2.1. Key chemicals
Wortmannin (Wort) was purchased from Toronto Research Chemicals, Canada (Cat. no. W499400). Minimum Essential Medium- alpha (MEMα) with nucleosides, Dolbecco’s Minimum Essential Medium (DMEM) with L-glutamine, Penicillin/streptomycin, fetal bovine serum (FBS), and trypsin-ethylenediamine tetra-acetic acid (Trypsin/EDTA) were from Lonza Pharma&Biotech, Switzerland. P-Akt, P-mTOR, and β-actin monoclonal antibodies were from Cell Signaling Technologies, Ma, USA. MSCs surface markers antibodies against CD105, CD90, CD45, CD34, and CD19 were from R&D Systems Inc. USA. Acridine orange (AO) was from Sigma.
2.2. Isolation of BM-MSCs and preparation of secretome
The study protocol was approved by the ethical committee, Faculty of Science, Tanta University (ECL: IACUC-SCI-TU-0214). Bone marrow-derived MSCs were isolated and passaged as previously described [22]. Briefly, bone marrow was taken from a male Lewis rat (250 g) by flushing the femoral and tibia bone cavities with PBS in MEMα complete media, containing 10% FBS,1% penicillin/streptomycin. After 24 h incubation at 37°C, and 5% CO2, the old media was replaced with fresh complete media, and MSCs were passaged to the 4th passage. MSCs phenotypical markers were assessed by flow cytometry to determine the expression of MSCs-characteristic clusters of differentiation, including CD105 and CD90, and the hematopoietic markers CD45, CD34, and CD19. Briefly, cells (with density 106 cells/ml) were incubated for 30 min at 4°C with mouse monoclonal antibodies against different CD markers. The labeled cells were analyzed on a FACSscan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) using CELLQuest Pro software (Becton-Dickinson). Well-authenticated and highly viable cells were used to prepare the secretome, where cells were incubated in serum-free media for 48 h, after which media were collected and centrifuged to remove any cell derbies.
2.3. Cancer cell lines, culture, and treatment
Breast cancer cell line (MCF-7) was obtained from VACSERA, Cairo, Egypt. Cells were maintained in DMEM with L-glutamine, supplemented with 10% heat-inactivated FBS and 1% Penicillin/Streptomycin. Cells were incubated in humidified conditions at 95% air, 5% CO2, and 37°C. Initially, cells were seeded with low cell density, subcultured with different cell densities, according to the experimental settings, and then treated with Wort or Wort-containing secretome.
2.4. Viability assay by sulforhodamine B assay
After their incubation with Wort or Wort-containing MSCs secretome, the sulforhodamine B (SRB) assay was used to measure cellular protein content [23]. Briefly, cells were cultured in DMEM complete media at a density of 2×104 cells/well in a 96-well plate. After overnight incubation of cells, for cell attachment, DMEM media was replaced with fresh one containing different concentrations of Wort and incubated at 37°C in 5% CO2 for 48 h or incubated in secretome-containing Wort for 24 h. Next, old media were decanted, cells were fixed with 10% (wt/vol) trichloroacetic acid, stained for 30 min with SRB, washed with 1% (vol/vol) acetic acid, and then the protein-bound dye was dissolved in 10 mM Tris base and wells absorbance was measured at 510 nm.
2.5. Apoptosis and autophagy assessments
The apoptosis assay was performed using Annexin-V FITC kit (Miltenyi Biotec, CA, USA) following the manufacturer’s guidelines. Briefly, MCF-7 cells were seeded in T25 flasks containing complete medium. After overnight incubation, cells were treated with Wort or Wort-containing secretome for 24 or 72 h after which cells were collected by Trypsin/EDTA and then centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in PBS and incubated with 0.25 µg/ml Annexin V in 1X binding buffer for 15 min, followed by two washes with Wash Buffer. Cells were resuspended again in a binding buffer containing 7-amino-actinomycin (7-AAD) and then subjected to flow cytometery. In parallel, macroautophagy was determined by measuring the abundance of LC3II protein by fluorescent antibody labeling of the microtubule-associated protein, using Rabbit anti-Homo sapiens MAP1LC3B Polyclonal antibody (MAP1LC3B Antibody, FITC conjugated) (CUSABIO, USA).
