Aspalathus linearis suppresses cell survival and proliferation of enzalutamide-resistant prostate cancer cells via inhibition of c-Myc and stability of androgen receptor.

Enzalutamide, a nonsteroidal antiandrogen, significantly prolonged the survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patients receiving enzalutamide frequently develop drug resistance. Rooibos (Aspalathus linearis) is a shrub-like leguminous fynbos plant endemic to the Cedarberg Mountains area in South Africa. We evaluated the possibility of using a pharmaceutical-grade green rooibos extract (GRT, containing 12.78% aspalathin) to suppress the proliferation and survival of enzalutamide-resistant prostate cancer (PCa) cells. Treatment with GRT dose-dependently suppressed the proliferation, survival, and colony formation of enzalutamide-resistant C4-2 MDV3100r cells and PC-3 cells. Non-cancerous human cells were more resistant to GRT treatment. GRT suppressed the expression of proteins involved in phosphoinositide 3-kinase (PI3K)-Akt signaling, androgen receptor (AR), phospho-AR (Ser81), cyclin-dependent kinase 1 (Cdk1), c-Myc and Bcl-2 but increased the expression of apoptotic proteins. Overexpression of c-Myc antagonized the suppressive effects of GRT, while knockdown of c-Myc increased the sensitivity of PCa cells to GRT treatment. Expression level of c-Myc correlated to resistance of PCa cells to GRT treatment. Additionally, immunofluorescence microscopy demonstrated that GRT reduced the abundance of AR proteins both in nucleus and cytoplasm. Treatment with cycloheximide revealed that GRT reduced the stability of AR. GRT suppressed protein expression of AR and AR's downstream target prostate specific antigen (PSA) in C4-2 MDV3100r cells. Interestingly, we observed that AR proteins accumulate in nucleus and PSA expression is activated in the AR-positive enzalutamide-resistant PCa cells even in the absence of androgen. Our results suggested that GRT treatment suppressed the cell proliferation and survival of enzalutamide-resistant PCa cells via inhibition of c-Myc, induction of apoptosis, as well as the suppression of expression, signaling and stability of AR. GRT is a potential adjuvant therapeutic agent for enzalutamide-resistant PCa.

Introduction

Prostate cancer (PCa) is the second most frequently diagnosed cancer of men and the fifth most common cancer overall in the world. Nearly 1,200,000 new cases were diagnosed every year in the world. More than 80% of patients died from PCa developed bone metastases. Compared to localized PCa, metastatic PCa has a poorer prognosis and higher mortality rate, with a 5-year survival less than 30%. Androgen deprivation therapy (ADT) is the standard treatment for metastatic prostate cancer (PCa). However, a majority of patients receiving ADT eventually develop castration-resistant prostate cancer (CRPC) within 1–3 years. Enzalutamide (formerly called MDV3100) is a nonsteroidal antiandrogen introduced in 2012 for treatment of metastatic CRPC. Enzalutamide significantly prolonged the survival of patients with metastatic CRPC. The enzalutamide treatment group has a longer median overall survival (18.4 vs. 13.6 months), a greater decrease in prostate-specific antigen (PSA) level by 50% or more (54% vs. 2%), and a longer time to radiographic progression-free survival (8.3 vs. 2.9 months) as compared to the placebo group [1]. However, most patients receiving enzalutamide treatment develop drug resistance after a median of 18 months [2]. New therapeutic agents are therefore needed.

Rooibos (Aspalathus linearis) is a shrub-like leguminous fynbos plant endemic to the Cedarberg Mountains area in the Western Cape Province of South Africa. Rooibos is becoming more and more popular and is consumed as daily beverage world-wide [3, 4]. The traditional rooibos tea product is produced by fermentation (oxidation) of the chopped grinded plant material with the leaf color changing from green to red-brown. Recently, more and more attentions are paid to preserve the antioxidant ingredient in rooibos. The “unfermented” or so-called green rooibos is produced by limiting oxidation of polyphenols during processing [3, 4]. Rooibos tea does not contain caffeine but it contains various types of polyphenols. Major compounds of rooibos include dihydrochalcone, aspalathin, orientin and isoorientin [5]. These compounds are C-glucosides which are poorly absorbed but exhibit strong anti-oxidative activity [6]. We previously reported that treatment with a pharmaceutical-grade green rooibos extract (GRT, containing 12.78% aspalathin) suppresses Akt signaling and therefore inhibits cell proliferation, cell survival, and tumor growth in CRPC cell line LNCaP-104-R1 [7]. Additionally, we observed that GRT treatment suppressed the migration and invasion of CRPC cells via inhibition of YAP signaling and paxillin [8]. PI3K/AKT signaling has been reported to play an essential role in the proliferation and survival of enzalutamide-resistant PCa cells [9], we therefore investigated if treatment with GRT can inhibit the proliferation and survival of enzalutamide-resistant PCa cells.

Materials and methods

Rooibos extract GRT

A pharmaceutical-grade aspalathin-rich green rooibos extract (GRTTM) was used in all experimental work from the same batch as previously described [7]. The phenolic component has previously been confirmed by high performance liquid chromatography (HPLC) [7] (S1 Fig). The major flavonoids of the extract (comprised 20.63% of the extract) include aspalathin (12.78%), nothofagin (1.97%), isoorientin (1.47%), orientin (1.26%) and bioquercetin (quercetin-3-O-robinobioside; 1.04%). Compounds which are less than 1% include luteoloside, vitexin, isovitexin rutin, hypersoide, isoquercitrin and the phenolic precursor, Z-2-(β-D-glucopyranosyloxy)-3-phenylpropenoic acid. The pinitol and glucose content of the extract were 1.92 and 0.50%, respectively (quantification by GC-MS by Central Analytical Facility, Stellenbosch University, Stellenbosch, South Africa).

