Comparison of Mitochondrial and Antineoplastic Effects of Amiodarone and Desethylamiodarone in MDA-MB-231 Cancer Line
Abstract
:1. Introduction
2. Results
2.1. Effects of AM and DEA on Mitochondrial Membrane Potential of MDA-MB-231 Cells
2.2. Effects of AM and DEA on Mitochondrial Network Dynamics in MDA-MB-231 Cells
2.3. Effects of AM and DEA on Mitochondrial Permeability Transition in MDA-MB-231 Cells
2.4. Effects of AM and DEA on Intracellular Calcium Levels ([Ca2+]i) of MDA-MB-231 Cells
2.5. Effect of AM and DEA on Energy Metabolism of MDA-MB-231 Cells
2.6. Anti-Neoplastic Effects of AM and DEA in MDA-MB-231 Cells
2.7. Apoptosis-Inducing Effects of AM and DEA in MDA-MB-231 Cells
2.8. Effects of AM and DEA on Wound-Healing in MDA-MB-231 Cells
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Cultures
4.3. Cell Viability Assay
4.4. Apoptosis Assay
4.5. Migration Assay
4.6. Subcellular Fractionation
4.7. Immunoblot Analysis
4.8. Bioenergetics Assay
4.9. ∆Ψm Assay
4.10. Mitochondrial Permeability Transition Pore Opening (MPTP)
4.11. Intracellular Calcium ([Ca2+]i)
4.12. D Spheroids
4.13. Analysis of Mitochondrial Network Dynamics
4.14. Statistical Analysis
5. Conclusions
6. Patents
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
7-AAD | 7-aminoactinomycin D |
AM | Amiodarone |
Bax | Bcl-2-like protein 4 |
BC | Breast cancer |
Bcl2 | B-cell lymphoma2 |
BH3 | Bcl homology domain-3 protein |
DEA | Desethylamiodarone |
ΔΨm | Mitochondrial membrane potential |
DRP1 | Dynamin-related protein 1 |
ECAR | Extracellular acidification rate |
EMT | Epithelial-to-mesenchymal transition |
FCCP | Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone |
Fis1 | Fission 1 protein |
FITC | Fluorescein isothiocyanate |
Her2 | Human epidermal growth factor receptor 2 |
HR+ | Hormone receptor positive |
MFN | Mitofusin |
OCR | Oxygen consumption rate |
OD | Optical density |
Opa1 | Optic atrophy1 |
PARP | Poly(ADP-ribose) polymerase |
PBS | Phosphate-buffered saline |
ROS | Reactive oxygen species |
SD | Standard deviation |
SEM | Standard error of the mean |
SRB | Sulforhodamine B |
TN | Triple negative |
References
- Mujovic, N.; Dobrev, D.; Marinkovic, M.; Russo, V.; Potpara, T.S. The role of amiodarone in contemporary management of complex cardiac arrhythmias. Pharmacol. Res. 2020, 151, 104521. [Google Scholar] [CrossRef] [PubMed]
- Nattel, S.; Talajic, M.; Quantz, M.; DeRoode, M. Frequency-dependent effects of amiodarone on atrioventricular nodal function and slow-channel action potentials: Evidence for calcium channel-blocking activity. Circulation 1987, 76, 442–449. [Google Scholar] [CrossRef]
- Nokin, P.; Clinet, M.; Schoenfeld, P. Cardiac beta-adrenoceptor modulation by amiodarone. Biochem. Pharmacol. 1983, 32, 2473–2477. [Google Scholar] [CrossRef]
- Varro, A.; Virag, L.; Papp, J.G. Comparison of the chronic and acute effects of amiodarone on the calcium and potassium currents in rabbit isolated cardiac myocytes. Br. J. Pharmacol. 1996, 117, 1181–1186. [Google Scholar] [CrossRef] [PubMed]
