The Effect of Bowman-Birk Inhibitor from Soybeans on the Sensitivity of Prostate Cancer Stem-like Cells to Anti-androgen Agent

Abstract

Prostate stem-like cells exhibit resistance to anti-androgen agents and contribute to the recurrence of this cancer. A novel approach to overcome the resistance of prostate cancer stem-like cells is thus essential. The present study investigated whether soybean derived Bowman-Birk inhibitor (BBI) could abrogate the resistance of prostate cancer stem-like cells from an androgen-dependent prostate cancer cell line (LNCaP cells) to an anti-androgen agent. The LNCaP stem-like cells exhibited stronger resistance to an androgen antagonist, flutamide, than LNCaP parental cells. Exposure of LNCaP stem-like cells to BBI treatment reduced their stemness and resistance to flutamide. In parallel, LNCaP stem-like cells showed a positive response based on changes in the typical androgen-governed gene, prostate-specific antigen. These results suggest the potential of BBI as a candidate to reduce the resistance of prostate cancer stem-like cells to anti-androgen agents.

Introduction

Among male-specific diseases, prostate cancer represents a major health risk worldwide, including in Japan (Siegel et al., 2011). Since prostate cancer cells grow and survive in an androgen-dependent manner at an early stage, androgen deprivation therapy (ADT) is the first-line treatment and is initially effective (Goktas and Crawford, 1999). However, the efficacy of therapy is short, with prostate cancer cells ultimately becoming androgen-independent and developing several malignant phenotypes, referred to as castration resistant prostate cancer (CRPC) (Zhang et al., 2015). Since the androgen-independent type of prostate cancer remains incurable due to its resistance to ADT, a novel treatment approach that overcomes this resistance is urgently required.

A minor cell population amongst cancer tissues is cancer stem-like cells, which have been proposed to be the primary mediators of tumor initiation, progression, recurrence, metastasis, and resistance to treatment (Lin et al., 2019; Ojo et al., 2015). Prostate cancer stem-like cells may be responsible for the androgen-independent and incurable phenotype in patients with advanced prostate cancer (Maitland et al., 2011). Therefore, although ADT may eliminate the majority of prostate cancer, it fails to kill cancer stem-like cells. To reduce the development of the androgen-independent phenotype in prostate cancer after ADT, the effective eradication of prostate cancer stem-like cells, whose undifferentiated status contributes to their resistance to ADT, is needed (Jin et al., 2017). Therefore, agent-mediated induction of stem-like cell differentiation may abrogate resistance.

Soybean derived Bowman-Birk inhibitor (BBI) has been reported as an anticarcinogenic protease inhibitor (Kennedy and Wan, 2002). Further, control of prostate cancer cell growth by BBI is closely associated with the induction of connexin (Cx)43, a tumor-suppressive gene in prostate cancer (Tang et al., 2009). Cx43 maintains the differentiation status of normal prostate cells via the formation of gap junctions, which are necessary for the passage of small growth-regulatory signal molecules between contacting cells (Hossain et al., 1999). Furthermore, we previously demonstrated that the BBI mediated expression of Cx43 in prostate cancer stem-like cells effectively induced the differentiation and eradication of some cancer stem-like phenotypes (Kaneko et al., 2017). Based on these findings, we speculated that BBI-induced differentiation in prostate cancer stem-like cells has the potential to overcome the resistance of prostate cancer cells to ADT and suppress the appearance of androgen-independent prostate cancer cells. The present study was performed to clarify this possibility.

Materials and Methods

All cultures and chemicals were obtained from Nacalai Tesque Inc. (Kyoto, Japan) unless otherwise indicated. Fetal bovine serum (FBS) and BBI were purchased from Sigma-Aldrich (St. Louis, MO). Flutamide and 5α-dihydrotestosterone (DHT) were supplied by Tocris Bioscience (Bristol, UK) and TCI (Tokyo, Japan), respectively. All antibodies were obtained from Cell Signaling Technology Inc. (Beverly, MA) unless otherwise stated.

LNCaP cells, a representative human androgen-dependent prostate cancer cell line (ATCC, Manassas, VA), were cultured in RPMI1640 supplemented with 10 % FBS, 50 IU/mL penicillin, and 50 µg/mL streptomycin at 37 °C in a humidified atmosphere with 5 % CO_2. We utilized the tumor sphere-forming capacity of cells in a three-dimensional (3D) culture system with a 96U-well low attachment culture plate (Thermo Fisher Scientific, Kanagawa, Japan) to concentrate cancer stem-like cells, as described previously (Kaneko et al., 2018). Since the tumor sphere formation capacity of LNCaP stem-like cells is maintained for at least four passages in two-dimensional culture, stem-like cells were used until the fourth passage. Exponentially growing cells were used in experiments. Stem-like cells were plated on culture plates and cultured for 24 h to permit cell adherence. After attachment, stem-like cells were cultured in DMEM/F12 (Wako Co., Osaka, Japan) supplemented with 2 % FBS containing each reagent (BBI, flutamide, and DHT) at the indicated concentration, and cell parameters were assessed.