Cell culture

LNCaP C4-2 MDV3100r cells were derived from parental androgen-independent LNCaP C4-2 cells (ATCC CRL-3314). LNCaP C4-2 and LNCaP C4-2 MDV3100r cells were maintained in HyClone RPMI-1640 phenol red free medium (Cytiva, Marlborough, MA, USA) supplemented with 10% hormone deprivation FBS (Corning, Corning, NY, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Merck Millipore, Burlington, MA, USA). Medium for C4-2 MDV3100r contained 10 μM enzalutamide plus 20 nM DHEA (dehydroepiandrosterone). PC-3 cells were maintained in HyClone DMEM medium with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml).

Cell proliferation assay and chemicals

LNCaP C4-2, LNCaP C4-2 MDV3100r, PC-3 cells were seeded at a density of 5 × 103 cells/well in 96-well plates with 100 μl culture medium containing 10% FBS and increasing concentration (0, 10, 25, 50, 75, 100 μg/ml) of GRT for 96 h. Relative cell number was analyzed by measuring the DNA content of cell lysates with Hoechst dye 33258-based 96-well proliferation assay (Sigma, St. Louis, MO, USA) as described previously [10]. All readouts were normalized to the average of the control condition in each individual experiment. The experiment was repeated at least three times. Eight wells were used for each condition.

Immunofluorescence staining

Immunofluorescence staining analysis has been described previously [11]. Briefly, LNCaP C4-2 MDV3100r cells were seeded on chamber slide overnight. The cells were treated with GRT for 96 h, washed by cold PBS, then fixed in 4% paraformaldehyde for 15 minutes at room temperature, followed by cold PBS wash for 5 minutes three times. Cells were incubated in blocking buffer (5% BSA, 0.1% triton X-100 in PBS) for an hour at room temperature, then washed by PBS for 5 minutes three times. Cells were incubated with primary antibodies overnight at 4°C. After cold PBS wash for 5 minutes three times, cells were incubated with secondary antibody (FITC) for an hour at room temperature. Following several washes, cells were stained using 4′,6-diamidino-2-phenylindole (DAPI) (5 mg/ml) for 15 minutes at room temperature. After staining, cells were washed, mounted and sealed. Images of the cells were captured at a magnification of 1000× fluorescent microscope with fluorescence secondary antibodies: goat anti mouse 488 (green) and goat anti rabbit 594 (red) (Sigma).

Soft agar colony formation assay

A total of 8,000 LNCaP C4-2 MDV3100r cells were suspended in 0.3% low melting agarose (Lonza) with 10% FBS in DMEM medium and then layered on top of 3 ml of 0.5% low melting agarose plus 10% FBS in DMEM medium in 6 cm dishes. Cells were allowed to grow at 37°C with 5% CO2 for 14 days. The plates were stained with 0.005% crystal violet in 30% ethanol for 6 h.

Western blot analysis

For determining the effect of GRT treatment on protein expression in C4-2 MDV3100r cells, cells were lysed in MCLB (Mammalian Cell Lysis Buffer) containing 50 mM Tris-HCl pH 8.0, 0.5% NP-40, 150 mM NaCl, 5 mM EDTA with Na3VO4, DTT, protease inhibitors and phosphatase inhibitors cocktail. The signal detection was performed using chemiluminescence (ECL) and Prime Western Blotting detection reagent (Fisher Scientific, Pittsburgh, PA, USA). Antibodies against caspase 3, Akt, phospho-Akt (T308), phospho-Akt (S473), CDK1, PDK1, phospho-PDK1 (S241), cyclin D1, PARP were purchased from Cell Signaling (Dancers, MA, USA). Antibodies detecting PI3K α, PI3K β, PI3K γ were purchased from Millipore (Burlington, MA, USA), while antibody against Skp2 was from Santa Cruz (Dallas, TX, USA). Antibody detecting BCL-2 was purchased from BD (Franklin Lakes, NJ, USA), while Novus (Littleton, CO, USA) supplied antibody detecting GAPDH and β-actin. Antibodies against AR and Myc were purchased from Abcam (Cambridge, MA, U.S.A), while antibody detecting phospho-AR (S81) was from Millipore. Relative expression level of proteins was quantified by ImageQuant LAS4000 (GE Healthcare-Biosciences, Pittsburgh, PA).

Comet assay

LNCaP C4-2 MDV3100r cells were seeded overnight in a 6 cm dish with a density of 1×105 cells per well in 5 ml of culture medium. Cells were treated with GRT (dissolved in 60% ethanol) at increasing concentrations (0, 50, 75, 100 μg/mL) for 48 h on the 2nd day. Cells were collected and re-suspended with ice-cold PBS without Mg2+ and Ca2+, followed by mixing with agarose (1:10) and transferred immediately to Comet Slides following the manufacture’s instruction. Comet Slides were kept at 4°C for 15 minutes protected from the light and were carefully transferred to ice-cold lysis buffer (Trevigen, Gaithersburg, MD, USA) at 4°C for 30–60 minutes in the dark. Lysis buffer was then discarded and re-filled with ice-cold alkaline solution at 4°C for 30 min protected from light. The alkaline solution was then decanted from the container and replaced with ice-cold TBE buffer and Comet Slides were immersed twice for 5 minutes. Comet Slides were carefully transferred to an electrophoresis chamber with ice-cold TBE buffer to approximately cover the Comet Slides, and then 35 V applied for 15 minutes. Comet Slides were carefully transferred from the chamber to ice-cold deionized water and immersed three times for 2 minutes. Ice-cold deionized water was then aspirated and replaced with ice-cold 70% ethanol for 5 minutes. After drying, 100 μl Vista Green DNA Dye was added to the agarose on the slide and the images were recorded by fluorescence microscopy.

