OBJECTIVES: Chemotherapeutic drugs induce senescence in cancer cells but, unlike replicative senescence or oncogene-induced senescence, do so rather inefficiently and depending on DNA damage. A thorough understanding of the biology of chemotherapy-induced senescent cells requires their isolation from a mixed population of adjacent senescent and non-senescent cancer cells. MATERIALS AND METHODS: We have developed and optimized a rapid iodixanol (OptiPrep)-based gradient centrifugation system to identify, isolate and characterize doxorubicin (DXR)-induced senescent hepatocellular carcinoma (HCC) cells (HepG2 and Huh-7) in vitro. RESULTS: After cellular exposure to DXR, we used iodixanol gradient-based centrifugation to isolate and re-plate cells on collagen-coated flasks, despite their low or null proliferative capacity. The isolated cell populations were enriched for DXR-induced senescent HCC cells, as confirmed by proliferation arrest assay, and β-galactosidase and DNA damage-dependent γH2A.X staining. CONCLUSIONS: Analysing pure cultures of chemotherapy-induced senescent versus non-responsive cancer cells will increase our knowledge on chemotherapeutic mechanisms of action, and help refine current therapeutic strategies.

Center for Translational Medicine International Clinical Research Center Brno Czech Republic

Zobrazit více v PubMed

Jeon OH, Kim C, Laberge R‐M, et al. Local clearance of senescent cells attenuates the development of post‐traumatic osteoarthritis and creates a pro‐regenerative environment. Nat Med. 2017;23(6):775‐781. PubMed PMC

Demaria M, O'Leary MN, Chang J, et al. Cellular senescence promotes adverse effects of chemotherapy and cancer relapse. Cancer Discov. 2017;7(2):165‐176. PubMed PMC

Wiley CD, Campisi J. From ancient pathways to aging cells‐connecting metabolism and cellular senescence. Cell Metab. 2016;23(6):1013‐1021. PubMed PMC

Bird TG, Muller M, Boulter L, et al. TGFbeta inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence. Sci Transl Med. 2018;10(454):pii eaan1230. PubMed PMC

Lowe D, Horvath S, Raj K. Epigenetic clock analyses of cellular senescence and ageing. Oncotarget. 2016;7(8):8524‐8531. PubMed PMC

Chen Y, Mao P, Snijders AM, Wang D. Senescence chips for ultrahigh‐throughput isolation and removal of senescent cells. Aging Cell. 2018;17(2):e12722. PubMed PMC

Matjusaitis M, Chin G, Sarnoski EA, Stolzing A. Biomarkers to identify and isolate senescent cells. Ageing Res Rev. 2016;29:1‐12. PubMed

Kirkland JL, Tchkonia T, Zhu Y, Niedernhofer LJ, Robbins PD. The clinical potential of senolytic drugs. J Am Geriatr Soc. 2017;65(10):2297‐2301. PubMed PMC

Kovacovicova K, Skolnaja M, Heinmaa M, et al. Senolytic cocktail Dasatinib+Quercetin (D+Q) does not enhance the efficacy of senescence‐inducing chemotherapy in liver cancer. Front Oncol. 2018;8:459. PubMed PMC

Chen J, Chen K‐H, Fu B‐Q, et al. Isolation and identification of senescent renal tubular epithelial cells using immunomagnetic beads based on DcR2. Exp Gerontol. 2017;95:116‐127. PubMed

Kim KM, Noh JH, Bodogai M, et al. Identification of senescent cell surface targetable protein DPP4. Genes Dev. 2017;31(15):1529‐1534. PubMed PMC

Frescas D, Roux CM, Aygun‐Sunar S, et al. Senescent cells expose and secrete an oxidized form of membrane‐bound vimentin as revealed by a natural polyreactive antibody. Proc Natl Acad Sci USA. 2017;114(9):E1668‐E1677. PubMed PMC

Wang D, Lu P, Liu Y, et al. Isolation of live premature senescent cells using FUCCI technology. Sci Rep. 2016;6:30705. PubMed PMC

Borghesan M, Fusilli C, Rappa F, et al. DNA hypomethylation and histone variant macroH2A1 synergistically attenuate chemotherapy‐induced senescence to promote hepatocellular carcinoma progression. Can Res. 2016;76(3):594‐606. PubMed PMC

Lo Re O, Fusilli C, Rappa F, et al. Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma. Hepatology. 2017;67(2):636-650. PubMed

Itahana K, Campisi J, Dimri GP. Methods to detect biomarkers of cellular senescence: the senescence‐associated beta‐galactosidase assay. Methods Mol Biol. 2007;371:21‐31. PubMed

Podrini C, Koffas A, Chokshi S, et al. MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat‐induced steatosis. FASEB J. 2015;29(5):1676‐1687. PubMed

Graham JM. Formation of self‐generated gradients of iodixanol. ScientificWorldJournal. 2002;2:1356‐1360. PubMed PMC

