Cellular Oncology & Therapeutics Georgia Institute of Technology In Vitro Wet-Lab Study

Dual-Pathway Targeted Cytotoxicity in Glioblastoma (U87EGFP)

Synergistic & Additive Induction of Caspase-Mediated Apoptosis via Combined Ursolic Acid & Erastin

Lead Investigator: Álvaro García Casas Affiliation: Georgia Institute of Technology

U87EGFP Cell Line CCK-8 Colorimetry EVOS FL Microscopy GraphPad Prism 9

Erastin Potency (IC₅₀)

0.50 mM

5.0 µL of 20 mM stock / 200 µL well

Tumor Cell Reduction

~40.0%

Viability drop from 100% to 60% (Additive)

Seeding Density

5,000 / well

U87EGFP glioblastoma cells (48h attachment)

Readout Spectrophotometry

450 nm

CCK-8 (WST-8) dehydrogenase reduction

Background & Motivation

Overcoming Glioblastoma Therapy Resistance

Erastin (Ferroptosis Induction)

Inhibits System Xc⁻ cystine/glutamate antiporter.
Depletes intracellular Glutathione (GSH).
Suppresses GPX4, triggering iron-dependent lipid peroxidation.

Ursolic Acid (Pro-Oxidative Stress)

Inhibits TGF-β signaling (blocks EMT & angiogenesis).
Elevates intracellular Reactive Oxygen Species (ROS).
Promotes mitochondrial outer membrane permeabilization.

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Core Hypothesis: Erastin-mediated antioxidant depletion will synergize with Ursolic Acid-induced ROS overload, amplifying caspase-mediated apoptosis in U87EGFP glioblastoma cells beyond either single-agent effect.

Axial T1-weighted contrast-enhanced MRI scan of Glioblastoma Multiforme

Axial T1+C MRI: Glioblastoma Multiforme

GBM remains an aggressive, lethal brain tumor (<5% 5-year survival) highly resistant to conventional pro-apoptotic regimens.

AIM 1 • Single-Agent Profiling Dose-Response

Cellular Toxicity & IC₅₀ Determination

Quantify U87EGFP toxicity of Erastin and Ursolic Acid (UA) individually.
Establish monotonic IC₁₀ (0.1 µL), IC₂₅ (1.0 µL), and IC₅₀ (5.0 µL) for Erastin.
Determine optimal fixed working dose for Ursolic Acid (5.0 µL static treatment).

AIM 2 • Combination & Phenotype Additive Efficacy

Dual Interaction & Morphological Impact

Test fixed UA (5.0 µL) combined with escalating Erastin (IC₁₀, IC₂₅, IC₅₀).
Quantify cell viability via CCK-8 assay: dropped to ~60% at IC₅₀ combination.
Characterize apoptotic phenotype vs. cobblestone control via EVOS fluorescence.

Figure 4 • Optical Phenotypic Validation

EVOS Fluorescent Microscopy: Control vs. IC₅₀ Dual-Treated U87EGFP

Nikon Eclipse / EVOS FL • 10× Phase • Scale = 300 µm

EGFP Reporter (Ex 488nm / Em 509nm) | 48h Drug Incubation

Control U87EGFP glioblastoma cells showing dense cobblestone morphology and cluster layering

Control U87 Cells (Untreated)

Morphology: Cobblestone Layers Viability: 100%

IC50 Erastin and Ursolic Acid treated U87 glioblastoma cells showing cell shrinkage and membrane blebbing

IC₅₀ E (5 µL) + UA (5 µL) Treated

Morphology: Apoptotic Blebbing Viability: ~60%

Normal U87 Morphology (Red Circles):

Cells maintain a rounded shape and organize in dense clusters with continuous cytoplasmic membranes and healthy nucleolar organization.

Apoptotic Morphology (Red Outlines):

Distinct apoptotic markers: visible shrinkage of the nucleus, central fragmentation spots, cytoskeletal collapse, and sharp membrane bulging/blebbing.

Quantitative Results

CCK-8 Colorimetric Dose-Response & Interaction Curves

Figure 1: CCK-8 dose-response curve for erastin in U87 cells

Erastin Dose-Response

Clear Monotonic Apoptotic Profile

Consistent with literature, Erastin demonstrated a clear, concentration-dependent reduction in U87 cell viability:

IC₁₀ Dose: 0.1 µL (0.01 mM)

IC₂₅ Dose: 1.0 µL (0.10 mM)

IC₅₀ Dose: 5.0 µL (0.50 mM)

Interactive Lab Bench

96-Well Plate Micro-Titration Explorer

Hover or click any well to inspect concentration, OD 450 nm, viability %, and phenotype.

High Mid Kill

Well Telemetry Well B3

Condition:

Untreated Control

Dose:

0 µL (0.05% DMSO)

OD 450nm:

1.482

Viability: 100.0%

Phenotype:

Dense cobblestone monolayer, intact membranes.

Experimental Infrastructure

Materials, Reagents & Laboratory Methodology

Cell Line & Reagents

U87EGFP Glioblastoma Ursolic Acid (UA) Erastin (System Xc⁻ Inhibitor) Complete DMEM Media Trypsin-EDTA & PBS Trypan Blue (>95% Viability)

Assay Kits & Readout

CCK-8 Cell Counting Kit Tecan Infinite 200Pro (OD 450nm) Formazan Colorimetry 4PL Nonlinear Curve Fitting

Optical & Lab Equipment

EVOS FL Auto Microscope Nikon Inverted Microscope C-Chip Hemocytometer 96-Well Flat-Bottom Plates BSL-2 Biosafety Cabinet

Conclusion • Translational Impact

Additive Cytotoxicity as a Therapeutic Strategy in GBM

Combined Erastin and Ursolic Acid treatment induces an additive reduction in U87 glioblastoma viability to ~60%. This bypasses the typical apoptosis resistance of glioblastoma via concurrent System Xc⁻/GPX4 ferroptosis and pro-oxidative mitochondrial stress.

Future directions will directly confirm pathway markers using selective inhibitors (Ferrostatin-1 for ferroptosis and Z-VAD-FMK for pan-caspases) and repeat flow cytometry (Annexin V / Caspase 3/7) across multiple glioblastoma lines.

Lead: Álvaro García Casas • Georgia Institute of Technology

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