title: Long-lasting reduction in clonogenic potential of colorectal cancer cells by sequential treatments with 5-azanucleosides and topoisomerase inhibitors.
abstract: The currently approved therapies fail in a substantial number of colorectal cancer (CRC) patients due to the molecular heterogeneity of CRC, hence new efficient drug combinations are urgently needed. Emerging data indicate that 5-azanucleosides are able to sensitize cancer cells to the standard chemotherapeutic agents and contribute to overcoming intrinsic or acquired chemoresistance.
  
  
  
  CRC cells with different genetic backgrounds (HCT116, DLD-1, HT-29) were sequentially treated with 5-azanucleosides and topoisomerase inhibitors. The combined effects of these two drug classes on cell viability, apoptosis, signaling pathways, and colony formation were investigated.
  
  
  
  Here, we demonstrate that pretreatment with DNA demethylating agents, 5-aza-2'-deoxycytidine and 5-azacytidine, sensitizes CRC cells to topoisomerase inhibitors (irinotecan, etoposide, doxorubicin, mitoxantrone), reducing cell viability and clonogenicity and increasing programmed cell death more effectively than individual compounds at the same or even higher concentrations. 5-Azanucleosides did not cause considerable immediate toxic effects as evaluated by analysis of cell viability, apoptosis, DNA damage (γH2A.X), and endoplasmic reticulum (ER) stress (CHOP). However, 5-azanucleosides exerted long-lasting effects, reducing cell viability, changing cell morphology, and affecting phosphoinositide 3-kinase (PI3-kinase)/Akt signaling pathway. We found that a single exposure to 5-azanucleosides is sufficient to induce long-lasting sensitization to topoisomerase inhibitors. The combinatorial, but not separate, treatment with low doses of 5-aza-2'-deoxycytidine (0.1 μM) and etoposide (0.5 μM) caused a long-lasting (almost 70 days) reduction in clonogenic/replating ability of DLD-1 cells.
  
  
  
  These results suggest that sequential treatments with DNA demethylating agents and topoisomerase inhibitors may exert clinically relevant anticancer effects.
authors.affiliation: Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Alicja
authors.initials: A
authors.lastname: Pawlak
authors.affiliation: Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Ziolo
authors.affiliation: Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Fiedorowicz
authors.affiliation: Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Klaudyna
authors.initials: K
authors.lastname: Fidyt
authors.affiliation: Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Leon
authors.initials: L
authors.lastname: Strzadala
authors.affiliation: Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland. kalas@iitd.pan.wroc.pl.|Jan Dlugosz University in Czestochowa, Waszyngtona 4/8, 42-200, Czestochowa, Poland. kalas@iitd.pan.wroc.pl.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Kalas
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000494:pharmacology*; D014408:Biomarkers, Tumor; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D060965:Clonal Evolution / Q000187:drug effects*; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D004249:DNA Damage / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects / Q000235:genetics; D006801:Humans; D009154:Mutation; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism; D059003:Topoisomerase Inhibitors / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D059003:Topoisomerase Inhibitors; D001374:Azacitidine
keywords: 5-azacytidine; Akt; DNA methylation; Decitabine; Doxorubicin; Etoposide; Irinotecan; Mitoxantrone; combinatorial therapy; epigenetics