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Table of Contents
Year : 2022  |  Volume : 9  |  Issue : 1  |  Page : 1-10

JM-17 Induces G0/G1 Cell cycle arrest in human breast cancer cells through the downregulation of androgen receptors and cyclin-dependent kinase 4 protein expression

1 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
2 Allianz Pharmascience Limited, Taipei, Taiwan
3 Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University and National Health Research Institutes, Taiwan
4 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University; Department of Laboratory Medicine, Taipei Medical University Hospital; TMU Research Center of Cancer Translational Medicine, Taipei Medical University; School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan

Date of Submission10-Apr-2021
Date of Decision25-Apr-2021
Date of Acceptance04-May-2021
Date of Web Publication07-Mar-2022

Correspondence Address:
Dr. Yuan-Soon Ho
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, No.250 Wu-Hsing Street, Taipei City 110
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JCRP.JCRP_11_21

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Background: Locally advanced breast cancer (BC) remains a clinical challenge for patients as many will eventually develop distant metastases despite receiving appropriate therapies. Materials and Methods: In this study, we have analyzed the expression of androgen receptors (AR) in a series of BC cell lines and found their expressions rather ubiquitous across many different cell lines. Moreover, we have demonstrated that JM-17 [(1E,4Z,6E)-4-(cyclobutylmethyl)-1,7-bis (3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one], a synthetic curcumin derivative, exhibited suitable antitumor activities on most of the BC cell lines tested. Results: Human MDA-MB-231 cells were treated with JM-17, and the results demonstrated that JM-17-induced cell cycle proliferation arrested at the G0/G1 phase in a dose-dependent manner. Cell cycle-regulated proteins, such as cyclin-dependent kinases 4 (CDK4), were downregulated and p21 was upregulated. We further demonstrated that JM-17 treatment reduced AR expressions in MDA-MB-231 cells. The AR/CDK4 protein complex was demonstrated for the first time using a fluorescence resonance energy transfer (FRET) activity assay and immunohistochemistry (IHC) staining. JM-17 reduced the FRET activity in vitro. An in vivo study further demonstrated that JM-17 (20 mg/kg) decreased considerably MDA-MB-231 xenograft tumor growth. Conclusion: AR-mediated BC formation is a factor that clinicians often neglect. Our study demonstrated that JM-17 could be a promising agent against specific targets in AR-positive BC patients.

Keywords: Cyclin-dependent kinases 4, cell-derived xenografts, G0/G1 cell cycle, JM-17, triple-negative breast cancer

How to cite this article:
Lai GY, Chan H, Chen TC, Lee WJ, Ho YS. JM-17 Induces G0/G1 Cell cycle arrest in human breast cancer cells through the downregulation of androgen receptors and cyclin-dependent kinase 4 protein expression. J Cancer Res Pract 2022;9:1-10

How to cite this URL:
Lai GY, Chan H, Chen TC, Lee WJ, Ho YS. JM-17 Induces G0/G1 Cell cycle arrest in human breast cancer cells through the downregulation of androgen receptors and cyclin-dependent kinase 4 protein expression. J Cancer Res Pract [serial online] 2022 [cited 2023 Jun 5];9:1-10. Available from: https://www.ejcrp.org/text.asp?2022/9/1/1/339172

  Introduction Top

Breast cancer (BC) is the most common cancer among women worldwide and among newly diagnosed cancers, the second most frequent one.[1] BCs are being subtyped based on the expression of hormone receptors (HRs), i.e. estrogen receptor (ER) and/or progesterone receptor (PR) (∼75% of cases), an overexpression/amplification of human epidermal growth factor receptor 2 (HER2) (∼20% of cases, half of which are also positive for HRs), etc.. Tumors without expressing the above receptors are commonly referred to as triple-negative BCs (TNBCs) (∼5%–10%).[2] Several broad classes of drugs for treating BCs are available. They were predicated on the tumor characteristics and disease extents. For instance, choices are made between systemic chemotherapy, endocrine therapy, or HER2-directed treatments. For early-stage BCs, the variables encompass ER, PR, and HER2 status; lymph node involvement; and also tumor sizes. As for stage IV diseases, receptor status and the locations of the metastatic sites are the main determining factors.[3] However, treatment of TNBC is particularly challenging due to the lack of druggable targets. Presently, chemotherapy remains the standard care and the identification of new therapeutic agents is, hence, an urgent priority.[4]

