title: The new esters derivatives of betulin and betulinic acid in epidermoid squamous carcinoma treatment - In vitro studies.
abstract: BACKGROUND
  Betulinic acid and betulin are triterpenes with documented cytotoxic properties toward various cell lines. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications.
  
  
  OBJECTIVE
  To investigate the specific anticancer role of the newly synthesized betulin derivatives in human epidermoid carcinoma cells.
  
  
  METHODS
  In the present study we synthesized five amino acid esters of betulin. For the synthesis we selected alanine (Boc-l-Ala-OH, negative control) and four basic amino acids - natural lysine (Boc-l-Lys(Boc)-OH) and three its unnatural derivatives (Boc-l-Dap(Boc)-OH, Boc-l-Dab(Boc)-OH, and Boc-l-Orn(Boc)-OH). Boc-protected amino acids were most convenient for the synthesis. All new esters have one (betulin-l-Ala-NH2) or two free amino groups which significantly increase their solubility in water and facilitate their transport through the cell membrane. It is worth noting that the biological activity of new esters of betulin is positive correlated with the length of the side chain of l-amino acid. The highest biological activity displayed compound containing lysine side chain (Lys, -CH2-CH2-CH2-CH2-NH2). Considering the biological activity, other derivatives can be set in the following series: Orn (-CH2-CH2-CH2-NH2)>Dab (-CH2-CH2-NH2)>Dap (-CH2-NH2)>Ala (CH3)>betulin. New betulin esters were tested in normal human keratinocytes (HaCaT) and human epidermoid carcinoma cells (A431). To assess cytotoxicity, MTT test was performed after 24, 48 and 72h of incubation with the test compounds at a concentration range of 0.75-100μM. In case of apoptotic activity, a TUNEL method and comet assay were performed. Additionally expression of caspase-3 and PARP1 was evaluated immunocytochemically.
  
  
  RESULTS
  The highest cytotoxicity in cells induced skin cancer new compounds, particularly compound containing a lysine side chain (IC50=7μM) and ornithine (IC50=10μM). The highest number of apoptotic cells was observed in case incubation with compound containing Orn, Dab and Dap side chain.
  
  
  CONCLUSIONS
  The new betulin ester derivatives display enhanced antitumor activity compared to their non-modified precursors. It is worth emphasizing their specific toxicity against epidermoid carcinoma cells.
authors.affiliation: Department of Histology and Embryology, Wroclaw Medical University, Chałubińskiego 6a, 50-368 Wroclaw, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Drąg-Zalesińska
authors.affiliation: Department of Histology and Embryology, Wroclaw Medical University, Chałubińskiego 6a, 50-368 Wroclaw, Poland.
authors.forename: Teresa
authors.initials: T
authors.lastname: Wysocka
authors.affiliation: Department of Histology and Embryology, Wroclaw Medical University, Chałubińskiego 6a, 50-368 Wroclaw, Poland.
authors.forename: Sylwia
authors.initials: S
authors.lastname: Borska
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drąg
authors.affiliation: Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Poręba
authors.affiliation: Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Choromańska
authors.affiliation: Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wroclaw, Poland. Electronic address: Julita.Kulbacka@umed.wroc.pl.
authors.forename: Julita
authors.initials: J
authors.lastname: Kulbacka
authors.affiliation: Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wroclaw, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Saczko
mesh_terms: D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D020552:Comet Assay; D004354:Drug Screening Assays, Antitumor; D004952:Esters / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D020128:Inhibitory Concentration 50; D015603:Keratinocytes / Q000187:drug effects; D053978:Pentacyclic Triterpenes; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012878:Skin Neoplasms / Q000188:drug therapy / Q000473:pathology; D014315:Triterpenes / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D000094062:Betulinic Acid
chemical_list: D004952:Esters; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; C002503:betulin; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D000094062:Betulinic Acid
keywords: Apoptosis; Betulin derivatives; Cytotoxicity; Skin cancer