Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. PRODH/POX Knock Uut CRISPR-cas9 DNA Plasmid Purification
4.2. Transfection into Melanoma Cell Line
4.3. Cell Culture and Treatment
4.4. Cell Viability
4.5. DNA Biosynthesis Assay
4.6. Cell Cycle Analysis
4.7. ROS Generation Assessment
4.8. Cell Membrane Integrity Assay
4.9. Preparation of Cell Lysates
4.10. Western Immunoblotting
4.11. Determination of Prolidase Activity
4.12. Collagen Biosynthesis Assay
4.13. LC-MS Analysis of Proline Concentration
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
AMP | 5′ adenosine monophosphate-activated protein |
AMPK | AMP kinase |
ATCC | American Type Culture Collection |
Atg7 | autophagy-related 7 protein |
ATP | adenozyno-5′-trifosforan |
C32 | type of melanoma cells |
CRISPR | clustered regularly interspaced short palindromic repeats |
DCFH-DA | 2′-7′dichlorofluorescin diacetate |
DMEM | Dulbecco’s Modified Eagle Medium |
DNA | deoxyribonucleic acid |
FBS | fetal bovine serum |
GAPDH | glyceraldehyde 3-phosphate dehydrogenase |
HILIC | hydrophilic interaction liquid chromatography |
HPLC | high-performance liquid chromatography |
MCF-7 | type of breast cancer cells |
MET | metformin |
MMP-2 | metaloproteinase 2 |
MMP-9 | metaloproteinase 9 |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
NAD+ | nicotinamide adenine dinucleotide |
NADH | nicotinamide adenine dinucleotide hydrogen |
NRU | neutral red uptake |
p53 | transcription factor p53 |
P5C | ∆1-pyrroline-5-carboxylate |
P5CR | P5C reductase |
PARP | poly (ADP-ribose) polymerase |
PBS | phosphate-buffered saline |
POX | proline oxidase |
PRODH | proline dehydrogenase |
QTOF | quadrupole time-of-flight mass spectroscopy |
ROS | reactive oxygen species |
TCA | tricarboxylic acid cycle |
THFA | tetrahydrofuroic acid |
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Oscilowska, I.; Rolkowski, K.; Baszanowska, W.; Huynh, T.Y.L.; Lewoniewska, S.; Nizioł, M.; Sawicka, M.; Bielawska, K.; Szoka, P.; Miltyk, W.; et al. Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line. Int. J. Mol. Sci. 2022, 23, 2354. https://doi.org/10.3390/ijms23042354
Oscilowska I, Rolkowski K, Baszanowska W, Huynh TYL, Lewoniewska S, Nizioł M, Sawicka M, Bielawska K, Szoka P, Miltyk W, et al. Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line. International Journal of Molecular Sciences. 2022; 23(4):2354. https://doi.org/10.3390/ijms23042354
Chicago/Turabian StyleOscilowska, Ilona, Karol Rolkowski, Weronika Baszanowska, Thi Yen Ly Huynh, Sylwia Lewoniewska, Magdalena Nizioł, Magdalena Sawicka, Katarzyna Bielawska, Paweł Szoka, Wojciech Miltyk, and et al. 2022. "Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line" International Journal of Molecular Sciences 23, no. 4: 2354. https://doi.org/10.3390/ijms23042354