In order to confirm the effect of BBI on the stemness of LNCaP cells, a tumor sphere formation assay was carried out based on previous reports (Zhifang et al., 2015; Zhang et al., 2016). LNCaP stem-like cells were treated with 200 µg/mL BBI or vehicle for 72 h, and the cells (1 000 cells/well) were subsequently seeded in the 3D culture system with a 24-well low attachment culture plate (Corning International, Tokyo, Japan) and cultured in serum-free DMEM/F12 (Wako Co.) supplemented with 4 µg/mL insulin, 2 % B27, 20 ng/mL EGF and 20 ng/mL FGF including 200 µg/mL BBI for 5 days. Next, tumor sphere formation in each group was counted using a Carl Zeiss light microscope (Tokyo, Japan). Also, LNCaP parental cells were utilized as a negative control.

Cell growth and the CD44-positive cell population were evaluated by a WST-8 assay and FACS analysis using a mouse anti-human FITC-conjugated CD44 antibody (Thermo), respectively.

Total RNA was isolated from each cell using a Tissue Total RNA Extraction Mini Kit (Favorgen Biotech Corp., Ping-Tung, Taiwan). Total RNA was used for cDNA synthesis with a ReverTra Ace qPCR RT Kit (Toyobo, Shiga, Japan), and the cDNA template was analyzed by real-time PCR using THUNDERBIRD SYBR qPCR Mix (Toyobo). Primer sets as described previously were used in the present study (Kaneko et al., 2018). Gene expression data were normalized to the expression of the reference gene ribosomal protein L32 (RPL32).

Differences among groups were analyzed by a one-way ANOVA followed by the Tukey-Kramer test, and differences between two groups were analyzed by a one-way ANOVA followed by the Student's t-test. All statistical analyses were performed using Ekuseru-Toukei software (Social Survey Research Information Co., Ltd., Tokyo, Japan). Differences with a p-value of 0.05 or less were considered to be significant.

Results and Discussion

The development of androgen independency in prostate cancer is an important clinical hallmark because of its association with progression to CRPC. Prostate cancer stem-like cells exhibit severe resistance to ADT, a critical property that can lead to the recurrence of androgen-independent prostate cancer post therapy (Maitland et al., 2011). Therefore, approaches that attenuate the resistance of prostate cancer stem-like cells to ADT may reduce the recurrence of prostate cancer. In the present study, we provide evidence that BBI shows the potential to overcome ADT resistance in prostate cancer stem-like cells.

LNCaP is a human androgen-sensitive cancer cell line that is widely used in investigating the molecular mechanisms responsible for changes in the androgen sensitivity and dependency of prostate cancer cells. LNCaP cells are a heterogeneous population, which contains various clonal lines with naturally occurring variability in androgen sensitivity caused by spontaneous mutations (Wan et al., 2003). Accordingly, we first generated prostate cancer stem-like cells, determined using several established cancer stem-like cell markers, from LNCaP parental cells. It has been demonstrated for many solid tumors including prostate cancer that cancer stem cells can be identified using the cell surface receptor CD44, alone, or in combination with other cell surface markers, for example CD133 (Lin et al., 2019). As shown in Fig. 1A, five typical markers of cancer stem-like cells in the LNCaP cell population isolated through 3D culture (CD24, CD44, CD133, SOX2, and Oct3/4) showed significantly higher levels than those in the LNCaP parental cell population. Furthermore, the CD44-positive cell population in the 3D culture system was approximately 3-fold higher than that of the usual culture system (Fig. 1B). These results indicate that the 3D culture system employed in the present study effectively concentrated LNCaP stem-like cells from LNCaP parental cells. We then investigated whether BBI reduced stemness and resistance to ADT in LNCaP stem-like cells. The treatment dose of BBI was fixed at 200 µg/mL, as the cell growth control effect of BBI on LNCaP reached a plateau at this dose in a prior study (Kaneko et al., 2019). The results obtained showed that the BBI treatment significantly suppressed the levels of three cancer stem cell markers (CD24, CD44, and CD133) and slightly reduced the levels of the two other markers (SOX2 and Oct3/4) (Fig. 2A). In order to further confirm the stemness of LNCaP stem-like cells, we examined whether BBI could reduce tumor sphere formation capability (a typical marker to show the stemness of cancer stem-like cells) in LNCaP stem-like cells. As shown in Fig. 2B, there was a significant increase in sphere formation capability in LNCaP stem-like cells compared with LNCaP parental cells, and BBI treatment suppressed the sphere formation capacity in LNCaP stem-like cells with a statistical significance. Similarly with the above observation, our previous report indicated that LNCaP stem-like cells, but not LNCaP parental cells, could exhibit resistance to flutamide (Kaneko et al., 2019). In this study, we demonstrated that concomitant treatment with a fixed dose of BBI almost abolished this resistance with a flutamide induced dose-dependent reduction in cell viability in LNCaP stem-like cells (Fig. 3). These results suggested that BBI could act as an effective agent to attenuate the resistance to ADT. We then attempted to confirm that BBI abrogates resistance to ADT in LNCaP stem-like cells. As shown in Fig. 4A, in LNCaP stem-like cells, the level of prostate-specific antigen (PSA), a typical target gene of androgens, was significantly increased by the combination of DHT and BBI, but not DHT alone; whereas, the PSA level in LNCaP parental cells was significantly increased by DHT alone, but not by the combination (Fig. 4B). This difference in the responses observed appear to support the different resistance of LNCaP stem-like cells and LNCaP parental cells to flutamide.