2.6. Immunoblotting of Akt and mTOR proteins
The ready Prep™ protein extraction kit was used to extract cell protein according to the manufacturer’s instructions. Bradford protein assay (Bio Basic Inc., Markham Ontario, Canada) was used to determine protein concentration following the manufacturer’s instructions. For blotting, 20 µg protein was mixed with an equal volume of 2x Laemmli sample buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl, pH 6.8), where the mixture was boiled at 95°C for 5 min before loading on the gel. The blot was run, followed by membrane blocking at room temperature for one hour. Primary antibodies against p-Akt and mTOR were diluted in TBST buffer and incubated overnight with each antibody at 4°C. The blot was rinsed 3–5 times for 5 min with TBST buffer and then incubated with the HRP-conjugated secondary antibody (Goat anti-rabbit IgG- HRP-1mg Goat mab-Novus Biologicals) for one hour at room temperature. After another wash with TBST, the chemiluminescent substrate (Clarity Western ECL substrate Bio-Rad) was applied, and the signals were captured. Image analysis software (Image J, Fiji) was used to measure band intensities.
2.7. Acridine orange staining
Acridine orange (AO) staining was used to assess the rate of cell death, where the nuclei of the living cells develop bright green fluorescence by binding AO with double stranded DNA, whereas AO causes red fluorescence in dead cells because of its affinity with single strand DNA (due to the existence of fragmented chromatin). This assay was performed by staining cells (4 × 106 cells/ml) with 5 µl of AO dye prepared in PBS separately) and incubation for 10 min at 37oC. Cells were examined under fluorescent microscopy (Lieca, Germany) under x20 and x40 magnification.
2.8. Wound healing assay
To investigate the effect of MSCs-secretome on cells in vitro migration, wound healing assay was performed. Briefly, after cells were plated in a 12-well plate and left to subconfluency, a scratch was made in the cells monolayer. After 2 washes with PBS, cells were incubated in MSCs secretome, Wort or both (Wort-containing secretome). The cell-free area was estimated, with Image J at day zero and after 48 h.
2.9. RNA isolation, cDNA synthesis and expression analysis
Quantitative real-time PCR was used to determine the fold-expression of the PI3KCa, protein kinase B (Akt), and mTOR genes at the mRNA levels using Qiagen Rotor-Gene Q PCR Cycler 5 Plex. Initially, total RNA was purified using GeneJET RNA purification kit, (ThermoFisher Scientific, USA), and its concentration and quality were assessed. Next, 200 ng RNA was used as a template for cDNA synthesis, using SensiFAST™ cDNA Synthesis Kit (Bioline Inc, USA), following the manufacturer’s guidelines. For RT-PCR quantitation, 50 ng/µl (2 µl) of cDNA was used as a template in 20 µl reactions containing 50 nmol/µl (2 µl) of the genes-specific primers (Table 1), the ready-to-use master mix of fluorescent dye QIAGEN SYBR green 1 and Hot Star Taq DNA polymerase. Reactions were subjected to a thermal cycling program consisting of a single denaturation step followed by 45 cycles (each consisted of a denaturation step at 94°C for 5 s, annealing at 62°C, 55°C and 62°C and 58°C, 56.8°C and 57.9°C (for PI3Kca, Akt, and mTOR, respectively) and an extension step at 72°C for 20 sec. Reactions were terminated with a single step at 99°C to produce melt curves that represent the changes in fluorescence observed when dsDNA with incorporated dye dissociates, or “melts” into single-stranded DNA as the reaction temperature is raised. In parallel, the expression of the β actin gene was used as an internal control to determine the relative quantification of the targeted genes. The critical threshold (Ct) of target genes was normalized with quantities (Ct) of GAPDH using the 2−ΔΔCt.
2.10. Computational prediction and statistical analysis
The candidate cellular targets of Wort were predicted using the Similarity ensemble approach (SEA) (https://sea.bkslab.org/) and TargetNet (http://targetnet.scbdd.com/) Also, Swiss Target Prediction (https://www.swisstargetprediction. ch) was employed to explore the interaction between Wort and major cellular regulatory proteins. Data analysis was performed using the SPSS.26.0 software package (IBM, Chicago, IL, USA). All data are presented as means of at least 3 experiments (± standard deviation). Statistical differences among means were performed by ANOVA and post hoc Tukey’s honestly significant difference test. Differences were considered significant at p < 0.05. Graphing was performed using Microsoft Excel, and illustrations were performed by BioRender (WWW.BioRender.com).