Caffeic Acid Derivative MPMCA Inhibits Prostate Cancer EMT and Metastasis by Regulating Transcription Factors Snail and Slug

TL;DR

This study shows that MPMCA, a caffeic acid derivative, inhibits prostate cancer metastasis by targeting EMT-related transcription factors Snail and Slug.

Contribution

MPMCA is identified as a novel therapeutic candidate for metastatic prostate cancer through its regulation of Snail and Slug.

Findings

MPMCA blocks prostate cancer cell migration and invasion without affecting cell viability.

MPMCA suppresses EMT by modulating mesenchymal and epithelial markers.

MPMCA reduces Snail and Slug expression and prostate cancer metastasis in vivo.

Abstract

Prostate cancer (PCa) is the most general cancer in men and is often linked with distant metastasis in its later stages. The caffeic acid (CA) derivative, N-(4-methoxyphenyl)methylcaffeamide (MPMCA), demonstrates superior liver-protective effects compared to CA. Nevertheless, the functions of MPMCA on prostate cancer metastasis remain unclear. Here, we demonstrate that MPMCA blocks migration and invasion in prostate cancer cells without affecting cell viability. By suppressing the production of mesenchymal markers Vimentin, N-cadherin and β-catenin and upregulating the production of the epithelial marker Zonula Occludens-1 (ZO-1), MPMCA also controls Epithelial–Mesenchymal Transition (EMT). The Phosphoinositide 3-kinase (PI3K), Protein kinase B (AKT) and mechanistic target of rapamycin (mTOR) pathway has been documented to regulate MPMCA-inhibited cell motility. Transfection with Snail and Slug cDNA reverses MPMCA’s suppression of EMT, migration, and invasion in prostate cancer cells. Importantly, our in vivo data indicates that MPMCA reduces Snail and Slug expression and prostate cancer metastasis. Our evidence suggests that MPMCA is a novel therapeutic candidate for treating metastatic prostate cancer.

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1. Introduction

Prostate cancer (PCa) is one of the most general malignancies and a leading cause of death for men worldwide. Over 300,000 males in the US are expected to be diagnosed with PCa in 2025, and over 35,000 will die from the illness [1]. PCa can be difficult to identify because it is initially asymptomatic, which emphasizes the significance of keeping an eye out for early symptoms. PCa is a highly varied illness, ranging from slowly progressing to highly aggressive and lethal forms [2]. The main cause of death from PCa is metastatic illness. While localized PCa generally has a favorable treatment outcome, metastatic PCa remains incurable [3]. Most men with advanced PCa present with multiple metastases. Bone is the most general organ of distant metastasis in PCa, with approximately 70% of patients with advanced disease exhibiting bone metastases at diagnosis [4].

Epithelial-mesenchymal transition (EMT) is a key pathway through which malignant epithelial cells acquire a mesenchymal phenotype, gaining invasive and metastatic properties [5]. During carcinogenesis, these cells transition into highly invasive mesenchymal-like cells. Malignant epithelial cells progressively lose adhesion and tight junction factors, including E-cadherin and fibronectin, while upregulating transcription activators, including Snail and Slug, and mesenchymal factors, for instance, vimentin and N-cadherin, promoting migratory and invasive abilities [6]. Among EMT transcription factors, Snail and Slug are critical drivers of EMT in PCa tissues [7,8]. Previous studies have demonstrated that EMT-related transcription factors contribute to therapeutic resistance, stemness, and tumor recurrence in PCa [9]. However, targeting these transcription factors remains a challenge in developing effective EMT inhibitors.