The most representative polyphenolic component extracted from the rhizomes of Curcuma longa (known as turmeric) is curcumin. The therapeutic benefits of curcumin have been alleged for multiple chronic diseases, including inflammation, arthritis, metabolic syndrome, liver disease, obesity, neurodegenerative diseases, and in a number of cancers.[5] In BC, curcumin has been reported to interfere with several signaling pathways involved, such as the nuclear factor-κB, epidermal growth factor receptor, HER2, and PI3K/Akt pathways. In particular, curcumin exhibited effective anti-invasive activities in the “triple-negative” MDA-MB-231 BC cell line.[6] Curcumin, however, is not free from side effects. It is often associated with nausea, diarrhea, headache, and yellow stool. It is also well-documented that curcumin is poor in bioavailability due to its low absorbability as well as its rapid systemic metabolism and elimination, limiting its efficacy in disease treatments.[7]

JM-17 [(1E,4Z,6E)-4-(cyclobutylmethyl)-1,7-bis (3,4-dimethoxyphenyl)-5-hydroxyhepta-1, 4, 6-trien-3-one] and ASC-J9 are synthetic analogs of curcumin. JM-17 has a structure, physicochemical properties, and toxicity similar to ASC-J9 but with higher metabolic stability and systemic bioavailability.[8] In previous studies, ASC-J9 was shown to inhibit the ligand-dependent activity of the AR in the spinal cord and also in bulbar muscular atrophy cell culture experiments.[9] It can lessen the deleterious effects of the spinal cord and bulbar muscular atrophy by reducing the amounts of polyglutamine-expanded variant AR proteins in mice.[10] JM-17, in the presence of dihydrotestosterone (DHT) or absence of AR ligands, can reduce the stability of normal AR and also the polyglutamine-expanded variant AR at a lower dose than ASC-J9. Moreover, the decreases in AR protein expression correlated well to the decrease in transcriptional activities. JM-17 was shown to reduce the half-life of normal AR and polyglutamine-expanded variant AR by promoting UPS, thereby increasing the speed of AR clearance.[8]

Independent but relevant analyses of AR-dependent cell cycle progression in prostate cancer cells have shown that androgens are critical regulators of the G1-S transition.[11] Prostate cancer cells deprived of androgen arrest in the early G1 phase, concomitant with the loss of cyclin D1 and cyclin D3 expression, the cyclin-dependent kinases 4 (CDK4) activity attenuation, and the retinoblastoma tumor suppressors' hypophosphorylated/activation.[12] Recent studies have revealed that androgen induces D-type cyclin expression via the mammalian target of rapamycin (mTOR)-dependent enhancement of translation.[13] The ability of androgens to modulate cyclin D translation is distinct from the mechanisms utilized by other hormones. For instance, estrogen induces cyclin D1 transcription in BC cells through the ability of its cognate receptor (the ER) to directly modulate cyclin D1 regulatory regions.[14] These observations have culminated in a postulation wherein androgen induces cyclin D1 accumulation through mTOR and promotes active CDK4/cyclin D1 assembly.[11] To date, only a small number of studies have directly examined the influence of cell cycles on AR activity. Suffice it to say, many proteins found to interact with or modulate AR are also regulated during the cell cycle. These include proteins whose expressions or activities were increased in G0, G1 to S phase, or G2. Thus, dissecting the mechanisms by which AR governs cell cycle progression could turn out to be critical for the design of new strategies to treat cancers.