Fig. 1.

The difference in mRNA level of each cancer stem cell marker (A) and CD44 protein level (B) between LNCaP parental cells and stem-like cells. (A) To assess the population of LNCaP stem-like cells from their parental cells, the mRNA levels of five cancer stem cell markers in the stem-like cells were compared with those in the parental cells by qRT-PCR, as described in the Materials and Methods. Each value is the mean of five assays, and vertical lines indicate SE. ** Significant difference from LNCaP parental cells (control). (B) CD44-positive cell population was evaluated by FACS analysis as described in Materials and Methods. The result is representative one of two assays.

Fig. 2.

Effects of BBI treatment on mRNA level of each cancer stem cell marker (A) and tumor sphere formation capability (B) in LNCaP stem-like cells. (A) LNCaP stem-like cells were treated with BBI 200 µg/mL for 72 h and the mRNA levels of the cancer stem cell markers were subsequently assessed, as described in Fig. 1(A). Each value is the mean of five assays, and vertical lines indicate SE. **Significant difference from control. (B) LNCaP stem-like cells were treated according to the procedure, as described in the Materials and Methods. Each value is the mean of three assays, and vertical lines indicate SE. **Significant difference from LNCaP parental cells (no treatment). ^##Significant difference from LNCaP parental cells (no treatment) and LNCaP stem-like cells (no treatment).

Fig. 3.

Effect of BBI treatment on cell viability in LNCaP stem-like cells. LNCaP stem-like cells were treated with BBI 200 µg/mL for 72 h and subsequently with flutamide at the indicated doses for 48 h. Cell viability was assessed by a WST-8 assay. Each value is the mean of five assays, and vertical lines indicate SE. **Significant difference from LNCaP stem-like cells (control).

Fig. 4.

Effect of BBI treatment on DHT-induced PSA mRNA level in LNCaP stem-like cells (A) and LNCaP parental cells (B). (A) and (B) Cells were treated with BBI 200 µg/mL for 72 h and subsequently with 50 nM DHT for 12 h. After the treatment, PSA mRNA level was assessed by qRT-PCR, as described in the Materials and Methods. **Significant difference from control. ^##Significant difference from DHT 50 nM tretament

As described above, the presence of prostate cancer stem cells in prostate cancer tissues induces resistance to ADT and promotes recurrence with the androgen-independent phenotype after treatment (Santer et al., 2015). A previous report clearly indicated that after ADT there is an increase in prostate cancer stem-like cells in the tumor tissues of prostate cancer patients, indicating the important role of stem-like cells in prostate cancer recurrence (Zhifang et al., 2013). Therefore, abrogating the resistance of prostate stem-like cells to ADT may reduce the risk of recurrence of prostate cancer post therapy. In this study, we demonstrated that LNCaP stem-like cells had stemness and resistance to a typical anti-androgen agent, flutamide, and that BBI treatment attenuated both of these. In addition, BBI treatment enhanced the response of LNCaP stem-like cells to androgen (DHT). Overall, BBI appears to be a promising agent to reduce the resistance of prostate cancer stem cells to ADT.

