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Supplementary figure 3 from HSP70 Inhibition Synergistically Enhances the Effects of Magnetic Fluid Hyperthermia in Ovarian Cancer

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posted on 2023-04-03, 14:46 authored by Karem A. Court, Hiroto Hatakeyama, Sherry Y. Wu, Mangala S. Lingegowda, Cristian Rodríguez-Aguayo, Gabriel López-Berestein, Lee Ju-Seog, Carlos Rinaldi, Eduardo J. Juan, Anil K. Sood, Madeline Torres-Lugo

(A) Lysosome permeability when cells were exposed to PES for 24 hours in 35mm dish plates. Permeability was determined by flow cytometry using acridine orange. Flow cytometry measurements were performed with an Accuri C6 flow cytometer (BD Biosciences) and a 488nm argon laser. 15,000 events were measured. A positive control consisted of cells treated with hydrogen peroxide (Sigma-Aldrich) 0.3 µM for 30 min. (B) Cathepsin B release when SKOV3 cells were exposed to PES for 24 hours. Nucleus was stained with Hoescht (blue) and Cathepsin B was stained with with Magic Red (red). cells were dyed with Magic Red{trade mark, serif} Cathepsin B Assay Kit in in glass bottom dishes (Mattek). (C) Representative histograms showing percent of pale cells as a result of exposure to PES at 10, 20 and 30 uM for 24 hours. (D) Cell viability of HeyA8 cells when exposed to MFH at 43˚C at various times and PES concentrations. (E) Cell viability when ovarian cancer cell lines were exposed to MFH at 41˚C (left) and 43˚C (Right) over time.

Funding

NIH

PR Institute for Functional Nanomaterials

Nanotechnology Center for Biomedical, Environmental and Sustainability Applications

Ovarian Cancer Research Fund

Cancer Prevention and Research Institute of Texas training

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ARTICLE ABSTRACT

Hyperthermia has been investigated as a potential treatment for cancer. However, specificity in hyperthermia application remains a significant challenge. Magnetic fluid hyperthermia (MFH) may be an alternative to surpass such a challenge, but implications of MFH at the cellular level are not well understood. Therefore, the present work focused on the examination of gene expression after MFH treatment and using such information to identify target genes that when inhibited could produce an enhanced therapeutic outcome after MFH. Genomic analyzes were performed using ovarian cancer cells exposed to MFH for 30 minutes at 43°C, which revealed that heat shock protein (HSP) genes, including HSPA6, were upregulated. HSPA6 encodes the Hsp70, and its expression was confirmed by PCR in HeyA8 and A2780cp20 ovarian cancer cells. Two strategies were investigated to inhibit Hsp70-related genes, siRNA and Hsp70 protein function inhibition by 2-phenylethyenesulfonamide (PES). Both strategies resulted in decreased cell viability following exposure to MFH. Combination index was calculated for PES treatment reporting a synergistic effect. In vivo efficacy experiments with HSPA6 siRNA and MFH were performed using the A2780cp20 and HeyA8 ovarian cancer mouse models. A significantly reduction in tumor growth rate was observed with combination therapy. PES and MFH efficacy were also evaluated in the HeyA8 intraperitoneal tumor model, and resulted in robust antitumor effects. This work demonstrated that HSP70 inhibition combination with MFH generate a synergistic effect and could be a promising target to enhance MFH therapeutic outcomes in ovarian cancer. Mol Cancer Ther; 16(5); 966–76. ©2017 AACR.

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