Dependance of doxorubicin efficacy of on p21 protein level in tumor cells

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Abstract

Introduction. The effectiveness of chemotherapy is determined by the percentage of tumor cells killed and the absence of tumor recurrence. The cell cycle regulator p21 plays a key role in these processes, although its role remains unclear.

Aim. To determine the effect of CDKN1A expression before and after exposure of tumor cells to doxorubicin on therapeutic efficacy.

Materials and methods. Flow cytometry was used for analysis of cell distribution per cell cycle phases, immunocytochemical staining was used for β-galactosidase activity measurement. Change in protein expression was evaluated using Western blot. For colony visualization, the cells were stained with crystal violet.

Results. It was found that overexpression of the p21 gene (CDKN1A) during cell exposure to doxorubicin reduces the number of cells that have stopped dividing in the G2/M phase of the cell cycle and increases their number in the G1 phase. This, in the absence of CDKN1A expression, subsequently leads to resumption of proliferation. Conversely, the absence of p21 during chemotherapy exposure results in a significant increase in the percentage of cells in the G2/M phase, followed by their death. Induction of CDKN1A expression after removal of doxorubicin, or throughout the experiment, leads to the development of a cellular senescence stage.

Conclusion. Increased CDKN1A expression during cell exposure to doxorubicin reduces its effectiveness. Maintaining high p21 levels after removal of the drug promotes the development of a cellular senescence phenotype.

About the authors

Maria A. Zamkova

Institute of Gene Biology, Russian Academy of Sciences; N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia

Author for correspondence.
Email: zamkovam@gmail.com
ORCID iD: 0000-0002-4687-7444
Russian Federation, 34/5 Vavilova St., Moscow 119334; 24 Kashirskoe Shosse, Moscow 115522

A. V. Bruter

Institute of Gene Biology, Russian Academy of Sciences; N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia

Email: zamkovam@gmail.com
ORCID iD: 0000-0002-2090-2488
Russian Federation, 34/5 Vavilova St., Moscow 119334; 24 Kashirskoe Shosse, Moscow 115522

M. V. Kubekina

Institute of Gene Biology, Russian Academy of Sciences

Email: zamkovam@gmail.com
ORCID iD: 0000-0002-8834-1111
Russian Federation, 34/5 Vavilova St., Moscow 119334

V. V. Tatarskiy

Institute of Gene Biology, Russian Academy of Sciences

Email: zamkovam@gmail.com
ORCID iD: 0000-0002-9080-5683
Russian Federation, 34/5 Vavilova St., Moscow 119334