Knockdown c-Myc with siRNA and c-Myc overexpression

Human c-Myc siRNA (Human c-Myc ON-TARGET plus SMART pool) and randomly scrambled sequence control were purchased from Dharmacon (Lafayette, CO, USA). The transfection procedure was performed using lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s recommended protocol. Cells were seeded at a density of 3x105 cells/well in 2.5ml complete medium in 6-well plates overnight. 40 nM siRNA were used for scramble or c-Myc knockdown. For overexpression of c-Myc, PC-3 cells were transfected with c-Myc plasmid, a generous gift from Shutsung Liao’s lab at the University of Chicago as previously described [12]. The PLNCX2 plasmid was used as controls for c-Myc overexpression. PolyJet™ in vitro DNA transfection reagent was used for the transfection (SL100688, SigmaGen Laboratories, Rockville, MD, USA). Western blotting was used to confirm the knockdown or overexpression of c-Myc.

Statistical analysis

Data are represented as the mean ± SD from three independent experiments. Student’s T-test was performed to test statistical significance. P < 0.05 was considered statistically significant.

Results

GRT treatment suppressed the proliferation of enzalutamide-resistant C4-2 MDV3100r and PC-3 PCa cells

We establish an enzalutamide (MDV3100)-resistant prostate cancer (PCa) cell line derived from LNCaP C4-2 by culturing the C4-2 cells in medium containing 10 μM enzalutamide and 20 nM dyhydroepiandrosterone (DHEA) for more than 3 months. The derived cell line, LNCaP C4-2 MDV3100r, was much more resistant to the enzalutamide treatment as compared to parental cell line (Fig 1A and 1B). The C4-2 MDV3100r cells were maintained in medium containing 10 μM enzalutamide for all the following experiments. Another commonly-used androgen receptor (AR)-negative PCa cell line, PC-3, was also very resistant to enzalutamide treatment (Fig 1C). The morphology of C4-2 MDV 3100r cells was slightly different from that of C4-2 cells (Fig 1D and 1E). C4-2 MDV 3100r cells were not as elongated as C4-2.

Fig 1

Characterizing the response of enzalutamide-resistant C4-2 MDV3100r and PC-3 cell lines to treatment of GRT and DHEA.

The enzalutamide-resistant C4-2 MDV3100r cell lines was derived from parental LNCaP C4-2 by culturing the cell in medium containing 10 μM enzalutamide and 20 nM DHEA for at least 3 months. The effect of enzalutamide (MDV-3100) treatment in the presence or absence of DHEA on the proliferation of C4-2 (A) and C4-2 MDV3100r (B) for 96 h was examined by Hoechst 33258 proliferation assay. (C) The proliferation of C4-2, C4-2 MDV3100r, and PC-3 under 0 or 10 μM MDV for 96 h was examined by proliferation assay. The morphology of C4-2 and C4-2 MDV3100r were show in (D) and (E) under light microscope. The magnification of the microscope is 1,000X and the yellow scale bar represented 100 μm.

We next examined if treatment with GRT can suppress the proliferation of C4-2 MDV3100r and PC-3 cells. Treatment with GRT dose-dependently reduced the proliferation of C4-2 MDV3100r cells (Fig 2A) and PC-3 cells (Fig 2B). The IC50 for GRT to suppress the proliferation of C4-2 MDV3100r and PC-3 cells was 98.5 and 226.6 (μg/ml), respectively. GRT treatment altered the morphology of C4-2 MDV3100r cells, C4-2 3100r cells became round-shape under GRT treatment (Fig 2C). For comparison, we examined if GRT exhibit suppressive effects on non-cancerous cells. We treated HUVEC (human umbilical vein endothelial cells) and HEK293 (human embryonic kidney cells 293) with increasing concentration of GRT (Fig 2D). Proliferation of HUVEC cells was not affected by 10–75 μg/ml GRT and was relatively resistant to 100 μg/ml GRT, while HEK293 was not affected by GRT at all. This observation suggested that enzalutamide-resistant PCa cells were more sensitive to GRT treatment than non-cancerous human cell lines.

Fig 2

Effects of GRT on proliferation of enzalutamide-resistant C4-2 MDV3100r, PC-3, and non-cancerous human cell lines.

Proliferation of LNCaP C4-2 MDV3100r (A) and PC-3 (B) being treated with increasing concentration of GRT (0–100 μg/ml for C4-2 MDV3100r cells and 0–200 μg/ml for PC-3 cells) for 96 h was determined by Hoechst 33258 proliferation assay. (C) Morphology of C4-2 MDV3100r cells under treatment of different concentrations of GRT (0, 50, 75, 100 μg/ml) was examined by microscope. The magnification of the microscope is 100X and the yellow scale bar represented 100 μm. (D) Proliferation of HUVEC and HEK293 cells being treated with increasing concentration of GRT (0–100 μg/ml) was determined by Hoechst 33258 proliferation assay. Asterisk *, ** and *** represents statistically significant difference p < 0.05, p < 0.01 and p < 0.001, respectively, between the two groups of cells being compared.

GRT treatment induced apoptosis in enzalutamide-resistant C4-2 MDV3100r PCa cells

To confirm the anti-cancer activity of GRT on enzalutamide-resistant PCa cells, we performed soft-agar colony formation assay. Treatment with GRT (0, 50, 75, 100 μg/ml) dose-dependently reduced the number and size of colonies of C4-2 MDV3100r cells in the soft agar plates (Fig 3A). We next investigated if GRT induced apoptosis in C4-2 MDV3100r cells. Comet assay revealed that treatment with GRT dose-dependently increased the DNA strand break in C4-2 MDV3100r cells (Fig 3B and 3C), indicating that GRT induced apoptosis in C4-2 MDV3100r cells.

Fig 3

GRT treatment reduced soft agar colony formation of C4-2 MDV3100r cells and induced apoptosis.

