Proscillaridin A slows the prostate cancer progression through triggering the activation of endoplasmic reticulum stress

ABSTRACT

Prostate cancer (PCa) is the second commonly diagnosed malignancy in men over the world. Although androgen deprivation therapy for advanced PCa patients has significantly improved their survival, the majority of these patients eventually develop castration-resistant prostate cancer (CRPC). Proscillaridin A (Pro A), a cardiac glycoside that is clinically used to treat various heart failure diseases, has been reported to have anticancer activity in several cancers. However, whether Pro A exerts an inhibitory effect on PCa progression remains unknown. In this study, we determined possible antitumor effects of Pro A on PCa cells and demonstrated the following: firstly, Pro A selectively inhibited androgen-independent PCa (including PC3 and DU145) cell growth and induced cell apoptosis in vitro; secondly, Pro A significantly decreased cell motility and invasion of androgen-independent PCa cells; thirdly, Pro A enhanced the sensitivity of PCa cells to docetaxel; fourthly, Pro A significantly inhibited the growth of PCa xenografts in vivo and patient-derived organoids (PDO). In addition, RNA-sequencing analysis revealed that the antitumor effects of Pro A on androgen-independent PCa appeared to be achieved via driving the activation of endoplasmic reticulum stress. The antitumor effects of Pro A could be ameliorated by reactive oxygen species scavenger and ER stress inhibitors. Therefore, these data suggest that Pro A may provide a potential therapeutic option for the treatment of PCa, particularly CRPC.

Introduction

Prostate cancer is the second commonly diagnosed cancer in males worldwide. According to the global cancer statistics in 2018, there will be estimated 1,276,106 new prostate cancer cases [1]. At present, the treatments for PCa patients are diverse, ranging from surveillance, radiotherapy, prostatectomy, and androgen deprivation therapy (ADT) to chemotherapy [2,3]. Among them, ADT is still the standard treatment for the PCa patients diagnosed at advanced tumor stages. However, the majority of these patients will eventually become castration-resistant prostate cancer (CRPC) [4]. Although several new treatment options for metastatic CRPC patients have been recently approved by the Food and Drug Administration (FDA), including docetaxel, cabazitaxel, abiraterone, enzalutamide and sipuleucel-T, the prognosis of CRPC patients is still poor and worrisome, as the most advanced PCa patients will develop chemoresistance, which eventually leads to deaths. Therefore, it is desirable to identify novel effective agents with little side effects to improve the prognosis of these patients.

An increased number of researchers and clinicians have now paid attention to natural agents and their derivatives as an alternative, because they serve as a safe, efficacious and inexpensive source of new drugs. From 1950 (earliest so far identified) to 2010, 48.6% antitumor drugs approved by the U.S. FDA were either natural products or extract from them [5]. Cardiac glycosides, a large family of natural compounds, can inhibit the sodium (Na+)/potassium (K+)-ATPase pump and enhance cardiac contractility; so, they have long been clinically used for the treatment of cardiac failures and cardiac arrhythmias. In 1979, scientists showed for the first report that cardiac glycosides exert anticancer effects on breast cancer [6]. Subsequently, the antitumor effect of cardiac glycosides has been also found on the various types of cancers. Proscillaridin A (Pro A), one of the cardiac glycosides, extracted from Urginea maritima, has been studied in several cancers. For example, Zhang et al. shows that Pro A and Digoxin reduce tumor growth by inhibiting HIF-1α synthesis [7]. Pro A also inhibits glioblastoma growth through GSK3β activation and alteration of microtubule dynamics [8]. Furthermore, Pro A suppresses non-small-cell lung cancer tumor growth by increasing calcium-induced DR4 expression and inducing cell apoptosis [9]. However, the possible functional role of Pro A in PCa progression remains unknown. More specifically, whether Pro A has a vital role in PCa growth and metastasis has not been well investigated.

In this study, we set forth to determine possible antitumor effects of Pro A on PCa cells, particularly the androgen-independent PCa cells by using not only cell proliferation, wound-healing, migration and invasion assays in vitro, but also androgen-independent PC3 xenografts in vivo and patient-derived organoids (PDO). We also performed RNA-sequencing, RT-PCR and Western blot analyses to explore possible mechanisms responsible for the Pro A’s effects.

