Exosome-Mediated Transfer of Cancer Cell Resistance to Antiestrogen Drugs
Abstract
:1. Introduction
2. Results
2.1. Development and Characteristics of MCF-7 Resistant Derivatives
2.2. Purification and Characterization of the Exosomes
2.3. Exosomes Influence on the Cell Response to Tamoxifen and Metformin
2.4. Protein Signature of the Exosome-Generated Resistant Cells
3. Discussion and Conclusions
4. Materials and Methods
4.1. Cell Cultures and Development of Drug Resistant Derivatives
4.2. Next Generation Sequencing
4.2.1. DNA Preparation
4.2.2. Library Preparation for NGS. Clonal Amplification
4.2.3. Sequencing
4.2.4. NGS Mapping
4.3. Transient Transfection and Measurement of Reporter Gene Activity
4.4. Western Blot Analysis of Cell Lysates
4.5. Exosomes Purification and Analysis
4.5.1. Exosomes Isolation by Ultracentrifugation
Western Blot Analysis of Exosomes
4.5.2. Transportation of Fluorescent-Labeled Compounds and Peptides
4.5.3. Extracellular Vesicles Size and Concentration Measurements
4.5.4. Transmission Electron Microscopy (TEM)
4.5.5. Cells Treatment with Exosome Preparations
4.6. Statistical Analysis
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
DMSO | Dimethylsulfoxide |
STR | Short Tandem Repeats |
DMEM | Dulbecco’s Modified Eagle Medium |
ERE | Estrogen-responsive elements |
FBS | Fetal Bovine Serum |
SDS | Sodium Dodecyl Sulfate |
TBS | Tris-Buffered Saline |
E2 | 17β-Estradiol |
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Sample Availability: Samples of the compounds are not available from the authors. |
Cell Line | MCF-7 | MCF-7/M | MCF-7/T | |
---|---|---|---|---|
Gene | ||||
PIK3CA | c.1633G > A Allele fraction: 66% (of 17,311 reads) | c.1633G > A Allele fraction: 67% (of 12,976 reads) | c.1633G > A Allele fraction: 65% (of 11,224 reads) | |
ALK | NO | NO | NO | |
EGFR | NO | NO | NO | |
EGFR-AS1 | NO | NO | NO | |
ESR1 | NO | NO | NO |
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Share and Cite
Semina, S.E.; Scherbakov, A.M.; Vnukova, A.A.; Bagrov, D.V.; Evtushenko, E.G.; Safronova, V.M.; Golovina, D.A.; Lyubchenko, L.N.; Gudkova, M.V.; Krasil’nikov, M.A. Exosome-Mediated Transfer of Cancer Cell Resistance to Antiestrogen Drugs. Molecules 2018, 23, 829. https://doi.org/10.3390/molecules23040829
Semina SE, Scherbakov AM, Vnukova AA, Bagrov DV, Evtushenko EG, Safronova VM, Golovina DA, Lyubchenko LN, Gudkova MV, Krasil’nikov MA. Exosome-Mediated Transfer of Cancer Cell Resistance to Antiestrogen Drugs. Molecules. 2018; 23(4):829. https://doi.org/10.3390/molecules23040829
Chicago/Turabian StyleSemina, Svetlana E., Alexander M. Scherbakov, Anna A. Vnukova, Dmitry V. Bagrov, Evgeniy G. Evtushenko, Vera M. Safronova, Daria A. Golovina, Ludmila N. Lyubchenko, Margarita V. Gudkova, and Mikhail A. Krasil’nikov. 2018. "Exosome-Mediated Transfer of Cancer Cell Resistance to Antiestrogen Drugs" Molecules 23, no. 4: 829. https://doi.org/10.3390/molecules23040829