A novel, synthesized, amphiphilic ethylene glycol squalene derivative suppresses BBN-induced bladder carcinogenesis

Abstract

Squalene, a natural triterpene with antioxidant, anti-inflammatory, and immunostimulatory properties, holds promise for cancer therapy. Here, we examined a previously developed, diethylene glycol derivative of squalene (SQ-diEG) and investigated its in vivo anti-carcinogenic effects in bladder cancer. C57BL/6 mice were treated with 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to induce bladder cancer, with SQ-diEG or PBS (control) administered orally from Week 0. SQ-diEG significantly reduced bladder cancer incidence to 3.7% after 8 weeks, compared to 21.4% in controls (p = 0.025). Transcriptomic analysis indicated that SQ-diEG may exert anti-carcinogenic effects by reducing ROS-mediated DNA damage, enhancing the immune microenvironment, and modulating cholesterol biosynthesis via SQLE downregulation. In vitro, SQ-diEG inhibited proliferation and induced apoptosis in bladder cancer cell lines. This study is the first to demonstrate that SQ-diEG significantly reduces bladder cancer in a BBN mouse model, highlighting potential for therapeutic development. Further research is needed to elucidate the mechanisms and long-term efficacy of SQ-diEG.

Keywords: Apoptosis; Bladder cancer; Cholesterol metabolism; Ethylene glycol derivative; SQLE; Squalene.

Fig. 1

Anti-tumor effect of SQ-diEG in a bladder cancer model. (A) Wild-type C57BL/6 mice treated with 0.025% BBN were orally administered SQ-diEG daily from Week 0 to Week 16 before sacrifice and bladder extraction at Week 16. (B) Representative image of the bladder stained with hematoxylin at 200 × magnification. (C) Percentage of bladder cancer in SQ-diEG treated and control groups. *p < 0.05 (chi-square test).

Fig. 2

Whole transcriptomic analysis using micro array in SQ-diEG-treated mice model. (A) Bar graphs showing the distribution of fold changes of the DEGs. The orange bars represent the numbers of upregulated DEGs. Volcano plots displaying differentially expressed genes (DEGs) between SQ-diEG-treated and nontreated BBN-induced mice (B) 8 weeks and (C) 16 weeks. The vertical axis (y-axis) corresponds to − log10 p value and the horizontal axis (x-axis) displays linear fold change. The orange dots represent the upregulated genes; the green dots represent the downregulated genes. The top 20 DEGs with the largest fold changes are shown. (D) 8-week SQ-diEG-treated and (E) 16-week SQ-diEG-treated timepoints. Clusters were computed using the Leiden algorithm and the points were plotted on the first two UMAP dimensions. The larger and black-outlined points represent significantly enriched terms. The greater the enrichment significance of a term, the larger and darker the corresponding point is. The countPlots from the GPSAdb web tool showing predicted top activated and inhibited Hallmark gene sets at (F) 8-week SQ-diEG-treated and (G) 16-week SQ-diEG-treated timepoints. Red bars represent activated and blue bars represent inhibited gene sets. The X-axis represents the net enrichment score (NES) and the Y-axis is ordered based on the NES score.

Fig. 3

Whole-transcriptome analysis targeted to bladder neoplasm-specific genes. (A) Heatmap showing the average signal intensity of ‘bladder neoplasm’-specific downregulated DEGs identified from the DisGeNET database. (B) Wheel graph showing important genes that showed similar expression patterns in both 8-week and 16-week conditions and the enriched functions. (C) Wheel graph showing important genes that showed pronounced downregulation in the 16-week conditions and the enriched functions.

Fig. 4

SQ-diEG inhibits cholesterol metabolism and exerts anti-carcinogenic effects in the BBN mouse model. (A) The representative top 10 downregulated biological functions in SQ-diEG-treated mice compared to those in control mice were analyzed by using IPA. (B) Extraction of the gene symbols that comprise the cholesterol biosynthesis pathway and the fold changes for each. (C) mRNA expression levels of Sqle in the bladders of SQ-diEG-treated and untreated mice were analyzed by qPCR.**p < 0.01(student’s t test).

Fig. 5

SQ-diEG modulates proliferation and apoptosis in bladder cancer cell lines by affecting SQLE expression. (A) CCK-8 assays were performed to examine cell proliferative potential in T24 and 253 J cell lines supplemented with SQ-diEG. (B) Analysis of cell apoptosis by Caspase-3 and Caspase-7 assay in SQ-diEG-treated cells from T24 cell lines. (C)The mRNA expression of proapoptotic genes was analyzed by qPCR. (D) mRNA expression of SQLE in T24 and 253 J cells supplemented with SQ-diEG was analyzed by qPCR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA followed by Dunnett’s post-hoc test).