Androgens, including testosterone and DHT, though often referred to as “male” hormones, are in fact involved in the functions of multiple female organs, including the reproductive tracts, bones, kidneys, and muscles, via binding directly or indirectly to the AR.[15] The binding of androgen in the circulation to AR triggers the transport of AR receptors to the nucleus, the binding to the target genes, and the activations of gene transcription activities.[16]. Preclinical studies have shown that the androgen signaling pathways play key roles in the development of normal and malignant breast tissues. Among them, animal models showed that androgen signaling could promote BC progressions.[17] Epidemiological studies have shown that the circulating androgen levels are related to an increased risk of BC s, mainly the ER/PR-positive ones.[18] Hence, ARs could be used as prognoses or predictors for BCs.[19] Previous reports have shown that ARs are expressed in 70%–90% of primary BCs, and this frequency is even higher than the ER and PR.[20],[21] Whereas, between TNBC and AR, studies have shown a significant variance in the frequency of AR expression.[22],[23] Researchers currently believe that TNBC is a heterogeneous disease comprised of many different molecular subtypes. Lehmann et al. used gene expression profiles to classify TNBC. They have identified six different types of TNBC, namely, (1) basal-like 1 (17%), which is characterized by the cell cycle, DNA repair, and proliferation to increase gene expression; (2) basal-like 2 (7%), which is characterized by the upregulation of genes in the growth factor signal transduction pathway; (3) immunomodulator (18%), a process rich in immune cells; (4) mesenchymal (17%) and mesenchymal stem cell-like (14%), which are rich in epithelial–mesenchymal transition and growth factor pathways; (5) unstable (14%); and (6) luminal androgen receptor (LAR, 12%).[24],[25] At present, the androgen receptor (AR) pathway is gaining increasing attention as a potential target for BC treatments. After defining the expression level of AR in LAR subtypes through gene expression profiling or immunohistochemistry (IHC), it was found that the prognosis of AR-dependent TNBC was better than that of non-AR-dependent TNBC. Unfortunately, in the study of Loibl et al., 22.5% of these patients relapsed after 5 years.[26] Therefore, it is worth considering the use of new therapies to further reduce the risk of recurrence in the chemotherapy-resistant population, and the development of alternative and better tolerated AR targeting agents for the LAR type of TNBC provides a more promising approach.

  Materials and Methods Top


JM17 was provided by Allianz Pharmascience Limited, Taipei, Taiwan (now part of AnnJi Pharmaceutical Co. Ltd).

Cell culture

AU565 (ATCC® CRL-2351TM), BT-474 (ATCC® HTB-20TM), HCC1419 (ATCC® CRL-2326™), HCC1954 (ATCC® CRL-2338TM), SKBR3 (ATCC® HTB-30TM), BT549 (ATCC® HTB-122TM), HCC1395 (ATCC® CRL-2324TM), HCC38 (ATCC® CRL-2314TM), Hs-578T (ATCC® HTB-126TM), MDA-MB-231 (ATCC® HTB-26TM), MDA-MB-436 (ATCC® HTB-130TM), MDA-MB-453 (ATCC® HTB-131TM), MDA-MB-468 (ATCC® HTB-132™), and MCF-10A (ATCC® CRL-10317TM) cells were purchased from American Type Culture Collection (ATCC, Northern Virginia, USA). The cells were cultured in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12, Gibco, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, CA, USA), and 50 U/mL penicillin/streptomycin/neomycin (Invitrogen, CA, USA) in a humidified (5% carbon dioxide [CO2], 37°C) incubator. MCF-10A cells were cultured in Dulbecco's modified Eagle's medium nutrient F-12 (DMEM/F12, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), 50 U/mL penicillin/streptomycin/neomycin (PSN, Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL epidermal growth factor (EGF, Thermo Fisher Scientific, Waltham, MA, USA), 10 μg/mL insulin (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 μg/mL hydrocortisone (Thermo Fisher Scientific, Waltham, MA, USA), and 1X nonessential amino acids (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified (5% CO2, 37°C) incubator.

Cell proliferation and viability assays

A total of 5 × 104/well cells were seeded onto 6-well plates and assessed using a 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium assay. The absorbance at 570 nm was measured using a Synergy™ HT Multi-Detection Microplate Reader (BioTek Instruments, Winooski, USA).

Flow cytometry cell cycle analysis

MDA-MB-231 cells were cultured to 80% confluence and then synchronized in DMEM/F12 (Gibco, CA, USA) supplemented with 0.04% heat-inactivated FBS (Gibco, CA, USA). Next, the synchronized cells (cultured in 0.04% FBS) were challenged with the addition of media containing 10% FBS. DMSO- and JM-17-treated groups were assessed via flow cytometry analysis to determine the cell cycle population. The cells were fixed with 70% cold ethanol for 1 h and then treated with RNase A (Sigma-Aldrich, MO, USA) in a 37°C water bath for 30 min. After RNase A treatment, all samples were stained with propidium iodide (Sigma-Aldrich, MO, USA) at room temperature for 15 min. A total of 10,000 cells from each sample were detected by a FACSCalibur flow cytometer (BD, NJ, USA).