(A) Anticancer activity of GRT on C4-2 MDV3100r cells was confirmed by measuring the formation of colonies on soft agar for C4-2 MDV3100r cells being treated with GRT (0, 50, 75 and 100 μg/mL, being added into the medium). The culture medium was changed twice a week for four weeks. (B) DNA damage caused by GRT treatment in C4-2 MDV3100r cells was determined using the Comet assay. Green fluorescent indicated broken DNA fragments in C4-2 MDV3100r cells being treated with increasing concentration of GRT (0, 50, 75, 100 μg/ml). (C) Tail extent moment of C4-2 MDV3100r cells in Comet assay was quantified. Data wa presented as mean ± SD in triplicate experiments. Asterisk ** and *** represents statistically significant difference p < 0.01 and p < 0.001, respectively, between the two groups of cells being compared.

GRT treatment suppressed c-Myc, AR, PI3K-AKT signaling proteins and cell cycle regulatory proteins

To determine how GRT repressed the proliferation and survival of enzalutamide-resistant cells, we studied the effects of GRT on expression of signaling proteins involved in regulation of cell cycle, apoptosis, cell survival and androgen receptor (AR) signaling (Fig 4). GRT treatment dose-dependently suppressed the protein expression level of Akt, PI3K p110α, PI3K p110γ, phosphoinositide-dependent kinase 1 (PDK1), phospho-PDK1 (S241), S phase kinase-associated protein 2 (Skp2), cyclin D1, AR, phospho-AR (S81), cyclin-dependent kinase 1 (Cdk1), pro-PARP, c-Myc and Bcl-2 but increased protein abundance of caspase 3 and cleaved-PARP in C4-2 MDV3100r cells (Fig 4).

Fig 4

Western blotting analysis of signaling proteins affected by GRT treatment in C4-2 MDV3100r cells.

The C4-2 MDV3100r cells were treated with increasing concentrations of GRT (0, 50, 75, and 100 μg/mL) for 48 h. Expression of Akt, PI3K p110α, PI3K p110β, PI3K p110γ, PDK1, phospho-PDK1 (S241), Skp2, cyclin D1, AR, phospho-AR (S81), Cdk1, Myc, Bcl-2, caspase 3 and PARP proteins were determined by Western blotting. Expression level of GAPDH and β-Actin were used as loading control.

GRT suppressed proliferation of enzalutamide-resistant PCa cells via inhibition of c-Myc

The c-Myc protein is essential in regulating tumor growth, metastasis, disease progression, and metabolism of PCa cells. Since GRT treatment significantly repressed the protein expression of c-Myc, we explored if c-Myc is one of the main targets of GRT. We overexpressed c-Myc in C4-2 MDV3100r cells (Fig 5A). C4-2 MDV3100r cells with c-Myc overexpression were more resistant to GRT treatment (Fig 5B). The IC50 for GRT to suppress the proliferation of C4-2 MDV3100r cells with control vector and C4-2 MDV3100r overexpressing c-Myc cells was 105.3 and 141.1 (μg/ml), respectively. On the other hand, knockdown of c-Myc with siRNA in PC-3 cells increased the sensitivity of PC-3 cells to GRT treatment (Fig 6A and 6B). We compared the c-Myc protein level in C4-2 MDV3100r and PC-3. PC-3, which was more resistant to GRT treatment, expressed higher level of c-Myc protein as compared to C4-2 MDV3100r (Fig 6C). GRT treatment caused a more dramatic reduction of c-Myc in C4-2 MDV3100r cells as compared to PC-3 cells (Fig 6C). These observations suggested that c-Myc is one of the main targets of GRT in enzalutamide-resistant PCa cells.

Fig 5

Overexpression of c-Myc enhanced resistance of C4-2 MDV3100r cells against GRT treatment.

(A) Overexpression of c-Myc in LNCaP C4-2 MDV3100r cells was confirmed by Western blotting. (B) LNCaP C4-2 3100r cells with mock vector or c-Myc overexpression were treated with increasing concentration of GRT (0, 10, 25, 50, 75, 100 μg/ml) and cell proliferation was examined by proliferation assay. Asterisk ** and *** represents statistically significant difference p < 0.01 and p < 0.001, respectively, between the group as compared to control group or the denoted two groups being compared.

Fig 6

Knockdown of c-Myc increased sensitivity of PC-3 cells to GRT treatment.

(A) Knockdown of c-Myc in PC-3 cells was confirmed by Western blotting. (B) PC-3 cells with control scramble or c-Myc knockdown were treated with increasing concentration of GRT (0, 10, 25, 50, 75, 100 μg/ml) and cell proliferation was examined by proliferation assay. Asterisk * and *** represents statistically significant difference p < 0.05 and p < 0.001, respectively, between the group as compared to control group or the denoted two groups being compared. (C) The protein expression of c-Myc in C4-2 MDV3100r or PC-3 cells being treated with or without 100 μg/ml was examined by Western blotting. The β-Actin was used as loading control.

GRT suppressed the stability, expression, and downstream signaling of AR in C4-2 MDV3100r cells

As we observed that GRT repressed the protein expression of AR and phospho-AR (S81) and AR plays an important role during the PCa progression, we used immunofluorescence microscopy to examine the effects of GRT on the distribution and abundance of AR protein in C4-2 MDV3100r cells. We did not examine PC-3 because PC-3 cells were AR-negative. The distribution of AR in LNCaP C4-2 MDV3100r cells was mainly located in the nucleus (Fig 7A). GRT treatment dose-dependently reduced the abundance of AR proteins in C4-2 MDV3100r cells (Fig 6A) as well as expression of AR mRNA (Fig 7B) in C4-2 MDV3100r cells. To determine if treatment with GRT reduces the stability of AR, we treated LNCaP C4-2 MDV3100r cells with cycloheximide in the presence or absence of GRT. GRT treatment accelerated the degradation of AR (Fig 7C). Additionally, GRT treatment repressed the protein expression of AR and AR’s target gene prostate specific antigen (PSA) both in the absence or presence of androgen (Fig 7D). Interesting, we observed that AR proteins accumulated in nucleus and PSA expression was abundant in the enzalutamide-resistant PCa cells even in the absence of androgen (Fig 7D). The androgen-independent activation of AR and PSA may be important for the proliferation and survival of enzalutamide-resistant PCa cells.

