title: Boronated protoporphyrin (BOPP): localization in lysosomes of the human glioma cell line SF-767 with uptake modulated by lipoprotein levels.
abstract: OBJECTIVE
  Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM.
  
  
  METHODS
  The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20-135.6 microg/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019-15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS).
  
  
  RESULTS
  BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 microg/ml, NS), the amount of boron retained was near 10(9) atoms per cell (15 microg B/g cells). Lysosomes containing high concentrations of BOPP were randomly distributed throughout the cytoplasm; however, larger lysosomes containing BOPP were concentrated around the cell nucleus. Little or no BOPP accumulated in the cell nucleus. At incubation concentrations of 20 and 40 microg/ml (24-h time point), BOPP uptake in SF-767 cells was reduced in LPDS compared with NS (66% reduction). A similar result was observed for normal human fibroblasts (AG-1522 cells, 40 microg/ml, 24 h). At 40 microg/ml, in both NS and LPDS at 24 h, BOPP accumulation in LDL receptor-deficient human fibroblasts (GM019-15C cells) was reduced relative to AG-1522 cells. BOPP accumulation in GM019-15C cells (40 microg/ml, 24 h) was not affected by serum lipoprotein levels.
  
  
  CONCLUSIONS
  In cell culture, BOPP is taken up by human glioma cells via the LDL pathway and is compartmentalized into cellular lysosomes. Knowledge of this mechanism of BOPP uptake and retention will be important in attempts to modify toxicity and efficacy of this drug.
authors.affiliation: Life Sciences Division, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Callahan
authors.forename: T M
authors.initials: TM
authors.lastname: Forte
authors.forename: S M
authors.initials: SM
authors.lastname: Afzal
authors.forename: D F
authors.initials: DF
authors.lastname: Deen
authors.forename: S B
authors.initials: SB
authors.lastname: Kahl
authors.forename: K A
authors.initials: KA
authors.lastname: Bjornstad
authors.forename: W F
authors.initials: WF
authors.lastname: Bauer
authors.forename: E A
authors.initials: EA
authors.lastname: Blakely
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D001896:Boron Compounds / Q000493:pharmacokinetics*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011524:Protoporphyrins / Q000493:pharmacokinetics*; D011973:Receptors, LDL / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001896:Boron Compounds; D008074:Lipoproteins; D011524:Protoporphyrins; D011973:Receptors, LDL