Ganoderiol F purified from Ganoderma leucocontextum retards cell cycle progression by inhibiting CDK4/CDK6

ABSTRACT

This study was designed to purify molecules possess anti-cancer cell activity from the fruit body of Ganoderma leucocontextum. Bio-activity-guided purification and chromatographic separation of Ganoderma leucocontextum extract led to the enrichment of bioactive fractions and isolation of a single compound. The purified compound was identified as Ganoderiol F, which induced cancer cell death. In the in vivo experiments, we founded ethanol extract and ethyl acetate fraction inhibited tumor growth in the mice injected with 4T1 cells. We found that Ganoderiol F-mediated suppression of breast cancer cell viability occurred through cell cycle arrest. Ganoderiol F down-regulated expression of cyclin D, CDK4, CDK6, cyclin E and CDK2 and inhibited cell cycle progression arresting the cells in G1 phase. In addition, Ganoderiol F up-regulated pro-apoptotic Foxo3, down-regulated anti-apoptotic c-Myc, Bcl-2 and Bcl-w leading to apoptosis in human breast cancer cells MDA-MB-231. These results showed that c-Myc, cyclin D-CDK4/CDK6 and cyclin E-CDK2 are the central components of Ganoderiol F regulation of cell cycle progression. Hence Ganoderiol F may serve as a potential CDK4/CDK6 inhibitor for breast cancer therapy.

Abbreviations: GLE: Ganoderma leucocontextum ethanol extract; GLEA: Ganoderma leucocontextum ethyl acetate fraction; GLPE: Ganoderma leucocontextum petroleum ether fraction; RP-HPLC: reversed-phase high-performance liquid chromatograph; DMEM: Dulbecco’s modified Eagle’s medium; FBS: fetal bovine serum; PAGE: polyacrylamide gel electrophoresis.

Introduction

Breast cancer is the most common malignancies among women globally. In clinical treatment, targeting the estrogen receptors (ERs) and estrogen action have been shown to be successful approaches for the prevention and intervention of breast cancer for decades. However, approximately 30% of breast cancer patients do not respond or develop resistance to estrogen receptor modulators [1]. Thus, new targeted therapies against the major signaling pathways of breast carcinogenesis have become one of the focuses in the breast cancer research field. A hallmark of cancers is the dysregulation of cell cycle progression, ultimately promoting abnormal cell proliferation and then cancer outgrowth. Numerous evidence has pointed to the important roles of dysregulated cyclins and cyclin-dependent kinases in many cancers, including breast cancer [2]. Central to these cell cycle control mechanisms is the cyclin D-CDK4/CDK6 and cyclin E-CDK2, which have been targeted by many drugs.

Ganoderma spp. is a well-known family of medicinal mushrooms, one of which is Ganoderma lucidum that has long been used as a traditional medicinal fungus and is probably the most well-studied species. The main bioactive compounds of Ganoderma lucidum are polysaccharides, triterpenoids, and sterols [3–8]. It has been found that many triterpenoids induced cell death, suppressed cell migration, and inhibited cell growth by regulating cell cycle progression, activating or inhibiting anti- or pro-apoptotic proteins [9–12].

Ganoderma leucocontextum, which was first found in the mountain of Linzhi, Tibet by us in 2011, when a group of scientists led by H.P. Hui were collecting Ganoderma lucidum in the area. This mushroom looks very different from the traditional Ganoderma lucidum and it was later identified as a new member of Ganoderma lucidum in 2014 [13]. The fruit bodies of Ganoderma leucocontextum have been used in folk medicine in the prevention and treatment of diseases. Bioactive compounds isolated from Ganoderma leucocontextum were mainly lanostane triterpenes, which have several biological activities, including anti-tumor growth, inhibition of HMG-CoA, inhibition of pancreatic lipase, and neuroprotective activities [14–18].

