Page 210 - EJMO-9-3

Eurasian Journal of
Medicine and Oncology FN3K–Nrf2 axis inhibition in breast cancer

hydrochloric acid (HCl). Absorbance was then recorded at 1 µL of SuperScript II Reverse Transcriptase (Invitrogen,
570 nm using a microplate spectrophotometer. Untreated USA) was added. The enzymatic reaction was carried
cells served as the negative control for determining relative out through a thermal program consisting of 10 min at
cell viability. 41 25°C, 90 min at 42°C, and 15 min at 70°C to inactivate the
enzyme. After a brief centrifugation step, 1 µL of RNase
2.2.2. qPCR evaluation H was added to degrade the RNA template, followed by
2.2.2.1. Cell culture and treatment incubation at 37°C for 30 min. The synthesized cDNA was
stored at −20°C until further use.
MCF-7, BT-474, and T-47D human breast cancer cell
lines, along with non-malignant Vero cells, were cultured 2.2.2.4. qPCR Analysis
as adherent monolayers in 25 cm tissue culture flasks. The The cDNA was diluted 1:1 with DEPC-treated water before
2
cells were maintained in DMEM supplemented with 10% amplification. Each 10 µL qPCR reaction consisted of
45
FCS and antibiotics, including penicillin and streptomycin,
under standard incubation conditions at 37°C with 5% 1 µL of diluted cDNA, 1 µL of gene-specific primer mix
CO and high humidity. Cultures were allowed to grow (forward and reverse), 5 µL of SYBR® Green Supermix
2
until approximately 70% confluency was achieved. Cells (Bio-Rad; Cat. No. 172-5270, USA), and nuclease-free
42
were gently rinsed with 1× PBS and subsequently detached Milli-Q water to reach the final volume.
using 0.2% trypsin-EDTA at a volume of 200 µL per flask qPCR was conducted using the QuantStudio 5 Real-
for 5 min at room temperature. The dissociated cells were Time PCR System (Applied Biosystems, USA) under a
then seeded into 6-well plates at a volume of 1.0 mL per thermal protocol comprising 40 amplification cycles.
well. The cells were allowed to adhere and stabilize for 48 h Cycle threshold (Ct) values were recorded for each target
before treatment. gene, and relative gene expression levels were quantified
using the ΔΔCt method. This approach ensures precise
2.2.2.2. Total RNA isolation
normalization and reliable detection of low-abundance
Cell pellets were resuspended in TRIzol reagent and transcripts, minimizing technical variability. 46,47
mixed thoroughly, followed by the addition of 300 µL
chloroform to each sample. After brief vortexing, the 2.2.2.5. Primer sequences
43
samples were incubated at room temperature for 15 min. The specific primer sequences used for the amplification
Phase separation was carried out by centrifugation at of FN3K, Nrf2, and ACTB (Beta actin) genes were
14,000 rpm for 10 min at 4°C, and the resulting aqueous summarized in Table 1.
phase was carefully transferred to a new microcentrifuge
tube. An equal volume of isopropanol was then added, 2.2.3. Western blot analysis
mixed gently, and incubated at room temperature for 2.2.3.1. Cell culture and treatment
5 min to precipitate RNA, followed by centrifugation
under the same conditions. The RNA pellet was washed MCF-7, BT-474, and T-47D human breast cancer cell lines,
with 70% ethanol, air-dried, and reconstituted in along with non-malignant Vero cells, were cultured as
100 µL of diethyl pyrocarbonate (DEPC)-treated water. adherent monolayers in 25 cm tissue culture flasks. The cells
2
RNA purity and concentration were then determined were maintained in DMEM supplemented with 10% FCS,
spectrophotometrically by measuring absorbance at penicillin, and streptomycin under standard incubation
260 nm. 44 conditions at 37°C with 5% CO in a humidified atmosphere,
2
until approximately 70% confluency was reached. 7
2.2.2.3. Complementary DNA (cDNA) synthesis
Cells were then rinsed with 1× PBS and detached by
First-strand cDNA synthesis was performed using 5 µg of treating with 0.2% trypsin-EDTA at a volume of 200 µL
total RNA according to standardized reverse transcription per flask for 5 min at room temperature. The detached
protocols. The reaction mixture was prepared by combining cells were resuspended in complete DMEM containing
2 µL of total RNA (~5 µg), 2 µL of random hexamer or 10% FCS and antibiotics, and subsequently seeded into
oligo(dT) primers, 2 µL of 10 mM dNTP mix, and 4 µL of 6-well plates at a volume of 1.0 mL per well. After a 48-h
DEPC-treated water. This mixture was heated at 65°C for adherence period, the cells were treated for 24 h with
2 min to denature secondary structures and then allowed selected compounds – amiloride, capivasertib, ritonavir,
to equilibrate to room temperature. Next, 2 µL of 10× oxaliplatin, lansoprazole, and 1-DMF.
reverse transcription buffer, 4 µL of 25 mM MgCl , 2 µL of
2
0.1 M DTT, and 1 µL of RNase inhibitor were added. The Following treatment, cells were harvested, washed once
mixture was pre-incubated at 25°C for 2 min, after which with 1× PBS, and resuspended in 500 µL of 1× PBS. To each

Volume 9 Issue 3 (2025) 202 doi: 10.36922/EJMO025150114