Diclofenac Enhances Docosahexaenoic Acid-Induced Apoptosis in Vitro in Lung Cancer Cells
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Co-Treatment of Lung Cancer Cells with NSAIDs and DHA Enhanced Cytotoxicity
2.2. Co-Treatment of Lung Cancer Cells with DHA and Diclofenac Decreased Clonogenic Cell Survival
2.3. Co-Treatment of Lung Cancer Cells with DHA and Diclofenac Induces Apoptosis
2.4. Co-Treatment of Lung Cancer Cells with DHA and Diclofenac Altered Expression of Cancer-Related Proteins in A549 and NCI-H1573 Cells
2.5. Co-Treatment of Lung Cancer Cells with DHA and Diclofenac Inhibits the Ras/MEK/ERK and PI3K/AKT Signaling Pathways
3. Discussion
4. Materials and Methods
4.1. Materials and Reagents
4.2. Cell Lines
4.3. Cell Viability Assay
4.4. Cell Apoptosis Assays
4.4.1. Acridine Orange/Ethidium Bromide (AO/EB) Double Staining Assay
4.4.2. Annexin-V-FITC Assay
4.4.3. Caspase-3/7 in situ Assay
4.5. Clonogenic Cell Survival and Anchorage-Independent Growth Assays
4.6. Effects of DHA with Diclofenac on the Expression of Cancer-Related Genes
4.7. Immunoblotting
RAS Pull-Down Assay
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
DHA | docosahexaenoic acid |
EPA | eicosapentaenoic acid |
COX | cyclooxygenase |
NSCLC | non-small cell lung cancer |
NSAIDs | non-steroidal anti-inflammatory drugs |
DCF | diclofenac |
PUFAs | polyunsaturated fatty acids |
PGs | prostaglandins |
CI | combination index |
GTPase | nucleotide guanosine triphosphate hydrolase |
NFκB | nuclear factor kappa-light-chain-enhancer of activated B |
TGF-β | transforming growth factor beta |
MEK | mitogen-activated protein kinase/extracellular signal-regulated kinase |
ERK | extracellular signal-regulated kinase |
PPARs | peroxisome proliferator-activated receptors |
MAPK | mitogen-activated protein kinase |
PI3K | phosphoinositide 3-kinase |
Akt | protein kinase B |
mTOR | mammalian target of rapamycin |
JAK | janus kinase |
STAT | signal transducer and activator of transcription |
FITC | fluorescein isothiocyanate |
RIPA | radioimmunoprecipitation assay |
BCA | bicinchoninic acid assay |
GADD45A | growth arrest and DNA damage-inducible alpha |
TNFAIP3 | tumor necrosis factor alpha-induced protein 3 |
CDC25C | M-phase inducer phosphatase 3 |
E2F1 | transcription factor |
PCNA | proliferating cell nuclear antigen |
CCNA2 | cyclin-A2 |
CCNB1 | G2/mitotic-specific cyclin-B1 |
CCNE2 | cyclin E2 |
HDAC2 | histone deacetylase 2 |
POLE2 | DNA polymerase epsilon subunit 2 |
UBE2T | ubiquitin-conjugating enzyme |
HIST1H3B | histone H3.1 |
CDKN2C | cyclin-dependent kinase 4 inhibitor C |
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Cell Line | IC50 of DHA (µM) | ||||
---|---|---|---|---|---|
NSAID | DHA Alone | DHA + NSAID | |||
25 µM | 50 µM | 100 µM | |||
NCI-H1573 | Diclofenac | 9.5 ± 1.3 | 4.5 ± 0.4 | 3.2 ± 0.4 | 3.0 ± 0.3 |
Piroxicam | 9.5 ± 0.6 | ~9.8 | 10.1 ± 1.3 | 8.3 ± 0.2 | |
Indomethacin | ~10.3 | 7.0 ± 0.2 | 7.0 ± 0.3 | 6.1 ± 0.1 | |
Naproxen | ~10.0 | ~9.8 | 11.3 ± 0.4 | 10.0 ± 0.3 | |
A549 | Diclofenac | 9.5 ± 1.1 | 4.3 ± 0.3 | 4.0 ± 0.3 | 3.7 ± 0.4 |
Piroxicam | 9.0 ± 1.5 | 4.9 ± 1.1 | 5.0 ± 0.6 | 5.3 ± 0.7 | |
Indomethacin | 10.3 ± 0.1 | 5.8 ± 0.1 | 5.7 ± 0.0 | 5.5 ± 0.1 | |
Naproxen | 9.4 ± 0.8 | 5.9 ± 0.6 | 7.3 ± 0.1 | 6.4 ± 0.2 | |
NCI-H1299 | Diclofenac | 9.8 ± 0.3 | 10.4 ± 0.4 | 11.3 ± 0.2 | 8.5 ± 0.5 |
Piroxicam | 10.2 ± 0.5 | 9.7 ± 0.2 | 8.7 ± 0.4 | 8.4 ± 0.1 | |
Indomethacin | 9.1 ± 0.1 | 5.9 ± 0.0 | 4.9 ± 0.3 | 3.1 ± 0.3 | |
NCI-H1975 | Diclofenac | ~11.2 | ~11.2 | 7.3 ± 0.4 | 5.8 ± 0.3 |
Piroxicam | 10.0 ± 1.0 | ~10.0 | 8.8 ± 0.1 | 8.2 ± 0.2 | |
Indomethacin | 11.9 ± 0.8 | 9.7 ± 0.6 | 9.2 ± 0.1 | 8.0 ± 0.2 | |
Naproxen | 9.8 ± 0.2 | 8.1 ± 0.3 | 9.6 ± 0.3 | 9.2 ± 0.3 |
Cell Line | IC50 of Diclofenac (µM) | |||
---|---|---|---|---|
Diclofenac Alone | Diclofenac + DHA | |||
2 µM | 5 µM | 10 µM | ||
NCI-H1573 | 87.5 ± 9.6 | 71.4 ± 9.0 | 48.3 ± 1.4 | 25.0 ± 3.4 |
A549 | 92.8 ± 9.9 | 83.1 ± 4.3 | 73.5± 6.5 | 11.3 ± 3.5 |
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Poku, R.A.; Jones, K.J.; Van Baren, M.; Alan, J.K.; Amissah, F. Diclofenac Enhances Docosahexaenoic Acid-Induced Apoptosis in Vitro in Lung Cancer Cells. Cancers 2020, 12, 2683. https://doi.org/10.3390/cancers12092683
Poku RA, Jones KJ, Van Baren M, Alan JK, Amissah F. Diclofenac Enhances Docosahexaenoic Acid-Induced Apoptosis in Vitro in Lung Cancer Cells. Cancers. 2020; 12(9):2683. https://doi.org/10.3390/cancers12092683
Chicago/Turabian StylePoku, Rosemary A., Kylee J. Jones, Megan Van Baren, Jamie K. Alan, and Felix Amissah. 2020. "Diclofenac Enhances Docosahexaenoic Acid-Induced Apoptosis in Vitro in Lung Cancer Cells" Cancers 12, no. 9: 2683. https://doi.org/10.3390/cancers12092683