References

  1. Bikinieva F.F., Dunaev P.D., Galembikova A.R. et al. Overexpression of ABC transporters in gastrointestinal stromal tumors as one of the mechanisms for the development of secondary chemoresistance. Uspekhi molekulyarnoy onkologii = Advances in Molecular Oncology 2025;12 (4):109–24. doi: 10.17650/2313-805X-2025-12-4-109-124
  2. Mijit M., Caracciolo V., Melillo A. et al. Role of p53 in the regulation of cellular senescence. Biomolecules 2020;10(3):420. doi: 10.3390/biom10030420
  3. Kumari R., Jat P. Mechanisms of cellular senescence: cell cycle arrest and senescence associated secretory phenotype. Front Cell Dev Biol 2021;9:645593. doi: 10.3389/fcell.2021.645593
  4. Zamkova M.A., Persiyantseva N.A., Tatarskiy V.V. et al. Therapy-induced tumor cell senescence: mechanisms and circumvention. Biochemistry (Mosc) 2023;88(1):86–104. doi: 10.1134/S000629792301008X
  5. Yosef R., Pilpel N., Papismadov N. et al. p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling. EMBO J 2017;36 (15):2280–95. doi: 10.15252/embj.201695553
  6. Yan J., Chen S., Yi Z. et al. The role of p21 in cellular senescence and aging-related diseases. Mol Cells 2024;47(11):100113. doi: 10.1016/j.mocell.2024.100113
  7. Chattopadhyay D., Ghosh M.K., Mal A. et al. Inactivation of p21 by E1A leads to the induction of apoptosis in DNA-damaged cells. J Virol 2001;75(20):9844–56. doi: 10.1128/JVI.75.20.9844-9856.2001
  8. Blagosklonny M.V., Robey R., Bates S. et al. Pretreatment with DNA-damaging agents permits selective killing of checkpoint-deficient cells by microtubule-active drugs. J Clin Invest 2000;105(4):533–39. doi: 10.1172/JCI8625
  9. Rohnalter V., Roth K., Finkernagel F. et al. A multi-stage process including transient polyploidization and EMT precedes the emergence of chemoresistent ovarian carcinoma cells with a dedifferentiated and pro-inflammatory secretory phenotype. Oncotarget 2015;6(37):40005–25. doi: 10.18632/oncotarget.5552
  10. Richter W.F., Nayak S., Iwasa J. et al. The Mediator complex as a master regulator of transcription by RNA polymerase II. Nat Rev Mol Cell Biol 2022;23(11):732–49. doi: 10.1038/s41580-022-00498-3
  11. Olszewska A., Borkowska A., Granica M. et al. Escape from cisplatin-induced senescence of hypoxic lung cancer cells can be overcome by hydroxychloroquine. Front Oncol 2021;11:738385. doi: 10.3389/fonc.2021.738385
  12. Hsu C.H., Altschuler S.J., Wu L.F. Patterns of Early p21 dynamics determine proliferation-senescence cell fate after chemotherapy. Cell 2019;178(2):361–73e312. doi: 10.1016/j.cell.2019.05.041
  13. Khamidullina A.I., Yastrebova M.A., Bruter A.V. et al. CDK8/19 inhibition attenuates G1 arrest induced by BCR-ABL antagonists and accelerates death of chronic myelogenous leukemia cells. Cell Death Discov 2025;11(1):62. doi: 10.1038/s41420-025-02339-6
  14. Persiyantseva N.A., Kazansky D.B., Tatarskiy V.V., Zamkova M.A. Genome editing by CRISPR/Cas to generate A549 human lung cancer subline knockout for the р21 gene. Meditsinskaya genetika = Medical Genetics 2023;22(11):35–9. (In Russ.). doi: 10.25557/2073-7998.2023.11.35-39
  15. Persiyantseva N.A., Vikhrova S.Y., Korotkova M.S. et al. Decreasing the ability of HCT116 cells to escape from therapy induced senescence by increasing the duration of doxorubicin treatment. Uspekhi molekulyarnoy onkologii = Advances in Molecular Oncology 2024;11(1):90–8. doi: 10.17650/2313-805X-202411-1-90-98
  16. Saleh T., Tyutyunyk-Massey L., Murray G.F. et al. Tumor cell escape from therapy-induced senescence. Biochem Pharmacol 2019;162:202–12. doi: 10.1016/j.bcp.2018.12.013
  17. Pluquet O., Abbadie C., Coqueret O. Connecting cancer relapse with senescence. Cancer Lett 2019;463:50–8. doi: 10.1016/j.canlet.2019.08.004