- Colunga Biancatelli, R.M.; Congedo, V.; Calvosa, L.; Ciacciarelli, M.; Polidoro, A.; Iuliano, L. Adverse reactions of Amiodarone. J. Geriatr. Cardiol. 2019, 16, 552–566. [Google Scholar] [CrossRef] [PubMed]
- Latini, R.; Tognoni, G.; Maggioni, A.P.; Cavalli, A.; Volpi, A. Amiodarone and desethylamiodarone: Plasma concentrations, therapeutic effects and side effects. J. Am. Coll. Cardiol. 1988, 11, 209–210. [Google Scholar] [CrossRef] [PubMed]
- Vorperian, V.R.; Havighurst, T.C.; Miller, S.; January, C.T. Adverse effects of low dose amiodarone: A meta-analysis. J. Am. Coll. Cardiol. 1997, 30, 791–798. [Google Scholar] [CrossRef]
- Latini, R.; Tognoni, G.; Kates, R.E. Clinical pharmacokinetics of amiodarone. Clin. Pharmacokinet. 1984, 9, 136–156. [Google Scholar] [CrossRef]
- Adams, P.C.; Holt, D.W.; Storey, G.C.; Morley, A.R.; Callaghan, J.; Campbell, R.W. Amiodarone and its desethyl metabolite: Tissue distribution and morphologic changes during long-term therapy. Circulation 1985, 72, 1064–1075. [Google Scholar] [CrossRef]
- Holt, D.W.; Tucker, G.T.; Jackson, P.R.; Storey, G.C. Amiodarone pharmacokinetics. Am. Heart J. 1983, 106, 840–847. [Google Scholar] [CrossRef]
- Serviddio, G.; Bellanti, F.; Giudetti, A.M.; Gnoni, G.V.; Capitanio, N.; Tamborra, R.; Romano, A.D.; Quinto, M.; Blonda, M.; Vendemiale, G.; et al. Mitochondrial oxidative stress and respiratory chain dysfunction account for liver toxicity during amiodarone but not dronedarone administration. Free Radic. Biol. Med. 2011, 51, 2234–2242. [Google Scholar] [CrossRef] [PubMed]
- Spaniol, M.; Bracher, R.; Ha, H.R.; Follath, F.; Krahenbuhl, S. Toxicity of amiodarone and amiodarone analogues on isolated rat liver mitochondria. J. Hepatol. 2001, 35, 628–636. [Google Scholar] [CrossRef] [PubMed]
- Waldhauser, K.M.; Torok, M.; Ha, H.R.; Thomet, U.; Konrad, D.; Brecht, K.; Follath, F.; Krahenbuhl, S. Hepatocellular toxicity and pharmacological effect of amiodarone and amiodarone derivatives. J. Pharmacol. Exp. Ther. 2006, 319, 1413–1423. [Google Scholar] [CrossRef] [PubMed]
- Takai, S.; Oda, S.; Tsuneyama, K.; Fukami, T.; Nakajima, M.; Yokoi, T. Establishment of a mouse model for amiodarone-induced liver injury and analyses of its hepatotoxic mechanism. J. Appl. Toxicol. 2016, 36, 35–47. [Google Scholar] [CrossRef] [PubMed]
- Card, J.W.; Lalonde, B.R.; Rafeiro, E.; Tam, A.S.; Racz, W.J.; Brien, J.F.; Bray, T.M.; Massey, T.E. Amiodarone-induced disruption of hamster lung and liver mitochondrial function: Lack of association with thiobarbituric acid-reactive substance production. Toxicol. Lett. 1998, 98, 41–50. [Google Scholar] [CrossRef]
- Li, Y.; Zhao, T.; Li, J.; Xia, M.; Li, Y.; Wang, X.; Liu, C.; Zheng, T.; Chen, R.; Kan, D.; et al. Oxidative Stress and 4-hydroxy-2-nonenal (4-HNE): Implications in the Pathogenesis and Treatment of Aging-related Diseases. J. Immunol. Res. 2022, 2022, 2233906. [Google Scholar] [CrossRef]
