Prophylactic role of artemisinin in modulating FGFR3, HRAS, and TP53 to prevent early-stage urothelial carcinoma in BBN-induced mouse models

Abstract

Purpose: Urinary bladder cancer remains a significant global health challenge, with effective early preventive strategies urgently needed to reduce incidence and progression. This study explores the prophylactic potential of artemisinin against N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced early-stage urothelial carcinoma in a mouse model.

Methods: A multidisciplinary approach was used to evaluate artemisinin’s molecular and physiological effects. Techniques included protein–protein interaction (PPI) network analysis, molecular docking, gene expression profiling, histopathological evaluation, and systemic biomarker assessment.

Results: PPI analysis revealed FGFR3, HRAS, and TP53 as central oncogenic drivers. Molecular docking confirmed strong binding affinities of artemisinin to these targets. Prophylactic artemisinin administration significantly downregulated FGFR3 and HRAS while upregulating TP53, indicating early correction of carcinogenic signaling. These molecular changes were associated with preserved bladder and renal histoarchitecture, normalized kidney function markers, and restored hematological profiles, reflecting systemic protection against BBN-induced toxicity.

Conclusions: Artemisinin effectively intercepts bladder carcinogenesis at multiple levels, modulating key genetic pathways and mitigating systemic damage. These findings provide compelling preclinical evidence supporting artemisinin as a promising prophylactic agent for bladder cancer prevention in high-risk populations.

Keywords: Artemisinin; Bladder carcinogenesis; Early-stage urothelial carcinoma (pT1); FGFR3; Gene expression profiling; HRAS; Molecular docking; N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN); Prophylactic agent; Protein–protein interaction network; TP53; Urinary bladder cancer; Urothelial carcinoma.

Fig. 1

Molecular Pathways in bladder cancer progression. This image illustrates the molecular pathways involved in bladder cancer progression highlighting the transition from normal urothelium to invasive tumors. It emphasizes key genetic alterations in oncogenes (HRAS, FGFR3) and tumor, suppressor genes (TP53, CDKN2A, RB), as well as critical signaling pathways (MAPK, p53, VEGF, ErbB) that regulate essential processes

Fig. 2

Schematic representation of the experimental design; W: week; n = number of mice

Fig. 3

Protein–protein interaction (PPI) network generated using STRING for FGFR3, HRAS, TP53, and their top predicted partners (FRS3, RPS6KA2, SHC2, RGL1, PPP1R13B) in Mus musculus. Each node represents a protein; filled nodes indicate proteins with known or predicted 3D structures, while edges represent protein–protein associations. Edge colors correspond to the type of supporting evidence: green = neighborhood, blue = co-occurrence, black = co-expression, and pink = experimental evidence,. HRAS, FGFR3, and TP53 appear as central, highly connected nodes, highlighting their key regulatory roles in oncogenic signaling and apoptosis

Fig. 4

Molecular Docking visualization of artemisinin’s binding poses within the FGFR3 Active Site Using CB-Dock. This figure illustrates the results of the molecular docking analysis of the top five predicted binding poses of Artemisinin within the highest-affinity binding site (C1, Vina Score = -8.4 kcal/mol) of FGFR3. (A) A composite view showing the overlay of all five poses within the binding pocket. (a-e) Detailed 2D and 3D representations of each individual pose, highlighting specific hydrogen bonds (dashed lines) and other interactions with key amino acid residues

Fig. 5

Molecular docking visualization of artemisinin’s binding poses within the HRAS active site using CB-Dock. This figure illustrates the results of the molecular docking analysis of the top five predicted binding poses of Artemisinin within the highest-affinity binding site (C1, Vina Score = -7.3 kcal/mol) of HRAS. (A) A composite view showing the overlay of all five poses within the binding pocket. (a-e) Detailed 2D and 3D representations of each individual pose, highlighting specific hydrogen bonds (dashed lines) and other interactions with key amino acid residues.

Fig. 6

Molecular docking visualization of artemisinin’s binding poses within the TP53 protein using CB-Dock. This figure illustrates the results of the molecular docking analysis of the top five predicted binding poses of Artemisinin within the highest-affinity binding site (C1, Vina Score = -8.6 kcal/mol) of TP53. (A) A composite view showing the overlay of all five poses within the binding pocket. (a-e) Detailed 2D and 3D representations of each individual pose, highlighting specific hydrogen bonds (dashed lines) and other interactions with key amino acid residues

Fig. 7

RT-qPCR was applied to detect the expressions of oncogenes (A) FGFR3, (B) HRAS and tumor suppressor genes (C) P53, in mouse urinary bladder tissues. Data are presented as mean ± SD from independent biological replicates (n = 10 per group) Statistical significance was determined by one-way ANOVA followed by LSD post-hoc test. Where, *P < 0.05 vs. Control, #P < 0.05 vs. BBN

Fig. 8

Histopathological examination of urinary bladder tissues across study groups. (A) Negative control group: Normal urothelial lining with no pathological changes or inflammation at 16 weeks (200×, scale bar = 200 μm). Inset: A high-power view showing regular maturation pattern. (B) BBN group: Carcinoma in situ with full-thickness dysplasia, nuclear pleomorphism, hyperchromasia, and invasive urothelial carcinoma (pT1) in the lamina propria at 12 weeks (100×, scale bar = 100 μm). Inset: A high-power view showing CIS. (C) Artemisinin group: Normal urothelial lining with mild inflammation in the lamina propria and submucosa at 16 weeks (200×, scale bar = 200 μm). Inset: a high-power view of the urothelium. and (D) Prophylactic group: Mild to moderate inflammation with preserved urothelial structure at 16 weeks (100×, scale bar = 100 μm). All sections were stained with H&E

Fig. 9

Histopathology of BBN-induced bladder cancer in mice. A, Schematic illustration of the BBN treatment timeline. B, Representative H&E-stained bladder sections showing progressive histological alterations: (a) Normal urothelium with no morphological changes (control) (1×, scale bar = 1 mm), (b) Moderate dysplasia with broad papillae lined by thickened urothelial epithelium at week 4 (100×, scale bar = 100 μm), (c) Full-thickness urothelial dysplasia consistent with carcinoma in situ (CIS) at week 8 (50×, scale bar = 50 μm) and (d) Invasive carcinoma nest (pathologic stage pT1) infiltrating the lamina propria at week 12 (100×, scale bar = 100 μm) (red arrow)

Fig. 10

Histological examination of kidney tissues across study groups. (A) Negative control group: Normal kidney architecture with no pathological changes at 16 weeks. (B) BBN group: Significant alterations including interstitial nephritis, tubular swelling, casts, and mesangial hypercellularity at 12 weeks. Inset: High-power view showing prominent mesangial hypercellularity and tubular epithelial cell degeneration. (C) Artemisinin group: Preserved kidney structure with no signs of inflammation or damage at 16 weeks. and (D) Prophylactic group: Normal kidney structure with mild interstitial inflammation at 16 weeks. All sections were stained with H&E and observed at 100× magnification (scale bar = 100 μm)

Fig. 11

Biochemical and hematological analysis in studied groups. (A) urea levels, (B) creatinine levels, (C) Hb concentrations, (D) RBCs count, (E) PLTs count and (F) WBCs count. Data are expressed as mean ± SD from independent biological replicates (n = 10 per group), Statistical significance was determined by one-way ANOVA followed by LSD post-hoc test, where *P < 0.05 vs. Control, #P < 0.05 vs. BBN