Fig 7

GRT treatment suppresses the protein expression, stability, and downstream signaling of AR in C4-2 MDV3100r cells.

(A) C4-2 MDV3100r cells being treated with or without 1 nM DHT or increasing concentration of GRT (0, 50, 100 μg/ml) for 48 h was observed by immunofluorescent microscopy. The column from left to right shows DAPI (blue), F-actin (green), and AR (androgen receptor) (red) images. The magnification of the microscope is 100X and the yellow scale bar represented 100 μm. (B) Expression of AR mRNA in C4-2 MDV3100r cells being treated with 0, 50, 100 μg/ml was examined by qRT-PCR. Asterisk * and *** represents statistically significant difference p < 0.05 and p < 0.001, respectively, between the two groups of cells being compared. (C) C4-2 MDV3100r cells were treated with 10 μg/ml cycloheximide with or without 100 μg/ml GRT for 0, 4, 8, 24, 48 h. Expression of AR protein was examined by Western blotting and the β-actin was used as loading control. (D) LNCaP C4-2 MDV3100r cells were treated with or without 1 nM DHT along with increasing concentration of GRT (0, 50, 100 μg/ml) for 96 h. Abundance of AR and PSA protein was determined by Western blotting. The β-actin was used as loading control.

Discussion

Prostate cancer (PCa) patients receiving androgen ablation therapy usually develop castration-resistant prostate cancer (CRPC) within 2–3 years. Enzalutamide, a nonsteroidal antiandrogen, is an USA FDA-approved drug for treatment of metastatic CRPC. However, a great portion of PCa patients who initially respond to enzalutamide treatment develop drug resistance after a few months [13]. Mechanisms of enzalutamide resistance include AR amplification and overexpression, AR mutation, altered steroidogenesis, AR splice variants, autophagy mediated resistance, and activation of Wnt signaling [14]. New therapy for enzalutamide-resistant PCa is undoubtedly needed. In this study, we examined the suppressive effects of GRT, an aspalathin-rich green rooibos extract containing 12.78% aspalathin as major flavonoid, on enzalutamide-resistant C4-2 MDV3100r and PC-3 cells. We observed that GRT effectively suppressed the proliferation, survival, and soft agar colony formation of enzalutamide-resistant C4-2 MDV3100r cells and PC-3 cells. The GRT treatment caused apoptosis in C4-2 MDV3100r cells within 48 h.

Phosphatase and tensin homolog (PTEN) is a negative regulator for phosphoinositide 3-kinase (PI3K)-Akt signaling pathway [15]. Deletion of PTEN was observed in 40–70% of PCa patients, resulting in upregulation of PI3K-Akt signaling. PI3K-Akt signaling plays an important role in the survival of PCa cells [16]. Phosphorylation of Akt is activated by PDK1 and mTOR kinase. We observed that GRT treatment dose-dependently suppressed the expression level of proteins involved in Akt signaling pathway, including Akt, PI3K α, PI3K γ, PDK1, phospho-PDK1 (S241) in C4-2 MDV3100r cells.

The oncoprotein c-Myc is commonly overexpressed in PCa [17]. Expression of c-Myc protein is elevated in CRPC [17] and promotes androgen-independent proliferation of CRPC cells [18]. Overexpression of c-Myc in PCa patients predicts biochemical recurrence of tumors [19]. The c-Myc protein regulates ribosome biogenesis as well as cooperates with the PI3K/AKT/mTOR pathway to promote the survival of metastatic CRPC cells [17]. Elevated expression of AR splice variant AR-V7 is an important mechanism for developing resistance to enzalutamide and abiraterone [13]. The c-Myc promotes the AR gene transcription and increases the stability of the full-length AR and AR-V7 [20]. Suppression of c-Myc restored sensitivity of enzalutamide-resistant PCa cells to enzalutamide treatment [20]. Pharmaceutical inhibition of c-Myc induces apoptosis in enzalutamide-resistant PCa cells [21, 22]. These observations suggested that c-Myc is essential for enzalutamide-resistant PCa cells. Our study suggested that GRT treatment significantly repressed the protein abundance of c-Myc in enzalutamide-resistant PCa cells. We observed that overexpression of c-Myc enhanced the resistance of C4-2 MDV3100r cells under GRT treatment while knockdown of c-Myc in PC-3 increased the sensitivity of PC-3 cells to GRT treatment. We also observed that level of c-Myc protein expression correlated to resistance of PCa cells to GRT treatment. These findings suggested that c-Myc is one of the main targets of GRT treatment in enzalutamide-resistant PCa cells. The expression level of c-Myc in enzalutamide-resistant prostate tumors may vary in different patients and we predict that those enzalutamide-resistant prostate tumors with lower expression level of c-Myc will be more sensitive to GRT treatment. We also predict that combined treatment of GRT with pharmaceutical inhibitor targeting c-Myc will enhance the regression of tumors.

Additionally, GRT treatment suppressed the expression of proteins involved in cell cycle regulation and cell survival, such as Skp2, cyclin D1, and Cdk1. Cdk1-cyclin complexes are important in the regulation of cell entry and progression to the S and G2/M phases during cell cycle [23]. Skp2 targets p27Kip1 by phosphorylating p27Kip1 at T187 for ubiquitination and degradation [24]. Skp2 forms a stable complex with the cyclin A-Cdk2 [25]. The reduction of c-Myc, Akt signaling proteins, Skp2, Cdk1, and cyclin D1 can explain the suppression of proliferation and survival of enzalutamide-resistant PCa cells by GRT.