One of the main indicators of carcinogenesis is alterations in several cellular signaling mechanisms. PCa development and progression are linked to dysregulation of pathways, for instance, NF-κB, Notch and Wnt [10]. Additionally, abnormalities in the PI3K, AKT, mTOR signaling pathway are linked to different human cancers, such as PCa [11]. The PI3K, AKT, mTOR signaling controls crucial cellular functions, such as growth, development, apoptosis, invasion, migration, and metastasis [12]. Investigations indicate that PI3K, AKT, mTOR signaling is elevated in approximately 30–50% of PCa cases [13]. Alterations in PI3K, AKT, mTOR mechanism components activate several downstream targets, many of which contribute to tumorigenesis. Given their therapeutic potential, natural bioactive compounds have garnered significant interest. To date, compounds such as afritaxel A, arctiin, vitexin and oridonin have been indicated to target the PI3K, AKT, mTOR mechanism, with a few presently being assessed in clinical studies [14].

Caffeic acid (CA), a phenolic acid compound, can be isolated from various sources, including coffee, tea and wine [15]. Numerous reports have demonstrated that CA and its derivatives exhibit anti-metastatic, anti-angiogenic and anti-proliferative functions across multiple cancer types [16,17,18]. CA also modulates reactive oxygen species (ROS), key mediators of EMT [19,20]. As a CA derivative, N-(4-methoxyphenyl)methylcaffeamide (MPMCA) demonstrates superior liver-protective effects compared to CA under oxidative stress conditions [21]. This study examined the functions of MPMCA on PCa metastasis and its underlying mechanisms to identify novel anti-metastatic treatment strategies. In vitro and in vivo experiments uncovered that MPMCA markedly diminished PCa cell migration, invasion, and EMT while also reducing metastasis in vivo. These findings suggest that MPMCA holds significant potential as a therapeutic agent for metastatic PCa.

2. Materials and Methods

2.1. Materials

MPMCA was synthesized by Dr. Yueh-Hsiung Kuo at China Medical University, Taiwan utilizing the process described in an earlier report (Figure 1A) [21]. The β-actin (SC-47778), β-Catenin (SC-133240), Vimentin (SC-6260), ZO-1 (SC-33725), Snail (SC-271977), Slug (SC-166476), p-PI3-kinase p85a (SC-12929), PI3-kinase p85a (SC-1637), AKT (SC-5298), PI3K activator (SC-3036), and AKT activator (Fumonisin B1; SC-201395) were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, TX, USA). p-mTOR (5536S), mTOR (2983S), N-cadherin (ab76057), p-AKT (4060S), and mTOR activator (MHY1485; CAS Number: 17949) were purchased from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine™ 2000 was purchased form Invitrogen (Carlsbad, CA, USA).

2.2. Cell Culture

The American Type Culture Collection (Manassas, VA, USA) provided the human PCa cell line PC3, while the Food Industry Research and Development Institute (Hsinchu, Taiwan) provided the LNCaP cell line. Cells were cultivated at 37 °C in a humidified 5% CO_2_ environment in RPMI-1640 media (Gibco BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Rockville, MD, USA).

2.3. MTT Assay

An MTT assay was used to examine cell viability, in accordance with our earlier publications [22,23]. 100 μL of culture media was used to seed PCa cells (5 × 10^3^) onto 96-well plates. Following a 24 h incubation period to promote cell adhesion, cells were incubated to varying doses of MPMCA (0–10 μM) for a 24 h period before undergoing a cell-viability test. Absorbance at 570 nm was measured using a Multiskan™ FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.4. Reverse Transcription-Quantitative PCR (RT-qPCR) Analysis

PC3 and LNCaP cells (~1 × 10^5^ cells/well) were seeded in 6-well plates and treated with MPMCA (0, 0.3, 1, or 3 μM) for 24 h at 37 °C. Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions. For each sample, 1 μg of RNA was reverse-transcribed into cDNA using oligo(dT) primers. Quantitative PCR was performed using SYBR Green Master Mix (A46012; Thermo Fisher Scientific, Waltham, MA, USA) on a StepOnePlus Real-Time PCR System (v2.4; 4444202; Thermo Fisher Scientific, Waltham, MA, USA). Each reaction contained 100 ng cDNA and gene-specific primers, with GAPDH used as an internal reference. Thermocycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Melt-curve analysis was conducted to confirm amplification specificity. Each sample was run in technical triplicates, and experiments were repeated in at least three independent biological replicates. Relative mRNA expression was calculated using the 2^−ΔΔCt^ method [24,25]. The primer sequences are presented in Supplementary Table S1.

