Exploring Darunavir, Rilpivirine and Etravirine as Potential Therapies for Bladder Cancer: Efficacy and Synergistic Effects

Abstract

This research explores the therapeutic efficacy of Darunavir (DRV), Rilpivirine (RPV), and Etravirine (ETV) against UM-UC-5 bladder cancer cells, addressing the critical need for innovative treatments in bladder cancer research. Through a comprehensive assessment of their individual and combined effects across diverse time intervals, ETV emerges as the most potent drug, with a lowest IC₅₀ of 5.9 µM, closely followed by RPV (lowest IC₅₀ of 9.6 µM), while DRV exhibits the least effectiveness (lowest IC₅₀ of 25.6 µM). Notably, a significant synergistic effect is evident in the ETV and RPV combination, especially at 48 and 72 h for low concentrations. Synergies are also observed with ETV and DRV, albeit to a lesser extent and primarily at 48 h. Conversely, the DRV and RPV combination yields minimal effects, predominantly additive in nature. In summary, this pre-clinical investigation underscores the promising therapeutic potential of ETV and RPV, both as standalone treatments and in combination, hinting at repurposing opportunities in bladder cancer therapy, which could give a new treatment method for this disease that is faster and without as severe side effects as anticancer drugs. These findings represent a substantial stride in advancing personalized medicine within cancer research and will be further scrutinized in forthcoming studies.

Keywords: antiretroviral drugs; bladder cancer; efficacy evaluation; etravirine; personalized medicine; repurposing possibilities; synergy.

Figure 1

Chemical structures of darunavir (DVR) (a), rilpivirine (RPV) (b) and etravirine (ETV) (c). Developed with ChemBioDraw^® Ultra version 13.0. A Chemical Drawing Software. Available online: https://chemdrawdirect.perkinelmer.cloud/js/sample/index.html (accessed on 10 July 2023).

Figure 2

Results of UM-UC-5 cell viability following exposure to DRV at escalating doses (0.01–100 µM) for 24 h (a), 48 h (b), and 72 h (c). A 0.1% DMSO was applied to negative control cells (vehicle). The MTT assay was used to determine cell viability, and the findings are shown as the mean ± SEM (n = 3). *** Statistically significant vs. negative control (vehicle) at p < 0.001; **** Statistically significant vs. negative control (vehicle) at p < 0.0001.

Figure 3

After being exposed to DRV at escalating concentrations (0.01–100 µM) for 24, 48, and 72 h, UM-UC-5 cell morphology was evaluated (n = 3). Negative control cells received the vehicle treatment (0.1% DMSO). The scale bar is 200 µm.

Figure 4

Dose-response curve and IC₅₀ of UM-UC-5 following exposure to DRV at increasing concentrations for 24 h (a), 48 h (b), and 72 h (c) (concentrations of 0.01–100 µM). A 0.1% DMSO was applied to negative control cells (vehicle). Using the MTT assay, cell viability was determined. The findings were normalized and are presented as the mean ± SEM (n = 3).

Figure 5

Results of UM-UC-5 cell viability following exposure to RPV at escalating doses (0.01–100 µM) for 24 h (a), 48 h (b), and 72 h (c). A 0.1% DMSO was applied to negative control cells (vehicle). The MTT assay was used to determine cell viability, and the findings are shown as the mean ± SEM (n = 3). * Statistically significant vs. negative control (vehicle) at p < 0.05; *** statistically significant vs. negative control (vehicle) at p < 0.001; **** statistically significant vs. negative control (vehicle) at p < 0.0001.

Figure 6

After being exposed to RPV at escalating concentrations (0.01–100 µM) for 24, 48, and 72 h, UM-UC-5 cell morphology was evaluated (n = 3). Negative control cells received the vehicle treatment (0.1% DMSO). The scale bar is 200 µm.

Figure 7

Dose–response curve and IC₅₀ of UM-UC-5 following exposure to RPV at increasing concentrations for 24 h (a), 48 h (b), and 72 h (c) (concentrations of 0.01–100 µM). A 0.1% DMSO was applied to negative control cells (vehicle). Using the MTT assay, cell viability was determined. The findings were normalized and are presented as the mean ± SEM (n = 3).

Figure 8

Results of UM-UC-5 cell viability following exposure to ETV at escalating doses (0.01–100 µM) for 24 h (a), 48 h (b), and 72 h (c). A 0.1% DMSO was applied to negative control cells (vehicle). The MTT assay was used to determine cell viability, and the findings are shown as the mean ± SEM (n = 3). * Statistically significant vs. negative control (vehicle) at p < 0.05; ** statistically significant vs. negative control (vehicle) at p < 0.01; **** statistically significant vs. negative control (vehicle) at p < 0.0001.

Figure 9

After being exposed to ETV at escalating concentrations (0.01–100 µM) for 24, 48, and 72 h, UM-UC-5 cell morphology was evaluated (n = 3). Negative control cells received the vehicle treatment (0.1% DMSO). The scale bar is 200 µm.

Figure 10

Dose–response curve and IC50 of UM-UC-5 following exposure to ETV at increasing concentrations for 24 h (a), 48 h (b), and 72 h (c) (concentrations of 0.01–100 µM). A 0.1% DMSO was applied to negative control cells (vehicle). Using the MTT assay, cell viability was determined. The findings were normalized and are presented as the mean ± SEM (n = 3).

