Disruption of pH Dynamics Suppresses Proliferation and Potentiates Doxorubicin Cytotoxicity in Breast Cancer Cells
Abstract
:1. Introduction
2. Material and Methods
2.1. Breast Tumor Samples
2.2. Cell lines and Culture Conditions
2.3. Drugs
2.4. Cell Survival Assays
2.5. Western Blotting
2.6. Immunofluorescence
2.7. Immunohistochemistry
2.8. Wound-Healing Assay
2.9. Invasion Assay
2.10. Effect of the pH Regulator Inhibitors on Doxorubicin Cytotoxicity
2.11. Zymography Assay
2.12. Statistical Analysis
3. Results
3.1. pH Regulators Are Strongly Expressed in Breast Cancer Cell Lines, but with a Distinct Expression Pattern
3.2. V-ATPase Overexpression Associates Significantly with High-Grade Invasive Breast Carcinomas
3.3. Extracellular Acidity Decreases Sensitivity to Doxorubicin and Increases the Migratory and Invasive Abilities of Breast Cancer Cells
3.4. Disruption of pH Regulation Decreases the Aggressive Behavior of MDA-MB-231 Cells
3.5. Treatment with pH Regulator Inhibitors Increased the Sensitivity of Breast Cancer Cell Lines to Doxorubicin
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Conflicts of Interest
Abbreviations
AZ | Acetazolamide |
CA | Carbonic anhydrase |
Conc. A | Concanamycin A |
CHC | α-cyano-4-hydroxycinnamate |
ER | Estrogen receptor |
GLUT1 | glucose transporter 1 |
HER2 | Human epidermal growth factor receptor 2 |
LN | lymph node |
MCT | Monocarboxylate transporter |
MMP | Metalloproteinase |
NHE1 | Sodium–hydrogen exchanger 1 |
PgR | Progesterone receptor |
pHe | Extracellular pH |
PM | Plasma membrane |
PRIs | pH regulator inhibitors |
TMAs | Tissue microarrays |
TNBC | Triple negative breast cancer |
V-ATPase | Vacuolar ATPase |
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Protein | Dilution | Species | Reference, Supplier |
---|---|---|---|
NHE1 | 1:200 | Rabbit | sc-28758, Santa Cruz Biotechnology |
V-ATPase | 1:200 | Mouse | sc-374475, Santa Cruz Biotechnology |
CAXII | 1:200 | Mouse | sc-374314, Santa Cruz Biotechnology |
MCT1 | 1:200 | Mouse | sc-365501, Santa Cruz Biotechnology |
MCT4 | 1:500 | Rabbit | sc-50329, Santa Cruz Biotechnology |
α-tubulin | 1:500 | Rabbit | ab15246, Abcam |
n | Positive (%) | P | PM (%) | P | |
---|---|---|---|---|---|
V-ATPase | 0.001 0.046 | 0.046 | |||
Normal breast epithelium | 44 | 4 (9.1) | 4 (9.1) | ||
Breast carcinoma | 203 | 197 (97.0) | 51 (25.9) | ||
CAXII | |||||
Normal breast epithelium | 40 | 0 (0.0) | 0.032 | 0 (0.0) | 0.001 |
Breast carcinoma | 196 | 98 (50.0) | 96 (98.0) |
n | MCT1 Positive (%) | P | n | MCT4 Positive (%) | P | |
---|---|---|---|---|---|---|
V-ATPase | 123 | 0.876 | 120 | 0.444 | ||
Negative | 87 | 18 (20.7) | 84 | 4 (4.8) | ||
Positive | 36 | 7 (19.4) | 36 | 3 (8.3) | ||
CAXII | 115 | 0.290 | 112 | 0.240 | ||
Negative | 55 | 9 (16.4) | 53 | 1 (1.9) | ||
Positive | 60 | 15 (25.0) | 59 | 4 (6.8) |
CI Values at | ||||||
---|---|---|---|---|---|---|
Dox IC25 | Dox IC50 | Dox IC75 | ||||
Drug combination | MDA | MCF-7 | MDA | MCF-7 | MDA | MCF-7 |
Conc. A + Dox | 1.23 ± 0.034 | 1.66 ± 0.023 | 0.68 ± 0.012 | 0.74 ± 0.011 | 0.56 ± 0.010 | 0.62 ± 0.022 |
Cariporide + Dox | 1.20 ± 0.022 | 1.34 ± 0.012 | 0.65 ± 0.003 | 0.84 ± 0.015 | 0.52 ± 0.007 | 0.76 ± 0.033 |
CHC + Dox | 0.79 ± 0.045 | 0.94 ± 0.003 | 0.45 ± 0.005 | 0.77 ± 0.027 | 0.41 ± 0.009 | 0.64 ± 0.008 |
AZ + Dox | N.A± | 0.71 ± 0.009 | N.A | 0.53 ± 0.007 | N.A | 0.51 ± 0.006 |
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Tavares-Valente, D.; Sousa, B.; Schmitt, F.; Baltazar, F.; Queirós, O. Disruption of pH Dynamics Suppresses Proliferation and Potentiates Doxorubicin Cytotoxicity in Breast Cancer Cells. Pharmaceutics 2021, 13, 242. https://doi.org/10.3390/pharmaceutics13020242
Tavares-Valente D, Sousa B, Schmitt F, Baltazar F, Queirós O. Disruption of pH Dynamics Suppresses Proliferation and Potentiates Doxorubicin Cytotoxicity in Breast Cancer Cells. Pharmaceutics. 2021; 13(2):242. https://doi.org/10.3390/pharmaceutics13020242
Chicago/Turabian StyleTavares-Valente, Diana, Bárbara Sousa, Fernando Schmitt, Fátima Baltazar, and Odília Queirós. 2021. "Disruption of pH Dynamics Suppresses Proliferation and Potentiates Doxorubicin Cytotoxicity in Breast Cancer Cells" Pharmaceutics 13, no. 2: 242. https://doi.org/10.3390/pharmaceutics13020242
APA StyleTavares-Valente, D., Sousa, B., Schmitt, F., Baltazar, F., & Queirós, O. (2021). Disruption of pH Dynamics Suppresses Proliferation and Potentiates Doxorubicin Cytotoxicity in Breast Cancer Cells. Pharmaceutics, 13(2), 242. https://doi.org/10.3390/pharmaceutics13020242