Zou

doi:10.1111/cpr.12732

Abstract

ObjectivesTemozolomide (TMZ) is one of the most commonly used clinical drugs for glioblastoma (GBM) treatment, but its drug sensitivity needs to be improved. Gamabufotalin (CS‐6), the primary component of the traditional Chinese medicine “ChanSu,” was shown to have strong anti‐cancer activity. However, more efforts should be directed towards reducing its toxicity or effective treatment doses.MethodsTarget fishing experiment, Western blotting, PCR, confocal immunofluorescence and molecular cloning techniques were performed to search for possible downstream signalling pathways. In addition, GBM xenografts were used to further determine the potential molecular mechanisms of the synergistic effects of CS‐6 and TMZ in vivo.ResultsMechanistic research revealed a negative feedback loop between ATP1A3 and AQP4 through which CS‐6 inhibited GBM growth and mediated the synergistic treatment effect of CS‐6 and TMZ. In addition, by mutating potential amino acid residues of ATP1A3, which were predicted by modelling and docking to interact with CS‐6, we demonstrated that abrogating hydrogen bonding of the amino acid Thr794 interferes with the activation of ATP1A3 by CS‐6 and that the Thr794Ala mutation directly affects the synergistic treatment efficacy of CS‐6 and TMZ.ConclusionsAs the main potential target of CS‐6, ATP1A3 activation critically depends on the hydrogen bonding of Thr794 with CS‐6. The combination of CS‐6 and TMZ could significantly reduce the therapeutic doses and promote the anti‐cancer efficacy of CS‐6/TMZ monotherapy.

Highlights

Glioma is the most common primary central nervous system (CNS) tumour and comprises approximately 60%-70% of all primary brain tumours
Target fishing experiment process: surface plasmon resonance (SPR) of target fishing was divided into two parts, and U87 cells and U118 cells were assessed in two groups of parallel control experiments. (a) Positive control: The U87/U118 cell lysate sample was flowed onto the chip saturated with CS-6 installed on the SPR instrument, which could monitor and detect the target protein in the cell lysate captured by the small molecule on the chip in real time
The results indicated that ATP1A3 and AQP4 colocalized in GBM cells, and both CS-6 and TMZ monotherapy promoted the activation of ATP1A3 and, together, AQP4 suppression

Summary

Introduction

Glioma is the most common primary central nervous system (CNS) tumour and comprises approximately 60%-70% of all primary brain tumours. More than 100 bufadienolides, including gamabufotalin (CS-6), bufalin, cinobufagin (CB), resibufogenin (RB) and telecinobufagin (CS-7), have been separated and identified as the major active components with anti-cancer activities in ChanSu.[3]. CS-6, a major bufadienolide of ChanSu, has been used for cancer therapy due to its good metabolic stability and few adverse effects.[4-7]. If the drug could be altered to selectively kill cancer cells or show increased efficacy at low concentrations, research on the clinical applications of CS-6 would be substantially accelerated. Temozolomide (TMZ) is one of the most commonly used clinical drugs in glioma treatment, but recurrent drug resistance limits the clinical application of this drug.

Objectives

Methods

Findings

Conclusion