Penicillin streptomycin

Cells of the MCF-7 human breast cancer cell line (a generous gift from Dan Levy, BGU) were maintained in Dulbecco's modified Eagle's medium (DMEM; Biological Industries, Beit-Haemek, Israel) supplemented with 10% fetal bovine serum (Thermo Fisher, MA, U.S.A.), 1% l-glutamine (Biological Industries) and 1% penicillin/streptomycin (Biological Industries). HT1080 cells (ATCC CCL-121), endogenously expressing both MMP9 and MMP2, were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with l-glutamine (Biological Industries) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were maintained at 37°C under 5% CO₂.

92-1, OMM2.5, and Mel270 UM cell lines [66 (link)–68 (link)] (kind gift of Prof. Martine Jager, MD, PhD, Leiden University) were grown in RPMI-1640 Dutch Modified medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Biological Industries, Kibbutz Beit-Haemek, Israel), 2 mml-glutamine (Biological Industries, Kibbutz Beit-Haemek, Israel), and 2% penicillin/streptomycin (Biological Industries, Kibbutz Beit-Haemek, Israel). MP46 UM cell line [69 (link)] (also a kind gift of Prof. Martine Jager, MD, PhD, Leiden University) was grown in IMDM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 20% FBS (Biological Industries, Kibbutz Beit-Haemek, Israel), 3 mml-glutamine (Biological Industries, Kibbutz Beit-Haemek, Israel), and 2% penicillin/streptomycin (Biological Industries, Kibbutz Beit-Haemek, Israel). The cells were incubated in a cell culture incubator at 37°C, 5% CO₂, and 60% humidity. The cell cultures were maintained by the replacement of media 2–3 times per week. Cells growing as a monolayer were subcultured by trypsin-EDTA once a week.

The cells were plated at a density of 3,000 cells/well in triplicate over 96-well plates in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 µg/ml penicillin/streptomycin (Biological Industries, Beit Ha-Emek, Israel) and allowed to attach and grow overnight at 37°C, in a 5% CO₂ atmosphere. The medium was replaced with a fresh treatment-containing medium, and the cells were propagated for an additional 48 h in the same conditions. In experiments with pyruvate and N-acetyl-cysteine (NAC), 1 mM pyruvate or 10 mM NAC were added to the medium together with botanical extracts, mixed and then added to the cells. An XTT viability test was performed by replacing the medium with fresh RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 µg/ml penicillin/streptomycin, to prevent interference of treatment color with XTT reagent signal, and adding XTT reagent (Biological Industries) for incubation for 2–3 h. The blank measurement was subtracted from each reading, and all values of treated cells were divided by control (PBS-treated cells) values in each experiment (control=1). The resulting signal was measured by an enzyme-linked immunosorbent assay reader. Each experiment was repeated at least three times.

HepG2 cells, were kindly provided by Prof. Yehiel Zick, Huh-6 cells were kindly provided by Prof. Yosef Shaul (both from the Weizmann Institute of Science, Rehovot, Israel). Primary human liver cells were kindly provided by Prof. Sara Ferber (Sheba Medical Center, Tel Hashomer, Israel) and isolated as previously described.^{51 (link), 52 (link)} Informed consent was obtained from each donor and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the Committee on Clinical Investigations (Institutional Review Boards of Sheba Medical Center and Rabin Medical Center, Petah Tikva, Israel). Non-small cell lung carcinoma (H1299) cells were obtained from the ATCC (Manassas, VA, USA) and MCF7 cells were provided by NCI-DCTD Repository (Bethesda, MD, USA). All cell lines were cultured in humidified incubator at 37 °C and 5% CO₂. Primary human liver cells, HepG2 and Huh-6 cell lines were cultured in DMEM supplemented with 10% FCS, 2 mM l-glutamine, 1 mM sodium pyruvate and 100 mg/ml penicillin–streptomycin (Biological Industries, Beit-Haemek, Israel). H1299 and MCF7 cell lines were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM l-glutamine, 1 mM sodium pyruvate and 100 mg/ml penicillin–streptomycin (Biological Industries).