Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Material
2.2. Cell Culture and Treatments
2.3. Preparation of Protein Lysates and Cell Fractionation
2.4. Co-Immunoprecipitation
2.5. Transient Transfection with siRNA
2.6. SDS-PAGE and Immunoblot Analysis
2.7. Preparation of RNA and Quantitative Real Time PCR
2.8. Confocal Immunofluorescence Microscopy of p53
2.9. Flow Cytometry-Based Analysis of Autophagy Induction
2.10. Cell Death Measurement by FLOW cytometry
2.11. Determination of Reactive Oxygen Species by Flow Cytometry
2.12. Assessment of Combination Effect
2.13. Statistics
3. Results
3.1. LA Leads to the Depletion of Wildtype and Mutant p53 in CRC Cell Lines
3.2. Supplementation with Antioxidants Does Not Rescue p53 Depletion
3.3. LA-Triggered Autophagy Is Not Involved in the Mechanism of p53 Depletion
3.4. LA Causes Nrf2 Induction, Which is Dispensible for p53 Degradation
3.5. p53 is Readily Ubiquitinated upon LA Treatment
3.6. MDM2 Inhibition Does Not Rescue LA-Triggered p53 Degradation
3.7. LA Synergizes with Standard Chemotherapeutics by Potentiating Cytotoxicity
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
5-FU | 5-fluorouracil |
ARE | antioxidant response element |
CDT | cytolethal distending toxin |
CI | combination index |
CRC | colorectal cancer |
CQ | chloroquine |
DBD | DNA binding domain |
DDR | DNA damage response |
DHLA | dihydrolipoic acid |
Doxo | doxorubicin |
DSF | disulfiram |
HO-1 | heme oxygenase 1 |
LA | ɑ-lipoic acid |
MDM2 | mouse double minute 2 homolog |
MGMT | O6-methylguanine-DNA methyltransferase |
NAC | N-Acetyl-cysteine |
NEM | N-ethylmaleimide |
NRF2 | nuclear factor erythroid 2-related factor 2 |
PI | propidium iodide |
ROS | reactive oxygen species |
RT | room temperature |
scr | scrambled |
SEM | standard error of the means |
Appendix A
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qPCR Primer | Sequence (5′–3′) |
---|---|
ACTB-real-up | TGGCATCCACGAAACTACC |
ACTB-real-low | GTGTTGGCGTACAGGTCTT |
FASR-real-up | TTATCTGATGTTGACTTGAGTAA |
FASR-real-low | GGCTTCATTGACACCATT |
GADD45a-real-up | ATCTCCCTGAACGGTGAT |
GADD45a-real-low | TGTAATCCTTGCATCAGTGT |
GAPDH-real-up | CATGAGAAGTATGACAACAG |
GAPDH-real-low | ATGAGTCCTTCCACGAT |
MDM2-real-up | ATCTTGATGCTGGTGTAA |
MDM2-real-low | AGGCTATAATCTTCTGAGTC |
NOXA-real-up | TCTTCGGTCACTACACAAC |
NOXA-real-low | CCAACAGGAACACATTGAAT |
p21-real-up | ACCATGTCAGAACCGGCTGGG |
p21-real-low | TGGGCGGATTAGGGCTTC |
PUMA-real-up | TAAGGATGGAAAGTGTAG |
PUMA-real-low | TTCAGTTTCTCATTGTTAC |
p53-real-up | AGCACTAAGCGAGCACTG |
p53-real-low | ACGGATCTGAAGGGTGAAA |
Cell Line/CI | LA + 5-FU | LA + Doxo |
---|---|---|
HCT116 | 0.67 | 0.55 |
HCT116-p53−/− | 1.22 | 1.29 |
RKO | 0.84 | 0.59 |
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Share and Cite
Neitzel, C.; Seiwert, N.; Göder, A.; Diehl, E.; Weber, C.; Nagel, G.; Stroh, S.; Rasenberger, B.; Christmann, M.; Fahrer, J. Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation. Cells 2019, 8, 794. https://doi.org/10.3390/cells8080794
Neitzel C, Seiwert N, Göder A, Diehl E, Weber C, Nagel G, Stroh S, Rasenberger B, Christmann M, Fahrer J. Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation. Cells. 2019; 8(8):794. https://doi.org/10.3390/cells8080794
Chicago/Turabian StyleNeitzel, Carina, Nina Seiwert, Anja Göder, Erika Diehl, Carina Weber, Georg Nagel, Svenja Stroh, Birgit Rasenberger, Markus Christmann, and Jörg Fahrer. 2019. "Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation" Cells 8, no. 8: 794. https://doi.org/10.3390/cells8080794