Protein extraction and western blotting

For protein extraction, the cells were washed twice with ice-cold PBS and lysed on ice in Golden lysis buffer (20 mM Tris-HCl [pH 8.0], 137 mM NaCl, 5.95 mM EDTA, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, and 10% glycerol) supplemented with protease inhibitors (Roche, Indianapolis, USA) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, USA). The proteins were separated via 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Specific antibodies against AR (sc-7305, Santa Cruz Biotechnology, CA, USA), p21 (GT8611, GeneTex, CA, USA), p27 (GT114, GeneTex, CA, USA), CDK4 (sc-23896, Santa Cruz Biotechnology, CA, USA), β-actin (GTX109639, GeneTex, CA, USA), and GAPDH (sc-47724, Santa Cruz Biotechnology, CA, USA) were diluted 1:2000 in Tris-buffered saline/Tween 20, and the membranes were incubated for 2 h at room temperature. Horseradish peroxidase-conjugated anti-mouse IgG (sc-2354, Santa Cruz Biotechnology, CA, USA) and anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology, CA, USA) secondary antibodies were diluted at 1:4000 and incubated with the membranes for 1 h at room temperature.


Cell lysates (0.8–1 mg) were precleared by adding 10 μL of protein A or G agarose beads for 1 h at 4°C. After centrifugation at 1200 × g, the supernatant was incubated with primary antibodies overnight at 4°C. IgG was used as a negative control. Then, 15 μL of protein A or G agarose beads were added, followed by incubation for 2 h at 4°C. After centrifugation at 1200 × g, the pellet was collected and washed twice with cell lysis buffer. The immunoprecipitated proteins were resolved by 12% SDS-PAGE and subsequently analyzed by Western blot.

Immunofluorescence staining and confocal microscopy

The cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The samples were blocked for 30 min in PBS with 2% bovine serum albumin (BSA). Primary antibodies were diluted 1/100 with 1% BSA in PBS and then incubated overnight at 4°C. The secondary antibody fluorescein (FITC) AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, USA), rhodamine (TRITC) AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch), and DyLight™ 405 AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch) were diluted at 1/50 and incubated with the cells for 1 h at room temperature. Coverslips were mounted with VECTASHIELD Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA), and a Leica FRET AB system measured the Förster resonance energy transfer (FRET) activity assay. All images were captured by confocal microscopy (Leica, Wetzlar, Germany).

Immunohistochemistry analysis

Paraffin-embedded breast tumor tissues were cut into 8-μm sections. The sections were preincubated in 3% H2O2 and 0.3% Triton X-100 before microwaving for antigen retrieval. For immunostaining, the sections were microwaved in Tris buffer (pH 6) for 30 min. Following this step, the antigenicity of the tumor cells in sections was blocked in 5% horse serum (Chemicon, Temecula, CA, USA) for 30 min and subsequently incubated with primary antibody (1:100 dilution) for 2 h. The slides were incubated with the secondary antibody for another 30 min. Then, the signals were detected and amplified using a biotinylated streptavidin-HRP and 3,3'-diaminobenzidine tetrahydrochloride system (Dako Corp K3468, CA, USA). The tissue specimens were stained with hematoxylin to locate the nuclei. All slides were dehydrated in an alcohol gradient and covered with coverslips and mounting medium.


All animals were purchased from the National Science Council Animal Center, Taipei, Taiwan. Five animals in each cage were fed and acclimatized at the Taipei Medical University experimental animal center for 2 weeks in rectangular cages, and a small wood strip served as environmental enrichment. The animals were fed a 5 k52 formulation (6% fat) lab diet, and water was accessible at all times. Our animal facility was maintained under standard specific-pathogen-free conditions and a 12 h/12 h light/dark cycle. The total number of animals used in this experiment was 16, and the animals were equally divided among the 2 groups based on tumor volume. The number of animals in each experimental group was 8. If the tumor size was more significant than 4 cm3 or the mouse weight was 15% below the original weight, euthanasia was performed by placing the mice in a chamber and piping in CO2 at increasing concentrations until the animal became unconscious and died.

