Fig. 5: Combining HMMR inhibition with ARSI treatment enhances suppression of prostate cell proliferation and tumour growth.

a Combination treatment with 4-MU + ENZ significantly inhibits LNCaP and V16D PCa cell viability compared to Vehicle, 4-MU or ENZ treatment alone. Cells were counted on days 3 and 6 post-treatment using Trypan blue dye exclusion. Data are presented as mean ± SD of triplicate wells, represent two independent experiments, and were analysed using two-way ANOVA with Tukey’s multiple comparison test (****p < 0.0001). b Combination treatment with siHMMR+ENZ significantly inhibits LNCaP PCa cell viability compared to Vehicle, 4-MU or ENZ treatment alone, as assessed by Trypan blue dye exclusion. Data are presented as mean ± SD of triplicate wells, represent two independent experiments, and were analysed using two-way ANOVA with Tukey’s multiple comparison test (****p < 0.0001). c, d Combination treatment with 4-MU+darolutamide (DARO) or 4-MU+apalutamide (APA) significantly inhibits V16D PCa cell viability compared to Vehicle, 4-MU or ENZ treatment alone, as determined by the Trypan blue dye exclusion. Data are presented as mean ± SD of triplicate wells, represent two independent experiments, and were analysed using two-way ANOVA with Tukey’s multiple comparison test (****p < 0.0001). e The Chou-Talalay method [17] was used to determine the combination indices of the growth curves (A, B, D, E). f Schematic diagram of treatment regimen for in vivo subcutaneous xenograft model. g Combination treatment of 4-MU + ENZ significantly inhibits the proliferation of V16D PCa xenograft tumours compared to 4-MU or ENZ treatment alone. At the completion of the in vivo experiment, xenograft tumours were analysed for the proliferative marker Ki67 by immunohistochemistry (IHC). Quantification of Ki67 staining on the left and representative IHC images on the right. Data are presented as mean ± SD of 7 mice in each treatment group and analysed using one-way ANOVA with Turkey’s multiple comparison test (*p < 0.05, **p < 0.01). h Combination treatment with 4-MU + ENZ inhibits proliferation in patient-derived prostate cancer explants (PDEs). PDEs (n = 7) were treated as indicated for 48 h, then paraffin-embedded and formalin-fixed prior to immunohistochemistry (IHC) with proliferation marker Ki67. Digital images were manually counted. Quantification of Ki67 staining on the left and representative IHC images on the right. Data are presented as mean ± SD of 7 patients and analysed using one-way ANOVA with Dunnett’s multiple comparison test (*p < 0.05).