Hydrocortisone hc

HEK-293T, MCF-7, MCF-10A, and MDA-MB-231 cell lines were obtained from the Cell Bank of the Iranian Biological Resource Centre (Tehran, Iran). MCF-7 and MDA-MB-231 were maintained in Dulbecco's modified Eagle medium (DMEM, Biosera, France) with 10 % fetal bovine serum (FBS) (Invitrogen, UK), penicillin (100 units/ml) and streptomycin (100 μg/ml). MCF-10A cells were cultured in mammary epithelial cell growth medium (MEGM; Lonza/Clonetics, Switzerland) supplemented with 100 ng/ml cholera toxin (CT) (Sigma-Aldrich, Germany), 10 μg/ml insulin (Sigma-Aldrich), 20 ng/ml epithelial growth factor (EGF) (Sigma-Aldrich), 0.5 μg/ml hydrocortisone (HC) (Sigma-Aldrich), 10 % FBS and 1 % penicillin-streptomycin. In the case of HEK-293T, cells were maintained in DMEM/F12 (Biosera, France) medium, supplemented with 1 % penicillin-streptomycin and 10 % FBS. All cell lines were kept in a 5 % CO₂ humidified incubator at 37 °C.

The non-malignant murine mammary epithelial cell line EPH-4, which was derived from spontaneously immortalized mouse mammary gland epithelial cells [18 (link)], was a gift of Dr. Calvin Roskelley (University of British Columbia, Vancouver, Canada). EPH-4 cells were cultured as previously described [13 (link),19 (link)]. EPH-4 cells stably transfected with H1-2 empty vector or shGR (see below) were maintained in serum-free media with 2 μg/mL puromycin (Sigma). Cell treatments were completed using media lacking serum and containing either 1 μg/mL hydrocortisone (HC) (Sigma), 10 μM RU-486 (Sigma), or ethanol vehicle for 48 hours.

The human brain EC line hCMEC/D3 was obtained at Tebubio. Cells were used up to passage 35. Cells were cultured in collagen coated (rat tail, type I, Sigma-Aldrich) culture flasks in growth medium (EGM-2MV medium [Lonza] supplemented with 2.5% FBS [Thermo Fisher Scientific]). Cells were collected using trypsin and cultured in 24-well plates or in Thincerts (both Greiner Bio-One) for migration assays as described further. After reaching near confluency (90%), cells were replenished with experimental medium (basal EBM2 [Lonza] supplemented with Gentamicin [10 μg/mL], Amphotericin B [1 μg/mL], fibroblast growth factor [FGF, 1 ng/mL], hydrocortisone [HC, 1.4 μM; all Sigma-Aldrich], 2.5% FBS [Thermo Fisher Scientific]). One day later, cells were treated with TNF-α (100 ng/mL) and IFN-γ (10 ng/mL, both from Peprotech) (72 (link)) for 24 hours in reduced medium (basal EBM2 [Lonza] supplemented with Gentamicin, Amphotericin, FGF, 0.25% FBS) (73 (link)).

Viable MNCs were resuspended and plated at a density of 1 × 10⁶ cells/mL on erythroid expansion media composed of basal medium and erythroid cytokines. The composition of the basal medium was 150 μg/mL transferrin, 50 μg/mL insulin, 90 ng/mL ferrous nitrate, and 160 μM monothioglycerol in Stemline II media (all from Sigma-Aldrich, Gillingham, UK). The erythroid expansion medium was prepared by adding 10 μg/mL hydrocortisone (HC) (Sigma-Aldrich), 10 μg/mL stem cell factor (SCF) (Sigma-Aldrich), 10 μg/mL erythropoietin (EPO) (Stem Cell Technologies, Vancouver, Canada), and 1 μg/mL interleukin-3 (IL-3) (Peprotech EC Ltd., London, UK) to the basal medium. MNCs were suspended at a density of 1 × 10⁷ cells/mL in 10 mL erythroid expansion medium in a 25T flask (Thermo Fisher Scientific, Waltham, MA, USA) for three days in a 37 °C incubator in a humidified environment containing 5% CO₂. On day 3, both adherent and non-adherent cells were collected into a 15-mL conical tube and centrifuged at 400× g for 5 min. The pellet was resuspended at a density of 1 × 10⁶ cells/mL in fresh erythroid expansion medium. Starting on day 5, microscopic morphological analysis was performed every day until the population of erythroid progenitor cells accounted for approximately 80% of the MNCs, at which point the cells were ready for transfection [26 (link)].

Poly(2-ethyl-2-oxazoline)s (50, 200, and 500 kDa; named as 50, 200 and 500 in the text, respectively) and hydrocortisone (HC) were purchased from Sigma-Aldrich Irvine, UK) and Carbopol 974 (highly cross-linked, 28,400–39,400 cP) and Carbopol 971 (moderately cross-linked, 4000–11,000 cP) were obtained from Lubrizol (Hazelwood, UK). HPLC grade acetonitrile (ACN) was purchased from Teknokroma (Barcelona, Spain). Sodium chloride, calcium chloride, potassium chloride, sodium hydrogen carbonate and potassium dihydrogen phosphate were obtained from Merck (Darmstadt, Germany) and used for preparation of artificial saliva fluids. A Milli-Q water purification system from Millipore (Bedford, MA, USA) was used. The drug hydrocortisone and internal standard dexamethasone (DXM) were purchased from Sigma-Aldrich (St Louis, MO, USA). Standard stock solutions of drug and internal standard were prepared in acetonitrile at a concentration of 1000 mg/L. Working solutions were prepared by dilution from stock solutions with the mix of excipients and polymers used in the pharmaceutical formulation of tablets (magnesium state, poly(2-ethyl-2-oxazoline) and Carbopols 974 and Carbopol 971) in artificial saliva.