Materials and methods

Cell and organoid culture

All cell lines were obtained from American Type Culture Collection. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 ug/mL streptomycin. Fresh PCa tissues were obtained from patients undergoing radical prostatectomy at the Department of Urology, Ren Ji Hospital (Shanghai, China). All samples were used with informed consent of the patients. After minced with scissors, tissues were enzymatically digested with collagenase type I for 4 ~ 5 hours and subsequently with TryLE for 30 min. The suspension was filtered through a 40 um cell strainer, then spun down and resuspended with culture medium/Matrigel (1:1) and plated into 48-well plates. The plates were placed in an incubator for 30 ~ 60 min to solidify the mixture. Then culture medium was added into each well. The organoid culture medium was made by using DMEM/f12 medium supplemented with 10 ng/mL epidermal growth factor, 10 μM Y-27632, 5% charcoal-stripped FBS (Gibco no. 12676), and 10 nM DHT. When the organoids were cultured for 5 ~ 7 days, they were treated with 2.5 nM Pro A or vehicle for 4 days. The medium was refreshed every 2 days.

Cell viability assay

PCa cells were seeded in 96-well plates (4,000 cells/well). After 24 hours, cells were treated with indicated concentrations of Pro A in the presence or absence of increasing concentrations of docetaxel, NAC, 4-PBA or TUDCA. The cell viability was measured by Cell Counting Kit-8(CCK-8) (Dojindo) according to manufacturer’s instructions after 24 or 48 hours treatment.

Colony formation assay

PC3 and DU145 cells were plated in 6-well plates (500 cells/well) in triplicates and treated with 0, 2.5, and 5 nM Pro A for 24 hours. After cultured for another 12 days, cell clones were fixed in 4% paraformaldehyde and stained with crystal violet for 20 min at room temperature. Afterward, colonies were counted under a microscopy.

Cell cycle distribution and apoptosis assays

After PCa cells were treated with 0 and 2.5 nM Pro A for 48 hours, they were collected and processed for cell cycle or apoptosis analysis. For cell cycle analysis, following cells were fixed with 70% ethyl alcohol and stained with propidium iodide for 30 min at 4°C in dark, they were analyzed by flow cytometry. For apoptosis analysis, cells were stained with both APC-Annexin V and 7-AAD according to manufacturer’s instructions, and analyzed by flow cytometry. All flow cytometry data were analyzed using the FlowJo software.

TUNEL staining assay

PCa cells were seeded at a 30 ~ 40% confluency in Chamber coverslips in 24-well plates. After 24 hours, the cells were treated with either vehicle or Pro A for 48 hours. Then, coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 for 10 min, and blocked in Equilibration Buffer for 30 min at room temperature. Slides were incubated in TdT buffer for 1 hour in 37°C. After they washed three times with PBS, the slides were observed under a fluorescent microscope.

Wound-healing assay

As Pro A inhibits PCa cells proliferation in a dose-dependent manner, the dose of Pro A selected for performing wound-healing assay was as low as 1 nM. After pretreated with Pro A or DMSO for 12 hours, cells were seeded into 6-well culture plates. When the cells reached 80 ~ 90% confluence, the culture medium was replaced with DMEM without serum to minimize cell proliferation for 24 hours. Then, a pipette tip was used to make a straight scratch. The cell scratch was examined and photographed under a light microscope at 0, 24 and 48 hours.

In vitro migration and invasion assays

The migration and invasion of PCa cells were examined using transwell plates (6.5 mm insert, 8.0 μm pores) coated without (migration) or with (invasion) matrigel. Generally, after pretreated with vehicle or 1 nM Pro A for 12 hours, 10⁴ PCa cells resuspended with 100 μL serum-free medium were plated in the upper chamber per well. Five hundred microliters of 10% FBS-supplemented medium was added to the lower chamber. Pro A or vehicle was added to the culture medium at indicated concentrations. After 8 hours, the migrated cells at the chamber bottom were fixed with 4% PFA, stained with crystal violent and examined under a microscope. The procedure of invasion assay was essentially the same as the migration assay. Only the bottom of up-class was coated with 20 µL matrigel (1:7 diluted with DMEM), and 24 hours were needed to guarantee the invasion of cells. The average number of migrating or invading cells was determined by randomly selecting three visual fields with triplicate cultures.

