Fig. 7: Combinatorial treatment induces a synergistic anti-tumor effect by suppressing mitochondrial function and triggers cell cycle arrest in 3D-cultured OV.

A Schematic illustrating the process of the Mito stress test using 3D-spheroids. B Left: Representative OCR patterns in 3D-cultured IGROV1 spheroids after treatment with GNE-617, disulfiram, and combinatorial therapy at the indicated doses for 48 h over time (min), normalized to spheroid area. Oligomycin, FCCP, rotenone, and antimycin A were added to measure Basal OCR, ATP-Linked OCR, maximal OCR, and non-mitochondrial OCR (n = 8 technical replicates). Middle: Maximal respiratory capacity in OCR (n = 8 technical replicates). Right: Maximal glycolytic capacity in ECAR (n = 8 technical replicates). C Changes in total ATP levels normalized to protein concentration in 3D-cultured A2780 and IGROV1 cells using the same cells as in (B) (n = 3 independent experiments). D Left: Representative figures showing changes in ROS production from mitochondria using MitoSOX Red (YFP) in the same cells as in (B). Cells within the rectangle represent MitoSOX Red-positive cells. Right: Comparison of MitoSOX Red using geometric MFI in 3D-cultured IGROV1 cells as described above (n = 3 independent experiments). E Relative fractions of live and dead cells in 3D-cultured A2780 and IGROV1 cells were analyzed using the AOPI assay after 96 h of treatment with either a DMSO control or the combination of GNE-617 (30 nM) and disulfiram (600 nM) (n = 6 independent experiments). F Left: Representative images of cell cycle analysis performed using Propidium Iodide (PI) staining and flow cytometry. 3D-spheroids were treated with either a DMSO control or a combination of GNE-617 and disulfiram for 96 h. Right: Quantitative comparison of the G2/M phase fraction between the control and combination drug treatment groups (n = 3 independent experiments, paired t-test). G Top: Immunoblot analysis of Cyclin B1 and Cdc2/CDK1 protein levels in 3D-cultured A2780 and IGROV1 cells treated with either DMSO control or the combination of GNE-617 and disulfiram. β-actin was used as a loading control. Bottom: Comparison of normalized protein levels relative to β-actin (n = 3 independent experiments). H Schematic representation illustrating how the combination treatment induces cell growth arrest by inhibiting the G2 to M phase transition. Graph data were presented as mean ± SD of multiple experiments.