Treatment of MDA-MB-231 cell-derived xenografts in vivo

The mice were handled and cared for with strict adherence to guidelines as established by the Animal Resource Center and following study protocols as approved by the Laboratory Animal Center and Use Committee at Taipei Medical University (IACUC protocol LAC-2018-0035). MDA-MB-231 cells were cultured in media as described above. The cells (5 × 106) were suspended in 0.1 mL of medium and injected into female mice. The animals were monitored for engraftment by routine palpation, and the tumors were harvested when they reached a volume of 180 mm3. The mice were randomly divided into two groups (n = 8 each group). One group was injected with JM-17 (20 mg/kg IV injection, twice per week). Corn oil-treated mice served as the treatment control. During the experiment, tumor size was measured using calipers, and tumor volume was estimated using the following formula: tumor volume (mm3) = ½ × L × W2, where L is the length and W is the width of the tumor.

Statistical analysis

For each analysis, data were represented as the mean ± standard error of the mean of at least three independent experiments. For comparison, statistical significance was tested using t-tests. All P values were based on two-sided statistical analyses, and P < 0.05 was considered statistically significant.

  Results Top

Inhibition of cell proliferation in JM-17-treated triple-negative breast cancer cells

As in a previous study,[8] JM-17, synthesized as shown in [Figure 1]a, mediated the degradation of ARs in both drosophila and mouse models of spinal and bulbar muscular atrophy. This study compared the protein expression levels of AR in different BC cell lines [Figure 1]b. Compared to the HER2-overexpressing cell line, a higher protein level of AR was detected in some of the TNBC cell lines. TNBC is defined by the lack of expressions of the ER/PR as well as the HER2. These cells are insensitive to antihormonal therapies or therapies targeting HER2. TNBCs, in general, are associated with poorer clinical outcomes when compared to other BC subtypes. To date, there are no approved targeted therapies due to the lack of any discerning biomarkers. Small molecular cytotoxic agents, hence, remain the only treatment options for TNBC patients.
Figure 1: JM-17 inhibits human breast cancer cell proliferation. (a) The chemical structures of JM-17 and curcumin. (b) Androgen receptor protein expression in human breast cancer and normal breast epithelial cells. The GAPDH protein level is shown as a loading control. (c) A cell proliferation assay was performed with JM-17-treated (0.5–5 μM for 0–48 h) human breast cancer (MDA-MB-231, MDA-MB-458, MDA-MB-436), and normal epithelial (MCF-10A) cells. Data are the mean ± standard error of the mean P value by two-tailed t-test. **: P <0.01, ***: P <0.001

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To test whether JM-17 will inhibit TNBC cell growth, three TNBC cell lines, MDA-MB-231, MDA-MB-468, and MDA-MB-436, were used with normal breast epithelial (MCF-10A) cells as a control. The cells were cultured for 48 h with or without the addition of JM-17 (0.5–5 μM), and harvested for the cell growth assay. The results indicated that a low (<2.5 μM) concentration of JM-17-induced cell growth inhibition in all TNBC cancer cells in a dose-dependent manner [Figure 1]c. Moreover, JM-17-induced cell death at high concentrations (>2.5 μM). JM-17-induced cell growth inhibition at low (<2.5 μM) concentrations was not observed in normal breast epithelial (MCF-10A) cells.

JM-17 induces G0/G1 phase cell cycle arrest in triple-negative breast cancer cells