Phosphorylation of AR has been reported to play critical roles in regulating AR function and AR stability. Phosphorylation at Ser81 on AR has been reported to stabilize AR and increase the protein expression of AR, the phosphorylation on Ser81 is regulated by Cdk1 [26] and Cdk5 [27]. For certain CRPC cells, elevation of CDK1 activity is a mechanism to increase AR expression and stability in response to low androgen levels in androgen-deprivation therapy [26]. Mutation of S81A on AR blocks its interaction with CDK5, reduces nuclear localization of AR, destabilizes protein level of AR, and decreases proliferation of PCa cells [27]. In our current study, we observed that GRT treatment suppressed protein expression of AR, phospho-AR (S81), Cdk1, as well as the protein expression of AR’s target gene PSA in AR-positive C4-2 MDV3100r cells. Immunofluorescent microscopy revealed that GRT treatment dose-dependently reduced the abundance of AR proteins both in nucleus and cytoplasm of C4-2 MDV3100r cells. Treatment with cycloheximide confirmed that GRT treatment reduced the stability of AR. The reduction of expression, downstream signaling and stability of AR by GRT will impair the survival and proliferation of C4-2 MDV3100r cells. According to these observations, we predict that enzalutamide-resistant prostate tumors expressing AR will be more sensitive to GRT treatment as compared to those AR-negative enzalutamide-resistant prostate tumors. Interesting, we observed that AR proteins accumulate in the nucleus and PSA expression is relatively high in the enzalutamide-resistant PCa cells even in the absence of androgen. The androgen-independent activation of AR and PSA may be an important mechanism for the proliferation and survival of enzalutamide-resistant PCa cells.

In conclusion, our study suggested that the green rooibos extract, GRT, suppressed the cell proliferation and survival of enzalutamide-resistant PCa cells via inhibition of c-Myc, PI3K-Akt signaling as well as suppressing the expression, stability, and downstream signaling AR. GRT is be a potential adjuvant therapy for PCa patients with enzalutamide-resistance.

HPLC analysis of components in GRT.

The GRT is the same production batch lot used in our previous study [7] and this figure is the same as Fig 1 in [7]. The components of GRT was analyzed at (A) 288 and (B) 350 nm. The content value of each numbered compounds (1, PPAG; 2, isoorientin; 3, orientin; 4, aspalathin; 5, bioquercetin; 6, vitexin; 7, hyperoside; 8, rutin; 9, isovitexin; 10, isoquercitrin; 11, luteoloside; 12, nothofagin) is indicated in brackets after the number on the chromatograms. Component is expressed as g/100 g GRT.

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12 Apr 2022

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Huang et describes the growth inhibitory effect of green rooibos extract on the proliferation of the castration resistant C4-2 prostate cancer cells, a derivative which is Enza resistant and the androgen receptor negative expressing PC3 cell line. Authors continue with the androgen receptor positive C4-2 cells and reveal that the extract induces apoptosis. Interestingly, some factors are reduced in protein level including c-Myc. Overexpression of c-Myc reduced extract-mediated growth inhibition.

This is a continuation of experiments of the group previously revealed that the rooibos extract inhibits castration resistance prostate cancer cells through inhibition of YAP and AKT signaling.

Therefore, some additional novelties and specificity must be added to the manuscript

Major points:

1. Authors show reduction of cell viability by the extract in prostate cancer cell lines. This may also indicate an unspecific effect by using 100microgram per ml. Authors should reveal a cell specific response by treatment with a primary explant or primary, non-immortalized, human cells with the same culture condition that is resistant to the treatment.

The general question is whether these high doses will lead to toxicity in non-cancerous cells?

2. Further, the data suggest that those cell lines with c-Myc overexpression are resistant to the treatment. Meaning that those cancer cells, which represent the more aggressive types, which overexpress c-Myc, are treatment resistant. Please discuss this issue.

3. Unclear is why authors focus on androgen receptor degradation, while they reveal that the androgen receptor-negative expressing PC3 cell line is also inhibited in growth by the extract. It would be more helpful to make a general statement to overexpress c-Myc in PC3 cells to reveal whether the rooibos extract targets c-Myc also in these cell lines and whether c-Myc overexpression can ameliorate the apoptotic effect. Thus, reveal in PC3 cells whether rooibos extract targets c-Myc downregulation and by overexpression whether c-Myc is the key factor for rooibos-mediated growth inhibition.

Error bars in Fig. 5B with no GRT are missing.

Label “Actin” with a capital “A”.

Reviewer #2: Enzalutamide is commonly used in treating prostate cancer (PCa) patients, but frequently results in resistance in PCa patients. In this manuscript, the authors examined the effect of a pharmaceutical-grade green rooibos extract (GRT, with 12.8% aspalathin) on the suppression of the survival and proliferation of enzalutamide-resistant prostate cancer (PCa) cells as well as the mechanism. By using two enzalutamide-resistant prostate cancer cell lines C4-2 MDV 3100r and PC-3, they demonstrated that both are resistant to 10uM MDV compared to the C4-2 cells, and identified the IC50 concentrations of GRT in C4-2 MDV 3100r and PC-3 both cell lines. Furthermore, GRT treatment in a dose-dependent manner can reduce cell proliferation, decrease colony formation in soft agar and induce apoptosis by comet assay in C4-2 MDV 3100r cells. In the mechanism study by examining a panel of signaling proteins in cell cycle, apoptosis, cell survival, and androgen receptor (AR) signaling in C4-2 MDV 3100r treated with different concentrations of GRT, the authors found the increasing dose of GRT reduces the levels of the proteins in cell cycle and cell survival as well as increases the proteins in apoptosis. In addition, they indicated that Myc overexpression elevates the resistance in C4-2 MDV 3100r cells under GRT treatments. Moreover, they showed that GRT treatment diminish AR expression, stability, and PSA expression in C4-2 MDV 3100r cells. The authors suggest that GRT may serve as a potential adjuvant therapeutic agent for enzalutamide-resistant prostate cancer. Overall, this is a nice and thorough work providing the mechanism study of GRT on enzalutamide-resistant prostate cancer cells.

However, there are some typos or grammatical errors as below need to be corrected.

1. In line 56, 280, and 347, it should be “Interestingly” not “Interesting”.

2. In line 78, delete “in” before every year.

3. Line 82, delete “will”.

4. Line 85, should be “patients”.

5. Line 108, play “an” essential role.

6. All the antibodies and special reagents or kits should be included with the catalogue number in the Materials and Methods.

7. The first paragraph in the first section of the results from line 208 to 217 can be moved to the Materials and Methods.

8. The more correct way of labeling should be “phospho-PDK1 (S241)” in line 253 and 308, and 427, similarly like “phosphor-AR (S81)” in line 254 and 428.

9. Line 276, “reduces”.

10. Line 292, “mediated”.

11. Line 432, “enhanced”.

In addition, the images of the figure 1D, 1E, 2C, and 6A should include the scale bar.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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27 May 2022

05/26/2022

Dear Editor,

We are here re-submitting the paper “Aspalathus linearis suppresses survival and proliferation of enzalutamide-resistant prostate cancer cells via inhibition of c-Myc and stability of androgen receptor” by Bi-Juan Wang and Shih-Han Huang et al. for consideration for publication as research paper. No related content of this research has been submitted or published elsewhere. We have revised the manuscript and added two experiments according to the suggestions made by the reviewer, as well as answered all the questions raised by the reviewers. We also included our original Western blot scans as well as provide a file of minimal data set as supplemental material for review purpose. We have revised the manuscript to meet PLOS ONE's style requirements. The pharmaceutical-grade green rooibos extract (GRT, containing 12.78% aspalathin) being used in this study was extracted and produce by the co-author Prof. Christo J.F. Muller from Medical Research Council of South Africa. We included the HPLC analysis of this batch of GRT as supplemental figure. We have corrected the funding information in the submission system so that the information is now consistent with that in the manuscript. We hope that the current revised manuscript fulfill the requirement of PLOS One.

The following is a response to the reviewers’ concerns and questions.

Reviewer #1: The manuscript by Huang et describes the growth inhibitory effect of green rooibos extract on the proliferation of the castration resistant C4-2 prostate cancer cells, a derivative which is Enza resistant and the androgen receptor negative expressing PC3 cell line. Authors continue with the androgen receptor positive C4-2 cells and reveal that the extract induces apoptosis. Interestingly, some factors are reduced in protein level including c-Myc. Overexpression of c-Myc reduced extract-mediated growth inhibition. This is a continuation of experiments of the group previously revealed that the rooibos extract inhibits castration resistance prostate cancer cells through inhibition of YAP and AKT signaling.

Therefore, some additional novelties and specificity must be added to the manuscript

Answer: We thank reviewer for the affirmative comment.

Major points:

1. Authors show reduction of cell viability by the extract in prostate cancer cell lines. This may also indicate an unspecific effect by using 100microgram per ml. Authors should reveal a cell specific response by treatment with a primary explant or primary, non-immortalized, human cells with the same culture condition that is resistant to the treatment. The general question is whether these high doses will lead to toxicity in non-cancerous cells?

Answer: We thank the reviewer for raising this important question. We now included the data of HUVEC (human umbilical vein endothelial cells) and HEK293 (human embryonic kidney cells 293). The HUVEC cells are normal human primary cells and HEK293 cells is an established cell line with mutant version of SV40 large T antigen. Proliferation of HUVEC cells was not affected by 10-75 μg/ml GRT and was relatively resistant to 100 μg/ml GRT, while HEK293 was not affected by GRT at all. This observation suggested that enzalutamide-resistant PCa cells were more sensitive to GRT treatment than non-cancerous human cell lines.

2. Further, the data suggest that those cell lines with c-Myc overexpression are resistant to the treatment. Meaning that those cancer cells, which represent the more aggressive types, which overexpress c-Myc, are treatment resistant. Please discuss this issue.

Answer: We thank the reviewer for raising this very interesting question. We performed a new Western blotting assay to compare the protein expression of c-Myc in C4-2 MDV3100r and PC-3 cells being treated with 0 or 100 μg/ml GRT. PC-3, which was more resistant to GRT treatment, expressed higher level of c-Myc protein as compared to C4-2 MDV3100r (new Fig. 6C). We also added a new c-Myc knockdown experiment on PC-3 cells to examine if knockdown of c-Myc increases the sensitivity of PC-3 cells to GRT treatment. Knockdown of c-Myc with siRNA in PC-3 cells increased the sensitivity of PC-3 cells to GRT treatment (new Fig. 6A, 6B). We added the following discussion in the Discussion, “Our study suggested that GRT treatment significantly repressed the protein abundance of c-Myc in enzalutamide-resistant PCa cells. We observed that overexpression of c-Myc enhanced the resistance of C4-2 MDV3100r cells under GRT treatment while knockdown of c-Myc in PC-3 increased the sensitivity of PC-3 cells to GRT treatment. We also observed that level of c-Myc protein expression correlated to resistance of PCa cells to GRT treatment. These findings suggested that c-Myc is one of the main targets of GRT treatment in enzalutamide-resistant PCa cells. The expression level of c-Myc in enzalutamide-resistant prostate tumors may vary in different patients and we predict that those enzalutamide-resistant prostate tumors with lower expression level of c-Myc will be more sensitive to GRT treatment. We also predict that combined treatment of GRT with pharmaceutical inhibitor targeting c-Myc will enhance the regression of tumors.”

3. Unclear is why authors focus on androgen receptor degradation, while they reveal that the androgen receptor-negative expressing PC3 cell line is also inhibited in growth by the extract. It would be more helpful to make a general statement to overexpress c-Myc in PC3 cells to reveal whether the rooibos extract targets c-Myc also in these cell lines and whether c-Myc overexpression can ameliorate the apoptotic effect. Thus, reveal in PC3 cells whether rooibos extract targets c-Myc downregulation and by overexpression whether c-Myc is the key factor for rooibos-mediated growth inhibition.

Answer: We thank the reviewer for raising this important question. A majority portion of CRPC tumors still express AR. We therefore examine if GRT may suppress the protein expression, phosphorylation, signaling transduction, and stability of AR in the AR-positive C4-2 MDV3100r cells. However, as the reviewer pointed out, PC-3 cells are AR-negative PCa cells. As PC-3 cells express much higher c-Myc protein as compared to C4-2 MDV3100r cells (new Fig. 6C), we chose to knock down c-Myc in PC-3 cells and to overexpress c-Myc in C4-2 MDV3100r cells to determine if c-Myc is one of the main targets of GRT in CRPC cells. We observed that overexpression of c-Myc enhanced the resistance of C4-2 MDV3100r cells under GRT treatment while knockdown of c-Myc in PC-3 increased the sensitivity of PC-3 cells to GRT treatment (new Fig. 6A, 6B). These observations suggested that c-Myc is one of the main targets of GRT in enzalutamide-resistant PCa cells.

Error bars in Fig. 5B with no GRT are missing.

Answer: We thank the reviewer for noticing this mistake. We now added the error bars into the condition with GRT treatment in Fig. 5B.

Label “Actin” with a capital “A”.

Answer: We thank the reviewer for noticing this mistake. We now corrected all “actin” to “Actin”.

Reviewer #2: Enzalutamide is commonly used in treating prostate cancer (PCa) patients, but frequently results in resistance in PCa patients. In this manuscript, the authors examined the effect of a pharmaceutical-grade green rooibos extract (GRT, with 12.8% aspalathin) on the suppression of the survival and proliferation of enzalutamide-resistant prostate cancer (PCa) cells as well as the mechanism. By using two enzalutamide-resistant prostate cancer cell lines C4-2 MDV 3100r and PC-3, they demonstrated that both are resistant to 10uM MDV compared to the C4-2 cells, and identified the IC50 concentrations of GRT in C4-2 MDV 3100r and PC-3 both cell lines. Furthermore, GRT treatment in a dose-dependent manner can reduce cell proliferation, decrease colony formation in soft agar and induce apoptosis by comet assay in C4-2 MDV 3100r cells. In the mechanism study by examining a panel of signaling proteins in cell cycle, apoptosis, cell survival, and androgen receptor (AR) signaling in C4-2 MDV 3100r treated with different concentrations of GRT, the authors found the increasing dose of GRT reduces the levels of the proteins in cell cycle and cell survival as well as increases the proteins in apoptosis. In addition, they indicated that Myc overexpression elevates the resistance in C4-2 MDV 3100r cells under GRT treatments. Moreover, they showed that GRT treatment diminish AR expression, stability, and PSA expression in C4-2 MDV 3100r cells. The authors suggest that GRT may serve as a potential adjuvant therapeutic agent for enzalutamide-resistant prostate cancer. Overall, this is a nice and thorough work providing the mechanism study of GRT on enzalutamide-resistant prostate cancer cells.

Answer: We thank reviewer for the affirmative comment.

However, there are some typos or grammatical errors as below need to be corrected.

1. In line 56, 280, and 347, it should be “Interestingly” not “Interesting”.

2. In line 78, delete “in” before every year.

3. Line 82, delete “will”.

4. Line 85, should be “patients”.

5. Line 108, play “an” essential role.

6. All the antibodies and special reagents or kits should be included with the catalogue number in the Materials and Methods.

7. The first paragraph in the first section of the results from line 208 to 217 can be moved to the Materials and Methods.

8. The more correct way of labeling should be “phospho-PDK1 (S241)” in line 253 and 308, and 427, similarly like “phosphor-AR (S81)” in line 254 and 428.

9. Line 276, “reduces”.

10. Line 292, “mediated”.

11. Line 432, “enhanced”.

Answer: We sincerely apologize for these typos in the manuscript and we appreciate the reviewer for pointing out these typos. We have now corrected all these typos.

In addition, the images of the figure 1D, 1E, 2C, and 6A should include the scale bar.

Answer: We thank the reviewer for finding these mistakes. We have now added scale bars into these figures.

Sincerely,

Chih-Pin Chuu, Ph.D., Associate Investigator/Associate Professor

Institute of Cellular and System Medicine, National Health Research Institutes, Taiwan

Submitted filename: Rebuttal letter PLOS One 20220525.docx

Click here for additional data file.

20 Jun 2022

Aspalathus linearis suppresses cell survival and proliferation of enzalutamide-resistant prostate cancer cells via inhibition of c-Myc and stability of androgen receptor

PONE-D-22-04289R1

Dear Dr. Chuu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Salvatore V Pizzo

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors addressed all crtical points in a satisfactory manner.

Authors addressed all crtical points in a satisfactory manner

Reviewer #2: The author has provided the additional data for the questions brought by the reviewers and corrected all the typos and grammatical errors, as well as included the scale bars. The manuscript has met the requirement now.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

23 Jun 2022

PONE-D-22-04289R1

Aspalathus linearis suppresses cell survival and proliferation of enzalutamide-resistant prostate cancer cells via inhibition of c-Myc and stability of androgen receptor

Dear Dr. Chuu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Salvatore V Pizzo

Academic Editor

PLOS ONE