Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells

Abstract

Aim: The development of chemotherapy resistance is the major obstacle in the treatment of advanced prostate cancer (PCa). Extracellular vesicles (EVs) secretion plays a significant role among different mechanisms contributing to chemoresistance. Hence, inhibition of EVs release may increase the efficacy of chemotherapeutic drugs against PCa. Methods: Paclitaxel (PTX) resistant PCa cells (PC3-R and DU145-R) were treated with GW4869, a known exosome biogenesis inhibitor. EVs were isolated from the conditioned media by ExoQuick-based precipitation method and characterized for concentration and size distribution by nanoparticle tracking analysis. The effect of GW4869 treatment on the survival and growth of PCa cells was assessed by MTT, and colony formation assays in vitro, and ectopic PC3-R xenografts in male athymic nude mice in vivo. The effect of other EV biogenesis inhibitors, imipramine and dimethyl amiloride (DMA), treatment was also analyzed on the survival of PC3-R cells. Results: GW4869 (10-20 µM) treatment of PTX resistant PCa cells significantly reduced the release of small EVs (50-100 nm size range) while increasing the release of larger EVs (> 150 nm in size), and inhibited their clonogenicity. Moreover, GW4869 (5-20 µM) treatment (24-72h) significantly inhibited the survival of PC3-R cells in a dose-dependent manner. We observed a similar growth inhibition with both imipramine (5-20 µg/mL) and DMA (5-20 µg/mL) treatment in PC3-R cells. Furthermore, GW4869 treatment (IP) in mice bearing PC3-R xenografts significantly reduced the tumor weight (65% reduction, P = 0.017) compared to the vehicle-treated control mice without causing any noticeable toxicity. Conclusion: Inhibiting the release of EVs could sensitize the resistant PCa cells to chemotherapy.

Keywords: GW4869; Prostate cancer; chemoresistance; extracellular vesicles; paclitaxel.

Figure 1

Characterization of EV concentration and size distribution following GW4869 treatment in PC3-R cells. EVs were isolated from the conditioned media of paclitaxel-resistant PC3-R cells following 24 h (upper panel) and 48 h (lower panel) of treatment with GW4869 (10-20 µM) and characterized for concentration and size distribution by NTA. (A) Concentration and size distribution of EVs isolated from DMSO (VC) or GW4869 (10 and 20 µM) treated cells are represented with green, orange, and red colors, respectively (n = 3). Each line represents the mean of three samples, and an average data of 5 videos of 30 sec each was used for each sample. (B-C) Total EV concentration per million cells and mean size are plotted. (D) Size distribution of EVs is presented as percent particles in the mentioned size range. Each bar represents mean ± SEM (n = 3). VC: Vehicle control; *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Figure 2

Effect of GW4869 treatment on the survival and clonogenicity of PC-R cells. (A) Paclitaxel-resistant PC3-R cells were treated with DMSO (VC) or GW4869 (5-20 µM), and cell viability was measured in MTT assay after 24, 48, and 72 h. Data are presented as mean ± SEM (n = 5 replicates per group). *P < 0.05, **P < 0.005. (B) Colony formation was measured in PC3-R cells after GW4869 treatment (10 and 20 μM) as described in the methods. Representative images are shown (left panel), and the colony number is presented as mean ± SEM (n = 3 replicates per group). ****P < 0.0001. VC: Vehicle control.

Figure 3

In vivo effect of GW4869 treatment on the growth of paclitaxel-resistant PC3-R cells’ xenografts. Male athymic nude mice were treated with vehicle or GW4869, and various study parameters were assessed as described in the methods. (A) Average body weight (mean ± SEM) at the start and day 40 of the study for control mice (n = 3) and GW4869 treated mice (n = 4) mice. (B) Average xenograft volume (mean ± SEM) in control and GW4869 treated mice survived at measured time points is presented. (C) In the end, mice were sacrificed, xenografts were excised, and their weight was measured. Average tumor weight in the control group (n = 6 xenografts) and the GW4869 group (n = 8 xenografts) is presented as mean ± SEM. Unpaired t test was used to calculate the statistical significance, *P = 0.017. (D) CD63 expression in xenograft tissues from the vehicle control (VC) group and GW4869 group (n = 6 each) was measured by IHC. For each image, ten random areas were analyzed for IHC scoring as described in the methods. Mean IHC scores are presented as mean ± SEM in the bar diagram. Representative images are shown. (E) EVs isolated from the plasma of control (n = 3) and GW4869 treated mice (n = 3) were analyzed by NTA, and the size distribution (mean ± SEM) of EVs was plotted as a percentage of total EVs. (F) EVs from VC (n = 3) and GW4869 treated (n = 4) mice were used for the analysis of syntenin, CD63, and GOLGA2 by Western blotting. Representative immunoblots are shown. (G) Densitometry analysis of syntenin and CD63 expression was performed and normalized with corresponding band intensity in Ponceau-stained membrane. The relative band intensity is presented as mean ± SEM.

Figure 4

Effect of DMA and imipramine treatment on the survival of paclitaxel-resistant PC3-R cells. PC3-R cells were treated with (A) DMA (5-20 µg/mL) or (B) imipramine (5-20 µg/mL) for 24-72 h and analyzed for cell viability in the MTT assay. Data are presented as mean ± SEM (n = 5 replicates per group). **P < 0.005, ***P < 0.0005; ****P < 0.0001.

Figure 5

Effect of GW4869 treatment on paclitaxel-resistant DU145-R cells. EVs were isolated from the conditioned media of paclitaxel-resistant DU145-R cells following 24 h (upper panel) and 48 h (lower panel) of treatment with GW4869 (10-20 µM) and characterized for concentration and size distribution by NTA. (A-B) Each colored line in the left panel represents the mean of three samples, and an average data of 5 videos of 30 s each was used for each sample. Concentration (particles/mL)-size distribution, average EV concentration per million cells, average size, and percent particles for various size ranges are presented. Each bar represents mean ± SEM (n = 3). *P < 0.05, **P < 0.005 ***P < 0.0005 ****P < 0.0001. (C) Colony formation was measured in DU145-R cells after GW4869 treatment (10 and 20 μM) as described in the methods. Representative images are shown (left panel), and the colony number is presented as mean ± SEM (n = 3 replicates per group). ***P < 0.0005, ****P < 0.0001.