In this study, we aimed to identify some active compounds in the ethanol extract of Ganoderma leucocontextum and to explore its antitumor mechanisms. We isolated and purified an active compound which was identified as Ganoderiol F. It has been reported that Ganoderiol F, a member of lanostane triterpenes from Ganoderma spp., has the cytotoxicity against liver and lung carcinoma cells [19,20]. We found that Ganoderiol F inhibited cell proliferation and induced cell death through cell cycle arrest in the G1-S phase. This occurred by down-regulating the cell cycle-associated proteins cyclin D1, CDK4, CDK6, cyclin E and CDK2. Ganoderiol F also down-regulated the expression of c-Myc. It has been reported that induction of c-Myc did not increase cyclin D1 expression, but c-Myc antisense decreased c-Myc levels leading to decreased cyclin D1 expression [21].

Material and methods

Fungal material

The fruit bodies of Ganoderma leucocontextum were cultivated and collected at Linzhi (Tibet Autonomous Region, China). The voucher specimen (GDGM40200) was firstly discovered and authenticated by Huiping Hu and deposited in the Fungal Herbarium of Guangdong Institute of Microbiology (GDGM).

Extraction, isolation, and purification of anti-tumor compounds

The dry fruit bodies of Ganoderma leucocontextum (5 kg) were ground to powder. The powder was soaked in 95% ethanol at a ratio of 1:15 (w/v) for 30 min, and then extracted by refluxing three times at 80°C. The extracted solution was filtered and concentrated under vacuum. Ganoderma leucocontextum ethanol extract (GLE, 350 g) was obtained.

The ethanol extract (GLE) was dispersed in distilled water and fractionated into petroleum ether, ethyl acetate, and water. After evaporation of the collection, petroleum ether fraction (GLPE), ethyl acetate fraction (GLEA) were obtained. The bioactive fraction was subjected to ODS-C18 chromatography using methanol-water in the gradient system (v/v, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to obtain 18 fractions (Fr. GL1 – GL18). Fraction 7 (GL7) eluted with methanol-water (v/v, 70:30) was further separated by reversed-phase high-performance liquid chromatograph (RP-HPLC) with acetonitrile-water (v/v, 71:29) to yield compound GL74 (35 mg). The detailed steps are provided in the results section. In the separation procedure of the ethanol extract, the antitumor activity of all fractions was tested with human breast cancer cell line MDA-MB-231.

MS and NMR analysis

For MS identification, GL74 was dissolved in methanol and recorded on HESI (Q Exactive Focus, Thermo Fisher, USA). For NMR measurements, GL74 was dissolved in CDCL₃. The NMR spectra were recorded on BRUKER AVANCEIIIT 600HD (Bruker BioSpin, Switzerland).

Cell proliferation assay

Human and mouse breast cancer cell lines (MDA-MB-231, MDA-MB-468, SK-BR-3, MCF-7 and 4T1) were used to test the anti-tumor effect of different extract and compounds of Ganoderma leucocontextum fruit body in inhibiting tumor cell proliferation. In brief, cancer cells (1x10⁵ cells/ml, 0.5 ml) were seeded in 24-well tissue culture plates in DMEM (10% FBS, 100 U/ml penicillin/streptomycin) and incubated at 37°C containing 5% CO₂. Four hours after cell inoculation, ethanol extract (GLE) and ethyl acetate fraction (GLEA) were added to the cultures at different concentrations (25, 50, 75, 100, 125, 150, 200 µg/mL). Similarly, GL74 were added to the cultures at concentrations of 2.2, 11, 22, 55, 88 µM (or 1, 5, 10, 15, 25, 50 µg/mL). The medium used to dissolve compounds also served as a control. We also examined the effects of the extracts on normal breast epithelial cell line MCF-10A (DMEM/f12 containing Horse serum, EGF, etc.). After being cultured at 37°C for 48 h in an incubator containing 5% CO₂, the cells were harvested and analyzed by trypan blue staining. Each experiment was repeated three times for statistical analysis. The data were analyzed and the IC₅₀ values were calculated by SPSS 21.0.