To ascertain whether JM-17 can induce cell growth cycle inhibition in TNBC cell lines, MDA-MB-231 cells were used as a test model. According to our previous studies, the cell growth cycle of MDA-MB-231 cells was first synchronized with 0.04% FBS medium according to our previous studies.[27] The synchronized cells were released and the cell cycle activated by switching to a 10% serum-supplemented medium containing JM-17 (0.5, 1, 2.5, and 5 μM) for 48 h. Flow cytometry analysis was performed, and the effects of JM-17 on different phases of the cell cycles were measured. The results in [Figure 2]a and [Figure 2]b indicate that JM-17 had significant effects on G0/G1 phase cell cycle arrest in MDA-MB-231 cancer cells [Figure 2]b (*P < 0.01, **P < 0.001). To investigate the underlying molecular mechanisms by which JM-17-induced G0/G1 arrest, MDA-MB-231 cells were switched to a medium with 0.04% FBS to render quiescent at the G0/G1 phase. Similar to the treatment described in [Figure 2]a, the cells were later returned to culture media supplemented with 10% FBS and with or without JM-17 (0.5–5 μM). After that, the cells were harvested for protein extraction and Western blot analysis [Figure 2]c. We demonstrated that JM-17 (0.5–5 μM for 24 h)-treated MDA-MB-231 cells were arrested at the G0/G1 phase of the cell cycle and that cyclin-dependent kinase inhibitors (CDKIs) (p21/Cip1) were upregulated (>1 μM). Interestingly, another CDKI, p27/Kip1, was not affected by JM-17 (0.5–2.5 μM)-induced cell growth arrest in MDA-MB-231 cells. The CDK4 protein was inhibited in cells treated with high concentrations of JM-17 (>2.5 μM). As shown in [Figure 1] and [Figure 2], significant cytotoxic effects were observed in TNBC cancer cells treated with high concentrations (>5 μM) of JM-17 and cell cycle-regulated proteins (p53, p27/Kip1, p21/Cip1, and CDK4) were altered [Figure 2]c.
Figure 2: Dose-dependent response of JM-17-induced G0/G1 phase arrest in MDA-MB-231 cells. (a) MDA-MB-231 cells were synchronized (denoted as starvation) with 0.04% FBS for 24 h as described in the Materials and Methods section. After synchronization, the cells were released into complete medium (10% FCS) containing 0.05% DMSO (denoted as re-serum) or 0.5–5 μM JM-17 in 0.05% DMSO (lower panel). (b) Percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle were determined using established CellFIT DNA analysis software. Three samples were analyzed in each group, and the values represent the mean ± standard error. (c) Protein extracts (100 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, probed with specific antibodies and detected using the NBT/BCIP system. In response to JM-17 treatment, the protein levels of p53, p27, p21, and cyclin-dependent kinases 4 were determined, and the b-actin protein was used as a loading control. *P < 0.01, **P < 0.001

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JM-17 inhibits the protein interaction between the androgen receptor and CDK4 in triple-negative breast cancer cells

To test whether JM-17 is a novel antitumor agent that targets the AR, MDA-MB-231 cells were treated with JM-17 (0.5–5 μM) for 24 h, and the protein levels of AR and CDK4 were evaluated by immunoblotting analysis [Figure 3]a. We found that JM-17-induced downregulation of AR protein expression in MDA-MB-231 cells in a dose-dependent manner (0.5–5 μM). Interestingly, CDK4 protein expression was also downregulated after the same treatments. We further found that the AR and CDK4 were associated in TNBC cells, as evidenced by an immunoprecipitation study [Figure 3]b. To confirm this observation, we performed fluorescence resonance energy transfer (FRET) assays in MDA-MB-231 cells, and the results indicated that 2.5 μM JM-17 significantly inhibited FRET activity. The statistical results revealed that the interaction between the AR and CDK4 significantly decreased in JM-17-treated cells [Figure 3]d, **P < 0.001].
Figure 3: Dose-dependent response of JM-17-induced androgen receptor/cyclin-dependent kinases 4-mediated signal inhibition in MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with JM-17 (0.5–5 μM) for 24 h. The protein extracts (100 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, probed with specific antibodies and detected using the NBT/BCIP system. In response to JM-17 treatment, the protein levels of androgen receptor and cyclin-dependent kinases 4 were determined, and the β-actin protein was used as a protein loading control. (b) MDA-MB-231 cell lysates were harvested for immunoprecipitation experiments with androgen recepto-and cyclin-dependent kinases 4-specific antibodies. The samples were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was then dried and subjected to autoradiography. (c and d) MDA-MB-231 cells were treated with JM-17 (0.5–5 μM) for 24 h. The cells were stained with androgen receptor-and cyclin-dependent kinases 4-specific antibodies labeled with FITC and rhodamine, respectively, as described in the Materials and Methods section. Androgen receptor/cyclin-dependent kinases 4 protein complex formation was detected by FRET assay. Scale bar = 20 μm. Data are the mean ± standard error of the mean P value by two-tailed t-test