  18. Montalto F.I., De Amicis F. Сyclin D1 in cancer: a molecular connection for cell cycle control, adhesion and invasion in tumor and stroma. Cells 2020;9(12):2648. doi: 10.3390/cells9122648
  19. Kalra R.S., Cheung C.T., Chaudhary A. et al. CARF (collaborator of ARF) overexpression in p53-deficient cells promotes carcinogenesis. Mol Oncol 2015;9(9):1877–89. doi: 10.1016/j.molonc.2015.07.003
  20. Sheldon L.A. Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein. Cell Cycle 2017;16(21):2058–72. doi: 10.1080/15384101.2017.1338221
  21. Ianari A., Gallo R., Palma M. et al. Specific role for p300/ CREB-binding protein-associated factor activity in E2F1 stabilization in response to DNA damage. J Biol Chem 2004;279(29):30830–35. doi: 10.1074/jbc.M402403200
  22. Fedorova N.E., Chernoryzh Y.Y., Vinogradskaya G.R. et al. Inhibitor of polyamine catabolism MDL72.527 restores the sensitivity to doxorubicin of monocytic leukemia Thp-1 cells infected with human cytomegalovirus. Biochimie 2019;158:82–9. doi: 10.1016/j.biochi.2018.12.012
  23. Luo D., Yu C., Yu J. et al. p53-mediated G1 arrest requires the induction of both p21 and Killin in human colon cancer cells. Cell Cycle 2022;21(2):140–51. doi: 10.1080/15384101.2021.2014249
  24. Hoeferlin L.A., Oleinik N.V., Krupenko N.I. et al. Activation of p21-dependent G1/G2 arrest in the absence of DNA damage as an antiapoptotic response to metabolic stress. Genes Cancer 2011;2(9):889–99. doi: 10.1177/1947601911432495
  25. Clay D.E., Fox D.T. DNA damage responses during the cell cycle: Insights from model organisms and beyond. Genes (Basel) 2021;2(12):1882. doi: 10.3390/genes12121882
  26. Zhou F.Y., Waterman D.P., Ashton M. et al. Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint. Elife 2024;13:RP94334. doi: 10.7554/eLife.94334
  27. Sandor V., Senderowicz A., Mertins S. et al. P21-dependent g(1)arrest with downregulation of cyclin D1 and upregulation of cyclin E by the histone deacetylase inhibitor FR901228. Br J Cancer 2000;83(6):817–25. doi: 10.1054/bjoc.2000.1327
  28. Liu Y., Chen H., Li X. et al. PSMC2 regulates cell cycle progression through the p21/cyclin D1 pathway and predicts a poor prognosis in human hepatocellular carcinoma. Front Oncol 2021;11:607021. doi: 10.3389/fonc.2021.607021
  29. Gao X., Zhang Y., Zhang R. et al. Cyclin-dependent kinase 1 disruption inhibits angiogenesis by inducing cell cycle arrest and apoptosis. Exp Ther Med 2019;18(4):3062–70. doi: 10.3892/etm.2019.7883
  30. Nakanishi M., Kaneko Y., Matsushime H. et al. Direct interaction of p21 cyclin-dependent kinase inhibitor with the retinoblastoma tumor suppressor protein. Biochem Biophys Res Commun 1999;263(1):35–40. doi: 10.1006/bbrc.1999.1296
  31. Chen J.Y., Lin J.R., Tsai F.C. et al. Dosage of Dyrk1a shifts cells within a p21-cyclin D1 signaling map to control the decision to enter the cell cycle. Mol Cell 2013;52(1):87–100. doi: 10.1016/j.molcel.2013.09.009
  32. Coleman M.L., Marshall C.J., Olson M.F. Ras promotes p21(Waf1/Cip1) protein stability via a cyclin D1-imposed block in proteasome-mediated degradation. EMBO J 2003;22(9):2036–46. doi: 10.1093/emboj/cdg189
  33. Stein G.H., Drullinger L.F., Soulard A. et al. Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol 1999;19(3):2109–17. doi: 10.1128/MCB.19.3.2109
  34. Putzer B.M. E2F1 death pathways as targets for cancer therapy. J Cell Mol Med 2007;11(2):239–51. doi: 10.1111/j.1582-4934.2007.00030.x
  35. Martinez L.A., Goluszko E., Chen H.Z. et al. E2F3 is a mediator of DNA damage-induced apoptosis. Mol Cell Biol 2010;30(2):524–36. doi: 10.1128/MCB.00938-09
  36. Meng P., Ghosh R. Transcription addiction: can we garner the Yin and Yang functions of E2F1 for cancer therapy? Cell Death Dis 2014;5(8):e1360. doi: 10.1038/cddis.2014.326

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