PSA is a serine protease and is expressed in both normal luminal epithelial cells of the prostate and prostate cancer cells. PSA transcription is stimulated by androgens via activation of androgen receptor (AR) mediated signaling (Kim and Coetzee, 2004). Recent studies have indicated that the AR level in prostate cancer stem-like cells is significantly lower than that in prostate cancer non-stem cells, and that the stem-like cells do not respond to DHT stimulation (Germann et al., 2012; Zhifang et al., 2015). Subsequently, the enforced expression of AR in prostate cancer stem-like cells caused a restoration of the DHT-induced response (Zhifang et al., 2015). Further, a recent report indicated that the activation of AR-governed signaling suppressed the stemness in prostate cancer stem cells via the inactivation of Akt signaling (Di Zazzo et al., 2016). Our study demonstrated that PSA levels in LNCaP derived stem-like cells were DHT stimulation insensitive and had a limited response to flutamide therapy. However, exposure to BBI restored their DHT responsiveness and significantly decreased their level of stemness. Therefore, we propose that BBI-induced activation of AR may relate to the reduction of stemness in LNCaP stem-like cells, and a restoration in both DHT and flutamide sensitivity.

We observed that BBI treatment suppressed DHT-mediated induction of PSA in LNCaP parental cells. The reason for the different response to BBI between LNCaP stem-like cells and parental cells is unclear at present. Other studies have reported Cx43 expression in LNCaP cells by BBI treatment and enforced expression resulted in cell growth inhibition with cellular differentiation and reduced tumorigenicity (Mehta et al., 1999; Tang et al., 2009). Since cell-cell communication is important in homeostatic control of normal cell growth and differentiation, and alteration in gap-junction-mediated intercellular communication (GJIC) has been found to be related to carcinogenesis (Yamasaki et al., 1999), the induction of Cx43-mediated GJIC by BBI may induce a variable differentiation status in LNCaP stem-like cells and parental cells. In general, prostate cancer stem cells have a relatively undifferentiated status compared to their parental cells (Adamowicz et al., 2017). Thus, when LNCaP stem-like cells and parental cells are treated with BBI, the former and the latter may have a different resultant differentiation status, i.e., LNCaP parental cells and non-tumorigenic prostate epithelial cells, respectively. Overall, differences in BBI treatment-induced differentiation status between the two cells after BBI treatment may contribute to differences in DHT-mediated stimulation of PSA.

In conclusion, BBI has potential as an agent to induce responses by prostate cancer stem-like cells to androgen deprivation therapy.

Acknowledgements    This research was supported by a research grant (Inoue Enryo Memorial Foundation of Toyo University).

