title: Enhanced anticancer properties of lomustine in conjunction with docosahexaenoic acid in glioblastoma cell lines.
abstract: OBJECTIVE
  Glioblastoma is a rapidly infiltrating tumor that consistently rematerializes despite various forms of aggressive treatment. Brain tumors are commonly treated with alkylating drugs, such as lomustine, which are chemotherapeutic agents. Use of these drugs, however, is associated with serious side effects. To reduce the side effects, one approach is to combine lower doses of chemotherapeutic drugs with other nontoxic anticancer agents. In this study, using glioblastoma cell lines, the authors investigated the anticancer effects of lomustine, alone and in combination with docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid normally abundant in the brain and known for its anticancer potential.
  
  
  METHODS
  Cells were cultured from 3 human-derived tumor cell lines (U87-MG, DB029, and MHBT161) and supplemented with either DHA or lomustine to determine the growth inhibitory potential using WST-1, a mitochondrial functional indicator. Human-derived cerebral cortex microvascular endothelial cells served as a normal phenotypic control. Cellular incorporation of DHA was analyzed by gas chromatography. Using flow cytometric analysis, the DHA and/or lomustine effect on induction of apoptosis and/or necrosis was quantified; subsequently, the DHA and lomustine effect on cell cycle progression was also assessed. Western blot analysis confirmed the role of downstream cellular targets.
  
  
  RESULTS
  U87-MG growth was inhibited with the supplementation of either DHA (ED50 68.3 μM) or lomustine (ED50 68.1 μM); however, growth inhibition was enhanced when U87-MG cells were administered equimolar doses of each compound, resulting in nearly total growth inhibition at 50 μM. Gas chromatography analysis of the fatty acid profile in DHA-supplemented U87-MG cells resulted in a linear dose-dependent increase in DHA incorporation (< 60 μM). The combination of DHA and lomustine potently induced U87-MG apoptosis and necrosis as indicated by flow cytometric analysis. Activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) was evident in lomustine-treated U87-MG cells, although this activation did not appear to be dependent on DHA supplementation. Additionally, lomustine-treated cells' growth arrested in the G2/M cell cycle stage, regardless of the presence of DHA. Similar to the U87-MG observations, the combination of DHA and lomustine resulted in growth inhibition of 2 additional human-derived glioblastoma cell lines, DB029 and MHBT161. Importantly, in primary human-derived cerebral cortex endothelial cells, this combination was only growth inhibitory (40.8%) at the highest dose screened (100 μM), which indicates a certain degree of selectivity toward glioblastoma.
  
  
  CONCLUSIONS
  Taken together, these data suggest a potential role for a combination therapy of lomustine and DHA for the treatment of glioblastomas.
authors.affiliation: Cellular Biochemistry Laboratory, Indiana University Health Methodist Research Institute;
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Harvey
authors.forename: Zhidong
authors.initials: Z
authors.lastname: Xu
authors.forename: M Reza
authors.initials: MR
authors.lastname: Saaddatzadeh
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wang
authors.forename: Karen
authors.initials: K
authors.lastname: Pollok
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Cohen-Gadol
authors.forename: Rafat A
authors.initials: RA
authors.lastname: Siddiqui
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004281:Docosahexaenoic Acids / Q000627:therapeutic use*; D004357:Drug Synergism; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005909:Glioblastoma / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D008130:Lomustine / Q000627:therapeutic use*
chemical_list: D000970:Antineoplastic Agents; D005230:Fatty Acids, Nonesterified; D004281:Docosahexaenoic Acids; D008130:Lomustine
keywords: BCA = bicinchoninic acid; CSC = Cell Systems Corporation; DHA = docosahexaenoic acid; DMEM = Dulbecco's modified essential medium; DPA = docosapentaenoic acid; ED50 = median effective dose; EMEM = Eagle's minimum essential medium; EPA = eicosapentaenoic acid; FBS = fetal bovine serum; HBMEC = human brain microvascular endothelial cell; HCCMEC = human cerebral cortex microvascular endothelial cell; OD = optical density; PARP = poly (ADP-ribose) polymerase; PBS = phosphate-buffered saline; PUFA = polyunsaturated fatty acid; brain cancer; docosahexaenoic acid; glioblastoma; lomustine; oncology