Immunofluorescence analysis

After permeabilized with 0.3% Triton X-100 for 10 min, the sections were blocked in 10% donkey serum for 1 hour at room temperature. Then, they were incubated with primary antibodies overnight at 4°C. The slides were washed with PBS for three times, and incubated with secondary antibodies (1:400, Thermo Fisher Scientific) for 1 hour at room temperature in dark. Sections were counterstained with DAPI, and examined using a microscope.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from cells using Trizol reagent (Invitrogen) according to the manufacturer’s instruction. Then, total cDNA was synthesized through reverse transcription using a Prime-Script RT kit (Takara) and amplified with SYBR Green Realtime PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific). β-actin was used to serve as a housekeeping gene control. Detailed information of primers is described in Table 1. Data were analyzed from three independent experiments.

Western blotting

The cells were lysed in RIPA buffer (Rockford, Prod#89901) supplemented with protease inhibitors (Thermo Fisher Scientific). The protein concentration of each sample was measured by the BCA Protein Assay Kit (Thermo, Prod#23227) respectively. Then, 20 micrograms of protein lysed buffer were subjected to a 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, #IPVH00010). After blocked with 5% nonfat milk for 1 hour at room temperature, membranes were incubated with specific primary antibody at 4°C overnight (GADD34, Proteintech, cat: 10449-1-AP; CHOP, Proteintech, cat: 15204-1-AP; TRIB3, Proteintech, cat: 13300-1-AP). After being washed with TBST for three times, membranes were incubated with HRP-conjugated secondary antibody (Cell Signaling Technology). Then, proteins were photographed using ECL detection reagents (Thermo Fisher Scientific).

Animal experiments

Three millions of PC3 cells suspended in 100 μL of PBS/Matrigel (1:1) were injected subcutaneously into the flanks of five-week-old BALB/c nude mice. When the volume of the tumor (calculated using the following formula: 0.5 × tumor length × tumor width²) reached 40 ~ 50 mm³, the mice were randomly separated into two groups. The two groups were injected intraperitoneally either with vehicle (DMSO in water) or with Pro A (5 mg/kg) every 2 days. The tumor volume was measured with a caliper every 2 days. At the end of the experiment, the animals were euthanized and the tumors were weighed after careful resection.

Gene set enrichment analysis (GSEA)

To interpret the function of regulated genes after Pro A treatment, GSEA (version 3.0) was performed using the 50 cancer hallmark gene sets.

Statistical analysis

All statistical diagrams were generated using GraphPad 5.0. Differences between groups were analyzed by a two-tailed Student’s t-test and presented as mean ± standard error mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001.

Results

Pro A selectively impedes androgen-independent PCa cell growth and induces cell apoptosis in vitro

To explore possible effects of Pro A on the growth of PCa cells, six commonly studied PCa lines including PC3, DU145, 22RV1, C4-2, LNcaP and LAPC4 cells were treated with various doses of Pro A for 48 hours and were evaluated using CCK-8 assays. The calculated IC50 values ranged from 2.127 to 10.99 nM. As shown in Figure 2(a), androgen-independent PCa cell lines (PC3 and DU145) were more sensitive than androgen-dependent LNcaP, and LAPC4 cells to Pro A compared with the other cell lines (Figure 2(a)). Thus, we chose the most susceptible PC3 and DU145 cell lines to carry out subsequent function experiments. To rule out the possibility of general toxicity effect of Pro A, we used mouse prostate epithelium cell derived organoid model as control cultures. At the similarity concentration, no apparent effect was observed (Figure 2(b)), thus suggesting that the effects of Pro A on PC3 and DU145 cells are specific. Furthermore, Pro A suppressed the proliferation of PC3 and DU145 cells in dose- and time-dependent manners (Figure 2(c)). Similar to CCK8 assays, Pro A remarkably inhibited PCa cells viability in colony-formation experiments (Figure 2(d)). These results indicate that Pro A exhibits anti-proliferative activity against PCa cells in vitro. In addition, consistent with the colony-formation assays, flow cytometry analysis using Annexin V-APC/7-AAD, also demonstrated that Pro A significantly induced PCa cells apoptosis (Figure 2(e)). TUNEL assays confirmed that Pro A significantly enhanced DNA fragmentation and PCa cells death (Figure 2(f)). Next, to determine whether Pro A influences PCa cell cycle, we examined the effect of Pro A on cell cycle progression in PC3 cells using propidium iodide staining. Cell cycle analysis demonstrated that there were more cells residing at the G1 phase and fewer cells at the S and G2 phases in Pro A treated PC3 cells compared with the control cells (Figure 2(g)). Taken together, these results suggest that Pro A has a potent antitumor effect in PCa cells.

Figure 2.

Pro A suppresses PCa cell metastatic potential. Wound-healing assay with representative images over time of PC3 (a) and DU145 (b) cells pretreated with 1 nM Pro A for 12 hours were shown. Bars = 100 μm. Migration (c) and invasion (d) of PC3 and DU145 cells exposed to Veh or Pro A. Bars = 100 μm. (Error bars represent SEM; *, p < 0.05; **, p < 0.01; *** p < 0.001; n = 3).

Figure 1.

Pro A selectively impedes androgen-independent PCa cell growth and induces cell apoptosis in vitro. (a) The viability of several PCa cell lines after treated with different concentration of Pro A for 48 hours. IC50 values were calculated and listed on the right. (b) Pro A had little effect on the proliferation of mouse prostate epithelium cell derived organoids. Bars = 100 μm. (c) Viability analysis of PC3 and DU145 cells treated with indicated concentrations of Pro A for different times. (d) Colonies formation of PC3 and DU145 cells treated with the indicated concentrations of Pro A for 24 hours. PC3 and DU145 cells were treated with 2.5 nM Pro A for 48 hours. (e) The above cells were subjected to apoptosis analysis using Annexin V/7-AAD staining by flow cytometry, the apoptotic rate represented the total percentage of early and late apoptosis, and tunel staining by fluorescent microscope (f) cell cycle analysis by PI staining (g). (Error bars represent SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 3).

Pro A suppresses metastatic potential of PCa cells

Invasion and metastasis are commonly used as indicators of the development and progression of PCa, which suggests an extremely poor prognosis [10]. We next wanted to explore whether Pro A could affect the metastatic potential of PCa cells. We performed wound-healing, migration and invasion assays in vitro. We pre-incubated PC3 and DU145 cells with Pro A at a dose of 1 nM for 12 hours. An equal number of cells were reseeded in the up-chamber in trans-well assays and then migration or invasion were assessed within 24 hours. As shown in Figure 2, wound-healing assay indicated that Pro A significantly inhibited the migratory potential of PCa cells. Consistent with wound-healing assay, transwell assays also showed that Pro A significantly reduced cell migration and invasion capability of PC3 and DU145 cells (Figure 2). Collectively, these results suggest that Pro A inhibits PCa cell migration and invasion in vitro.

Pro A reduces the growth of PCa cell in nude mice and PDO

In order to further evaluate Pro A’s antitumor effect on PCa in vivo, we established subcutaneous xenografts in a nude mice model. PC3 cells were subcutaneously injected into nude mice (3.0 × 10⁶ cells/mouse). When the tumor volume reached approximately 50 mm³, the mice were randomly separated into two groups (control and experimental, seven mice/group). The two groups received neither 5 mg/kg of Pro A nor the same volume of vehicle every 2 days. Throughout the study, body weight and tumor volume were recorded every 2 days. At the end of the study, the Pro A treatment group displayed significantly smaller tumor volumes then the control group (p < 0.001) (Figure 3(a)). Moreover, the mean tumor weight was also significantly lower in the mice treated with Pro A (p < 0.001) (Figure 3(b,c)). The results suggest that Pro A treatment group significantly inhibited the growth of PC3 xenografts compared to the control group. In addition, the average body weights of the control group mice were slightly reduced relative to the experimental group at the end of the study (data not show). In contrast to the tumor xenografts of control group, a reduction in Ki-67 expression was observed in the tumor xenografts derived from Pro A treatment group (Figure 3(d)). Furthermore, we verified Pro A’s antitumor effect on PCa in a PDO model. As shown in Figure 3(e), Pro A also led to a significantly fewer and smaller size of the PDO (Figure 3(e)).

