Botryococcus braunii (B. braunii) is a green microalga primarily found in freshwater, reservoirs, and ponds. Photosynthetic pigments from algae have shown many bioactive molecules with therapeutic potential. Herein, we report the purification, characterization, and anticancer properties of photosystem I light-harvesting complex I (PSI-LHCI) from the green microalga B. braunii UTEX2441. The pigment–protein complex was purified by sucrose density gradient and characterized by its distinctive peaks using absorption, low-temperature (77 K) fluorescence, and circular dichroism (CD) spectroscopic analyses. Protein complexes were resolved by blue native-PAGE and two-dimensional SDS-PAGE. Triple-negative breast cancer MDA-MB-231 cells were incubated with PSI-LHCI for all of our experiments. Cell viability was assessed, revealing a significant reduction in a time- and concentration-dependent manner. We confirmed the internalization of PSI-LHCI within the cytoplasm and nucleus after 12 h of incubation. Cell death mechanism by oxidative stress was confirmed by the production of reactive oxygen species (ROS) and specifically superoxide. Furthermore, we monitored autophagic flux, apoptotic and necrotic features after treatment with PSI-LHCI. Treated MDA-MB-231 cells showed positive autophagy signals in the cytoplasm and nucleus, and necrotic morphology by the permeabilization of the cell membrane. Our findings demonstrated for the first time the cytotoxic properties of B. braunii PSI-LHCI by the induction of ROS and autophagy in breast cancer cells.

Botryococcus braunii (B. braunii) is a green photosynthetic microalga that is highly investigated due to its capacity of hydrocarbon accumulation, secretion, and synthesis. However, its photosynthetic apparatus has not been elucidated. Photosystem I (PSI) is a membrane‐bound pigment‐protein complex that mediates the electron transfer from plastocyanin on the lumenal side to ferredoxin on the cytoplasmic side. In this study, our purpose was to isolate, purify and characterize PSI core subunits and light harvesting proteins. After the initial isolation via differential ultracentrifugation and solubilization of thylakoid membranes with 1% of n‐dodecyl β‐D‐maltoside, the resulting supernatant was loaded onto a Toyopearl DEAE‐650 ion exchange chromatography column. The purified PSI fraction obtained was pooled and concentrated using an Amicon centrifugal filter unit. The resulting protein concentration was 1 mg/ml, and the PSI characteristic 677 nm absorption maximum was attained. The biophysical characterization was performed via UV/Vis and fluorescence spectroscopy, native and SDS‐polyacrylamide gel electrophoresis, and MALDI‐MS, with the characteristic peaks and molecular weight results of the proteins and subunits found in PSI. Our characterization results show that PSI has been successfully purified, which may provide the fundamental knowledge of the function and possible applications of this integral membrane protein complex in B. braunii.Support or Funding InformationThis research was funded by NIH Research Initiative for Scientific Enhancement (RISE) Program Grant No. 5R25GM061151‐14 and DOD Contract No. W911NF‐11‐1‐0218.

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 (λmax=660 ± 10 nm) at a distance of 10 cm and a fluence rate of 50 mW/cm2 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 InformationRISE Program: 5R25GM061151‐16This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.