Exploring the “Cascade Effect” of AEBSF on Reduced Glio-blastoma Proliferation

: Glioblastomas (GBMs


INTRODUCTION
Glioblastomas (GBM) are grade IV gliomas and are the most lethal primary malignancy of the central nervous system (CNS).Despite comprising 54% of all gliomas and 16% of all primary brain tumors, GBM lacks a cure and only has a handful of treatments [1].The lack of curative treatment for GBM can be attributed to several factors, some of which being high GBM resistance rates, rapid angiogenesis, and vast spread [10].
This acidic extracellular pH has recently been demonstrated to activate proteases (such as serine proteases, plasminogen activators, and metalloproteinases).These proteases degrade the GBM extracellular environment, explaining why they have been indicated to be involved in GBM growth, invasion, angiogenesis and metastasis [2,4].Given the importance of pH in GBM progression, this study aimed to focus on pH regulation as a means of GBM treatment.
ABSTRACT: Glioblastomas (GBMs) are one of the deadliest cancers, and yet still lack treatment.In this study, serine protease inhibitor AEBSF is shown to decrease GBM viability and proliferation through a series of intertwined steps.By decreasing the expression of ATP6V1A--the first step of the "cascade effect"--AEBSF creates an unfavorable environment for protease activity.Such "unfavorability" is proven by a decline in expression of proteases MMP9 and ELANE upon GBM treatment with AEBSF.Increasingly higher concentrations of AEBSF are shown to decrease the percentage of cell viability, proliferation, and metastasis.This is explained through biological process enrichment analyses, which indicate that MMP9 and ELANE play a role in the disassembly of the extracellular protein network.This disassembly of the extracellular environment enables tumorigenic spread and viability.The results of the viability and proliferation assays, in combination with those of the indirect ELISAs, enrichment analyses, and scratch assays, underscore an intricate and profound sequence of events that constitute the "cascade effect".endosomes, secretory vesicles, clathrin-coated vesicles, and the plasma membrane [4].Consisting of 13 subunits in mammalian cells, the primary function of these proton pumps is to regulate both intracellular and extracellular acidification [5].V-ATPases comprise two domains: the V0 and V1 domains.The peripheral V1 domain consists of eight subunits (A-H), all of which assist in ATP hydrolysis.The membrane-embedded V0 domain--consisting of subunits a, c, c″, d, and e--is responsible for proton translocation [6].In this study, subunit A of the V1 domain (ATP6V1A) is closely examined due to its cytoplasmic location and ubiquitous nature across the body [14,15].This study explores the sequence of events in the "cascade effect" perpetuated by 4-benzenesulfonyl fluoride hydrochloride (AEBSF).AEBSF, also known as AEBSF hydrochloride, is an irreversible serine protease inhibitor that has previously been indicated to inactivate serine proteases such as chymotrypsin and proteinase K [11].AEBSF has been shown in literature to have a potential regulatory effect on the secretion of antitumor effector molecules in leukemia [12].AEBSF has also been shown to reduces dengue virus infection via decreased cholesterol synthesis [13].
In this study, AEBSF was studied under the context of GB, proliferation and metastasis.The question sought to be answered is, "What are the chronological events that occur in the reduction of GBM metastasis perpetuated by AEBSF?" The findings in this study may: 1) provide more insight into the mechanics of GBM metastasis, 2) highlight the role of serine proteases in GBM progression, and 3) provide greater insight into such proteases' mechanisms of action, thereby clarifying the link between ECM disassembly and GBM metastasis.

Preparation of lysates for enzyme-linked immunosorbent assay (ELISA)
Phosphate buffered saline (PBS) (Ther-moFisher, Waltham, MA, USA), 1X cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) were used to lyse 10^6 cells/mL from each treatment group.Cells were incubated at 37°C with 5% CO2 and 100% humidity with trypsin for 3 minutes until detachment.Cells were subsequently neutralized with DMEM and transferred to 15mL centrifuge tubes.Tubes were gently inverted and cells were counted using ViCell XR (Beckman Coulter, Indianapolis, IN, USA).Tubes were subsequently centrifuged at 1000 rpm for 7 minutes at room temperature, after which the pellets were poured off and the pellet was broken.Chilled PBS was added to the tubes, which were subsequently re-centrifuged at 1000 rpm for 7 minutes at 25°C.The residual supernatant was poured out and the pellet was broken up.The appropriate volume of lysis buffer, calculated using the following equation, was added to each tube and incubated on ice for 10 minutes: [Viable cell count (cells/mL) × volume of cell suspension prior to first round of centrifugation (mL)] ÷ 106 cells/mL Lysates from the tubes were subsequently transferred to chilled Eppendorf tubes.
The tubes were micro-centrifuged at 13000 rpm for 15 minutes at 4°C.The supernatants were stored at -80°C.

Indirect ELISA
100µL/well of lysates were transferred at 4 replicates per treatment group to a 96-well plate.Lysates were left to incubate for 24 hours at 4°C and then removed by blotting.200µL/ well of blocking buffer (ThermoFisher, Waltham, MA, USA) with 1% bovine serum albumin (BSA) (Seracare, Milford, MA, USA) was added and left to incubate for 10 minutes at 25°C.

Cell proliferation assay
Cells were seeded at 62,500 cells/well 24 hours prior to treatment in a 6-well plate.Cells were subsequently treated for 48 hours, supernatants were collected, and cell proliferation was analyzed using a synergy machine.

Trypan Blue Exclusion Viability Assay
Cells were seeded at ~40,000 cells/ flask 24 hours prior to treatment.Cells were subsequently treated for 48 hours, supernatants were collected, and cells were trypsinized and counted using ViCell XR (Beckman Coulter, Indianapolis, IN, USA).

Reverse Docking
Reverse docking tool ChemMapper was used to predict the protein targets of AEBSF.The SMILES (Simplified Molecular Input Line Entry System) notation of AEBSF, obtained from PubChem, was inputted into ChemMapper.Subsequently, the "Target Navigator" service was selected and the 3D similarity method SHAFT was chosen.The default similarity threshold of 1.2 was selected, and the Bioactivity Database ChEMBL was then chosen [7][8][9].

Data Analysis
Statistical and computational biology analyses were performed using Microsoft Excel.An unpaired, two-tailed T-test was performed in Excel, and a p-value < 0.05 was considered statistically significant.Means and standard deviations are taken for all data and compared as % of control.

AEBSF reduces T98G proliferation
AEBSF was first demonstrated to reduce proliferation in T98G cells.As hypothesized, AEBSF reduces proliferation in a statistically significant manner, demonstrating the efficacy of this treatment (Fig. 1A).

AEBSF decreases T98G viability
AEBSF was also demonstrated to reduce viability in T98G cells.Specifically, higher concentrations of AEBSF were shown to decrease the number of viable cells and increase the number of dead cells (Fig. 2).As hypothesized, AEBSF reduces viability in a statistically significant manner, once again demonstrating the efficacy of this treatment (Fig. 2).

Plasma membrane V-ATPase subunit A (AT-P6V1A) protein expression is enhanced in glioblastomas and decreased upon AEBSF treatment
V-ATPase subunit A (ATP6V1A) plays a vital role in the primary function of the overall proton pump--acidification of the extracellular environment.Through an indirect ELISA, it is demonstrated that ATP6V1A protein expression decreases with increasing AEBSF concentration (Fig. 3).

AEBSF targets serine proteases and metalloproteinases
AEBSF is a well known serine protease inhibitor.Although it is known that AEBSF inhibits serine proteases such as trypsin and chymotrypsin, shape-screening tool ChemMapper was utilized to predict its protein targets with the highest confidence scores.ChemMapper is fundamentally rooted in the "chemical similarity principle", which states that structurally similar compounds have similar bioactivies [7].Thus, it can be said that a higher similarity score between the query ligand (AEBSF in this study) and the database ligands indicates a higher likelihood that a given protein target of the database ligand is also a target of AEBSF, the query ligand.Thus, as previously discussed, a similarity threshold of 1.2 (on a scale of 2.0) was set in order to filter out protein targets of database ligands with low-similarity scores.Using ChemMapper, it was predicted that AEBSF targets 6 serine proteases (Fig. 4A) and 2 metalloproteinases (Fig. 4B).

Serine proteases and metalloproteinases targeted by AEBSF primarily play a role in extracellular matrix disassembly and cell migration in glioblastomas
The eight protein targets predicted by ChemMap were plugged into "Search Tool for the Retrieval of Interacting Genes/Proteins" (STRING) to carry out an enrichment analysis of the biological processes that they are involved in.The top five biological processes, along with their false discovery rates (FDRs) and strength values, were analyzed (Fig. 5).

AEBSF downregulates ELANE and MMP9 protein expression
Following the reverse docking and biological processes enrichment analyses, neutrophil elastase (ELANE) and matrix metalloproteinase-9 (MMP9) expression were selected to be analyzed.These two proteins were selected to be studied, as in the reverse docking analysis, ELANE and MMP9 were demonstrated to have the highest similarity scores, which was previously established to be a good indicator of the likelihood that a given protein target of the database ligand is also a target of AEBSF, the query ligand (Fig. 4A, 4B).Hence, serine protease ELANE and metalloproteinase MMP9 expression were analyzed through indirect ELISAs.Both ELANE and MMP9 protein expression were demonstrated to decrease with increasing AEBSF concentrations (Fig. 6A, 6B).

Figure 1 .
Created by author

Figure 1 .
Figure 1.48-hour treatment with 4-benzenesulfonyl fluoride hydrochloride (AEBSF) reduces proliferation in T98G cells.A: Bars are means ± STDEV (n=4).*Significantly different by t-test at p<0.05 comparing data to that at vehicle control (cells and H2O).**Significantly different by t-test at p<0.01 comparing data to that at vehicle control.***Significantly different by t-test at p<0.001 comparing data to that at vehicle control.B: Light microscopy images of T98G treated with 125µM, 500µM, or 2000µM AEBSF at 100x.