Lutein

The MDA-MB-231 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque, Kyoto, Japan). The media was supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Shanghai, China), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific, Shanghai, China).
The cells were treated with 100 µg/mL F3, which was sourced from the Universiti Sains Malaysia Centre for Drug Research. The F3 was prepared from pulverized freeze-dried leaves of S. crispus as previously reported [29 (link),30 (link)]. MDA-MB-231 cells were also treated with 20 µM lutein, 25 µM β-sitosterol, or 90 µM stigmasterol (Cayman, MI, USA), based on the IC₅₀ concentrations obtained from the MTT assay [32 (link)]. Apigenin was used as a positive control for the detection of GLUT1 because it has been shown to inhibit both GLUT1 mRNA and protein expression [106 (link),107 (link)]. Meanwhile, tamoxifen was used as a positive control in all subsequent experiments because it has previously been reported to decrease PKC activity and affect the PI3K/AKT pathway in MDA-MB-231 cells [108 (link),109 (link)].

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). (R)-(+)-limonene and tricine were used as received (Sigma-Aldrich, St. Louis, MO, USA). Nitrogen-purged hexane, dichloromethane (DCM) and chloroform (CHCl₃) were passed through an activated alumina column, dried with CaSO₄, and stored over 4Å molecular sieves. All kinetic experiments were performed with the same batch of samples. Lutein and violaxanthin were purchased from Cayman Chemicals (Ann Arbor, MI, USA), and zeaxanthin (65%) from Toronto Research Chemicals (Toronto, ON, Canada).

Lutein, purchased from Cayman Chemical Co. (Catalog No. 10010811, Ann Arbor, MI, USA), was dissolved in dimethyl sulfoxide (DMSO) according to the product information (Cayman Chemical Co, Ann Arbor, MI, USA), aliquoted, and stored at −80 °C. Final concentrations of 5, 10, or 20 μM were applied to the cells. NADPH oxidase 1 inhibitor ML171 and etoposide were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO. Antioxidant NAC (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water. For each experiment, cells incubated with DMSO alone (less than 0.3%) served as the control.

The analyzed samples were −80°C stored K3-EDTA fasting plasma that had been drawn at baseline. Handling of all samples and standards was always done under cool conditions and avoiding exposure to light. Carotenoid standards: astaxanthin, canthaxanthin, E-β-apo-8′-carotenal, α-carotene, β-carotene, fucoxanthin, and lycopene were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lutein was purchased from Cayman Chemical (Ann Arbor, MI, USA), zeaxanthin and β-cryptoxanthin were provided by Extrasynthese (Genay, Lyon, France). 13-Z-β-carotene and 9-Z-β-carotene were purchased from Carbosynth (Newbury, Berkshire, UK).
Methanol of LC-MS grade, n-hexane, ethanol, and methyl tert-butyl ether (MTBE) of HPLC grade, blank human plasma, and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich. Ammonium acetate (AMAC) and acetic acid of HPLC grade were purchased from Panreac Quimica SLU (Barcelona, Spain). Ultrapure water (Milli-Q) was generated by a Millipore system (Bedford, MA, USA).

K3-EDTA fasting plasma samples from the baseline blood extractions were analyzed. These samples were drawn in the first visit of the study, just after being randomly assigned to an intervention group and stored at −80 °C until use. All samples and standards were always handled avoiding exposure to light and under cool conditions. Carotenoid standards: astaxanthin, canthaxanthin, E-β-apo-8′-carotenal, α-carotene, β-carotene, fucoxanthin, and lycopene were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lutein was provided by Cayman Chemical (Ann Arbor, MI, USA), zeaxanthin and β-cryptoxanthin were purchased from Extrasynthese (Genay, Lyon, France). 13-Z-β-carotene and 9-Z-β-carotene were purchased from Carbosynth (Newbury, Berkshire, UK). Standards were stored in powder form at −20 °C and protected from light.
Methanol of LC-MS grade, n-hexane, ethanol and methyl tert-butyl ether (MTBE) of HPLC grade, blank human plasma and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich. Ammonium acetate (AMAC) and acetic acid of HPLC grade were purchased from Panreac Quimica SLU (Barcelona, Spain). Ultrapure water (Milli-Q) was generated by a Millipore system (Bedford, MA, USA).

The NMR spectra using standard pulse programs were recorded at room temperature on a Bruker Avance DPX-400 spectrometer (Bruker, Billerica, MA, USA) operating at 400 (¹H) and 100 (¹³C) MHz. The chemical shift (δ, ppm) values were calibrated using the residual NMR solvent and coupling constant (J) was reported in Hertz (Hz). Column chromatography was done on normal-phase silica gel (230 × 400 mesh, J. T. Baker, Center Valley, PA, USA) or reversed-phase silica gel (C₁₈, 40 μm, J. T. Baker). Silica gel 60 F₂₅₄ TLC plates (Merck, Darmstadt, Germany) and reversed-phase TLC plates (C₁₈, Merck, Darmstadt, Germany) were used for analytical TLC. The plates were visualized by spraying 10% H₂SO₄ followed by heating. The authentic samples pheophorbide a (>95%) and lutein (>90%) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA) and Acros Organics (Pittsburgh, PA, USA), respectively. Their structure and purity were further confirmed by NMR and HPLC in our laboratory. The 37 standards of fatty acid methyl esters (Supelco® 37 components FAME mix) used for GC-MS analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA).