Anticancer Activity of Methyl Protodioscin against Prostate Cancer by Modulation of Cholesterol-Associated MAPK Signaling Pathway via FOXO1 Induction

Abstract

Methyl protodioscin (MPD), a furostanol saponin found in the rhizomes of Dioscoreaceae, has lipid-lowering and broad anticancer properties. However, the efficacy of MPD in treating prostate cancer remains unexplored. Therefore, the present study aimed to evaluate the anticancer activity and action mechanism of MPD in prostate cancer. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound healing, transwell, and flow cytometer assays revealed that MPD suppressed proliferation, migration, cell cycle, and invasion and induced apoptosis of DU145 cells. Mechanistically, MPD decreased cholesterol concentration in the cholesterol oxidase, peroxidase and 4-aminoantipyrine phenol (COD-PAP) assay, disrupting the lipid rafts as detected using immunofluorescence and immunoblot analyses after sucrose density gradient centrifugation. Further, it reduced the associated mitogen-activated protein kinase (MAPK) signaling pathway protein P-extracellular regulated protein kinase (ERK), detected using immunoblot analysis. Forkhead box O (FOXO)1, a tumor suppressor and critical factor controlling cholesterol metabolism, was predicted to be a direct target of MPD and induced by MPD. Notably, in vivo studies demonstrated that MPD significantly reduced tumor size, suppressed cholesterol concentration and the MAPK signaling pathway, and induced FOXO1 expression and apoptosis in tumor tissue in a subcutaneous mouse model. These results suggest that MPD displays anti-prostate cancer activity by inducing FOXO1 protein, reducing cholesterol concentration, and disrupting lipid rafts. Consequently, the reduced MAPK signaling pathway suppresses proliferation, migration, invasion, and cell cycle and induces apoptosis of prostate cancer cells.

INTRODUCTION

Among the types of cancers affecting humans in the United States, the most prevalent malignancy is prostate cancer (PCa), with 248530 new cases in men in 2021, leading to 34130 deaths in the same year.¹⁾ Although several treatments may benefit patients with localized PCa, metastatic PCa remains lethal. Consequently, patients with metastatic PCa receive limited benefits from these treatments.²⁾ Additional therapeutic agents should be evaluated to improve the survival of patients with PCa.

PCa cells have higher cholesterol concentrations than normal cells, often twice the cholesterol concentration in their nuclei.³⁾ Cholesterol reportedly plays a multi-potent role in PCa⁴⁾ and has gained attention in PCa research in recent years. Increased cholesterol levels reportedly promote tumor growth and epithelial-to-mesenchymal transition (EMT), reducing the apoptosis of PCa cells through the cyclin E/AKT signaling pathway and extracellular regulated protein kinases 1/2 (ERK1/2).^5–7) Prolonged use of cholesterol-lowering statins is associated with reduced cancer-related mortality,⁸⁾ including a reduced risk of advanced PCa.⁹⁾ In addition, cholesterol maintains the stability of cholesterol and sphingolipid-rich membrane microdomains known as lipid rafts, which act as a platform for recruiting receptors and their downstream targets in signal transduction. Many important regulators of cell growth, cell adhesion, migration, and apoptosis are located in lipid rafts.³⁾ The interaction of transient receptor potential melastatin 8 with androgen receptors in lipid rafts enhances the migration of PCa cells.¹⁰⁾ The increasing cholesterol efflux and subsequent disruption of lipid rafts by Liver X receptors downregulate the AKT survival pathway, thus inducing apoptosis of PCa cells.¹¹⁾ These findings suggest cholesterol could serve as a diagnostic marker and therapeutic target for PCa.^12,13) Traditional Chinese medicine (TCM), a comprehensive efficacious therapy in treating most diseases worldwide, has attracted much research attention.^14,15) Thus, discovering novel TCM drugs that inhibit PCa cell cholesterol synthesis may be a promising approach for PCa treatment.

Methyl protodioscin (MPD), one of the main bioactive components of TCM Dioscoreaceae included in the Pharmacopoeia of the People’s Republic of China, is used for treating cardiovascular diseases and cancer.^16,17) MPD can prevent cardiovascular diseases by improving the expression of ATP-binding cassette transporter A1 (ABCA1) and inhibiting sterol regulatory element-binding proteins (SREBPs), leading to induced cholesterol efflux and reduced cholesterol synthesis, respectively.¹⁸⁾ MPD exhibits anticancer effects by suppressing proliferation and inducing apoptosis of HepG2 liver cancer,¹⁹⁾ A549 lung cancer,²⁰⁾ cervical cancer,²¹⁾ osteosarcoma,²²⁾ and pancreatic cancer cells.²³⁾ However, research on the anticancer effects of MPD in PCa remains limited. Additionally, whether the anticancer effect of MPD is associated with its cholesterol-lowering ability requires further research.

Therefore, this study aimed to investigate the inhibitory effects and molecular mechanisms of MPD against PCa to facilitate the discovery and development of anti-PCa drugs from TCM compounds.

MATERIALS AND METHODS

Cell Culture and Treatment

Castration-resistant prostate cancer (CRPC) cell line DU145 and murine prostate cancer cell line RM-1 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Before experiments, cell lines used in this study were tested for mycoplasma contamination using a Mycoalert™ kit (Lonza, Cat#LT07–218, Basel, Switzerland). DU145 cells were cultured in minimum essential medium (MEM) medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. RM-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. MPD was sourced from Chengdu HerbSubstance Biotechnology Co., Ltd. (Cat#54522-52-0, Chengdu, China), with a purity of up to 98%. To determine cell viability and subsequent mechanisms, DU145 and RM-1 cells were treated with different concentrations of MPD for 24 and 48 h to determine the role of MPD in PCa. FOXO1 inhibitor AS1842856 (98% purity) was purchased from MedChemExpress (Cat#HY-100596, Brea, CA, U.S.A.). Caspase 3 inhibitor Z-VAD-FMK (99.42% purity) was purchased from SparkJade (Cat#SJ-BP0022, Shandong, China). Cholesterol was sourced from Solarbio (Cat#C8280, Beijing, China).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

Cell proliferation was evaluated using an MTT kit (M1020, Solarbio). Briefly, cells were cultured in 96-well plates, with 5 × 10³ cells in each well. After 24 h, cells were treated with MPD for 24 or 48 h. MTT solution was added to the culture for 4 h, and absorbance was measured at 570 nm.

Cell Apoptosis Assay and Cell Cycle Evaluation

The fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kits (BB-4101, Bestbio Co., Ltd., Shanghai, China) were used to assess apoptosis in DU145 cells after treatment. Briefly, after treatment, DU145 cells (1 × 10⁶) were carefully digested with trypsin and stained with FITC/PI cell apoptosis kit. Immediately afterward, the cell apoptosis levels were determined using flow cytometer.

For cell cycle evaluation, DU145 cells (1 × 10⁶) were collected, washed twice with phosphate buffered saline (PBS), and fixed in 1 mL cold 70% ethanol for 12 h. Next, the cells were collected, washed, and stained with a cell cycle and apoptosis analysis kit (BL114A, Biosharp, Shanghai, China). Finally, cell cycle analysis was performed using flow cytometer.

Scratch Wound Healing and Transwell Assays

DU145 cells (1 × 10⁵) were seeded and cultured in a 24-well plate. After 24 h, a 10 µL tip was used to draw scratches perpendicular to the horizontal line on the cell plate, followed by PBS washing twice. Three different areas were selected under the microscope and analyzed using Image J software to calculate the healing rate of the cell scratches.

We used the transwell assay (8 µm well, #3422, Corning, NY, U.S.A.) to detect cell invasion. Twenty microliters Matrigel (#356234, Corning, NY, U.S.A.) was applied to the transwell upper chamber and preincubated at 37 °C for half an hour to gel. Cells (1 × 10⁵) were incubated in a matrix gel-coated chamber with 100 µL MEM and the lower chamber with Dulbecco’s modified Eagle’s medium (DMEM) (containing 80% serum). After 48 h of MPD treatment, the cells were fixed in methanol and stained with a crystal violet solution. Images were counted under a microscope in three randomly selected areas.

Immunoblot Analysis, Isolation of Lipid Rafts, and Immunofluorescence Staining

Cells or tissues were homogenized in radio-immunoprecipitation assay (RIPA) buffer. After centrifugation at 15000 × g for 15 min at 4 °C, total protein was collected and prepared for immunoblot analysis. Briefly, proteins (30 µg/lane) were loaded onto sodium dodecyl sulfate (SDS) gels and transferred onto polyvinylidene fluoride membranes. After blocking with 5% (w/v) skim milk, the membranes were probed with the primary antibodies listed in Supplementary Table S1 at 4 °C overnight and incubated with HRP-conjugated secondary antibody for 1 h at 37 °C. The signals on the membrane were visualized using an enhanced chemiluminescence detection kit. The Image J software was used to analyze the target bands.

The method described in the literature was optimized for separating lipid rafts.^24,25) DU145 cells (1 × 10⁷) with different treatments were lysed in 1.5 mL of lysis buffer and homogenized for 30 min on ice. After 5 min of centrifugation at 500 × g, the supernatant was collected. An equal amount of lysate was adjusted to a final concentration of 40% using a 60% OptiPrep solution (D1556-250ML, Sigma-Aldrich, St. Louis, MO, U.S.A.) and then overlaid with 30% OptiPrep in lysis buffer. Samples were centrifuged at 100000 × g for 4 h at 4 °C (TLS55 rotor, Beckman Coulter, Brea, CA, U.S.A.). Fractions (F1–F9) from the top of the gradient to the end were collected. Western blot was used to test for lipid rafts.

For immunofluorescence staining, DU145 cells were seeded onto climbing tablets in plates, fixed with 4% paraformaldehyde, and permeabilized with a staining buffer containing 0.05% Triton X-100. Cells were then incubated with the rabbit anti-Flotillin1 primary antibody overnight at 4 °C and incubated for 1 h with the Alexa-594 mouse anti-rabbit immunoglobulin G (IgG) at 20 µg/mL. After washing three times, 4′-6-diamidino-2-phenylindole (DAPI) was applied for 5 min to stain the nuclei. The slides were examined using confocal microscopy (FV3000, Olympus, Tokyo, Japan).

Detection of the mRNA Expression Level

Total RNA in the cell line was extracted with the TRIzol reagent (Omega, Norcross, GA, U.S.A.). Total RNA (2 µg) was used to synthesize cDNA. A reaction mixture (20 µL) of PrimeScriptTM first strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan) was added for quantitative real-time PCR (qRT-PCR). Relative gene expression was analyzed using the 2-^ΔΔCt method. The primer sequences used in this experiment are listed in Supplementary Table S2.

Animal Models and Cholesterol Concentration Measurement

Male C57BL/6 mice (4–6-week old, 18–20 g) were used for in vivo experiments to study the effects of MPD on tumor-generating capacity. RM-1 cells were injected subcutaneously into the right armpit of C57BL/6 mice and allowed to form tumors. The mice were randomly divided into the following groups: control (saline), low MPD (0.5 mg/kg, dissolved in saline solution), and high MPD (1 mg/kg, dissolved in saline solution). Mice were administered with saline or MPD via tail vein injection for 15 d. Tumor length and width were measured every 2 d to record tumor growth. Mice were sacrificed after the administration protocol, and tumor tissues were divided into fixed tumors for hematoxylin–eosin (H&E) staining or TdT-mediated dUTP nick-end labeling (TUNEL) assay and frozen tumors for protein extraction or cholesterol detection. All studies were approved by the Institutional Animal Care and Use Committee of the Anhui University of Chinese Medicine. All animals were treated according to institutional guidelines. Paraformaldehyde (4%) was used for fixing tumor tissues for H&E staining. Stained images were observed and photographed under an optical microscope (Olympus).

For TUNEL assay, the tumor tissue sections (5 µm thick) in each group were stained with the TUNEL Apoptosis Detection kit (S1086, Shanghai, China). The slides were visualized using confocal microscopy. Cells with green fluorescence were apoptotic cells.

The cholesterol concentration was measured using the cholesterol oxidase, peroxidase and 4-aminoantipyrine phenol (COD-PAP) assay (A111-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). DU145 cells were collected and treated with RIPA buffer. The cell suspensions were added to plates and incubated with COD-PAP working buffer for 10 min at 37 °C. The absorbance of the plates was measured at 510 nm. For the cholesterol test in serum, mice sera were transferred to plates and incubated with COD-PAP working buffer for 10 min at 37 °C. The absorbance at 510 nm was determined using a microplate reader. For the cholesterol test in tumor tissue, weighed tissue was crushed in RIPA buffer, and the homogenate was added to plates and incubated with COD-PAP working buffer for 10 min at 37 °C. The absorbance at 510 nm was measured.

Molecular Docking

We searched the PubChem database for the MPD molecular structure and downloaded the file in SDF format. The X-ray crystal structure of FOXO1 was retrieved from the RCSB. Proteins were hydrotreated to remove water molecules using AutoTool, and energy grid calculations were performed using AutoGrid. Autodock Vina was used for docking the ligand with the protein. The conformation with the highest score was used for analysis and drawing.

RESULTS

MPD Suppresses the Proliferation of DU145 and RM-1 Cells

Cells from different species were exposed to different MPD concentrations (1, 2, 3, 4, 5, and 10 µM MPD and control group) and incubated for 24 and 48 h. Based on MTT assay results, MPD significantly inhibited the viability of DU145 cells, a castration-resistant PCa cell line, in a time- and dose-dependent manner (Fig. 1A). After 48 h of MPD treatment, the IC₅₀ value was about 4 µM. MPD also suppressed RM-1 cells, a prostate cancer cell line from C57BL/6 mice, in a dose- and time-dependent manner, as determined by the MTT assay (Fig. 1B). After 48 h, MPD inhibited RM-1 cells growth with IC₅₀ values of 6 µM. Thus, MPD inhibited the proliferation of PCa cells in humans and mice. Therefore, concentrations of 1, 2, and 4 µM MPD were chosen for treating DU145 cells in the subsequent experiments. In addition to inhibiting the proliferation of DU145 cells, MPD also caused cell atrophy, as observed by microscopy (Fig. 1C), possibly indicating apoptotic cell death.

Fig. 1. Anti-proliferative Effects of Methyl Protodioscin (MPD) on DU145 and RM-1 Cells

(A) Cell viability of human prostate cancer DU145 cells was analyzed using MTT assay after treatment with different concentrations of MPD for 24 or 48 h. (B) Cell viability of mouse prostate cancer RM-1 cells was analyzed using MTT assay after treatment with different concentrations of MPD for 24 or 48 h. (C) Representative phenotypes of MPD-treated DU145 cells and the control group. Scale bars, 10 µM. Data are presented as the mean ± standard deviation (S.D.) of three independent experiments. Significant differences from the control group (0 µM MPD) were determined using Student’s t-test, * p < 0.05, ** p < 0.01.

MPD Induces Apoptosis and Inhibits Cell Cycle in DU145 Cells

DU145 cells were stained with FITC and PI, which indicate viable and non-viable non-apoptotic cells, respectively, and subsequently analyzed using flow cytometry to confirm the induction of cell apoptosis by MPD. After treating DU145 with MPD and placebo (untreated control), respectively, for 48 h, 0% of the untreated cells exhibited early apoptosis in the Q4 region, whereas 0.02% of the untreated cells exhibited late apoptosis in the Q2 region. After treatment with 4 µM MPD for 48 h, the corresponding quantities increased significantly to 85.8 and 11.4%, respectively (Figs. 2A, B). These results indicated that DU145 cells underwent apoptosis after MPD treatment. Bax, a pro-apoptotic protein in the family of apoptosis, is abundantly and selectively expressed in the process of apoptosis and promotes cell death.²⁶⁾ Bcl-2 is an anti-apoptotic protein that downregulates the apoptotic pathway and prevents cell death.²⁷⁾ In the caspase family, the activation of cleaved caspase-3 is the main mechanism of apoptosis.²⁸⁾ Cleaved poly(ADP-ribose) polymerase (PARP), an important substrate cleaved by activated caspase 3, is used as a hallmark of apoptosis together with caspase 3.²⁹⁾ These apoptosis-related proteins are widely used to evaluate apoptosis in cells. Therefore, the expression of Bax, Bcl2, cleaved caspase-3, and cleaved PARP proteins was determined using a Western blot to analyze apoptosis in DU145 elicited by MPD treatment. The level of Bcl2 decreased, while that of Bax, cleaved caspase-3, and cleaved PARP increased gradually in a dose-dependent manner after MPD treatment (Figs. 2C, D). We used caspase 3 inhibitor Z-VAD-FMK to confirm MPD-induced apoptosis in DU145 cells. Our results showed that Z-VAD-FMK suppressed the MPD-induced cleaved caspase-3 accumulation (Figs. 2E, F). These results illustrate that MPD could induce the apoptosis of DU145.

Fig. 2. Effect of MPD on Apoptotic Induction and Cell Cycle Arrest in DU145 Cells

(A) DU145 cells were treated with different concentrations of MPD for 48 h, cultured, and stained with FITC/PI. Cell apoptosis was determined using flow cytometry. Q1 region, high PI staining representing non-viable non-apoptotic cells; Q2 region, high FITC and PI staining representing non-viable apoptotic cells; Q3 region, low FITC and PI staining representing living cells; and Q4 region, high FITC staining representing viable apoptotic cells. The numbers in red color represent the proportion of cells in each region. (B) Histograms represent the apoptosis ratio (Q2 region + Q4 region) of DU145 cells from (A) after treatment with MPD for 48 h. (C) Western blot was used to detect the expression levels of Bcl2, Bax, cleaved caspase-3, and cleaved PARP proteins in DU145 cells after MPD treatment. The cells were treated with MPD at doses of 0, 1, 2, and 4 µM MPD. GAPDH was used as an internal reference. (D) Histograms represent the quantification of the Bcl2, Bax, cleaved caspase-3, and cleaved PARP bands from (C). Bax and Bcl2 protein levels are expressed as the ratio of total protein to GAPDH. (E) Effects of Z-VAD-FMK (caspase inhibitor) on the protein expression of cleaved caspase 3 after treatment with 4 µM MPD. (F) Histograms represent the quantification of the cleaved caspase-3 bands from (E). Cleaved caspase-3 protein levels are expressed as total protein ratio to GAPDH. (G) Cell cycle detection using flow cytometry after DU145 cells were cultured with different concentration of MPD for 48 h and stained with PI. (H) Histograms represent the percentage of DU145 cells in each phase from (G) after treatment with MPD for 48 h. Data from three independent experiments (mean standard error n = 3) analyzed using ANOVA presented significant differences indicated by letters (p < 0.05).

Flow cytometer was used to assess whether MPD also inhibited PCa cell cycle progression. With MPD treatment, the percentage of DU145 cells in the G2/M phase increased remarkably, indicating that MPD induced cell cycle arrest at the G2/M phase (Figs. 2G, H).

MPD Suppresses DU145 Cell Migration and Invasion

Wound healing and transwell experiments were used to assess the anti-migration activity of MPD on DU145 cells. As expected, MPD substantially suppressed the migration of DU145 cells (Figs. 3A, B). Next, the transwell assay revealed that treatment with 4 µM MPD remarkably reduced the number of DU145 cells moving through the membrane (Figs. 3C, D). The above results indicate that MPD substantially inhibited the migration and invasion of DU145 cells in vitro.

Fig. 3. Effect of MPD on the Migration and Invasion of DU145 Cells

(A) Cell scratch test was used to detect the anti-migration activity of MPD on DU145 cells, applied at different concentrations for 0 or 48 h. Scale bars, 100 µm. (B) Scratch healing ratio of DU145 cells from (A). (C) Transwell assay was performed to detect the invasion of DU145 cells after culture with different concentrations of MPD for 48 h. (D) Number of invaded cells from (C). Each independent experiment was performed in triplicate. Bars, mean ± standard error (n = 3). Different letters indicate significant differences (p < 0.05), determined using ANOVA.

MPD Inhibits Cholesterol Synthesis in DU145 Cells

MPD, the main component of Dioscoreaceae, has been used to treat dyslipidemia and is a source of active components for anticancer drugs. During dyslipidemia treatment, the main component, MPD, increased cholesterol efflux through increased levels of ABCA1 mRNA and protein and inhibited the transcription of SREBP1 and SREBP2, leading to an increase in ABCA1 levels in THP-1 macrophages and HepG2 liver cancer cells.¹⁸⁾ MPD exerted anticancer effects by inducing G2/M cell cycle arrest and apoptosis in HepG2 liver,¹⁹⁾ A549 lung,²⁰⁾ and cervical cancer cells.²¹⁾ MPD also induced apoptosis in osteosarcoma cells via caspase-dependent and mitogen-activated protein kinase (MAPK) signaling pathways.²²⁾ Further, it suppressed proliferation and inhibited glycolysis in pancreatic cancer cells.²³⁾ Considering that increased cholesterol levels reportedly promote tumor growth^5,6) and MPD exhibited both cholesterol-lowering and anticancer functions, we investigated whether MPD exhibited anticancer function by lowering cholesterol levels. We quantified total cholesterol concentration in DU145 cells after treatment with MPD to investigate the mechanism by which MPD inhibits DU145 cells and the cholesterol involvement. The total cholesterol concentration significantly decreased (Fig. 4A). SREBPs, regulators of cholesterol and lipogenesis, can transcribe and activate a series of enzymes needed for the synthesis of endogenous cholesterol, fatty acid (FA), triacylglycerol, and phospholipid synthesis,³⁰⁾ whereas an aberrant SREBP-dependent lipogenic program reportedly promotes metastatic prostate cancer.³¹⁾ Among the SREBP family, SREBP1 is involved in FA synthesis and insulin-induced glucose metabolism (particularly lipogenesis), whereas SREBP2 is relatively specific to cholesterol synthesis.³⁰⁾ Here, the qRT-PCR analysis revealed that MPD decreased the mRNA expression of SREBP1 and SREBP2. The effect was more pronounced for SREBP2 (Fig. 4B). This result indicates that although MPD has a negative effect on both FA and cholesterol synthesis, the inhibitory effect on cholesterol synthesis is more potent. The expression of the hydroxymethylglutaryl-CoA (HMG-CoA) reductase (HMGCR) gene, encoding the rate-limiting enzyme of cholesterol synthesis, downstream of SREBP2, decreased after MPD treatment (Fig. 4C). As SREBP2 contains an intronic microRNA (miR-33) that inhibits the expression of ABCA1, decreased SREBP2 expression induced transcription of the cholesterol export pump ABCA1 after MPD treatment, leading to a further decrease in cholesterol levels (Fig. 4C).

Fig. 4. Effect of Methyl Protodioscin on Cholesterol Synthesis in DU145

(A) Total cholesterol content in DU145 cells after MPD treatment for 48 h. (B) Expression levels of SREBP1 and SREBP2 relative to GAPDH in DU145 cells after MPD treatment for 48 h. (C) Expression levels of ABCA1 and HMGCR in DU145 cells after MPD treatment for 48 h. (D) Total cholesterol content in DU145 cells after treatment with different concentrations of MPD alone or with cholesterol (CHOL) for 48 h. (E) Cell viability of DU145 cells after treatment with different concentrations of MPD alone or with cholesterol (CHOL) for 48 h. (F) Expression levels of SREBP1 and SREBP2 relative to that of GAPDH in DU145 cells after treatment with various concentrations of MPD alone or with cholesterol (CHOL) for 48 h. (G) Effects of cholesterol on the protein expression of Bcl2 and cleaved caspase 3 after treatment with 4 µM MPD. (H) Histograms represent the quantification of the Bcl2 and cleaved caspase 3 bands from (G). Bcl2 and cleaved caspase-3 protein levels are expressed as the ratio of total protein to GAPDH. (I) Effects of cholesterol on the MPD-induced cell cycle arrest at G2/M phase in DU145.(J) Cell scratch test was used to detect the migration activity of DU145 cells after treatment with various concentrations of MPD alone or with cholesterol (CHOL) for 48 h. Scale bars, 100 µm. (K) S cratch healing ratio of DU145 cells from (J). (L) Transwell assay was performed to detect the invasion of DU145 cells after treatment with various concentrations of MPD alone or with cholesterol (CHOL) for 48 h. (M) The number of invading cells from (C). Bars, mean ± standard error (n = 3). Different letters indicate significant differences (p < 0.05), determined using ANOVA.

Additionally, cholesterol supplementation after MPD treatment rescued the MPD-inhibited cholesterol concentration (Fig. 4D), SREBP1/2 transcription (Fig. 4E) and proliferation (Fig. 4F) of DU145 cells. The anti-apoptotic protein Bcl2 inhibited by MPD was recovered, and cleaved caspase-3 induced by MPD was also suppressed after cholesterol treatment (Figs. 4G, H). The flow cytometer, wound healing, and transwell experiments showed that MPD inhibited cell cycle, invasion, and migration effects were attenuated when treatment with MPD was combined with cholesterol (Figs. 4I–M). The above data show that the disincentive effect of MPD on DU145 cells is mediated by the regulation of cholesterol synthesis.

Lipid Raft Modulation by MPD Results in the Disruption of MAPK Signaling Components

Lipid rafts are critical cell membrane platforms rich in sphingolipids and cholesterol. They are involved in diverse cellular processes that modulate proliferation, death, and metastasis, suggesting that they might be promising targets in cancer therapy.³²⁾ MPD treatment decreased cholesterol, the main component of lipid rafts. We detected lipid rafts in DU145 cells using immunofluorescence and immunoblot analysis after sucrose density-gradient centrifugation. Flotillin1, a lipid raft protein, regulates cell proliferation and signal transduction and is extensively used as a lipid raft protein marker.³³⁾ Immunofluorescence analysis suggested that Flotillin1 localization was not uniform and exhibited a patchy distribution in the PM of DU145 cells, especially on the contact surface between two cells (Fig. 5A, top left, white arrow), similar to another report.³⁴⁾ After MPD treatment, Flotillin1 was mainly distributed intracellularly as vesicles (Fig. 5A, middle left, yellow arrow), similar to the modulation of lipid rafts by 10-gingerol,³⁵⁾ indicating the disruption of lipid rafts by cholesterol reduction. Cholesterol supplementation after MPD treatment rescued the patchy distribution of Flotillin1 in the plasma membrane (PM) of DU145 cells (Fig. 5A, bottom left, yellow arrow). For immunoblot analysis, the lipid rafts could be extracted using a density gradient; nine layers in total were isolated after Triton X-100 treatment, with the lipid rafts in the first three layers corresponding to 0–30% of OptiPrep.^25,35) Flotillin1 was detected in the first three fractions, indicating the distribution of Flotillin1 in lipid rafts (Fig. 5B, upper panel). Compared to that in the untreated control, Flotillin1 after MPD treatment moved from the raft to non-raft areas (Fig. 5B, middle; Fig. 5C), further confirming that disruption of lipid rafts by cholesterol reduction contributed to Flotillin1 intracellular localization. As expected, cholesterol supplementation after MPD treatment rescued the distribution of Flotillin1 in lipid rafts (Fig. 5B, bottom, and Fig. 5C). These results illustrate that MPD modulates lipid rafts in prostate cancer, mediated through cholesterol reduction.

Fig. 5. MPD Modulates Lipid Rafts and Related MAPK Signaling Pathway in DU145 Cells

(A) Distribution of lipid raft marker Flotillin1 in cell membranes examined using immunofluorescence after subjecting DU145 cells to various treatments for 48 h. Scale bars, 10 µm. The white arrows indicate the accumulation of Flotillin1 in the plasma membrane. The yellow arrow indicates the intracellular distribution of flotillin1. (B) The distribution of Flotillin1 in lipid rafts (fractions 1–3) and non-raft fractions (fractions 4–9) of DU145 cells. (C) Percentage of Flotillin1 protein in lipid rafts of DU145 cells after different treatments in (B). (D) Immunoblot analysis of ERK and P-ERK levels in DU145 cells using anti-ERK and anti-P-ERK antibodies. (E) Histograms represent the quantification of the ERK and P-ERK bands from (D). ERK and P-ERK protein levels are expressed as the ratio of each protein relative to GAPDH. (F) Immunoblot analysis of ERK and P-ERK expression in DU145 cells using anti-ERK and anti-P-ERK antibodies. (G) Histograms represent the quantification of the ERK and P-ERK bands from (F). ERK and P-ERK protein levels are expressed as the ratio of each protein relative to GAPDH. Each independent experiment was performed in triplicate. Bars, mean ± standard error (n = 3). Different letters indicate significant differences (p < 0.05), determined using ANOVA.

Cholesterol reportedly induces EMT in prostate cancer cells through activation of the ERK1/2 pathway; epidermal growth factor receptor and adipocyte PM-associated proteins mediate this process by accumulation in lipid rafts.⁷⁾ Therefore, we examined the MAPK signaling pathway, a downstream signal of cholesterol regulation. Immunoblot analysis revealed decreased phosphorylation of ERK1/2 in DU145 cells after MPD treatment but no change in ERK1/2 (Figs. 5D, E). Cholesterol supplementation after MPD treatment rescued ERK1/2 phosphorylation (Figs. 5F, G). These results indicate that modulation of cholesterol in DU145 cells by MPD treatment leads to the disruption of lipid rafts and the MAPK signaling pathway, inhibiting cell growth, metastasis, and invasion while inducing cell apoptosis.

MPD Significantly Upregulates FOXO1 in DU145 Cells

Although MPD has multiple effects, including anti-dyslipidemic and anticancer effects, research on the direct targets of MPD remains limited. FOXO transcription factors are involved in cell death and multiple signaling pathways necessary for angiogenesis, cell proliferation, and tumor suppressor gene function. Hence, the FOXO family is a target for cancer regulation.^36,37) FOXO1, as a critical negative regulatory transcription factor in tumors, leads to growth, metastasis, and reduced survival of cancer cells.³⁸⁾ Furthermore, FOXO1 is directly associated with the SREBP1 and SREBP2 promoters. It negatively regulates the transcriptional level of SREBP1 and SREBP2 via multiple mechanisms to regulate lipid metabolism.^39,40) The similarity of both the lipid-lowering and anticancer functions of FOXO1 to MPD led us to speculate that FOXO1 may be the direct target of MPD.

To explore the target of MPD and its effect on FOXO1, we predicted the interaction of FOXO1 with MPD using the Discovery Studio software (Fig. 6A). The affinity between FOXO1 and MPD is approximately −7 kcal/mol, lower than the required value of −1.435 kcal/mol, indicating strong binding between FOXO1 and MPD. After MPD treatment, FOXO1 protein levels increased in a concentration-dependent manner, whereas cholesterol supplementation inhibited the expression of FOXO1 protein (Figs. 6B, C). As the transcription of FOXO1 was unaltered after MPD treatment (Fig. 6D), the inhibitory effect of MPD on FOXO1 likely occurred at the post-translational level. We treated DU145 cells with AS1842856, a FOXO1 inhibitor regarded as a valuable tool for examining the function of FOXO1 in animals,⁴¹⁾ to confirm the specific role of FOXO1 in MPD anticancer function. The cholesterol concentration (Fig. 6E) and P-ERK protein (Figs. 6F, G) reduced by MPD significantly increased after AS1842856 treatment. Moreover, AS1842856 supplementation after MPD treatment rescued the distribution of Flotillin1 in lipid rafts (Figs. 6H, I). The mRNA expression of SREBP1, SREBP2, and HMGCR decreased by MPD treatment, and ABCA1 induced by MPD was reversed with AS1842856 supplementation (Fig. 6J). The above data shows that the reduction of cholesterol and the associated MAPK signaling pathway by MPD is mediated by FOXO1 induction.

Fig. 6. FOXO1 Protein Is Induced in MPD-Treated DU145 Cells

(A) Prediction of the interaction structure of FOXO1 with MPD. The blue helix represents the amino acids of FOXO1. The green grid represents MPD. The yellow dotted lines represent the hydrogen bonds between MPD and amino acids of FOXO1. In MPD, the red line represents an oxygen atom, and the white line represents a hydrogen atom. (B) Immunoblot analysis of FOXO1 expression in DU145 cells using anti-FOXO1 antibody after different treatments. (C) Histograms represent the quantification of the FOXO1 bands from (B). FOXO1 protein levels are expressed as the ratio of FOXO1 relative to GAPDH. (D) Relative expression levels of FOXO1 in DU145 cells after MPD treatment for 48 h. (E) Total cholesterol content in DU145 cells after treatment with MPD alone or with FOXO1 inhibitor AS1842856 (100 nM) for 48 h. (F) Immunoblot analysis of ERK and P-ERK expression in DU145 cells using anti-ERK and anti-P-ERK antibodies. Total protein was extracted from DU145 cells treated with MPD alone or with FOXO1 inhibitor AS1842856 (100 nM) for 48 h. (G) Histograms represent the quantification of the ERK and P-ERK bands from (F). ERK and P-ERK protein levels are expressed as the ratio of each protein relative to GAPDH. (H) The distribution of flotillin1 in lipid rafts (fractions 1–3) and non-raft fractions (fractions 4–9) of DU145 cells after treatment with MPD alone or with FOXO1 inhibitor AS1842856 (100 nM) for 48 h. (I) Percentage of flotillin1 protein in lipid rafts of DU145 cells after the different treatments in (H). (J) Expression levels of SREBP1, SREBP2, ABCA1, and HMGCR relative to GAPDH in DU145 cells after treatment with MPD alone or with FOXO1 inhibitor AS1842856 (100 nM) for 48 h. Independent experiments were performed in triplicates. Bars, mean ± standard error (n = 3). Different letters indicate significant differences (p < 0.05), determined using ANOVA.

MPD Inhibits Tumor Growth and Cholesterol Concentration in Vivo

We used a subcutaneous prostate cancer mouse model to assess whether MPD suppresses the growth of prostate cancer cells in vivo. Administration of low (0.5 mg/kg injection per day) and high MPD (1 mg/kg injection per day) concentrations significantly inhibited the tumor growth capacity of RM-1 cells in vivo (Fig. 7A), including tumor weight (Fig. 7B) and volume (Fig. 7C). However, the weight of the mice was unaffected (Fig. 7D). These results demonstrated the inhibitory effect of MPD on prostate cancer in vivo. To validate the effect of MPD on cholesterol in vivo, we measured cholesterol concentration in the serum and tumor tissue of mice subcutaneously injected with RM-1. MPD treatment remarkably decreased the cholesterol content in the tumor tissue (Fig. 7E). However, it did not influence serum cholesterol (Fig. 7F), illustrating that MPD inhibition of cholesterol occurs mainly in tumor tissue.

Fig. 7. MPD Reduced Tumor Growth, Cholesterol Synthesis, and MAPK Signaling Pathway and Induced FOXO1 Expression in Vivo

(A) Representative image of tumor samples from C57BL/6 mice bearing subcutaneous PCa RM-1 cells after MPD treatment. Mice bearing subcutaneous RM-1 cells were administered 0 (control), 0.5 (low MPD), and 1 mg/kg (high MPD) MPD for 15 d. Tumor samples were then collected and imaged using a high-definition digital camera. (B) Weight of tumor samples from C57BL/6 mice bearing subcutaneous PCa RM-1 cells. (C) Daily changes in the volume of tumor samples. Results represent mean ± S.E.M. from seven mice. Significant differences from the control group were determined using Student’s t-test; * p < 0.05. (D) Daily changes in the weight of mice. (E) Total cholesterol content in tumor tissue. (F) Total cholesterol content in mice serum. (G) Pathological changes in the tumors in different groups of mice as visualized after staining with hematoxylin–eosin (H&E) and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling). Blue in TUNEL staining is DAPI, and green in TUNEL staining is FITC. Scale bar, 100 µm. (H) Immunoblot analysis of the expression of FOXO1, P-ERK, ERK, Bcl2, Bax, cleaved caspase-3, and cleaved PARP in tumors using an anti-FOXO1 antibody, anti-ERK antibody, anti-P-ERK antibody, anti-Bcl2 antibody, anti-Bax antibody, anti-cleaved caspase-3 antibody, and anti-cleaved PARP antibody, respectively. GAPDH was used as an internal reference. (I) Histograms represent quantification of the FOXO1, P-ERK, ERK, Bcl2, Bax, cleaved caspase-3, and cleaved PARP bands from (H). FOXO1, P-ERK, ERK, Bcl2, Bax, cleaved caspase-3, and cleaved PARP protein levels are expressed as the ratio of each protein relative to GAPDH. Independent experiments were performed in five replicates. Bars, mean ± standard error (n = 5). Different letters indicate significant differences (p < 0.05), determined using ANOVA.

H&E staining was used to observe the pathological tissue (Fig. 7G). The cells grew well in untreated tumors and possessed blue, large, and intensely stained nuclei. After MPD treatment, the nuclear staining was weaker in the tumor cells than in normal cells, and nuclei size was reduced or fragmented. The TUNEL Apoptosis Detection kit was used to detect the cell apoptosis of tumor tissue (Fig. 7G); apoptosis cells were stained green. The MPD-treated group showed obvious apoptosis. Similar to protein expression changes in vitro, MPD treatment in a subcutaneous prostate cancer mouse model induced the expression of FOXO1, Bax, cleaved caspase-3, and cleaved PARP and inhibited the expression of P-ERK and Bcl2 (Figs. 7H, I), indicating the MAPK signaling pathway disruption and apoptosis induction function of MPD in vivo. The targeted induction of FOXO1 via MPD impaired the MAPK signaling pathway and tumor cell proliferation in prostate cancer in vivo.

DISCUSSION

Cholesterol is a steroid lipid that participates in membrane and steroid synthesis.⁴²⁾ It mediates cell proliferation, inflammation, and steroid production. In addition, it participates in the ab-initio synthesis of androgens, affecting androgen levels in prostate cancer cells and playing an important role in prostate cancer.^42,43) The cholesterol homeostasis disorder is considered one of the manifestations of cancer.⁴⁴⁾ In the field of prostate cancer research, it is crucial to find drugs and treatment options targeting cholesterol.

In this study, we report on a natural compound, MPD, which displayed anti-prostate cancer activity by reducing cholesterol levels and the associated MAPK signaling pathway in CRPC DU145 cells. Lipid rafts, the cholesterol-enriched platform acting as a signaling hub in cancer cell invasion, survival, and death, were disrupted by MPD. The effect of MPD on prostate cancer may be pleiotropic, except for the MAPK signaling pathway. Ediriweera et al. showed that gingerol damaged the production of lipid rafts in radiation-resistant triple-negative breast cancer cells, which then disrupted the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, thereby inhibiting the progression of cancer.³⁵⁾ The PI3K/Akt pathway has a role in prostate cancer metastasis and tumor growth.⁴⁵⁾ As expected, MPD significantly decreased P-Akt protein levels compared with the control. In contrast, the Akt protein level did not change (Supplementary Fig. S1). In addition, vascular endothelial growth factor receptor 2 (VEGFR2), which regulates the production of tumor blood vessels, is colocalized with lipid rafts, and by destroying lipid rafts, it can increase lysosomal degradation of VEGFR2, thereby inhibiting tumor angiogenesis in PCa.⁴⁶⁾ The CD44 is involved in cancer metastasis when contacted with the enzyme processing required for its activity. It must migrate outward from the lipid raft. Destroying lipid rafts, which stops CD44 from recruiting to lipid rafts, promotes CD44 shedding and inhibits CD44-dependent cancer cell motility.⁴⁷⁾ The potassium channel SK3, which controls constitutive Ca²⁺ entry to induce cancer cell migration, also localizes in lipid raft, exhibiting its function through interaction with the orai1 Ca²⁺ channel in lipid rafts.⁴⁸⁾ Whether these signaling pathways in lipid rafts are also affected by MPD requires further research. Moreover, caveolin and Flotillin-containing structures of lipid rafts promoted the localization of membrane proteins on the lipid rafts and regulated cell proliferation and signal transduction.⁴⁹⁾ Our results show that the affected localization of Flotillin1 indicates that the lipid raft is damaged (Fig. 5). Similarly, the destruction of the lipid raft could also affect the localization of caveolin-1. A high level of intracellular caveolin-1 expression is linked with metastatic progression of human prostate cancer.⁵⁰⁾ Therefore, caveolin-1 shows considerable promise in the treatment of PCa.

FOXO1 is considered the direct target of MPD, and MPD induces its protein expression. Considering the lack of change of FOXO1 mRNA after MPD treatment, MPD may affect protein stability. Further research will be required to confirm the interaction of MPD with FOXO1 using surface plasmon resonance (SPR) or NMR, the methodology used to confirm the epigallocatechin gallate (EGCG)-P53 interaction contributing to the stabilization of p53 protein.⁵¹⁾ In addition to acting as a critical negative regulatory transcription factor for tumors, FOXO1 could negatively regulate the transcriptional level of SREBP2 by specifically recognizing the sterol regulatory element (IRE) sequence, a key factor determining cholesterol synthesis.⁴⁰⁾ Furthermore, cholesterol metabolism affects the immune response and the ability of organisms to clear away infection and tumor cells; hence, its proper metabolism is an important factor for maintaining a balanced and healthy body.⁵²⁾ However, high cholesterol level promotes the expression of T cell immune checkpoints, making T cells more easily inhibited and thus losing anti-tumor function.⁵³⁾ Cancer cells have higher cholesterol levels than normal cells. Therefore, in addition to the lipid raft-regulated signaling pathway, MPD could also play an anti-tumor role by regulating the tumor function mediated by FOXO1 or reducing cholesterol content to regulate inflammation.

Natural products have been used in several clinical applications since ancient times.⁵⁴⁾ Dioscoreaceae, is well known for its unique medical and nutritional properties. The active components of Dioscoreaceae reportedly exhibited therapeutic effects on autoimmune thyroiditis in a rat model,⁵⁵⁾ lung ischemia/reperfusion injury,⁵⁶⁾ and breast cancer.⁵⁷⁾ Based on the in vitro and in vivo profiles reported here, MPD may be an essential compound for the in vivo therapeutic effects of Dioscoreaceae extract in patients with prostate cancer. Our findings indicate that MPD may be considered a putative natural anti-prostate cancer agent in the form of functional foods or medicinal products.

In conclusion, MPD, an active component of the TCM herb Dioscoreaceae, displays anti-prostate cancer activity by inducing FOXO1 protein, leading to a decrease in cholesterol concentration and remolding of lipid rafts in cancer cells. In addition, the reduced signaling in the associated MAPK pathway suppressed proliferation, migration, and invasion and induced apoptosis of PCa cells.

Acknowledgments

This work was supported by the following Grants: Key Natural Science Research Projects in Anhui Universities under Grant KJ2021A0540; the Talent Support Program of Anhui University of Chinese Medicine under Grant 2020rcyb004; the Foundation of Anhui Province Key Laboratory of Research & Development of Chinese Medicine under Grant AKLPDCM202008; and the Natural Science Foundation of Anhui Province under Grants 2208085QH278 and 2108085QH374.

