Prodigiosin Sensitizes Sensitive and Resistant Urothelial Carcinoma Cells to Cisplatin Treatment

Abstract

Cisplatin-based treatment is the standard of care therapy for urothelial carcinomas. However, complex cisplatin resistance mechanisms limit the success of this approach. Both apoptosis and autophagy have been shown to contribute to this resistance. Prodigiosin, a secondary metabolite from various bacteria, exerts different biological activities including the modulation of these two cellular stress response pathways. We analyzed the effect of prodigiosin on protein levels of different autophagy- and apoptosis-related proteins in cisplatin-sensitive and -resistant urothelial carcinoma cells (UCCs). Furthermore, we investigated the effect on cell viability of prodigiosin alone or in combination with cisplatin. We made use of four different pairs of cisplatin-sensitive and -resistant UCCs. We found that prodigiosin blocked autophagy in UCCs and re-sensitized cisplatin-resistant cells to apoptotic cell death. Furthermore, we found that prodigiosin is a potent anticancer agent with nanomolar IC₅₀ values in all tested UCCs. In combination studies, we observed that prodigiosin sensitized both cisplatin-sensitive and -resistant urothelial carcinoma cell lines to cisplatin treatment with synergistic effects in most tested cell lines. These effects of prodigiosin are at least partially mediated by altering lysosomal function, since we detected reduced activities of cathepsin B and L. We propose that prodigiosin is a promising candidate for the therapy of cisplatin-resistant urothelial carcinomas, either as a single agent or in combinatory therapeutic approaches.

Keywords: apoptosis; autophagy; chemoresistance; cisplatin; prodigiosin; urothelial carcinoma.

Figure 1

Chemical structure of prodigiosin.

Figure 2

Prodigiosin is cytotoxic for cisplatin-sensitive and -resistant bladder carcinoma cells. RT-112 and RT-112^res cells were treated with different concentrations of prodigiosin for 24 h (A) or 72 h (B). After treatment, cell viability was measured using an thiazolyl blue (MTT) assay. Results are shown as the mean ± SEM of three independent experiments performed in triplicates for each treatment.

Figure 3

Prodigiosin modulates autophagy in RT-112 and RT-112^res in a concentration-dependent manner. RT-112 and RT-112^res cells were treated with the indicated concentrations of prodigiosin or 2.5 µM STS ± 10 µM QVD, 10 nM bafilomycin A₁ (BafA₁) or 250 nM Torin 2. After 6 h, the cells were lysed and cellular lysates were immunoblotted for the indicated proteins. (A,C) One representative immunoblot is shown for each experiment. (B,D) The densities of the bands of each protein of at least three independent experiments were quantified and normalized to actin. The mean of the solvent control of RT-112 (black bars) and RT-112^res (white bars) was set as 1 for each protein. Bars represent the means + SD. p values were determined by ordinary one-way ANOVA with Dunnett´s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. PARP: poly (ADP-ribose) polymerase; SQSTM1: sequestosome 1; LC3: light chain 3.

Figure 4

Prodigiosin blocks autophagy in RT-112 and RT-112^res and induces apoptosis in RT-112^res in a time-dependent manner. (A) RT-112 and RT-112^res cells were treated with 100 nM prodigiosin for the indicated periods of time or with 2.5 µM STS ± 10 µM QVD, 10 nM BafA1 or 250 nM Torin 2 for 24 h. After the incubation, cells were lysed, and cellular lysates were immunoblotted for the indicated proteins. One representative immunoblot is shown for each experiment. (B) The densities of bands of each protein of at least three independent experiments were quantified and normalized to actin. The mean of the solvent control of RT-112 (black bars) and RT-112^res (white bars) was set as 1 for each protein. Bars represent the means + SD. p values were determined by ordinary one-way ANOVA with Dunnett´s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. PARP: poly (ADP-ribose) polymerase; SQSTM1: sequestosome 1; LC3: light chain 3.

Figure 5

Prodigiosin increases cisplatin-mediated cytotoxicity in RT-112 and RT-112^res cells after 24 h. RT-112 and RT-112^res cells were treated with different concentrations of (A) cisplatin (CP) alone or (B,C) prodigiosin (PG) or CP alone or in combination for 24 h. For combinatory analysis, 0.25x, 0.5x or 1x of the IC₅₀ values of the single substances in RT-112 (B) and RT-112^res (C) were used. After treatment, cell viability was measured using an MTT assay. Results are shown as the mean ± SEM of three independent experiments performed in triplicates for each treatment. The combination index (CI) for different fractions affected of RT-112 (D) and RT-112^res (E) was calculated using the software CompuSyn (black dots). CompuSyn uses algorithms to extrapolate CI values for any effect level from the CI values of actual experiment points (blue dots). Synergism (CI < 1), additivism (CI = 1) and antagonism (CI > 1) can thereby be determined.

Figure 6

The combination of prodigiosin and cisplatin modulates apoptosis and autophagy in RT-112 and RT-112^res. RT-112 and RT-112^res cells were treated with IC₅₀ concentrations of prodigiosin and cisplatin alone or in combination in absence or presence of 10 µM QVD. After 24 h, the cells were lysed and cellular lysates were immunoblotted for the indicated proteins. (A) One representative immunoblot is shown for each experiment. The densities of bands of cleaved PARP (B) or LC3-II and SQSTM1 (C) of at least three independent experiments were quantified and normalized to actin. The mean of the solvent control of each cell line was set as 1 for each protein. Bars represent the means + SD. p values were determined by ordinary one-way ANOVA with Dunnett´s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. PARP: poly (ADP-ribose) polymerase; SQSTM1: sequestosome 1; LC3: light chain 3.

Figure 7

Prodigiosin treatment reduces cathepsin B and L activity in RT-112 and RT-112^res. (A,B) RT-112 and RT-112^res cells in chambered coverslips were treated with 100 nM LysoTracker^TM Deep Red for 30 min. (A) Representative sections are depicted and (B) the number and area of lysosomes of 10 representative images from two biological replicates for each cell line were quantified using ImageJ 1.53c. p values were determined by Student´s t-test. Scale bar: 20 µm. (C) RT-112 and RT-112^res cells were treated with 100 nM prodigiosin. After 24 h, the cells were lysed and cathepsin assays were performed according to the manufacturer’s instructions. For cathepsin B and L assays, 20 µM Z-Phe-Phe-FMK and for cathepsin D assays, 0.1 µM pepstatin A were used as inhibitor control. The fluorescence of duplicates for each treatment of three independent experiments was measured and the mean of the DMSO control of RT-112 was set as 100%. Bars represent the means + SD. p values were determined by two-way ANOVA with Tukey´s multiple comparisons post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 8

Prodigiosin increases cisplatin-mediated cytotoxicity in sensitive and -resistant bladder carcinoma cells. RT-112, RT-112^res, T24, T24^res, 253J, 253J^res, J82 and J82^res cells were treated with different concentrations of prodigiosin and cisplatin alone or in combination for 24 or 72 h. After treatment, cell viability was measured using an MTT assay. (A) IC₅₀ values were calculated using GraphPadPrism using the results of three independent experiments performed in triplicates for each treatment. (B) For combinatory analysis, cells were treated with multiples of the IC₅₀ concentrations of prodigiosin and cisplatin alone or in combination. CI values were calculated for different effective doses (ED) using the software CompuSyn. CompSyn uses algorithms to extrapolate CI values for any effect level from the CI values of actual experiment points. Synergism (CI < 1; green), additivism (CI = 1; black) and antagonism (CI > 1; blue) can thereby be determined.