Priming with HDAC Inhibitors Sensitizes Ovarian Cancer Cells to Treatment with Cisplatin and HSP90 Inhibitors
Abstract
:1. Introduction
2. Results
2.1. HDACi and HSP90i Mediated Biological Effects in Ovarian Cancer Cells
2.2. Treatment Order of HDACi and HSP90i Affects Increase in Cytotoxic Activity and Apoptosis Induction in A2780 and A2780CisR
2.3. Effects of HDACi and HSP90i on Cisplatin Induced Cytotoxicity and Apoptosis
2.4. Effects of HDACi and/or HSP90i Plus Cisplatin Incubation on mRNA and Protein Expression of Pro-/Antiapoptotic Key Genes
2.5. Effects of Panobinostat or HSP990 Plus Cisplatin on Cell Viability in High Grade Serous Ovarian Cancer Cell Lines CaOV3 and OVCAR3 and Their Cisplatin-Resistant Sub-Cell Lines
2.6. Effects of HDACi or HSP90i and Cisplatin on the Non-Cancer Cell Line HEK293
2.7. Long Term Treatment with Low-Dose HDACi or HSP90i can Overcome and Prevent the Development of Cisplatin Resistance in A2780 Ovarian Cancer Cells.
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Lines
4.3. Cell Viability Assay
4.4. Doubling Time
4.5. Analysis of Apoptosis Induction
4.6. Immunoblotting
4.7. RNA Extraction and PCR
4.8. Caspase 3/7 Activation Assay
4.9. Statistical Methods
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
(p)Akt/AKT | (phosphorylated) protein kinase B |
APAF-1 | apoptotic protease activating factor 1 |
BAK | Bcl-2 homologous antagonist killer |
BAX | Bcl-2-associated X protein |
Bcl-xL | B-cell lymphoma-extra large |
cDDP | cis-diamminedichloroplatinum(II) (cisplatin) |
coinc | coincubation |
HDAC | histone deacetylase |
HDACi | histone deacetylase inhibitor |
HSP90 | heat shock protein 90 |
HSP90i | heat shock protein 90 inhibitor |
HSP990 | NVP-HSP990 |
lumi | luminespib; NVP-AUY922 |
pano | panobinostat |
PCR | polymerase chain reaction |
preinc | preincubation |
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Cell Line | HDACi | HSP90i | ||||||
---|---|---|---|---|---|---|---|---|
Panobinostat | LMK235 | Luminespib | HSP990 | |||||
IC50 [nM] | pIC50 ± SEM | IC50 [nM] | pIC50 ± SEM | IC50 [nM] | pIC50 ± SEM | IC50 [nM] | pIC50 ± SEM | |
A2780 | 28.0 | 7.55 ± 0.02 | 847 | 6.07 ± 0.03 | 11.2 | 7.95 ± 0.06 | 20.1 | 7.70 ± 0.06 |
A2780CisR | 28.2 | 7.55 ± 0.03 | 644 | 6.19 ± 0.02 | 16.4 | 7.78 ± 0.04 | 29.8 | 7.53 ± 0.02 |
A – Dual Combination (inhibitor + cDDP) | |||||||||
cell line | cDDP IC50 [µM] | ||||||||
Control (cDDP only) | HSP90i | HDACi | |||||||
Luminespib | HSP990 | Panobinostat | LMK235 | ||||||
Coinc | preinc | coinc | preinc | coinc | preinc | coinc | preinc | ||
A2780 | 3.34 | 2.41 | 0.71 | 2.22 | 0.50 | 1.63 | 0.57 | 0.69 | 0.57 |
A2780CisR | 19.7 | 24.9 | 13.2 | 25.7 | 11.8 | 16.6 | 6.53 | 11.0 | 7.05 |
B – Triple Combination I (HSP90i prior to HSP90i + HDACi + cDDP) | |||||||||