2.5. Western Blot Analysis

Protein samples were transferred onto PVDF membranes (Merck; Darmstadt, Germany) following electrophoretic separation on SDS-PAGE gels. The membranes were blocked with 5% nonfat milk prior to being incubated with primary antibodies for a full night at 4 °C. The membranes were then exposed to specific secondary antibodies for an hour at room temperature. The expression of the target protein was detected using an ECL kit (Merck Millipore, Billerica, MA, USA) and visualized using an ImageQuant^TM^ LAS 4000 (GE Healthcare, Chicago, IL, USA) biomolecular imager [26,27]. Protein band intensities were quantified using ImageJ software (version 1.49; National Institutes of Health, Bethesda, MD, USA). For each sample, the densitometric value of the target protein was normalized to the corresponding internal loading control. The resulting ratios were subsequently normalized to the untreated control group, which was defined as 1, and expressed as fold changes relative to control. Immunoblot quantification was performed from at least three independent experiments.

2.6. Migration and Invasion Assay

Cell migration and invasion were evaluated using 24-well Transwell chambers with 8-μm pore size polycarbonate membrane inserts (Costar, Corning, NY, USA). For the migration assay, 1 × 10^4^ cells suspended in 200 μL serum-free medium were seeded into the upper chamber. For the invasion assay, the upper inserts were precoated with Matrigel (BD Biosciences, Bedford, MA, USA) and incubated at 37 °C for 1 h to allow polymerization. Subsequently, 5 × 10^4^ cells in 200 μL serum-free medium were added to the Matrigel-coated inserts. In both assays, the lower chamber was filled with 300 μL complete medium containing 10% FBS as a chemoattractant. Cells were treated with MPMCA (0, 0.3, 1, or 3 μM) or 0.1% DMSO as a vehicle control and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO_2_. After incubation, cells remaining on the upper surface of the membrane were gently removed using cotton-tipped swabs. Cells that migrated or invaded to the underside of the membrane were fixed with 3.7% formaldehyde for 5 min, stained with 0.05% crystal violet in PBS, and washed thoroughly with PBS to remove excess stain. Migrated or invaded cells were quantified by counting at least five randomly selected fields per insert under a light microscope [28,29].

2.7. Cell Treatment and Transfection

PC3 and LNCaP cells were seeded in 6-well plates at a density of 5 × 10^5^ cells per well and allowed to adhere overnight. For pathway activation experiments, cells were pre-treated with PI3K, AKT, or mTOR activators (10 μM each) for 30 min prior to MPMCA treatment. Subsequently, cells were treated with MPMCA (3 μM) for 24 h before protein extraction and downstream analyses. For overexpression experiments, cells were transfected with Snail or Slug cDNA plasmids (1 μg/well) using Lipofectamine 2000 transfection reagent (11668019; Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After 24 h of transfection, cells were treated with MPMCA (3 μM) for an additional 24 h prior to analysis.

2.8. Metastatic Prostate Cancer Model

Six-week-old male BALB/c nude mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). PC3 cells stably expressing luciferase (2 × 10^6^ cells suspended in 50 μL Matrigel) were orthotopically injected into the anterior prostate using a 22-gauge needle. After tumor implantation, mice were randomly assigned to three groups (n = 7 per group): vehicle control, MPMCA 5 mg/kg, and MPMCA 15 mg/kg. MPMCA was first dissolved in DMSO to prepare a stock solution and then diluted with sterile 1× PBS to the desired working concentration prior to administration (final DMSO concentration 0.1%). One week after tumor implantation, mice received intraperitoneal injections of MPMCA (5 or 15 mg/kg) three times per week for four consecutive weeks. Control mice received an equal volume of the vehicle (0.1% DMSO diluted in sterile 1× PBS) on the same schedule. Tumor growth and distant metastasis were monitored weekly using an IVIS Spectrum imaging system (Xenogen, Tucson, AZ, USA). For imaging, mice were anesthetized with isoflurane and injected intraperitoneally with D-luciferin (150 mg/kg). Bioluminescent images were acquired 10 min after luciferin administration. Mice were monitored daily and humanely sacrificed at week 4, after which distant organs were harvested [28]. Mice that died during the experimental period were excluded from analysis. The final number of animals analyzed in each group is reported in the figure legends. Each individual mouse was considered an independent experimental unit. All animal procedures were approved by the Institutional Animal Care and Use Committee of China Medical University (Approval No. CMUIACUC-2019-079, approved on 14 December 2018).

2.9. Immunohistochemistry (IHC) Staining

Paraffin-embedded primary tumor tissues and distant organs collected from the metastatic prostate cancer model described above were sectioned at 5 μm thickness. Sections were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, 85%, and 75%) to distilled water. Antigen retrieval was performed in citrate buffer by heat-induced epitope retrieval (boiling for 12 min followed by 1 min at medium heat), and sections were cooled/rinsed in running tap water for 10 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 min at room temperature. Sections were blocked with 4% bovine serum albumin (BSA) in PBS for 30 min at room temperature and incubated overnight at 4 °C with primary antibodies against Snail and Slug. Immunoreactivity was visualized using the Leica Novolink Polymer Detection System (Leica Biosystems, Buffalo Grove, IL, USA) and DAB substrate, followed by hematoxylin counterstaining. Negative controls were performed by omitting the primary antibody. Staining was quantified as described in our previous study [29,30]. To indicate positive expression, IHC staining was given scores between 0 and 5 (from weak to strong).

2.10. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8.2 (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation (SD) from at least three independent biological replicates. Statistical comparisons among more than two groups were analyzed by one-way ANOVA with Bonferroni’s post hoc test. Differences between groups were considered significant if the p-value was <0.05.

3. Results

3.1. MPMCA Blocks EMT, Migration and Invasion in PCa

To investigate the effects of MPMCA on prostate cancer cell migration and invasion, we first determined the non-cytotoxic concentration range of MPMCA in PC3 and LNCaP cells. Treatment with MPMCA (0.3–10 μM) did not significantly affect cell viability (Figure 1B,C), indicating that the subsequent functional changes were not attributable to general growth inhibition. Consistently, MPMCA significantly inhibited both migration and invasion in PC3 and LNCaP cells in a concentration-dependent manner after 24 h of treatment (Figure 2A,B).

Because EMT is a critical step in tumor metastasis [5], we next examined whether MPMCA alters EMT-associated markers. MPMCA increased the epithelial marker ZO-1 at both the protein and mRNA levels, whereas mesenchymal markers, including Vimentin, N-cadherin, and β-catenin, were decreased (Figure 3A,B). Collectively, these results demonstrate that MPMCA suppresses EMT-associated molecular changes and reduces the migratory and invasive capacities of PCa cells.

3.2. The PI3K, AKT, mTOR Pathway Controls MPMCA-Promoted Inhibition of PCa Migration and Invasion

To explore the mechanism underlying MPMCA-mediated suppression of migration and invasion, we investigated PI3K/AKT/mTOR signaling, which is frequently hyperactivated in advanced PCa and contributes to metastatic progression [31]. MPMCA treatment resulted in a pronounced reduction in PI3K, AKT, and mTOR phosphorylation in PCa cells (Figure 4A), suggesting that MPMCA inhibits this pro-metastatic signaling axis.

To further establish the functional relevance of PI3K/AKT/mTOR inhibition, we pharmacologically activated PI3K, AKT, and mTOR signaling during MPMCA treatment. Notably, pathway activation significantly antagonized the inhibitory effects of MPMCA on cell migration and invasion (Figure 4B,C). These findings indicate that suppression of PI3K/AKT/mTOR activity is a critical contributor to MPMCA-induced inhibition of the migratory and invasive phenotypes of PCa cells.

3.3. Transcription Factors Snail and Slug Control MPMCA’s Inhibitory Effects on EMT, Migration, and Invasion

Because the EMT-inducing transcription factors Snail and Slug are key regulators of metastatic competence [32], we next evaluated whether MPMCA modulates Snail/Slug expression. MPMCA significantly decreased both mRNA and protein levels of Snail and Slug in PCa cells (Figure 5A–C), consistent with the observed reversal of EMT marker expression. To determine whether Snail and Slug are required for MPMCA-mediated inhibition of EMT and motility, PCa cells were transfected with Snail or Slug expression plasmids. Forced expression of Snail or Slug markedly rescued the migratory and invasive abilities suppressed by MPMCA (Figure 5D,E). Moreover, ectopic Snail and Slug expression reversed the MPMCA-induced changes in EMT marker expression (Figure 5F,G). Together, these results demonstrate that downregulation of Snail and Slug is a key mechanism through which MPMCA suppresses EMT, migration, and invasion in PCa cells.

3.4. MPMCA Inhibits Prostate Cancer Metastasis in Vivo

To validate the anti-metastatic effects of MPMCA in vivo, we established an orthotopic prostate cancer model by injecting luciferase-expressing PC3 cells into the anterior prostate of male nude mice, thereby allowing longitudinal monitoring of primary tumor growth and metastatic dissemination [33]. IVIS imaging and tumor size measurements demonstrated that MPMCA treatment significantly inhibited tumor progression (Figure 6A–D). Importantly, MPMCA did not significantly affect body weight throughout treatment (Figure 6E), suggesting that the anti-tumor effects were not associated with overt systemic toxicity. Consistent with the in vitro results, ex vivo IVIS quantification revealed that MPMCA substantially reduced the metastatic burden in distant organs, including the lungs, liver, and bones (Figure 6F,G). Histological analysis further confirmed fewer metastatic lesions in these organs following MPMCA treatment (Figure 7A). In parallel, IHC staining showed decreased expression of Snail and Slug in tumor tissues from MPMCA-treated mice (Figure 7B,C), supporting suppression of EMT-related signaling in vivo. Collectively, these findings indicate that MPMCA inhibits PCa metastasis in vivo, at least in part, through downregulation of Snail and Slug.

4. Discussion

PCa cases are diagnosed at a localized stage and generally have a favorable prognosis [34]. Metastatic and advanced PCa has limited therapy options, resulting in poor patient outcomes [35,36]. Researchers have been actively exploring more effective therapeutic strategies and medicines for PCa, such as targeted therapy, immunotherapy and hormone treatment [37]. In the last few years, several traditional Chinese herbal medicine components have demonstrated antitumor effects [38,39,40]. CA, a versatile molecule for synthesizing derivatives, and its derivatives have garnered significant attention for their diverse biological and pharmacological properties, including antioxidant [41,42], antibacterial [43], and anticancer activities [15]. However, research on the antimetastatic effects of MPMCA, a CA derivative, remains limited, with no prior studies investigating its impact on PCa metastasis. In this study, we evaluated MPMCA’s effects on PCa cell motility. Our findings demonstrate that MPMCA inhibits PCa cell migration, invasion, and metastasis in vitro and in vivo by suppressing EMT through the downregulation of Snail and Slug.

Importantly, we employed two biologically distinct PCa cell models: LNCaP cells, which represent androgen-sensitive [35,36], relatively low-migratory adenocarcinoma cells, and PC3 cells, which are androgen-independent and highly metastatic with neuroendocrine-like characteristics [44,45]. Although LNCaP cells exhibit lower basal migratory capacity compared with PC3 cells, MPMCA consistently suppressed EMT markers and motility in both models. This suggests that the anti-metastatic effects of MPMCA are not limited to highly aggressive PCa but may also be effective in earlier-stage or less invasive disease contexts.

The majority of PCa-associated deaths are caused by metastatic disease rather than the primary tumor. Metastasis is a complex, multi-step biological process involving tumor cell detachment, invasion, intravasation, survival in circulation, extravasation, and colonization at distant sites [46]. It is believed that cancer cells activate EMT, which helps them separate from the main tumor and enter blood arteries [47]. Epithelial cells lose their apical-basal polarity and adherens junctions during EMT, acquiring a mesenchymal phenotype with increased motility. Therefore, EMT inhibition is an attractive therapeutic strategy. The results of this study showed that MPMCA therapy in PCa prevents EMT in PCa cells by increasing the expression of epithelial cell markers (ZO-1) and reducing mesenchymal cell markers (N-cadherin, Vimentin, and β-Catenin). EMT advancement during PCa metastasis is linked to transcription factors of the Snail family, which includes Snail and Slug [46]. Our study indicated that MPMCA inhibited Snail and Slug mRNA and protein expression. Overexpression of Snail and Slug cDNA abolished MPMCA-controlled restriction of EMT, migration, and invasion in PCa. In vivo studies further confirmed that MPMCA treatment attenuated Snail and Slug expression and reduced PCa metastasis. Thus, MPMCA reduces EMT and metastasis in PCa by suppressing Snail and Slug expression.

The PI3K/AKT/mTOR mechanism is a key modulator of malignancy and drug resistance in patients with solid cancers [48]. Clinical studies have shown that enhanced phosphorylation and activation of key components of the PI3K, AKT, mTOR pathway are linked with PCa progression [49,50,51,52,53]. Additionally, genomic and transcriptome analyses show that up to 42% of main PCa samples and 100% of metastatic PCa samples had genetic changes and dysregulated gene expression of PI3K pathway components, which are frequent in PCa patients [54,55,56,57,58]. The PI3K, AKT, mTOR mechanism is emerging as a potential therapeutic target for cancer and related diseases. According to the existing literature, few reports describe the effects of single phytochemicals or a limited number of natural compounds on the PI3K, AKT, mTOR mechanism in tumors [59]. Our results indicated that MPMCA markedly inhibited the phosphorylation levels of PI3K, AKT, and mTOR in PCa cells. Activators of PI3K, AKT, and mTOR rescued MPMCA-inhibited cell motility, indicating that the PI3K/AKT/mTOR cascade controls MPMCA-mediated suppression of PCa motility and metastasis.

Caffeic acid (CA) has been reported to exert antiproliferative and pro-apoptotic effects in prostate cancer models, primarily through suppression of the IL-6/JAK/STAT3 signaling axis in PC3 and LNCaP cell lines [60]. However, evidence regarding its role in regulating EMT and metastatic progression remains limited. Structural modification of CA may enhance cellular uptake, stability, and biological potency compared with the parent compound. In the present study, we demonstrate that the CA derivative MPMCA significantly suppresses EMT, migration, and invasion in both androgen-sensitive LNCaP cells and highly metastatic PC3 cells. These effects are accompanied by inhibition of the PI3K/AKT/mTOR signaling pathway and downregulation of the EMT-associated transcription factors Snail and Slug. Collectively, our findings suggest that chemical modification of CA confers enhanced anti-metastatic activity. Nevertheless, direct comparative analyses between CA and MPMCA are required in future studies to more precisely delineate their relative efficacy and structure–activity relationships.

In conclusion, this study demonstrated for the first time that the CA derivative MPMCA inhibits EMT, migration, and invasion in PCa cells. Preclinical animal experiments also demonstrated that MPMCA reduces PCa growth and distant metastasis in vivo. MPMCA restricts EMT, migration, and invasion by inhibiting the expression of the transcription factors Snail and Slug through suppression of the PI3K/AKT/mTOR pathway (Figure 8). These results suggest that MPMCA may be a promising candidate drug for the treatment of metastatic PCa.