Figure 11

Results of UM-UC-5 cell cytotoxicity following exposure to single drugs and a combination of DRV and RPV for 24 h (a), 48 h (b), and 72 h (c). Both drugs were added at the same time. A 0.1% DMSO was applied to negative control cells (vehicle). The MTT assay was used to determine cell viability, and the findings are shown as the mean ± SEM (n = 3). * Statistically significant vs. drug alone at p < 0.05; ** statistically significant vs. drug alone at p < 0.01; *** statistically significant vs. drug alone at p < 0.001; **** statistically significant vs. drug alone at p < 0.0001.

Figure 12

Morphological evaluation of UM-UC-5 cells after exposure to combinations of DRV and RPV at increasing concentrations for 24 h, 48 h, and 72 h. Both drugs were added at the same time. Negative control cells were treated with the vehicle (0.1% DMSO). These images are representative of three independent experiments. The scale bar is 200 μm.

Figure 13

Results of UM-UC-5 cell cytotoxicity following exposure to single drugs and a combination of ETV and RPV for 24 h (a), 48 h (b), and 72 h (c). A 0.1% DMSO was applied to negative control cells (vehicle). The MTT assay was used to determine cell viability, and the findings are shown as the mean ± SEM (n = 3). * Statistically significant vs. drug alone at p < 0.05; ** statistically significant vs. drug alone at p < 0.01; *** statistically significant vs. drug alone at p < 0.001; **** statistically significant vs. drug alone at p < 0.0001.

Figure 14

Morphological evaluation of UM-UC-5 cells after exposure to combinations of ETV and RPV at increasing concentrations for 24 h, 48 h, and 72 h. Both drugs were added at the same time. Negative control cells were treated with the vehicle (0.1% DMSO). These images are representative of three independent experiments. The scale bar is 200 μm.

Figure 15

Results of UM-UC-5 cell cytotoxicity following exposure to single drugs and a combination of ETV and DRV for 24 h (a), 48 h (b), and 72 h (c). A 0.1% DMSO was applied to negative control cells (vehicle). The MTT assay was used to determine cell viability, and the findings are shown as the mean ± SEM (n = 3). * Statistically significant vs. drug alone at p < 0.05; ** statistically significant vs. drug alone at p < 0.01; *** statistically significant vs. drug alone at p < 0.001; **** statistically significant vs. drug alone at p < 0.0001.

Figure 16

Morphological evaluation of UM-UC-5 cells after exposure to combinations of ETV and DRV at increasing concentrations for 24 h, 48 h, and 72 h. Both drugs were added at the same time. Negative control cells were treated with the vehicle (0.1% DMSO). These images are representative of three independent experiments. The scale bar is 200 μM.

Figure 17

DECREASE output. The cell inhibition values of DRV alone (first column), RPV alone (bottom row), and in combination at the same concentrations (diagonal) were input into the DECREASE web application to form the incomplete dose–response matrices for 24, 48, and 72 h (a). The predicted full matrices of all combinations for the three time points were then generated by DECREASE using the Non-negative Matrix Factorization cNMF algorithm (b).

Figure 18

DECREASE output. The cell inhibition values of RPV alone (first column), ETV alone (bottom row), and in combination at the same concentrations (diagonal) were input into the DECREASE web application to form the incomplete dose–response matrices for 24, 48, and 72 h (a). The predicted full matrices of all combinations for the three time points were then generated by DECREASE using the Non-negative Matrix Factorization cNMF algorithm (b).

Figure 19

DECREASE output. The cell inhibition values of ETV alone (first column), DRV alone (bottom row), and in combination at the same concentrations (diagonal) were input into the DECREASE web application to form the incomplete dose–response matrices for 24, 48, and 72 h (a). The predicted full matrices of all combinations for the three time points were then generated by DECREASE using the Non-negative Matrix Factorization cNMF algorithm (b).

Figure 20

SynergyFinder scores and 2D synergy maps for the combination of DRV and RPV for 24, 48, and 72 h. The red areas indicate synergism, while the green areas indicate antagonism. Bliss–Loewe scores lower than −10 are indicative of the combination being antagonistic, between −10 and 10 are additive, and above 10, the combination is synergistic. The most synergistic areas (three-by-three concentration windows) for each time are highlighted.

Figure 21

SynergyFinder scores and 2D synergy maps for the combination of RPV and ETV for 24, 48, and 72 h. The red areas indicate synergism, while the green areas indicate antagonism. Bliss–Loewe scores lower than −10 are indicative of the combination being antagonistic, between −10 and 10 are additive, and above 10, the combination is synergistic. The most synergistic areas (three-by-three concentration windows) for each time are highlighted.

Figure 22

SynergyFinder scores and 2D synergy maps for the combination of ETV and DRV for 24, 48, and 72 h. The red areas indicate synergism, while the green areas indicate antagonism. Bliss–Loewe scores lower than −10 are indicative of the combination being antagonistic, between −10 and 10 are additive, and above 10, the combination is synergistic. The most synergistic areas (three-by-three concentration windows) for each time are highlighted.