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Characterization and Evaluation of ROS‐Containing Photosystem I Light‐Harvesting Complex I (PSI‐LHCI) Isolated from the Green Microalga Botryococcus braunii as a Potential Anticancer Drug
Author(s) -
Ovalle Freisa Milagros Joaquín,
Guihurt Grace,
BarcelóBovea Vanessa Celeste,
RamirezPaz Josell,
Doble Katerina,
Saba Andraous Hani,
Griebenow Kai
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb82
Subject(s) - viability assay , reactive oxygen species , photosystem i , hela , chemistry , singlet oxygen , biophysics , photosynthesis , photosystem ii , thylakoid , chlorophyll , photosystem , oxygen , biochemistry , cell , biology , chloroplast , organic chemistry , gene
Reactive oxygen species (ROS) are generated during normal metabolism; however, at high levels, they can promote cell damage and death. In green microalgae, such as Botryococcus braunii ( B. braunii ), the thylakoid membrane (TM) and particularly photosystem I (PSI) are the main contributors to the overall production of ROS. B. braunii PSI‐LHCI was purified by differential centrifugation in a 15–35% sucrose gradient for 16.5 hours, 4 ºC at 37,500 rpm and characterized by visible absorption spectroscopy, low temperature (77K) fluorescence emission spectroscopy and LC‐MS/MS. PSI can act as a light‐sensitive molecule called photosensitizer; that upon photoactivation by exposure to LED light (λ max =660 ± 10 nm) at a distance of 10 cm and a fluence rate of 50 mW/cm 2 for 40 minutes triggered the formation of ROS species. ROS generation in TM and PSI, specifically singlet oxygen was monitored spectrophotometrically using the p‐nitrosodimethylaniline (RNO) assay at 440 nm, which produced RNO dye bleaching upon singlet oxygen generation. To test PSI‐LHCI use as a prospective anticancer drug, two cancer cell lines, HeLa and MDA‐MB‐231, and one normal cell line, NIH‐3T3 were utilized for in vitro cell viability studies. Each cell line was incubated for 24 h with various concentrations of PSI‐LHCI (3.125, 6.25, 12.5, 25, and 50 μg/ml) which demonstrated a concentration‐dependent reduction in cell viability. At a 12.5 μg/ml PSI‐LHCI concentration, cell viability experienced a steep drop to less than 5% in HeLa cells, and a 47% and 53% decreased cell viability in NIH‐3T3 and MDA‐MB‐231 cell lines. Confocal microscopy demonstrated PSI‐LHCI internalization, and apoptosis induction studies confirmed cellular damage due to morphological change hallmarks such as condensed nuclear chromatin, decreased and fragmented nucleus after DAPI staining and PI counterstaining positively identified dead or necrotic cells represented by the intense, condensed nucleus staining. Support or Funding Information RISE Program: 5R25GM061151‐16 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .