Hsp70 Negatively Regulates Autophagy via Governing AMPK Activation, and Dual Hsp70-Autophagy Inhibition Induces Synergetic Cell Death in NSCLC Cells
Abstract
:1. Introduction
2. Results
2.1. Autophagy Is Differentially Regulated upon Heat Shock Exposure or Hsp70 Chemical Hyperactivation in NSCLC Cells
2.2. HSF1 Knockdown and Genetic or Chemical Inhibition of Hsp70 Activate Autophagy, While Genetic Hsp70 Overexpression Suppresses Autophagy in NSCLC Cells
2.3. Rapamycin-Induced Autophagy Downregulates Hsp70 Levels, While Inhibition of Autophagy by SAR405 Does Not Influence the Levels of Hsp70 in NSCLC Cells
2.4. Hsp70 Orchestrates the Activation of Autophagy by Upregulating mTOR, Suppressing AMPK and ULK1/Beclin1 Activation
2.5. The Combined Inhibition of Hsp70 and Autophagy Synergistically Enhances Cisplatin-Induced Apoptotic Cell Death in NSCLC Cells
2.6. Dual Targeting of Hsp70 and Autophagy Provokes Substantial Cell Death in Three-Dimensional (3D) Spheroids of NSCLC Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Plasmids
4.3. Chemicals and Drug Treatments
4.4. Heat Shock Treatment
4.5. Western Blot Analysis
4.6. Hsp70 Substrate-Binding Assay
4.7. Cell Viability Assay (MTT Assay)
4.8. Analysis of Drugs Combination Index
4.9. Real-Time PCR
4.10. Colony Formation Assay
4.11. Annexin V/PI Staining Apoptotic Assay
4.12. CellTiter-Glo 3D-Spheroid Viability Assay
4.13. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AMPK | Adenosine monophosphate-activated protein kinase |
ATG5 | Autophagy-related protein 5 |
ATG7 | Autophagy-related protein 7 |
CMLA | Carboxymethylated lactalbumin |
CQ | Chloroquine |
HS | Heat-shock |
HSF1 | Heat-shock factor 1 |
Hsp70 | Heat-shock protein 70 kDa |
HSR | Heat-shock response |
LC3 I/II | Microtubule-associated protein 1A/1B-light chain 3 I/II |
mTOR | Mechanistic target of rapamycin |
NSCLC | Non-small cell lung cancer |
p62 | Sequestosome-1/ubiquitin-binding protein 62 kDa |
PSR | Proteotoxic-stress response |
PIK3C3/VPS34 | Phosphatidylinositol 3-kinase catalytic subunit type 3/vacuolar protein-sorting 34 |
ULK1 | Unc-51-like autophagy-activating kinase 1 |
UPR | Unfolded protein response |
UPS | Ubiquitin–proteasome system |
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Gene of Interest | Forward | Reverse |
---|---|---|
Hsp70 | 5′-AGAAGGACATCAGCCAGAACAA-3′ | 5′-AGAAGTCGATGCCCTCAAACA-3′ |
ULK1 | 5′-CTGCTGGGGAAGGAAATCAAAAT-3′ | 5′-AACCAGGTAGACAGAATTAGCCAT-3′ |
ATG7 | 5′-AAGCCATGATGTCGTCTTCCTAT-3′ | 5′-GCATTGATGACCAGCTTTCTCTT-3′ |
p62 | 5′-TCAGGAGGAGATGATGACTGGA-3′ | 5′-TTGGCCCTTCGGATTCTGG-3′ |
Actin | 5′-CCATCATGAAGTGTGACGTGC-3′ | 5′-GTCCGCCTAGAAGCATTTGCG-3′ |
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Alhasan, B.; Gladova, Y.A.; Sverchinsky, D.V.; Aksenov, N.D.; Margulis, B.A.; Guzhova, I.V. Hsp70 Negatively Regulates Autophagy via Governing AMPK Activation, and Dual Hsp70-Autophagy Inhibition Induces Synergetic Cell Death in NSCLC Cells. Int. J. Mol. Sci. 2024, 25, 9090. https://doi.org/10.3390/ijms25169090
Alhasan B, Gladova YA, Sverchinsky DV, Aksenov ND, Margulis BA, Guzhova IV. Hsp70 Negatively Regulates Autophagy via Governing AMPK Activation, and Dual Hsp70-Autophagy Inhibition Induces Synergetic Cell Death in NSCLC Cells. International Journal of Molecular Sciences. 2024; 25(16):9090. https://doi.org/10.3390/ijms25169090
Chicago/Turabian StyleAlhasan, Bashar, Yana A. Gladova, Dmitry V. Sverchinsky, Nikolai D. Aksenov, Boris A. Margulis, and Irina V. Guzhova. 2024. "Hsp70 Negatively Regulates Autophagy via Governing AMPK Activation, and Dual Hsp70-Autophagy Inhibition Induces Synergetic Cell Death in NSCLC Cells" International Journal of Molecular Sciences 25, no. 16: 9090. https://doi.org/10.3390/ijms25169090