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In vivo antitumor effects of JM-17 in MDA-MB-231 xenograft tumor mice

We further examined the antitumor effects of JM-17 in vivo by treating BALB/c nude mice bearing MDA-MB-231 tumor xenografts. After the growth of palpable tumors reaching a mean tumor volume of 180 mm3, the animals received JM-17 at a dose of 20 mg/kg or DMSO control every 2 days by intravenous injection. The tumor size of the JM-17-treated group decreased significantly as compared with that of the control group from the 4th week [Figure 4]a and [Figure 4]b. No gross signs of toxicity were observed based on body weight measurements [Figure 4]c. To confirm the involvement of CDK4 and AR in the JM-17-induced suppression of tumor growth, protein expressions in the tumor tissues were determined by Western blot analysis [Figure 4]d. As shown in [Figure 4]d, downregulations of AR and CDK4 were detected in JM-17-treated MDA-MB-231 tumor-bearing mice compared to control mice. These findings demonstrated that the JM-17-induced antitumor effects in MDA-MB-231 tumor-bearing mice were due to the downregulation of the interaction between CDK4 and the AR.
Figure 4: JM-17 treatment of MDA-MB-231-derived xenografts in vivo. (a) MDA-MB-231 cells (5 × 106) in 0.1 mL of RPMI 1640 were injected subcutaneously between the scapulae of each nude mouse. Once the tumor reached a volume of 200 mm3, the animals received intraperitoneal injections of DMSO (25 μL) and JM-17 (20 mg/kg) three times per week for 4 weeks. (b) The gross morphology of the tumors dissected from the mice. (c) The body weights were measured during the experiments. (d) Androgen receptor and cyclin-dependent kinases 4 protein expression was detected by immunoblotting analysis in tumor tissues dissected from the mice. GAPDH was used as a protein loading control. Data are the mean ± standard error of the mean P value by two-tailed t-test

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  Discussion Top

Curcumin is a polyphenol component extracted from C. longa. It has been reported to provide health benefits in multiple chronic diseases, such as inflammation, metabolic syndrome, neurodegenerative disease, liver disease, and some cancers;[28] previous studies indicated that curcumin targets different cell signaling pathways. Curcumin, however, as a therapeutic agent, suffers from its rapid metabolism and poor absorption. In this study, the antitumor effect of JM-17 was tested in human BC cells. As shown in a number of previous studies, JM-17 effectively mediated the degradation of the AR. A mouse model[8] was established to determine whether the antitumor effect of JM-17 correlates with the AR expression in different BC cell line subtypes. The results shown in [Figure 1]b indicated that AR expression preferentially occurred in HER2+ and TNBCs. The antiproliferative abilities of JM-17 in three TNBC cell lines were evaluated, as shown in [Figure 1]c. A low concentration of JM-17 (<2.5 μM) inhibited cell growth, whereas a high concentration (>2.5 μM) caused cell death in a normal breast cell line.

Previous studies have pointed out that curcumin and its derivatives inhibit cancer cell growth in breast, prostate,[29] and colorectal cancers.[30] To explore the signaling pathway of JM-17-mediated regulation of cancer cell growth inhibition, we found that JM-17 effectively inhibited the MDA-MB-231 cell growth cycle and arrested the cells at the G0/G1 phase. Therefore, we believe that the role of JM-17 may be related to proteins that regulate G0/G1 in the cell cycle. G0/G1 phase regulatory proteins, such as cyclin D-CDK4/6 and cyclin E-CDK2, phosphorylate Rb, thereby dissociating the HDAC-repressor complex, which in turn enables the transcription of genes required for DNA replication. This study found that high concentrations of JM-17 (>2.5 μM) significantly inhibited CDK4 protein expression in MDA-MB-231 cells. The antitumor mechanisms of JM-17 were similar to those of the clinically used therapeutic drugs palbociclib[31] and ribociclib[32] (CDK4/6 inhibitors) as a new treatment strategy in combination with letrozole for HR-positive, advanced-stage BC, showing promising results for these patients. Palbociclib (PD0332991, Ibrance®, Pfizer Inc, New York, USA) is an oral, reversible small-molecule inhibitor of CDK4/6. While CDK4/6 can bind cyclin D1, resulting in Rb hyperphosphorylation,[33] it also effectively separates CDK4/6-cyclin D1 complexes, blocking Rb phosphorylation and preventing E2F1 release, thus leading to G1 phase arrest and tumor growth suppression.