Abbreviations

BBI

Bowman-Birk inhibitor

DHT

androgen

PSA

prostate-specific antigen

AR

androgen receptor

References

•  Adamowicz,  J.,  Pakravan,  K.,  Bakhshinejad,  B.,  Drewa,  T., and  Babashah,  S. (2017). Prostate cancer stem cells: from theory to practice. Scand. J. Urol., 51, 95-106.
•  Di Zazzo,  E.,  Galasso,  G.,  Giovannelli,  P.,  Di Donato,  M.,  Di Santi,  A.,  Cernera,  G.,  Rossi,  V.,  Abbondanza,  C.,  Moncharmont,  B.,  Sinisi,  A.A.,  Castoria,  G., and  Migliaccio,  A. (2016). Prostate cancer stem cells: the role of androgen and estrogen receptors. Oncotarget, 7, 193-208.
•  Germann,  M.,  Wetterwald,  A.,  Guzmán-Ramirez,  N.,  van der Pluijm,  G.,  Culig,  Z.,  Cecchini,  M.G.,  Williams,  E.D., and  Thalmann,  G.N. (2012). Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer. Stem Cells, 30, 1076-086.
•  Goktas,  S. and  Crawford,  E.D. (1999). Optimal hormonal therapy for advanced prostatic carcinoma. Semin. Oncol., 26, 162-173.
•  Hossain,  M.Z.,  Jagdale,  A.B.,  Ao,  P.,  LeCiel,  C.,  Huang,  R.P., and  Boynton,  A.L. (1999). Impaired expression and posttranslational processing of connexin43 and downregulation of gap junctional communication in neoplastic human prostate cells. Prostate, 38, 55-59.
•  Jin,  X.,  Jin,  X., and  Kim,  H. (2017). Cancer stem cells and differentiation therapy. Tumour Biol., 39, 1010428317729933
•  Kaneko,  S.,  Sato,  C.,  Ota,  M., and  Yano  T. (2017). Preventive effect of Bowman-Birk inhibitor from soybeans on prostate cancer based on regulation of prostate cancer stem cells. J. Med. Use Func. Food, 11, 216 (in Japanese)
•  Kaneko,  S.,  Sato,  C.,  Shiozawa,  N.,  Sato,  A.,  Sato,  H.,  Virgona,  N., and  Yano,  T. (2018). Suppressive effect of delta-tocotrienol on hypoxia adaptation of prostate cancer stem-like cells. Anticancer Res., 38, 1391-1399.
•  Kaneko,  S.,  Yamazaki,  T.,  Kohno,  K.,  Sato,  A.,  Kato,  K., and  Yano,  T. (2019). Combination effect of Bowman-Birk inhibitor and α-tocopheryl succinate on prostate cancer stem-like cells. J. Nutr. Sci. Vitaminol (Tokyo), 65, 272-277.
•  Kennedy,  A.R. and  Wan,  X.S. (2002). Effects of Bowman-Birk inhibitor on growth, invasion, and clonogenic survival of human prostate epithelial cells and prostate cancer cells. The Prostate, 50, 125-133.
•  Kim,  J. and  Coetzee,  G.A. (2004). Prostate specific antigen gene regulation by androgen receptor. J. Cell Biochem., 93, 233-241.
•  Lin,  C.J.,  Lo,  U.G., and  Hsieh,  J.T. (2019). The regulatory pathways leading to stem-like cells underlie prostate cancer progression. Asian J. Androl., 21, 233-240.
•  Maitland,  N.J.,  Frame,  F.M.,  Polson,  E.S.,  Lewis,  J.L., and  Collins,  A.T. (2011). Prostate cancer stem cells: do they have a basal or luminal phenotype? Horm. Cancer, 2, 47-61.
•  Mehta,  P.P.,  Perez-Stable,  C.,  Nadji,  M.,  Mian,  M.,  Asotra,  K., and  Roos,  B.A. (1999). Suppression of human prostate cancer cell growth by forced expression of connexin genes. Dev. Genet., 24, 91-110.
•  Ojo,  D.,  Lin,  X.,  Wong,  N.,  Gu,  Y., and  Tang,  D. (2015). Prostate cancer stem-like cells contribute to the development of castration-resistant prostate cancer. Cancers (Basel), 7, 2290-2308.
•  Santer,  F.R.,  Erb,  H.H., and  McNeill,  R.V. (2015). Therapy escape mechanisms in the malignant prostate. Semin. Cancer Biol., 35, 133-144.
•  Siegel,  R.,  Ward,  E.,  Brawley,  O., and  Jemal,  A. (2011). Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA. Cancer J. Clin., 61, 212-236.
•  Tang,  M.,  Asamoto,  M.,  Ogawa,  K.,  Naiki-Ito,  A.,  Sato.  S.,  Takahashi.  S., and  Shirai,  T. (2009). Induction of apoptosis in the LNCaP human prostate carcinoma cell line and prostate adenocarcinomas of SV40T antigen transgenic rats by the Bowman-Birk inhibitor. Pathol. Int., 59, 790-796.
•  Wan,  X.S.,  Zhou,  Z.,  Steele,  V.,  Kopelovich,  L., and  Kennedy,  A.R. (2003). Establishment and characterization of sublines of LNCaP human prostate cancer cells. Oncol Rep., 10, 1569-1575.
•  Yamasaki,  H.,  Omori,  Y.,  Zaidan-Dagli,  M.,  Mironov,  N.,  Mesnil,  M., and  Krutovskikh  V. (1999). Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis. Cancer Detect. Prev., 23, 273-279.
•  Zhang,  W.,  Meng,  Y.,  Liu,  N.,  Wen,  X.F., and  Yang,  T. (2015). Insights into chemoresistance of prostate cancer. Int. J. Biol. Sci., 11, 1160-1170.
•  Zhang,  Y.,  Cabarcas,  S.M.,  Zheng,  J.I.,  Sun,  L.,  Mathews,  L.A.,  Zhang,  X.,  Lin,  H., and  Farrar,  W.L. (2016). Cryptotanshinone targets tumor-initiating cells through down-regulation of stemness genes expression. Oncol Lett., 11, 3803-3812.
•  Zhifang,  M.,  Zhaoliang,  X.,  Dongwen,  W.,  Liang,  Y.,  Rui,  T., and  Bin,  H. (2013). The different role of androgen receptor in the stem/progenitor cells and none stem/progenitor cells isolated from human prostate cancer cell line. Chin. J. Clinicians, 23, 10788-10792.
•  Zhifang,  M.,  Liang,  W.,  Wei,  Z.,  Bin,  H.,  Rui,  T.,  Nan,  W., and  Shuhai,  Z. (2015). The androgen receptor plays a suppressive role in epithelial-mesenchymal transition of human prostate cancer stem progenitor cells. BMC Biochem., 16, 13.