Page 216 - EJMO-9-3

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

may be effective at lower concentrations in reducing cell 3.2.2. qPCR analysis
viability and warrant further investigation as potential 3.2.2.1. FN3K and Nrf2 expression in MCF-7 Cells
anti-cancer agents targeting FN3K pathways.
qPCR was conducted to assess the modulatory effects of
Capivasertib showed moderate cytotoxicity, with IC six tested compounds on the gene expression levels of
50
values ranging from 100 – 125 µg/mL across the tested FN3K and Nrf2 in MCF-7 breast cancer cells. The integrity
breast cancer cell lines. On the other hand, ritonavir, of the isolated RNA was validated through agarose gel
amiloride, and 1-DMF exhibited higher IC values electrophoresis (Supplementary Figure S5).
50
(125 – 150 µg/mL), suggesting relatively lower cytotoxic
potential. Interestingly, the non-malignant Vero cells The expression patterns of FN3K and Nrf2 were
showed markedly higher IC values (>150 µg/mL for evaluated across different treatment conditions, revealing
50
most compounds), indicating selective cytotoxicity toward distinct regulatory effects. In the untreated control, FN3K
cancer cells. This selective response enhances the potential and Nrf2 exhibited baseline expression levels of 1.000
therapeutic index of the effective compounds. Although and 1.020, respectively. Among the tested compounds,
the IC values observed for oxaliplatin and lansoprazole oxaliplatin, lansoprazole, and capivasertib emerged as
potent FN3K inhibitors. Oxaliplatin exhibited the most
50
(~90 – 110 µg/mL) and for capivasertib (100 – 125 µg/mL), significant suppression, reducing FN3K expression by
indicate promising in vitro potency, their pharmacological nearly 99.99% (2.0 × 10 ), along with a moderate Nrf2
-5
relevance must be interpreted with caution. Drug
concentrations required to achieve cytotoxic effects in suppression (0.032). Similarly, lansoprazole showed
strong FN3K inhibition, with a 99.90% reduction (0.001),
monolayer culture systems are often higher than those accompanied by a mild decrease in Nrf2 expression
effective in vivo, due to differences in drug metabolism, (0.015). Capivasertib also demonstrated substantial
microenvironmental factors, and tumor tissue architecture. FN3K suppression (0.000), along with moderate Nrf2
Moreover, in vitro IC values do not account for sustained inhibition (0.063), making its efficacy comparable to that
50
exposure, tissue retention, or intracellular accumulation – of the other top inhibitors. These findings confirm that
all of which significantly influence therapeutic outcomes in oxaliplatin, lansoprazole, and capivasertib act as effective
clinical settings. FN3K inhibitors, capable of significantly reducing FN3K
For instance, oxaliplatin forms DNA adducts that persist expression with varying degrees of Nrf2 suppression. This
long after plasma clearance, contributing to prolonged is illustrated in Supplementary Figure S6, which presents
53
anti-tumor activity. Capivasertib has demonstrated qPCR gel images of FN3K and Nrf2 expression.
efficacy in breast cancer clinical trials with intermittent Conversely, amiloride, ritonavir, and 1-DMF did not
oral dosing (400 – 480 mg BID), showing favorable exhibit strong FN3K inhibition, with some compounds
toxicity profiles. Lansoprazole, although not classified showing unexpected regulatory effects. Amiloride led
54
as an anti-cancer drug, has exhibited synergistic effects to a 157.0% increase in FN3K expression (2.570), while
with chemotherapeutics in pre-clinical models due to its simultaneously inducing marked suppression of Nrf2 (7.4 ×
modulation of lysosomal pH and autophagy. 55 10 ), suggesting a potential inverse regulatory mechanism.
-5
Therefore, while the IC values provide a Similarly, ritonavir induced a slight upregulation of FN3K
50
useful benchmark for early-stage screening, in vivo (1.220), along mild Nrf2 suppression (0.001), indicating no
pharmacokinetic and tumor accumulation studies are inhibitory effect on FN3K. Meanwhile, 1-DMF displayed
essential for confirming therapeutic relevance. Our future moderate FN3K suppression (0.176), but only weak
animal studies will evaluate FN3K–Nrf2 axis modulation, inhibition of Nrf2 (0.022), making it less potent compared
assess systemic toxicity, and establish optimal dosing to oxaliplatin, lansoprazole, and capivasertib.
strategies to bridge the current gap between in vitro and Since FN3K inhibition was the primary objective of
in vivo findings. this study, only compounds demonstrating significant
To further assess treatment-induced effects, microscopic FN3K downregulation in MCF-7 cells were selected for
images of both treated and untreated cells were captured. further qPCR analysis. Among the six tested compounds,
These images, provided in Supplementary Figures S3 and oxaliplatin, lansoprazole, and capivasertib exhibited potent
S4, depict the cellular morphological changes observed FN3K suppression (≥99.6%), whereas amiloride and
post-treatment. However, as this study primarily focuses ritonavir failed to inhibit FN3K, and 1-DMF showed only
on the molecular downregulation of FN3K expression, moderate inhibition.
further analyses were conducted using qPCR and Western To substantiate these observations and examine the
blot techniques. consistency of FN3K inhibition across distinct breast

Volume 9 Issue 3 (2025) 208 doi: 10.36922/EJMO025150114