Although CDK4/6 inhibitors offer HR-positive BC patients new options, not all patients respond to these drugs, and most patients whose tumors respond to CDK4/6 inhibitors eventually develop acquired resistance.[34] The early and late adaptation mediated by persistent G1–S-phase cyclin expression and other bypass signaling limits the effectiveness of CDK4/6 inhibitors. Besides, various other mechanisms also exist responsible for intrinsic or acquired resistance to CDK4/6 inhibitors.[35] Evidence collected from preclinical studies has suggested that various mechanisms may contribute to intrinsic or acquired resistance to CDK4/6 inhibitors. Multiple factors involved in the regulation of the cell cycle are associated with resistance to CDK4/6 inhibitors. Loss of drug target genes, such as RB and FZR1,[36] and the overexpression of various genes that are directly or indirectly involved in the progression of the cell cycle are responsible for resistance to CDK4/6 inhibitors. Overexpression of the levels of various proteins upstream of the cell cycle, such as FGFR, PI3K/AKT/mTOR, and AP-1, acts as bypass pathways for the progression of the cell cycle, resulting in a decreased efficacy of CDK4/6 inhibitors.[37] In addition, inhibition of cyclin D activates autophagy, leading to the reversal of cell cycle arrest mediated by CDK4/6 inhibitors. CDK4/6 inhibitors may activate various immune-related genes, which may also play a role in developing tumor resistance. Because there are some disadvantages of recent CDK4/6 inhibitors, the role of JM-17 might act as a new cell cycle deregulation treatment, as it targets different proteins or hormones from recent CDK4/6 inhibitors.

JM-17 can stabilize AR performance by inhibiting the UPS in the presence of testosterone. In Steven's 2008 review, it was mentioned that the analysis of AR-dependent cell cycle progression in prostate cancer cells showed that androgen is the key regulator of G1-S transition. Based on these reports, the results shown in [Figure 2] and [Figure 3] revealed that JM-17 may inhibit the expression of CDK4 through AR-mediated signals. The protein and protein interaction between AR and CDK4 was significantly inhibited by JM-17 [Figure 3], as evidenced by the FRET assay. We suggest that AR/CDK4 protein complex formation may stabilize carcinogenic signals and that the JM-17-induced degradation of both proteins will diminish cell growth proliferation through G0/G1 phase cell cycle regulation [Figure 5].
Figure 5: Schematic presentation of the molecular mechanisms induced by JM-17 treatment in breast cancer cellsby JM-17 treatment in breast cancer cells

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  Conclusion Top

This study demonstrated a low (<2.5 μM) concentration of JM-17-induced cell growth inhibition in all TNBC cancer cells in a dose-dependent manner. Moreover, JM-17-induced cell death at high concentrations (>2.5 μM). JM-17-induced cell growth inhibition at low (<2.5 μM) concentrations were not observed in normal breast epithelial (MCF-10A) cells. The results indicated that JM-17 induces G0/G1 phase cell cycle arrest in triple-negative breast cancer cells. The results also indicated that JM-17 inhibits the protein interaction between the androgen receptor and CDK4 in triple-negative breast cancer cells. In vivo study, JM-17-induced antitumor effects in MDA-MB-231 tumor-bearing mice were due to the downregulation of the interaction between CDK4 and the AR.


This study was supported by the Health and Welfare Surcharge of Tobacco Products grant (MOHW109-TDU-B-212-134014), Ministry of Science and Technology, Taiwan (MOST108-2320-B-038-033-MY3 and MOST109-2320-B-038-028), and industry-academia collaboration between Allianz Pharmascience Limited and Taipei Medical University (A-106-069), awarded to Dr. Ho.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]


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