Original article
Serum exosomal P-glycoprotein is a potential marker to diagnose docetaxel resistance and select a taxoid for patients with prostate cancer

https://doi.org/10.1016/j.urolonc.2015.04.019Get rights and content

Highlights

  • P-gp was higher in docetaxel-resistant PC-3 prostate cancer cells than in PC-3 cells.

  • P-gp was also higher in exosomes derived from docetaxel-resistant PC-3 cells.

  • P-gp knockdown increased the sensitivity to docetaxel but not cabazitaxel.

  • P-gp level in blood exosomes increased in docetaxel-resistant patients.

  • Serum exosomal P-gp could be a marker for docetaxel-resistance.

Abstract

Objectives

Docetaxel is used as the first-line chemotherapy for castration-resistant prostate cancer (CRPC), but docetaxel resistance occurs in part owing to induction of P-glycoprotein (P-gp) encoded by multidrug resistance protein 1 (MDR1) gene. A recently developed taxane—cabazitaxel—has poor affinity for P-gp and is thereby effective in docetaxel-resistant CRPC. It has been recently demonstrated that exosomes in the body fluids could serve as a diagnostic marker because they contain proteins and RNAs specific to the cells from which they are derived. In this study, we aimed to investigate if P-gp in blood exosomes could be a marker to diagnose docetaxel resistance and select a taxoid for patients with CRPC.

Methods and materials

Exosomes were isolated by differential centrifugation from docetaxel-resistant prostate cancer (PC-3) cells (PC-3R) and their parental PC-3 cells and from the serum of patients. Silencing of P-gp was performed by small interfering RNA transfection. Protein expression was examined by Western blot analysis. Viability of cells treated with docetaxel or cabazitaxel was determined by water soluble tetrazolium salt (WST) assay.

Results

The level of P-gp was higher in exosomes as well as cell lysates from PC-3R cells than in those from PC-3 cells. Cabazitaxel effectively killed PC-3R cells, and MDR1 knockdown improved the sensitivity of PC-3R cells to docetaxel but not to cabazitaxel. The P-gp level in blood exosomes was relatively higher in clinically docetaxel-resistant patients than in therapy-naïve patients.

Conclusions

Our results suggest that detection of P-gp in blood exosomes, which is involved in resistance to docetaxel but not to cabazitaxel, could be useful to diagnose docetaxel resistance and select an appropriate taxoid for patients with CRPC—docetaxel or cabazitaxel.

Introduction

Prostate cancer is one of the most common male cancers and is the second leading cause of cancer death among men in the United States [1]. Although many patients with prostate cancer have disease control after primary therapy, 34% of them developed metastatic disease [2]. Androgen deprivation therapy is considered as the most appropriate intervention for metastatic or recurrent prostate cancer [3]. However, 10% to 20% of patients with prostate cancer develop castration-resistant prostate cancer (CRPC) [4]. Docetaxel that is currently used as the first-line chemotherapy for CRPC offers an overall survival benefit for patients, but there is a finite amount of time before acquiring resistance [5].

P-glycoprotein (P-gp) encoded by multidrug resistance protein 1 (MDR1) gene is a member of the superfamily of adenosine triphosphate–binding cassette transporters, which acts as a drug efflux pump and contributes to the development of resistance against chemotherapy [6], [7], [8], [9]. Docetaxel resistance is also caused in part by P-gp [10], [11], [12]. A novel taxane-based chemotherapeutic agent cabazitaxel is one of the second-line treatments for CRPC [13], [14]. Although cabazitaxel improved overall survival among patients with docetaxel-resistant CRPC because of its poor affinity for P-gp, adverse events occurred at a higher rate. Especially, the incidence of febrile neutropenia is higher in patients treated with cabazitaxel when compared with those treated with docetaxel or mitoxantrone after docetaxel therapy [13], [14]. In addition, several studies have recently shown the efficacy of docetaxel rechallenge in patients who were diagnosed as docetaxel refractory as well as in those who were previously docetaxel sensitive and in those who had an interval from the first treatment [15], [16], [17]. Although cabazitaxel chemotherapy and docetaxel rechallenge are effective therapeutic options for some patients with docetaxel-refractory CRPC, there are no available biomarkers to diagnose docetaxel resistance and select an appropriate taxoid for patients with CRPC—docetaxel or cabazitaxel.

Exosomes are microvesicles with a diameter of 40 to 150 nm that are secreted from cells [18], [19], [20]. Exosomes are present in the body fluids such as blood and urine and could serve as diagnostic markers for various diseases including cancer, because they contain proteins and RNAs specific to the cells from which they are derived [21], [22], [23]. Given that molecular information within the cells is available by examining exosomes in the body fluids, one could select appropriate treatment for patients. This is especially the case for patients with prostate cancer, because serial biopsy of the prostate is not usually performed because of its invasiveness.

In this study, we hypothesized that the P-gp level in blood exosomes could reflect that in prostate cancer cells and be used as a marker to diagnose docetaxel resistance and select a taxoid—docetaxel or cabazitaxel. We first confirmed that the P-gp level in exosomes secreted from docetaxel-resistant prostate cancer cells was higher than that from sensitive cells. We then characterized the docetaxel and cabazitaxel sensitivity of docetaxel-resistant cells and analyzed the effects of MDR1 knockdown on the sensitivity. Lastly, we demonstrated that serum exosomal P-gp level was relatively higher in patients with clinically docetaxel-resistant prostate cancer than in therapy-naïve patients.

Section snippets

Reagents and antibodies

Docetaxel and cabazitaxel were purchased from Sigma-Aldrich (St. Louis, MO) and Selleck Chemicals (Houston, TX), respectively. Anti–P-gp and anti-CD9 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-GAPDH and anti–prostate-specific membrane antigen (PSMA) antibodies were from Cell Signaling Technology (Beverly, MA).

Cell culture

Human castration-resistant prostate cancer cell line PC-3 cells were obtained from the American Type Cell Collection (Manassas, VA) and cultured in Roswell

Expression of P-gp in cell lysates and exosomes derived from docetaxel-sensitive PC-3 and docetaxel-resistant PC-3R cells

We previously demonstrated that P-gp expression was higher in PC-3R cells when compared with that in parental PC-3 cells [24], [26], [27]. To determine if the P-gp level is also higher in exosomes, we isolated exosomes from the cell culture medium by differential centrifugation and performed Western blot analysis. As shown in Fig. 1, CD9, an exosomal marker, was detected at a similar level in PC-3 and PC-3R cells. The P-gp levels in exosomes as well as cell lysates were higher in PC-3R cells

Discussion

In prostate cancer as well as other types of cancer, resistance to docetaxel and paclitaxel has been reported to be caused in part owing to P-gp expression [7], [9], [10]. We have previously shown higher P-gp expression in docetaxel- and paclitaxel-resistant PC-3 cells when compared with that in parental PC-3 cells [24], [26], [27]. The P-gp level was reported to be elevated in the tissues of patients with prostate cancer and correlate with higher tumor grade, stage, and prostate-specific

Conclusion

Our results suggest that detection of P-gp in blood exosomes, which is involved in resistance to docetaxel but not to cabazitaxel, could be useful to diagnose docetaxel resistance and select an appropriate taxoid for patients with CRPC—docetaxel or cabazitaxel. The development of diagnostic systems to specifically detect P-gp in exosomes derived from prostate cancer cells and their evaluation in large-scale clinical studies remains to be done.

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    This work was supported in part by Grant-in-Aid for Scientific Research, Japan Society for the Promotion of Science, Japan (Grant no. 15K10582).

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