- Varbiro, G.; Toth, A.; Tapodi, A.; Veres, B.; Sumegi, B.; Gallyas, F., Jr. Concentration dependent mitochondrial effect of amiodarone. Biochem. Pharmacol. 2003, 65, 1115–1128. [Google Scholar] [CrossRef]
- Varbiro, G.; Toth, A.; Tapodi, A.; Bognar, Z.; Veres, B.; Sumegi, B.; Gallyas, F., Jr. Protective effect of amiodarone but not N-desethylamiodarone on postischemic hearts through the inhibition of mitochondrial permeability transition. J. Pharmacol. Exp. Ther. 2003, 307, 615–625. [Google Scholar] [CrossRef]
- Bognar, Z.; Fekete, K.; Antus, C.; Hocsak, E.; Bognar, R.; Tapodi, A.; Boronkai, A.; Farkas, N.; Gallyas, F., Jr.; Sumegi, B.; et al. Desethylamiodarone-A metabolite of amiodarone-Induces apoptosis on T24 human bladder cancer cells via multiple pathways. PLoS ONE 2017, 12, e0189470. [Google Scholar] [CrossRef]
- Bognar, Z.; Fekete, K.; Bognar, R.; Szabo, A.; Vass, R.A.; Sumegi, B. Amiodarone’s major metabolite, desethylamiodarone, induces apoptosis in human cervical cancer cells. Can. J. Physiol. Pharmacol. 2018, 96, 1004–1011. [Google Scholar] [CrossRef]
- Bognar, Z.; Cseh, A.M.; Fekete, K.; Antus, C.; Bognar, R.; Tapodi, A.; Ramadan, F.H.J.; Sumegi, B.; Gallyas, F., Jr. Amiodarone’s major metabolite, desethylamiodarone inhibits proliferation of B16-F10 melanoma cells and limits lung metastasis formation in an in vivo experimental model. PLoS ONE 2020, 15, e0239088. [Google Scholar] [CrossRef] [PubMed]
- Gallyas, F., Jr.; Ramadan, F.H.J.; Andreidesz, K.; Hocsak, E.; Szabo, A.; Tapodi, A.; Kiss, G.N.; Fekete, K.; Bognar, R.; Szanto, A.; et al. Involvement of Mitochondrial Mechanisms and Cyclooxygenase-2 Activation in the Effect of Desethylamiodarone on 4T1 Triple-Negative Breast Cancer Line. Int. J. Mol. Sci. 2022, 23, 1544. [Google Scholar] [CrossRef] [PubMed]
- Ramadan, F.H.J.; Szabo, A.; Kovacs, D.; Takatsy, A.; Bognar, R.; Gallyas, F., Jr.; Bognar, Z. Involvement of Mitochondrial Mechanisms in the Cytostatic Effect of Desethylamiodarone in B16F10 Melanoma Cells. Int. J. Mol. Sci. 2020, 21, 7346. [Google Scholar] [CrossRef] [PubMed]
- Zorova, L.D.; Popkov, V.A.; Plotnikov, E.Y.; Silachev, D.N.; Pevzner, I.B.; Jankauskas, S.S.; Babenko, V.A.; Zorov, S.D.; Balakireva, A.V.; Juhaszova, M.; et al. Mitochondrial membrane potential. Anal. Biochem. 2018, 552, 50–59. [Google Scholar] [CrossRef]
- Lugli, E.; Troiano, L.; Ferraresi, R.; Roat, E.; Prada, N.; Nasi, M.; Pinti, M.; Cooper, E.L.; Cossarizza, A. Characterization of cells with different mitochondrial membrane potential during apoptosis. Cytom. A 2005, 68, 28–35. [Google Scholar] [CrossRef]
- Chazotte, B. Labeling mitochondria with JC-1. Cold Spring Harb. Protoc. 2011, 2011, pdb.prot065490. [Google Scholar] [CrossRef]
- Adebayo, M.; Singh, S.; Singh, A.P.; Dasgupta, S. Mitochondrial fusion and fission: The fine-tune balance for cellular homeostasis. FASEB J. 2021, 35, e21620. [Google Scholar] [CrossRef]
- Chan, D.C. Mitochondrial Dynamics and Its Involvement in Disease. Annu. Rev. Pathol. 2020, 15, 235–259. [Google Scholar] [CrossRef]