Figure 3.

Pro A reduces the growth of PCa cell in nude mice and patient-derived-organoids. Tumor volumes (a), tumor weights (b) and the picture of tumors (c) were shown (Pro A group: n = 7, Veh group: n = 7). (d) Ki-67 stain was performed to analyze proliferation in veh- and Pro A-treated tumors shown in Figure 3c. Pro A repressed the proliferation of patient-derived-organoids (e). (f) PC3 and DU145 cells were treated with Pro A and docetaxel as indicated combinational concentrations. The cell viability was measured via cell counting kit-8 (n = 6). (Error bars represent SEM; *, p < 0.05; **, p < 0.01; *** p < 0.001).

Given that docetaxel is the first-line chemotherapy in advanced CRPC patients with a lot of side effects for CRPC patients and that Pro A selectively suppresses androgen-independent PCa cell growth and induces cell apoptosis in vitro and in vivo, we wondered whether Pro A enhances the cytotoxicity of docetaxel in PCa cells. By performing a cell viability experiment using CCK8 assays, we found showed that Pro A significantly augmented the effect of docetaxel both in PC3 and DU145 cells (Figure 3(f)). Collectively, these results demonstrate that Pro A possesses an antitumor effect in in vivo PC3 xenograft and PDO models, and significantly augments the effect of docetaxel in PCa cells.

The inhibition effects of Pro A on PCa cell growth are achieved through driving the activation of ER stress

To further elucidate the molecular mechanism underlying the inhibition effects of Pro A on PCa cell growth, we performed RNA-sequencing analysis of PC3 cells treated with Pro A or vehicle for 36 hours to identify specific signaling pathways. As expected, GSEA of the transcriptome data showed that prominent alterations about the cell death and proliferation processes were significantly enriched in Pro A-treated cells, which include activation of the apoptosis, and unfolded protein response (UPR signaling), and up-regulation of TP53 signaling and TNF-α/NF-kB signaling (Figure 4(a) & Supplementary Figure(a)). In order to verify this observation, we chose several key genes implicated in the processes of the cell apoptosis and UPR signaling. Real-time PCR assays demonstrated that the changes of the above-mentioned key genes were consistent with the RNA-seq data, especially the pivotal genes promoting cell death in UPR signaling (such as ATF4, CHOP, GADD34, and TRIB3) were remarkably upregulated in the PC3 and DU145 cells (Figure 4(b) & Supplementary Figure(b)). Western blot assays also confirmed that the protein levels of CHOP, GADD34 and TRIB3 were significantly increased in the Pro A treated PC3 and DU145 cells compared to the control cells (Figure 4(c)). Similarly, immunofluorescence assays also supported the above outcome (Figure 4(d)). Notably, a significant increase in cell viability was observed after a combined treatment of increasing concentrations of ROS scavenger (NAC) and ER stress inhibitors (4-PBA and TUDCA) (Figure 4(e) & Supplementary Figure(c)).Taken together, these results indicate that the Pro A’s antitumor effects on PCa are mediated through driving the activation of the ER stress and UPR signaling.

Figure 4.

Pro A inhibited PCa cell growth through driving the activation of UPR signaling. (a) GSEA analysis of RNA-seq data indicates increased expression of genes in the UPR, apoptosis, TNF and P53 signaling. (b) Realtime-PCR demonstrated the activation of UPR signaling in PC3 cell. Pro A-treated PC3 and DU145 cells display a higher expression of TRIB3, GADD34 and CHOP by immunoblotting (c) and immunofluorescence (d). Bars = 100 μm. (e) PC3 and DU145 cells were incubated with 10nM Pro A in the presence or absence of increasing concentrations of NAC, 4-PBA. Cell viability was measured by the CCK8 assay and results are reported as percent relative to untreated cells. (Error bars represent SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; n = 3).

