Cationic amphiphilic drugs as potential anticancer therapy for bladder cancer

Abstract

More effective therapy for patients with either muscle-invasive or high-risk non-muscle-invasive urothelial carcinoma of the bladder (UCB) is an unmet clinical need. For this, drug repositioning of clinically approved drugs represents an interesting approach. By repurposing existing drugs, alternative anticancer therapies can be introduced in the clinic relatively fast, because the safety and dosing of these clinically approved pharmacological agents are generally well known. Cationic amphiphilic drugs (CADs) dose-dependently decreased the viability of a panel of human UCB lines in vitro. CADs induced lysosomal puncta formation, a hallmark of lysosomal leakage. Intravesical instillation of the CAD penfluridol in an orthotopic mouse xenograft model of human UCB resulted in significantly reduced intravesical tumor growth and metastatic progression. Furthermore, treatment of patient-derived ex vivo cultured human UCB tissue caused significant partial or complete antitumor responses in 97% of the explanted tumor tissues. In conclusion, penfluridol represents a promising treatment option for bladder cancer patients and warrants further clinical evaluation.

Keywords: bladder cancer; cationic amphiphilic drugs; ex vivo culture; penfluridol; preclinical in vivo model.

Fig. 1

CADs reduce viability and clonogenicity in a panel of human bladder cancer cells. Assessment of the viability of T24 (A) and RT‐112 (B) cells after treatment with eight different CADs for 40 h (n = 3; six replicates each). Panels C–E represent dose–response experiments with astemizole, terfenadine, and penfluridol, respectively, on the viability of multiple subconfluent bladder cancer cells. Viability was measured 48 h after a 2‐h treatment. Mean normalized to vehicle‐treated cells (n = 3; six replicates each). Clonogenic assay: Multiple bladder cancer cells were treated for 2 h with a dose range of penfluridol. (F) Representative image of a clonogenic assay UM‐UC‐3 cells. (G) The number of colonies was measured using imagej after 10–14 days of culture (n = 3; three replicates each).

Fig. 2

Penfluridol induces cell death in human bladder cancer cells. (A) Real‐time apoptosis (luminescence measurement of phosphatidylserine on outer leaflet of cell membranes with annexin V) and (B) cell death (fluorescence measurement of a DNA‐binding dye) after administration of a dose range of penfluridol. Data presented are mean ± SE (n = 3; six replicates each). One‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Penfluridol targets lysosomal structures in UM‐UC‐3 cells (n = 3, 3 replicates each). Representative images of LAMP‐1 (green)‐ and LGALS1 (red)‐/DAPI (blue)‐stained UM‐UC‐3 cells treated for 2 h with a dose range of penfluridol.

Fig. 3

The antitumor effects of penfluridol in an orthotopic murine xenograft model with stable firefly luciferase‐2 UM‐UC‐3 human bladder cancer cells. Female BALB/c nude mice were inoculated with firefly luciferase‐2‐labeled UM‐UC‐3 bladder cancer cells and intravesically treated with vehicle (n = 8) or penfluridol (n = 9; 100 µm once weekly; equivalent to 130 µg·kg⁻¹·week⁻¹). (A) Quantification of the whole‐body total tumor burden in real time (relative light units, RLU). Data are presented as mean ± SE. Mann–Whitney U‐test. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Representative bioluminescent images of vehicle‐ and penfluridol‐treated mice at day 29. Representative images of vehicle (C)‐ and penfluridol (D)‐treated bladders stained with hematoxylin and eosin. Scale bar: 500 and 20 µm, respectively. (E) The percentage of mice with detectable metastases in the listed organs. (F) Number of metastatic foci per mouse per experimental group. (G) Metastatic tumor burden per experimental group (RLU). Mann–Whitney U‐test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4

Ex vivo treatment of cultured human bladder cancer slices with penfluridol. Heat maps showing median cumulative scores of the bladder cancer slices per condition per patient. Multiple explanted tissue slices were cultured for each condition. Cumulative score is based on histological evaluation of entire tissue slice (TS; see Fig S4). TS were scored in four categories: (1) overall quality based on H&E staining, (2) the presence of apoptotic cells [cleaved caspase 3⁺(c‐CASP‐3⁺)/keratin⁺ (KRT⁺) cells], (3) cancer cell debris (fragmented keratin), and (4) nuclear proliferation based on proliferation cell nuclear antigen (PCNA). TS received a score of either 0 or 1 for each category. For category 1, TS received a score of 1 when > 50% of tissue was fragmented/degraded. For categories 2 and 3, TS received a score of 1 when multiple clusters were observed. For category 4, TS received a score of 1 when < 50% of KRT+ cells in the TS displayed nuclear PCNA. Cumulative score was calculated as the sum of the 4 categories; median cumulative scores are shown (see also Fig. S4). Explanted tumor slices were ex vivo cultured with vehicle solution or penfluridol (100 µm for A–B and a dose range for C–D) for 3 days. Chi‐squared test for categorical data with more than two categories. P value = 4.36424E⁻¹¹. (B) Representative images of bladder cancer slices obtained from a patient diagnosed with NMIBC stage T1 grade 2 (#28) after 3 days in the presence of vehicle solution or penfluridol (100 µm). Complete loss of tumor cells was observed in five patient‐derived tumor explants (#9, 28, 29, 31, and 35). Significant reduction was found in 30 patient‐derived tumor explants (#1–8, 10–15, 17, 20, 22–27, 30, 32–34, and 36–39). A partial response was observed in 3 patient‐derived tumor explants (#18, 19, and 21). No response was observed in one patient‐derived tumor explant (#16). (D) Representative images of bladder cancer tissue slices obtained from a patient diagnosed with NMIBC stage T1 grade 2 (#9) after 3 days in the presence of vehicle solution or a dose range of penfluridol. c‐CASP‐3 and PCNA: green; panKRT: red; and DAPI: blue. Scale bar: 25 µm.

Fig. 5

Evaluation of the effect of penfluridol on normal murine or human urothelium integrity. (A) Average body weight (mean ± SE) of non‐tumor‐bearing female BALB/c nude mice treated intravesically with vehicle (n = 5) or penfluridol solution (n = 5; 100 µm once weekly for 4 weeks; equivalent to 130 µg·kg⁻¹·week⁻¹). One‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Representative overview images of H&E‐stained murine bladders of vehicle‐treated (B) and 100 µm penfluridol‐treated (C) mice. (D) Representative images of vehicle‐ and penfluridol‐treated, non‐tumor‐bearing murine bladders. c‐CASP‐3 and PCNA: green; panKRT: red; and DAPI: blue. Scale bar: H&E: 20 µm; IF staining: 25 µm. (E) Representative images of nontransformed ‘normal’ urothelium in tissue slices obtained from a patient with prostate cancer with no history of bladder carcinoma, treated either with vehicle solution or with 100 µm penfluridol for 3 days (#40). c‐CASP‐3 and PCNA: green; panKRT: red; and DAPI: blue. Scale bar: 25 µm.