title: Wild carrot pentane-based fractions suppress proliferation of human HaCaT keratinocytes and protect against chemically-induced skin cancer.
abstract: BACKGROUND
  Previous studies in our laboratory showed that the Lebanese Daucus carota ssp. carota (wild carrot) oil extract possesses in vitro and in vivo anticancer activities. The present study aims to examine the cytotoxic effect of Daucus carota oil fractions on human epidermal keratinocytes and evaluate the chemopreventive activity of the pentane diethyl ether fraction on DMBA/TPA induced skin carcinogenesis in mice.
  
  
  METHODS
  Wild carrot oil extract was chromatographed to yield four fractions (F1, 100% pentane; F2, 50:50 pentane:diethyl ether; F3, 100% diethyl ether; F4 93:7 chloroform:methanol). The cytotoxic effect of fractions (10, 25, 50 and 100 μg/mL) was tested on human epidermal keratinocytes (non-tumorigenic HaCaT cells and tumorigenic HaCaT-ras variants) using WST a ssay. Cell cycle phase distribution of tumorigenic HaCaT-ras variants was determined by flow cytometry post-treatment with F2 fraction. Apoptosis related proteins were also assessed using western blot. The antitumor activity of F2 fraction was also evaluated using a DMBA/TPA induced skin carcinoma in Balb/c mice.
  
  
  RESULTS
  All fractions exhibited significant cytotoxicity, with HaCaT cells being 2.4-3 times less sensitive than HaCaT-ras A5 (benign tumorigenic), and HaCaT-ras II4 (malignant) cells. GC-MS analysis revealed the presence of a major compound (around 60%) in the pentane/diethylether fraction (F2), identified as 2-himachalen-6-ol. Treatment of HaCaT-ras A5 and HaCaT-ras II4 cells with F2 fraction resulted in the accumulation of cells in the sub-G1 apoptotic phase and decreased the population of cells in the S and G2/M phases. Additionally, F2 fraction treatment caused an up-regulation of the expression of pro-apoptotic (Bax) and down-regulation of the expression of anti-apoptotic (Bcl2) proteins. A decrease in the phosphorylation of AKT and ERK was also observed. Intraperitoneal treatment with F2 fraction (50 or 200 mg/kg) in the DMBA/TPA skin carcinogenesis mouse model showed a significant inhibition of papilloma incidence (mice with papilloma), yield (number of papilloma/mouse) and volume (tumor relative size) at weeks 15, 18 and 21.
  
  
  CONCLUSIONS
  The present data reveal that F2 fraction has a remarkable antitumor activity against DMBA/TPA-induced skin carcinogenesis, an effect that may be mediated through inhibition of the MAPK/ERK and PI3K/AKT pathways.
authors.affiliation: Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, P.O. Box 36, Byblos, Lebanon.
authors.forename: Wassim N
authors.initials: WN
authors.lastname: Shebaby
authors.affiliation: School of Pharmacy, Department of Pharmaceutical Sciences, Lebanese American University, P.O. Box 36, Byblos, Lebanon.
authors.forename: Mohamad A
authors.initials: MA
authors.lastname: Mroueh
authors.affiliation: Deutsches Krebsforschungszentrum DKFZ, German Cancer Research Center, Genetics of Skin Carcinogenesis, Im Neuenheimer Feld 280, Heidelberg, 69120, Germany.|IUF-Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Boukamp
authors.affiliation: Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, P.O. Box 36, Byblos, Lebanon.
authors.forename: Robin I
authors.initials: RI
authors.lastname: Taleb
authors.affiliation: Faculty of Health and Medical Sciences, Department of Microbial and Cellular Sciences, University of Surrey, Guildford, UK.
authors.forename: Kikki
authors.initials: K
authors.lastname: Bodman-Smith
authors.affiliation: Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, P.O. Box 36, Byblos, Lebanon.
authors.forename: Mirvat
authors.initials: M
authors.lastname: El-Sibai
authors.affiliation: Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, P.O. Box 36, Byblos, Lebanon. cdaher@lau.edu.lb.
authors.forename: Costantine F
authors.initials: CF
authors.lastname: Daher
mesh_terms: D015127:9,10-Dimethyl-1,2-benzanthracene / Q000633:toxicity; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism; D018552:Daucus carota / Q000737:chemistry*; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D020011:Protective Agents / Q000008:administration & dosage*; D012878:Skin Neoplasms / Q000139:chemically induced / Q000378:metabolism / Q000503:physiopathology / Q000517:prevention & control*; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D020011:Protective Agents; D051028:bcl-2-Associated X Protein; D019938:Cyclin D1; D015127:9,10-Dimethyl-1,2-benzanthracene; D013755:Tetradecanoylphorbol Acetate
keywords: Daucus carota; HaCaT; TPA/DMBA skin cancer; Wild carrot