Author Contributions

JC: Conceptualization, investigation, methodology, and data analysis; PQ: investigation, methodology, and data analysis. ZT: investigation, methodology, data analysis, and contribution to manuscript writing. WD: investigation and methodology; YY: investigation and methodology; WX: methodology and data analysis; DY: conceptualization, data analysis, and funding acquisition. ST: conceptualization, methodology, data analysis, contribution to manuscript writing, and funding acquisition. All authors have read and approved the final version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2021. CA Cancer J. Clin., 71, 7–33 (2021).
• 2) Wu JN, Fish KM, Evans CP, DeVere White RW, Dall’Era MA. No improvement noted in overall or cause-specific survival for men presenting with metastatic prostate cancer over a 20-year period. Cancer, 120, 818–823 (2014).
• 3) Yang J, Wang L, Jia R. Role of de novo cholesterol synthesis enzymes in cancer. J. Cancer, 11, 1761–1767 (2020).
• 4) Wilson RL, Taaffe DR, Newton RU, Hart NH, Lyons-Wall P, Galvao DA. Obesity and prostate cancer: a narrative review. Crit. Rev. Oncol. Hematol., 169, 103543 (2022).
• 5) Zhuang L, Kim J, Adam RM, Solomon KR, Freeman MR. Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts. J. Clin. Invest., 115, 959–968 (2005).
• 6) Singh G, Sankanagoudar S, Dogra P, Chandra NC. Interlink between cholesterol & cell cycle in prostate carcinoma. Indian J. Med. Res., 146 (Suppl.), S38–S44 (2017).
• 7) Jiang S, Wang X, Song D, Liu X, Gu Y, Xu Z, Wang X, Zhang X, Ye Q, Tong Z, Yan B, Yu J, Chen Y, Sun M, Wang Y, Gao S. Cholesterol induces epithelial-to-mesenchymal transition of prostate cancer cells by suppressing degradation of EGFR through APMAP. Cancer Res., 79, 3063–3075 (2019).
• 8) Van Rompay MI, Solomon KR, Nickel JC, Ranganathan G, Kantoff PW, McKinlay JB. Prostate cancer incidence and mortality among men using statins and non-statin lipid-lowering medications. Eur. J. Cancer, 112, 118–126 (2019).
• 9) Pon D, Abe A, Gupta EK. A review of statin use and prostate cancer. Curr. Atheroscler. Rep., 17, 474 (2015).
• 10) Grolez GP, Gordiendko DV, Clarisse M, Hammadi M, Desruelles E, Fromont G, Prevarskaya N, Slomianny C, Gkika D. TRPM8-androgen receptor association within lipid rafts promotes prostate cancer cell migration. Cell Death Dis., 10, 652 (2019).
• 11) Pommier AJ, Alves G, Viennois E, Bernard S, Communal Y, Sion B, Marceau G, Damon C, Mouzat K, Caira F, Baron S, Lobaccaro JM. Liver X Receptor activation downregulates AKT survival signaling in lipid rafts and induces apoptosis of prostate cancer cells. Oncogene, 29, 2712–2723 (2010).
• 12) Liu YP, Zhang YX, Li PF, Cheng C, Zhao YS, Li DP, Du C. Cholesterol levels in blood and the risk of prostate cancer: a meta-analysis of 14 prospective studies. Cancer Epidemiol. Biomarkers Prev., 24, 1086–1093 (2015).
• 13) Platz EA, Clinton SK, Giovannucci E. Association between plasma cholesterol and prostate cancer in the PSA era. Int. J. Cancer, 123, 1693–1698 (2008).
• 14) He YH. General survey of traditional Chinese medicine and Western medicine researches on tumor metastasis. Chin. J. Integr. Med., 12, 75–80 (2006).
• 15) Layne K, Ferro A. Traditional Chinese medicines in the management of cardiovascular diseases: a comprehensive systematic review. Br. J. Clin. Pharmacol., 83, 20–32 (2017).
• 16) Zhang R, Gilbert S, Yao X, Vallance J, Steinbrecher K, Moriggl R, Zhang D, Eluri M, Chen H, Cao H, Shroyer N, Denson L, Han X. Natural compound methyl protodioscin protects against intestinal inflammation through modulation of intestinal immune responses. Pharmacol. Res. Perspect., 3, e00118 (2015).
• 17) Lee JH, Lim HJ, Lee CW, Son KH, Son JK, Lee SK, Kim HP. Methyl protodioscin from the roots of Asparagus cochinchinensis attenuates airway inflammation by inhibiting cytokine production. Evid. Based Complement. Alternat. Med., 2015, 640846 (2015).
• 18) Ma W, Ding H, Gong X, Liu Z, Lin Y, Zhang Z, Lin G. Methyl protodioscin increases ABCA1 expression and cholesterol efflux while inhibiting gene expressions for synthesis of cholesterol and triglycerides by suppressing SREBP transcription and microRNA 33a/b levels. Atherosclerosis, 239, 566–570 (2015).
• 19) Wang G, Chen H, Huang M, Wang N, Zhang J, Zhang Y, Bai G, Fong WF, Yang M, Yao X. Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells. Cancer Lett., 241, 102–109 (2006).
• 20) Bai Y, Qu XY, Yin JQ, Wu L, Jiang H, Long HW, Jia Q. Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells. Pharmacogn. Mag., 10, 318–324 (2014).
• 21) Ma YL, Zhang YS, Zhang F, Zhang YY, Thakur K, Zhang JG, Wei ZJ. Methyl protodioscin from Polygonatum sibiricum inhibits cervical cancer through cell cycle arrest and apoptosis induction. Food Chem. Toxicol., 132, 110655 (2019).
• 22) Tseng SC, Shen TS, Wu CC, Chang IL, Chen HY, Hsieh CP, Cheng CH, Chen CL. Methyl protodioscin induces apoptosis in human osteosarcoma cells by caspase-dependent and MAPK signaling pathways. J. Agric. Food Chem., 65, 2670–2676 (2017).
• 23) Chen L, Cheng CS, Gao H, Zhan L, Wang F, Qu C, Li Y, Wang P, Chen H, Meng Z, Liu L, Chen H, Chen Z. Natural compound methyl protodioscin suppresses proliferation and inhibits glycolysis in pancreatic cancer. Evid. Based Complement. Alternat. Med., 2018, 7343090 (2018).
• 24) Colin D, Limagne E, Jeanningros S, Jacquel A, Lizard G, Athias A, Gambert P, Hichami A, Latruffe N, Solary E, Delmas D. Endocytosis of resveratrol via lipid rafts and activation of downstream signaling pathways in cancer cells. Cancer Prev. Res. (Phila), 4, 1095–1106 (2011).
• 25) Tan S, Zhang P, Xiao W, Feng B, Chen LY, Li S, Li P, Zhao WZ, Qi XT, Yin LP. TMD1 domain and CRAC motif determine the association and disassociation of MxIRT1 with detergent-resistant membranes. Traffic, 19, 122–137 (2018).
• 26) Chen F, Chen Y, Kang X, Zhou Z, Zhang Z, Liu D. Anti-apoptotic function and mechanism of ginseng saponins in Rattus pancreatic beta-cells. Biol. Pharm. Bull., 35, 1568–1573 (2012).