- del Campo, A.; Parra, V.; Vasquez-Trincado, C.; Gutierrez, T.; Morales, P.E.; Lopez-Crisosto, C.; Bravo-Sagua, R.; Navarro-Marquez, M.F.; Verdejo, H.E.; Contreras-Ferrat, A.; et al. Mitochondrial fragmentation impairs insulin-dependent glucose uptake by modulating Akt activity through mitochondrial Ca2+ uptake. Am. J. Physiol. Endocrinol. Metab. 2014, 306, E1–E13. [Google Scholar] [CrossRef]
- Bernardi, P.; Rasola, A.; Forte, M.; Lippe, G. The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology. Physiol. Rev. 2015, 95, 1111–1155. [Google Scholar] [CrossRef]
- Bonora, M.; Pinton, P. The mitochondrial permeability transition pore and cancer: Molecular mechanisms involved in cell death. Front. Oncol. 2014, 4, 302. [Google Scholar] [CrossRef] [PubMed]
- Priber, J.; Fonai, F.; Jakus, P.B.; Racz, B.; Chinopoulos, C.; Tretter, L.; Gallyas, F., Jr.; Sumegi, B.; Veres, B. Cyclophilin D disruption attenuates lipopolysaccharide-induced inflammatory response in primary mouse macrophages. Biochem. Cell Biol. 2015, 93, 241–250. [Google Scholar] [CrossRef] [PubMed]
- Marchi, S.; Giorgi, C.; Galluzzi, L.; Pinton, P. Ca(2+) Fluxes and Cancer. Mol. Cell 2020, 78, 1055–1069. [Google Scholar] [CrossRef] [PubMed]
- Patergnani, S.; Danese, A.; Bouhamida, E.; Aguiari, G.; Previati, M.; Pinton, P.; Giorgi, C. Various Aspects of Calcium Signaling in the Regulation of Apoptosis, Autophagy, Cell Proliferation, and Cancer. Int. J. Mol. Sci. 2020, 21, 8323. [Google Scholar] [CrossRef] [PubMed]
- NavaneethaKrishnan, S.; Rosales, J.L.; Lee, K.Y. mPTP opening caused by Cdk5 loss is due to increased mitochondrial Ca(2+) uptake. Oncogene 2020, 39, 2797–2806. [Google Scholar] [CrossRef]
- Lee, J.; Yesilkanal, A.E.; Wynne, J.P.; Frankenberger, C.; Liu, J.; Yan, J.; Elbaz, M.; Rabe, D.C.; Rustandy, F.D.; Tiwari, P.; et al. Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism. Nature 2019, 568, 254–258. [Google Scholar] [CrossRef]
- Weinberg, S.E.; Chandel, N.S. Targeting mitochondria metabolism for cancer therapy. Nat. Chem. Biol. 2015, 11, 9–15. [Google Scholar] [CrossRef]
- Mookerjee, S.A.; Gerencser, A.A.; Nicholls, D.G.; Brand, M.D. Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements. J. Biol. Chem. 2017, 292, 7189–7207. [Google Scholar] [CrossRef]
- Mookerjee, S.A.; Goncalves, R.L.S.; Gerencser, A.A.; Nicholls, D.G.; Brand, M.D. The contributions of respiration and glycolysis to extracellular acid production. Biochim. Biophys. Acta 2015, 1847, 171–181. [Google Scholar] [CrossRef]
- Desousa, B.R.; Kim, K.K.; Jones, A.E.; Ball, A.B.; Hsieh, W.Y.; Swain, P.; Morrow, D.H.; Brownstein, A.J.; Ferrick, D.A.; Shirihai, O.S.; et al. Calculation of ATP production rates using the Seahorse XF Analyzer. EMBO Rep. 2023, 24, e56380. [Google Scholar] [CrossRef]
- Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 2006, 1, 1112–1116. [Google Scholar] [CrossRef] [PubMed]