A2780 cDDP IC50 [µM] | A2780CisR cDDP IC50 [µM] | ||||||||
cDDP | HDACi/cDDP | cDDP | HDACi/cDDP | ||||||
pano | LMK235 | pano | LMK235 | ||||||
HSP90i | lumi | 0.71 | 0.65 | 0.63 | 13.2 | 12.3 | 9.69 | ||
HSP990 | 0.50 | 0.70 | 0.95 | 11.8 | 7.78 | 7.07 | |||
C – Triple Combination II (HDACi prior to HDACi + HSP90i + cDDP) | |||||||||
A2780 cDDP IC50 [µM] | A2780CisR cDDP IC50 [µM] | ||||||||
cDDP | HSP90i/cDDP | cDDP | HSP90i/cDDP | ||||||
lumi | HSP990 | lumi | HSP990 | ||||||
HDACi | pano | 0.57 | 0.91 | 0.84 | 6.53 | 6.07 | 5.91 | ||
LMK235 | 0.57 | 0.70 | 0.84 | 7.05 | 9.23 | 9.26 |
Cell Line | Panobinostat | HSP990 | ||
---|---|---|---|---|
IC50 [nM] | pIC50 ± SEM | IC50 [nM] | pIC50 ± SEM | |
CaOV3 | 15.9 | 7.80 ± 0.02 | 40.9 | 7.39 ± 0.03 |
CaOV3CisR | 16.9 | 7.77 ± 0.02 | 55.6 | 7.26 ± 0.02 |
OVCAR3 | 41.3 | 7.38 ± 0.02 | 27.6 | 7.56 ± 0.03 |
OVCAR3CisR | 30.5 | 7.52 ± 0.01 | 25.0 | 7.60 ± 0.02 |
Cell Line | Control (Cisplatin Only) | + 48h Pretreatment | |||
---|---|---|---|---|---|
HSP990 | Panobinostat | ||||
Cisplatin IC50 [µM] | SF | Cisplatin IC50 [µM] | SF | ||
CaOV3 | 1.92 | 0.79 | 2.4 *** | 1.04 | 1.8 *** |
CaOV3CisR | 4.80 | 3.20 | 1.5 ** | 1.38 | 3.5 *** |
OVCAR3 | 3.94 | 2.77 | 1.4 ns | 1.50 | 2.6 ** |
OVCAR3CisR | 37.7 | 12.6 | 3.0 * | 8.03 | 4.7 *** |
Gene. | Primer Forward | Primer Reverse | Size [bp] |
---|---|---|---|
BAK | GAACAGGAGGCTGAAGGGGT | TCAGGCCATGCTGGTAGACG | 307 |
survivin | CGAGGCTGGCTTCATCCACT | ACGGCGCACTTTCTTCGCA | 283 |
Bcl-xL | CTGAATCGGAGATGGAGACC | TGGGATGTCAGGTCACTGAA | 211 |
BAX | GATGCGTCCACCAAGAAGCT | CGGCCCCAGTTGAAGTTG | 170 |
β-tubulin (TUBB) | TCTACCTCCCTCACTCAGCT | CCAGAGTCAGGGGTGTTCAT | 161 |
Mcl-1 | GGACATCAAAAACGAAGACG | GCAGCTTTCTTGGTTTATGG | 154 |
APAF-1 | ACAATGCTCTACTACATGAAGGATATAAAGA | CACTGGAAGAAGAGACAACAGGAA | 81 |
p21 | CCTAATCCGCCCACAGGAA | AAGATGTAGAGCGGGCCTTTG | 68 |
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Rodrigues Moita, A.J.; Bandolik, J.J.; Hansen, F.K.; Kurz, T.; Hamacher, A.; Kassack, M.U. Priming with HDAC Inhibitors Sensitizes Ovarian Cancer Cells to Treatment with Cisplatin and HSP90 Inhibitors. Int. J. Mol. Sci. 2020, 21, 8300. https://doi.org/10.3390/ijms21218300
Rodrigues Moita AJ, Bandolik JJ, Hansen FK, Kurz T, Hamacher A, Kassack MU. Priming with HDAC Inhibitors Sensitizes Ovarian Cancer Cells to Treatment with Cisplatin and HSP90 Inhibitors. International Journal of Molecular Sciences. 2020; 21(21):8300. https://doi.org/10.3390/ijms21218300
Chicago/Turabian StyleRodrigues Moita, Ana J., Jan J. Bandolik, Finn K. Hansen, Thomas Kurz, Alexandra Hamacher, and Matthias U. Kassack. 2020. "Priming with HDAC Inhibitors Sensitizes Ovarian Cancer Cells to Treatment with Cisplatin and HSP90 Inhibitors" International Journal of Molecular Sciences 21, no. 21: 8300. https://doi.org/10.3390/ijms21218300