• 27) Jiang J, Li L, Xie M, Fuji R, Liu S, Yin X, Li G, Wang Z. SPATA4 counteracts etoposide-induced apoptosis via modulating Bcl-2 family proteins in HeLa cells. Biol. Pharm. Bull., 38, 1458–1463 (2015).
• 28) Chang HK, Shin MS, Yang HY, Lee JW, Kim YS, Lee MH, Kim J, Kim KH, Kim CJ. Amygdalin induces apoptosis through regulation of Bax and Bcl-2 expressions in human DU145 and LNCaP prostate cancer cells. Biol. Pharm. Bull., 29, 1597–1602 (2006).
• 29) Han H, Qiu L, Wang X, Qiu F, Wong Y, Yao X. Physalins A and B inhibit androgen-independent prostate cancer cell growth through activation of cell apoptosis and downregulation of androgen receptor expression. Biol. Pharm. Bull., 34, 1584–1588 (2011).
• 30) Eberlé D, Hegarty B, Bossard P, Ferre P, Foufelle F. SREBP transcription factors: master regulators of lipid homeostasis. Biochimie, 86, 839–848 (2004).
• 31) Chen M, Zhang J, Sampieri K, et al. An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer. Nat. Genet., 50, 206–218 (2018).
• 32) Codini M, Garcia-Gil M, Albi E. Cholesterol and sphingolipid enriched lipid rafts as therapeutic targets in cancer. Int. J. Mol. Sci., 22, 726 (2021).
• 33) Jang D, Kwon H, Choi M, Lee J, Pak Y. Sumoylation of Flotillin-1 promotes EMT in metastatic prostate cancer by suppressing Snail degradation. Oncogene, 38, 3248–3260 (2019).
• 34) Jang D, Kwon H, Jeong K, Lee J, Pak Y. Essential role of Flotillin-1 palmitoylation in the intracellular localization and signaling function of IGF-1 receptor. J. Cell Sci., 128, 2179–2190 (2015).
• 35) Ediriweera MK, Moon JY, Nguyen YT, Cho SK. 10-Gingerol targets lipid rafts associated PI3K/Akt signaling in radio-resistant triple negative breast cancer cells. Molecules, 25, 3164 (2020).
• 36) Farhan M, Wang H, Gaur U, Little PJ, Xu J, Zheng W. FOXO signaling pathways as therapeutic targets in cancer. Int. J. Biol. Sci., 13, 815–827 (2017).
• 37) Kim CG, Lee H, Gupta N, Ramachandran S, Kaushik I, Srivastava S, Kim SH, Srivastava SK. Role of Forkhead Box Class O proteins in cancer progression and metastasis. Semin. Cancer Biol., 50, 142–151 (2018).
• 38) Xing YQ, Li A, Yang Y, Li XX, Zhang LN, Guo HC. The regulation of FOXO1 and its role in disease progression. Life Sci., 193, 124–131 (2018).
• 39) Deng X, Zhang W, O-Sullivan I, Williams JB, Dong Q, Park EA, Raghow R, Unterman TG, Elam MB. FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c. J. Biol. Chem., 287, 20132–20143 (2012).
• 40) Li Y, Wu S. Epigallocatechin gallate suppresses hepatic cholesterol synthesis by targeting SREBP-2 through SIRT1/FOXO1 signaling pathway. Mol. Cell. Biochem., 448, 175–185 (2018).
• 41) Hu H, Zhang XW, Li L, Hu MN, Hu WQ, Zhang JY, Miao XK, Yang WL, Mou LY. Inhibition of autophagy by YC-1 promotes gefitinib induced apoptosis by targeting FOXO1 in gefitinib-resistant NSCLC cells. Eur. J. Pharmacol., 908, 174346 (2021).
• 42) Kuzu OF, Noory MA, Robertson GP. The role of cholesterol in cancer. Cancer Res., 76, 2063–2070 (2016).
• 43) Yamaguchi T, Ishimatu T. Effects of cholesterol on membrane stability of human erythrocytes. Biol. Pharm. Bull., 43, 1604–1608 (2020).
• 44) Yano Y. Effects of membrane cholesterol on stability of transmembrane helix associations. Chem. Pharm. Bull., 70, 514–518 (2022).
• 45) Ye J, Gao M, Guo X, Zhang H, Jiang F. Breviscapine suppresses the growth and metastasis of prostate cancer through regulating PAQR4-mediated PI3K/Akt pathway. Biomed. Pharmacother., 127, 110223 (2020).
• 46) Huang S, Tang Y, Peng X, Cai X, Wa Q, Ren D, Li Q, Luo J, Li L, Zou X, Huang S. Acidic extracellular pH promotes prostate cancer bone metastasis by enhancing PC-3 stem cell characteristics, cell invasiveness and VEGF-induced vasculogenesis of BM-EPCs. Oncol. Rep., 36, 2025–2032 (2016).
• 47) Murai T. The role of lipid rafts in cancer cell adhesion and migration. Int. J. Cell Biol., 2012, 763283 (2012).
• 48) Chantôme A, Potier-Cartereau M, Clarysse L, Fromont G, Marionneau-Lambot S, Gueguinou M, Pages JC, Collin C, Oullier T, Girault A, Arbion F, Haelters JP, Jaffres PA, Pinault M, Besson P, Joulin V, Bougnoux P, Vandier C. Pivotal role of the lipid Raft SK3-Orai1 complex in human cancer cell migration and bone metastases. Cancer Res., 73, 4852–4861 (2013).
• 49) Lin CJ, Yun EJ, Lo UG, Tai YL, Deng S, Hernandez E, Dang A, Chen YA, Saha D, Mu P, Lin H, Li TK, Shen TL, Lai CH, Hsieh JT. The paracrine induction of prostate cancer progression by caveolin-1. Cell Death Dis., 10, 834 (2019).
• 50) Thompson TC, Tahir SA, Li L, Watanabe M, Naruishi K, Yang G, Kadmon D, Logothetis CJ, Troncoso P, Ren C, Goltsov A, Park S. The role of caveolin-1 in prostate cancer: clinical implications. Prostate Cancer Prostatic Dis., 13, 6–11 (2010).
• 51) Zhao J, Blayney A, Liu X, Gandy L, Jin W, Yan L, Ha JH, Canning AJ, Connelly M, Yang C, Liu X, Xiao Y, Cosgrove MS, Solmaz SR, Zhang Y, Ban D, Chen J, Loh SN, Wang C. EGCG binds intrinsically disordered N-terminal domain of p53 and disrupts p53-MDM2 interaction. Nat. Commun., 12, 986 (2021).
• 52) Cardoso D, Perucha E. Cholesterol metabolism: a new molecular switch to control inflammation. Clin. Sci. (Lond), 135, 1389–1408 (2021).
• 53) Ma X, Bi E, Huang C, Lu Y, Xue G, Guo X, Wang A, Yang M, Qian J, Dong C, Yi Q. Cholesterol negatively regulates IL-9-producing CD8⁺ T cell differentiation and antitumor activity. J. Exp. Med., 215, 1555–1569 (2018).
• 54) Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J. Nat. Prod., 83, 770–803 (2020).
• 55) Zhang C, Qin L, Sun B, Wu Y, Zhong F, Wu L, Liu T. Transcriptome analysis of the effect of diosgenin on autoimmune thyroiditis in a rat model. Sci. Rep., 11, 6401 (2021).
• 56) Dong L, Yin L, Li R, Xu L, Xu Y, Han X, Qi Y. Dioscin alleviates lung ischemia/reperfusion injury by regulating FXR-mediated oxidative stress, apoptosis, and inflammation. Eur. J. Pharmacol., 908, 174321 (2021).
• 57) Liu Y, Zhou Z, Yan J, Wu X, Xu G. Diosgenin exerts antitumor activity via downregulation of Skp2 in breast cancer cells. Biomed. Res. Int., 2020, 8072639 (2020).