- Nath, S.; Devi, G.R. Three-dimensional culture systems in cancer research: Focus on tumor spheroid model. Pharmacol. Ther. 2016, 163, 94–108. [Google Scholar] [CrossRef] [PubMed]
- Silva-Gomez, J.A.; Galicia-Moreno, M.; Sandoval-Rodriguez, A.; Miranda-Roblero, H.O.; Lucano-Landeros, S.; Santos, A.; Monroy-Ramirez, H.C.; Armendariz-Borunda, J. Hepatocarcinogenesis Prevention by Pirfenidone Is PPARgamma Mediated and Involves Modification of Nuclear NF-kB p65/p50 Ratio. Int. J. Mol. Sci. 2021, 22, 1360. [Google Scholar] [CrossRef]
- Millan, A.; Huerta, S. Apoptosis-inducing factor and colon cancer. J. Surg. Res. 2009, 151, 163–170. [Google Scholar] [CrossRef]
- Schafer, M.; Werner, S. Cancer as an overhealing wound: An old hypothesis revisited. Nat. Rev. Mol. Cell Biol. 2008, 9, 628–638. [Google Scholar] [CrossRef]
- Shayeganpour, A.; Hamdy, D.A.; Brocks, D.R. Pharmacokinetics of desethylamiodarone in the rat after its administration as the preformed metabolite, and after administration of amiodarone. Biopharm. Drug Dispos. 2008, 29, 159–166. [Google Scholar] [CrossRef] [PubMed]
- Roth, F.C.; Mulder, J.E.; Brien, J.F.; Takahashi, T.; Massey, T.E. Cytotoxic interaction between amiodarone and desethylamiodarone in human peripheral lung epithelial cells. Chem. Biol. Interact. 2013, 204, 135–139. [Google Scholar] [CrossRef]
- Perou, C.M.; Sorlie, T.; Eisen, M.B.; van de Rijn, M.; Jeffrey, S.S.; Rees, C.A.; Pollack, J.R.; Ross, D.T.; Johnsen, H.; Akslen, L.A.; et al. Molecular portraits of human breast tumours. Nature 2000, 406, 747–752. [Google Scholar] [CrossRef]
- Icard, P.; Shulman, S.; Farhat, D.; Steyaert, J.M.; Alifano, M.; Lincet, H. How the Warburg effect supports aggressiveness and drug resistance of cancer cells? Drug Resist. Updat. 2018, 38, 1–11. [Google Scholar] [CrossRef]
- Li, X.; Jiang, Y.; Meisenhelder, J.; Yang, W.; Hawke, D.H.; Zheng, Y.; Xia, Y.; Aldape, K.; He, J.; Hunter, T.; et al. Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis. Mol. Cell 2016, 61, 705–719. [Google Scholar] [CrossRef]
- LeBleu, V.S.; O’Connell, J.T.; Gonzalez Herrera, K.N.; Wikman, H.; Pantel, K.; Haigis, M.C.; de Carvalho, F.M.; Damascena, A.; Domingos Chinen, L.T.; Rocha, R.M.; et al. PGC-1alpha mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis. Nat. Cell Biol. 2014, 16, 992–1003. [Google Scholar] [CrossRef] [PubMed]
- Hirpara, J.; Eu, J.Q.; Tan, J.K.M.; Wong, A.L.; Clement, M.V.; Kong, L.R.; Ohi, N.; Tsunoda, T.; Qu, J.; Goh, B.C.; et al. Metabolic reprogramming of oncogene-addicted cancer cells to OXPHOS as a mechanism of drug resistance. Redox Biol. 2019, 25, 101076. [Google Scholar] [CrossRef] [PubMed]
- Lin, C.S.; Liu, L.T.; Ou, L.H.; Pan, S.C.; Lin, C.I.; Wei, Y.H. Role of mitochondrial function in the invasiveness of human colon cancer cells. Oncol. Rep. 2018, 39, 316–330. [Google Scholar] [CrossRef]
- Ashton, T.M.; McKenna, W.G.; Kunz-Schughart, L.A.; Higgins, G.S. Oxidative Phosphorylation as an Emerging Target in Cancer Therapy. Clin. Cancer Res. 2018, 24, 2482–2490. [Google Scholar] [CrossRef]