Discussion

Although cardiac glycosides such as Pro A have been reported to have an anticancer activity in several types of cancers [7,11], their potential effect on PCa is undetermined. The present study demonstrates clearly that Pro A also has an anticancer effect on PCa cells, including inhibition of PCa cell proliferation, migration, and invasion and induction of apoptosis in vitro. In addition, we also show that Pro A possesses an inhibitory effect in PC3 xenografts and PDO models. Organoid is one of novels in vitro 3D culture technologies, which is believed to recapitulate better the disease’s properties and serves as a more physiological disease model [12]. Furthermore, Pro A shows an enhanced inhibitory effect on PCa cell growth when combined with docetaxel. Therefore, our findings were consistent with previous studies on other types of cancers, including glioblastoma, non-small-cell lung cancer and lung adenocarcinoma, supporting a notion that Pro A has a potential therapeutic value for the treatment of advanced PCa, especially for CRPC.

It is important to point out that our current findings that Pro A selectively inhibits androgen-independent PCa cell growth are therapeutically significance. ADT is the main treatment for the advanced PCa patients, but the majority of these patients will eventually develop into CRPC. Furthermore, our data showed that Pro A significantly augments the effect of docetaxel in the PCa cells. Docetaxel has been approved by the FDA to treat metastatic castration-resistant PCa patients [13]. The present finding suggests that Pro A might have a vital role in facilitating determination of docetaxel treatment outcome in PCa and points to potential strategies for improving docetaxel efficacy.

The current study provides mechanistic insight into the potential mechanism for the tumor growth-inhibitory effects of Pro A. Our data show that Pro A inhibits PCa cell growth and induces cell apoptosis through driving the activation of the UPR signaling. Our real-time PCR and western blot assays also provide supporting evidence that Pro A significantly upregulates expression levels of ATF4, CHOP, GADD34, and TRIB3 in the PC3 and DU145 cells, which are pivotal genes promoting cell death in UPR signaling pathway. Furthermore, the antitumor effects of Pro A can be significantly ameliorated by ER stress inhibitors (4-PBA and TUDCA) and a ROS scavenger (NAC). The endoplasmic reticulum (ER) has been well-known to play a vital role in many essential biological functions, such as the synthesis, folding and translocation of most secretory and membrane proteins. Multiple stimuli, including drugs, hypoxia, oxidative stress, as well as altered glycosylation, can perturb the ER function and cause the accumulation of unfolded proteins, which can be called as ER stress [14]. In addition, the UPR pathway is activated to counterbalance this stress and restore the homeostasis of cells [15]. However, when the events are protracted and drastic, the ER stress will induce cell death [16]. Reactive oxygen species is one of main mechanism by which the UPR contributes to cell death [17,18]. Various reports have indicated that the ER stress participates in the process of oxidative stress, apoptosis and plays an important role in drug-induced toxicity. In this regard, our results have discovered a novel mechanism of Pro A for its antitumor activity, which is different from what have been reported previously for other types of cancers. Whether such as mechanism via activation of the UPR signaling is also responsible for the antitumor effects of Pro on other types of cancers awaits further in-depth studies.

In summary, we for the first time demonstrate that Pro A significantly inhibits PCa growth, migration and invasion. Pro A reduces the growth of PCa cells in nude mice and PDO. Furthermore, the antitumor effects by Pro A on PCa cells are achieved through driving the activation of UPR signaling. These findings also indicate a therapeutic potential of Pro A for the treatment of PCa, particularly CRPC in a combination with docetaxel.

Funding Statement

The study is supported by funds to W-Q Gao from the National Key R&D Program of China (2017YFA0102900), the National Natural Science Foundation of China (NSFC, 81630073 and 81872406), the Science and Technology Commission of Shanghai Municipality (16JC1405700), the Education Commission of Shanghai Municipality (for the High Peak IV subject on Stem Cells and Translational Medicine Research) and the KC Wong foundation, and by funds to YF Liu from the NSFC (81572832 and 81874174), Shanghai Rising-Star Program (18QA1402600), Shanghai Municipal Commission of Health and Family Planning (2018YQ12), and School of Medicine, Shanghai Jiao Tong University (Excellent Youth Scholar Initiation Grant 17XJ11015 and 18XJ11006).

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.