Htb 22tm

Colorimetric MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assays were carried out to assess the cell viability of the samples against a panel of five different cancer cell lines (i.e., human lung carcinoma A549 ATCC® CCL-185TM, human skin melanoma A2058 ATCC® CRL-11147TM, hepatocyte carcinoma HepG2 ATCC® HB-8065TM, breast adenocarcinoma MCF7 ATCC® HTB-22TM, and pancreas carcinoma MiaPaca-2 ATCC® CRL-1420TM). All cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were grown in Ham’s F12K medium with 2 mM glutamine, 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. A2058 and HepG2 were grown in ATCC formulated Eagle’s M essential medium (MEM) with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, and 100 μM MEM nonessential amino acids. MCF-7 cells were grown in the previous medium supplemented with 0.01 mg/mL of bovine insulin. MiaPaca-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100U/mL penicillin, and 100 μg/mL streptomycin. The bioassays were performed as reported by Audoin et al. [20 (link)]. The cytotoxic activity was assessed after 72 h of treatment of the compound at the concentrations 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.250, 12.5, 25, 50, and 100 μM.

The MCF-7 human breast cancer cell line was purchased from the ATCC (HTB-22^TM) (transcriptome sequence data were deposited in the ArrayExpress database https://www.ebi.ac.uk/biostudies/arrayexpress/ (accessed on 21 May 2022) by Chiang et al. with accession number E-MTAB-609) [24 (link)]. The cells were grown in Eagle’s minimum essential medium (EMEM) supplemented with 1% penicillin/streptomycin and 10% heat-inactivated fetal bovine serum in a 96-well plate (37 °C, 5% CO₂, 20 h). Then, the cells were treated with various concentrations of B. cernua stem extract (200, 400, 800, and 1600 ppm) for 3, 6, 8, and 24 h. Treatment with solvent (DMSO 0.5% v/v) was also used as a negative control. The cell viability was estimated by measuring absorbance at λ570 nm using a Quant ELISA plate reader (Agilent, CA, USA). Trypan blue staining was used to measure the level of cell death. The morphology of cells was observed using inverted microscopy (Agilent).

MDA-MB-231 (HTB-22TM, ATCC, USA), a human breast adenocarcinoma cell line, was used in the formation of cancer cell clusters. SW480 (ATCC CCL-227), a colorectal epithelial cell line was used to characterise cluster formation. Cell lines were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) and 1% penicillin-streptomycin (Invitrogen, USA). Cultures were maintained under normoxia at 37 °C in a humidified atmosphere and 5% (v/v) CO₂. Cells were cultured in sterile 25 cm² flasks (BD Bioscience, USA) and sub-cultivated two times a week, with media refreshed every 48 h.

Cytotoxicity evaluation was performed using normal human skin fibroblast (BJ cell line, ATCC^® CRL-2522^TM). In turn, anti-proliferative activity was assessed towards cancer cells: human lung adenocarcinoma (A549, ATCC^® CCL-185^TM), human hepatocellular carcinoma (HepG2 cell line, ATCC^® HB-8065^TM), and human breast adenocarcinoma (MCF-7, ATCC^® HTB-22^TM) as well as towards normal cells (BJ cell line was used as model of normal cells). All cell lines were purchased from ATCC, United Kingdom and cultured according to manufacturer recommendations.

The cell culture media used were: high glucose Dulbecco’s Modified Eagle’s Medium (DMEM), Eagle’s Minimum Essential Medium (EMEM), and Dulbecco’s Modified Eagle’s Medium and Ham’s F12 medium (1:1 mixture; DMEM:F12), acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). Of the cell lines tested in this study, two genotypically distinctive breast adenocarcinoma cell lines (MDA-MB-231—ATCC^® HTB-26 TM, MCF-7—ATCC^® HTB-22 TM) and one nontumorigenic cell line (MCF-10—ATCC^® CRL-10317) were obtained from ATCC (Manassas, VA, USA) as frozen items.