Jueliger S, Lyons J, Cannito S, et al. Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI‐110) in preclinical models of hepatocellular carcinoma. Epigenetics. 2016;1–12. PubMed PMC

Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;92(20):9363‐9367. PubMed PMC

Kilic Eren M, Kilincli A, Eren O. Resveratrol induced premature senescence is associated with DNA damage mediated SIRT1 and SIRT2 down‐regulation. PLoS One. 2015;10(4):e0124837. PubMed PMC

Boothe T, Hilbert L, Heide M, et al. A tunable refractive index matching medium for live imaging cells, tissues and model organisms. eLife. 2017;6:e27240. PubMed PMC

Mita A, Ricordi C, Messinger S, et al. Antiproinflammatory effects of iodixanol (OptiPrep)‐based density gradient purification on human islet preparations. Cell Transplant. 2010;19(12):1537‐1546. PubMed PMC

Holtzclaw JD, Jiang M, Yasin Z, Joiner CH, Franco RS. Rehydration of high‐density sickle erythrocytes in vitro. Blood. 2002;100(8):3017‐3025. PubMed

Inaba N, Hiruma T, Togayachi A, et al. A novel I‐branching beta‐1,6‐N‐acetylglucosaminyltransferase involved in human blood group I antigen expression. Blood. 2003;101(7):2870‐2876. PubMed

Gorgoulis VG, Halazonetis TD. Oncogene‐induced senescence: the bright and dark side of the response. Curr Opin Cell Biol. 2010;22(6):816‐827. PubMed

Ewald JA, Desotelle JA, Wilding G, Jarrard DF. Therapy‐induced senescence in cancer. J Natl Cancer Inst. 2010;102(20):1536‐1546. PubMed PMC

Abou‐Alfa GK, Johnson P, Knox JJ, et al. Doxorubicin plus sorafenib vs doxorubicin alone in patients with advanced hepatocellular carcinoma: a randomized trial. JAMA. 2010;304(19):2154‐2160. PubMed

Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol. 1999;57(7):727‐741. PubMed

Pang B, Qiao X, Janssen L, et al. Drug‐induced histone eviction from open chromatin contributes to the chemotherapeutic effects of doxorubicin. Nat Commun. 2013;4:1908. PubMed PMC

Denard B, Lee C, Ye J. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1. eLife. 2012;1:e00090. PubMed PMC

d'Adda di Fagagna F Living on a break: cellular senescence as a DNA‐damage response. Nat Rev Cancer. 2008;8(7):512‐522. PubMed

von Zglinicki T, Saretzki G, Ladhoff J, d'Adda di Fagagna F, Jackson SP. Human cell senescence as a DNA damage response. Mech Ageing Dev. 2005;126(1):111‐117. PubMed

Wang S, Konorev EA, Kotamraju S, Joseph J, Kalivendi S, Kalyanaraman B. Doxorubicin induces apoptosis in normal and tumor cells via distinctly different mechanisms. intermediacy of H(2)O(2)‐ and p53‐dependent pathways. J Biol Chem. 2004;279(24):25535‐25543. PubMed

Di Biase S, Shim HS, Kim KH, et al. Fasting regulates EGR1 and protects from glucose‐ and dexamethasone‐dependent sensitization to chemotherapy. PLoS Biol. 2017;15(3):e2001951. PubMed PMC

Vinciguerra M, Carrozzino F, Peyrou M, et al. Unsaturated fatty acids promote hepatoma proliferation and progression through downregulation of the tumor suppressor PTEN. J Hepatol. 2009;50(6):1132‐1141. PubMed

Jung H‐R, Kang HM, Ryu J‐W, et al. Cell spheroids with enhanced aggressiveness to mimic human liver cancer in vitro and in vivo. Sci Rep. 2017;7(1):10499. PubMed PMC

Bhardwaj B, Bhardwaj G, Lau JY. Expression of p21 and p27 in hepatoma cell lines with different p53 gene profile. J Hepatol. 1999;31(2):386. PubMed

Jackson J, Pant V, Li Q, et al. p53‐mediated senescence impairs the apoptotic response to chemotherapy and clinical outcome in breast cancer. Cancer Cell. 2012;21(6):793‐806. PubMed PMC

Kang T‐W, Yevsa T, Woller N, et al. Senescence surveillance of pre‐malignant hepatocytes limits liver cancer development. Nature. 2011;479(7374):547‐551. PubMed

Yevsa T, Kang TW, Zender L. Immune surveillance of pre‐cancerous senescent hepatocytes limits hepatocellular carcinoma development. Oncoimmunology. 2012;1(3):398‐399. PubMed PMC

Histone Variant macroH2A1.1 Enhances Nonhomologous End Joining-dependent DNA Double-strand-break Repair and Reprogramming Efficiency of Human iPSCs

Nociceptin/orphanin FQ opioid receptor (NOP) selective ligand MCOPPB links anxiolytic and senolytic effects

Senescence-like phenotype in post-mitotic cells of mice entering middle age