- Leung, E.; Cairns, R.A.; Chaudary, N.; Vellanki, R.N.; Kalliomaki, T.; Moriyama, E.H.; Mujcic, H.; Wilson, B.C.; Wouters, B.G.; Hill, R.; et al. Metabolic targeting of HIF-dependent glycolysis reduces lactate, increases oxygen consumption and enhances response to high-dose single-fraction radiotherapy in hypoxic solid tumors. BMC Cancer 2017, 17, 418. [Google Scholar] [CrossRef]
- Palominos, C.; Fuentes-Retamal, S.; Salazar, J.P.; Guzman-Rivera, D.; Correa, P.; Mellado, M.; Araya-Maturana, R.; Urra, F.A. Mitochondrial bioenergetics as a cell fate rheostat for responsive to Bcl-2 drugs: New cues for cancer chemotherapy. Cancer Lett. 2024, 594, 216965. [Google Scholar] [CrossRef]
- Vyas, S.; Zaganjor, E.; Haigis, M.C. Mitochondria and Cancer. Cell 2016, 166, 555–566. [Google Scholar] [CrossRef]
- Zhang, Y.; Yan, H.; Wei, Y.; Wei, X. Decoding mitochondria’s role in immunity and cancer therapy. Biochim. Biophys. Acta Rev. Cancer 2024, 1879, 189107. [Google Scholar] [CrossRef] [PubMed]
- Guerra, F.; Arbini, A.A.; Moro, L. Mitochondria and cancer chemoresistance. Biochim. Biophys. Acta Bioenerg. 2017, 1858, 686–699. [Google Scholar] [CrossRef]
- Finley, L.W.S. What is cancer metabolism? Cell 2023, 186, 1670–1688. [Google Scholar] [CrossRef]
- Burke, P.J. Mitochondria, Bioenergetics and Apoptosis in Cancer. Trends Cancer 2017, 3, 857–870. [Google Scholar] [CrossRef] [PubMed]
- Folkerts, H.; Wierenga, A.T.; van den Heuvel, F.A.; Woldhuis, R.R.; Kluit, D.S.; Jaques, J.; Schuringa, J.J.; Vellenga, E. Elevated VMP1 expression in acute myeloid leukemia amplifies autophagy and is protective against venetoclax-induced apoptosis. Cell Death Dis. 2019, 10, 421. [Google Scholar] [CrossRef] [PubMed]
- Kinnel, B.; Singh, S.K.; Oprea-Ilies, G.; Singh, R. Targeted Therapy and Mechanisms of Drug Resistance in Breast Cancer. Cancers 2023, 15, 1320. [Google Scholar] [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Ramadan, F.H.J.; Koszegi, B.; Vantus, V.B.; Fekete, K.; Kiss, G.N.; Rizsanyi, B.; Bognar, R.; Gallyas, F., Jr.; Bognar, Z. Comparison of Mitochondrial and Antineoplastic Effects of Amiodarone and Desethylamiodarone in MDA-MB-231 Cancer Line. Int. J. Mol. Sci. 2024, 25, 9781. https://doi.org/10.3390/ijms25189781
Ramadan FHJ, Koszegi B, Vantus VB, Fekete K, Kiss GN, Rizsanyi B, Bognar R, Gallyas F Jr., Bognar Z. Comparison of Mitochondrial and Antineoplastic Effects of Amiodarone and Desethylamiodarone in MDA-MB-231 Cancer Line. International Journal of Molecular Sciences. 2024; 25(18):9781. https://doi.org/10.3390/ijms25189781
Chicago/Turabian StyleRamadan, Fadi H. J., Balazs Koszegi, Viola B. Vantus, Katalin Fekete, Gyongyi N. Kiss, Balint Rizsanyi, Rita Bognar, Ferenc Gallyas, Jr., and Zita Bognar. 2024. "Comparison of Mitochondrial and Antineoplastic Effects of Amiodarone and Desethylamiodarone in MDA-MB-231 Cancer Line" International Journal of Molecular Sciences 25, no. 18: 9781. https://doi.org/10.3390/ijms25189781