title: 1,3-Bis(2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of resveratrol on 5-fluorouracil sensitive/resistant colon cancer cells.
abstract: OBJECTIVE
  To study the mechanism of 5-fluorouracil (5-FU) resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment.
  
  
  METHODS
  We established and characterized a 5-FU resistance (5-FU-R) cell line derived from continuous exposure (25 μmol/L) to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells. The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting, respectively, after treatment with Resveratrol (Res) and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU). Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis, respectively. The extent of DNA damage was measured by the Comet assay. We measured the visible changes in the DNA damage/repair cascade by Western blotting.
  
  
  RESULTS
  The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner. Combined application of BCNU and Res caused more apoptosis in 5-FU sensitive cells in comparison to individual treatment. In addition, the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells. We established a 5-FU resistance cell line (5-FU-R) from 5-FU-sensitive HCT-116 (mismatch repair deficient) cells that was not resistant to other chemotherapeutic agents (e.g., BCNU, Res) except 5-FU. The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay. There was no significant cell death noted in 5-FU-R cells in comparison to 5-FU sensitive cells after 5-FU treatment. This resistant cell line overexpressed anti-apoptotic [e.g., AKT, nuclear factor κB, FLICE-like inhibitory protein), DNA repair (e.g., DNA polymerase beta (POL-β), DNA polymerase eta (POLH), protein Flap endonuclease 1 (FEN1), DNA damage-binding protein 2 (DDB2)] and 5-FU-resistance proteins (thymidylate synthase) but under expressed pro-apoptotic proteins (e.g., DAB2, CK1) in comparison to the parental cells. Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells. BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells. Fifty percent cell death were noted in parental cells when 18 μmol/L of Res was associated with fixed concentration (20 μmol/L) of BCNU, but a much lower concentration of Res (8 μmol/L) was needed to achieve the same effect in 5-FU resistant cells. Interestingly, increased levels of adenomatous polyposis coli and decreased levels POL-β, POLH, FEN1 and DDB2 were noted after the same combined treatment in resistant cells.
  
  
  CONCLUSIONS
  BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance.
authors.affiliation: Dipon Das, Ranjan Preet, Purusottam Mohapatra, Shakti Ranjan Satapathy, Chanakya Nath Kundu, Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Bhubaneswar, Odisha 751024, India.
authors.forename: Dipon
authors.initials: D
authors.lastname: Das
authors.forename: Ranjan
authors.initials: R
authors.lastname: Preet
authors.forename: Purusottam
authors.initials: P
authors.lastname: Mohapatra
authors.forename: Shakti Ranjan
authors.initials: SR
authors.lastname: Satapathy
authors.forename: Chanakya Nath
authors.initials: CN
authors.lastname: Kundu
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002330:Carmustine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004357:Drug Synergism; D005472:Fluorouracil / Q000494:pharmacology; D045325:HCT116 Cells; D006801:Humans; D000077185:Resveratrol; D013267:Stilbenes / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; D018797:Cell Cycle Proteins; D013267:Stilbenes; D000077185:Resveratrol; D005472:Fluorouracil; D002330:Carmustine
keywords: 1,3-Bis(2-chloroethyl)-1-nitrosourea; 5-fluorouracil; Colon cancer; Combination therapy; Resveratrol