Cell survival assay

The cell survival assay was performed as described [22]. Cells (1 × 10⁵ cells) were cultured in DMEM/10% FBS medium in 24-well tissue culture plates and maintained at 37°C containing 5% CO₂. After 24 h, the cultures were gently washed with PBS twice, followed by the addition of serum-free DMEM. The GL74 was added to the wells at the concentrations of 0, 2.2, 11, 22, 55, 88 µM (or 0, 1, 5, 10, 15, 25, 50 µg/mL), the medium used to dissolve the compound also served as a control. After 48 h, the cells were harvested and the cell number was counted by trypan blue staining to estimate cell survival.

Cell migration assay

Cell migration was tested by scratch assay as described [23]. In brief, MDA-MB-231 cells were inoculated onto six-well plates at a density of 4 x10⁵ cells/well and cultured overnight. The cultures were scraped linearly with micropipette tips, then treated with or without GLE, GLEA and GL74 at different concentrations. Cell migration patterns were recorded by light microscopy at 0, 12, and 24 h. Migration distance was measured and quantified.

Colony formation assay

Colony formation was performed as previously described [23,24]. In brief, 0.66% agarose gel was loaded on each well of the 6-well tissue culture plates. 4T1 cells (1000 cells, 1 ml) treated with GLE (25, 50 µg/mL), GLEA (10, 25 µg/mL) and GL74 (11 µM, 22 µM) were mixed with 0.66% low melting agarose in ratio 1:1, the medium used to dissolve the samples also served as a control. The mixtures were plated on the top of the 0.66% agarose gel plates. Two weeks after inoculation, colonies were counted, fixed with 100% cold methanol, stained with 0.25% Coomassie Blue, and photographed under a light microscope.

Western blot

Protein assays on Western blot were performed as described [25,26]. In brief, MDA-MB-231 cells were inoculated onto 6-well plate at 2 × 10⁵ cells per well overnight. The cells were treated with GL74 at concentrations of 0, 11, 22 μM (or 0, 5,10 µg/mL) for 48 h. After the treatment, the cells were lysed by lysis buffer supplemented protease inhibitor cocktail. The plates were placed on ice for 30 min. The lysates were collected and centrifuged to obtain supernatant. Samples with equal amounts of proteins were objected to SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocking with 5% milk for 30 min, followed by incubation with primary antibodies at 4°C overnight. Anti-β-actin antibody or anti-GAPDH antibody were used as controls. After three rinses with TBST buffer, the membranes were incubated with secondary antibodies for 2 h at room temperature. After three rinses with TBST buffer, the membranes were incubated in ECL (Millipore) and imaged using a ChemiDoc^TM XRS+ System (Bio-Rad).

Cell cycle measurement

Propidium iodide staining was used to distinguish the distribution of cells in cell cycle phases with or without treatment with GL74 at the concentrations of 0, 22, 44 μM (or 0, 10, 20 µg/mL). Flow cytometric analysis (FCM) was performed as follows. MDA-MB-231 cells were inoculated at 2 × 10⁵ cells/well and cultured for 4 h. The cultures were treated with different concentrations of GL74 for 48 h. The cells were harvested, washed and fixed with ice-cold 75% ethanol for 1 h on ice. After a wash with PBS twice, the cells were stained with propidium iodide solution (50 µg/mL) at room temperature. Stained cells were assessed by FCM using a BD FACSCanto II (Becton Dickinson, San Jose, CA, USA) with the BD FACSDive software. The cell cycle distribution was calculated using software of ModFit LT.

Animal assay

Six-week-old BALB/c mice (20 ± 2) were obtained from Guangdong Medical Laboratory Animal Center. The mice were kept in the Animal Core Facility of Guangdong Institute of Microbiology for one week before experimentation. The mice were randomly divided into four groups. Forty mice were subcutaneously injected with 4T1 cells (0.5 × 10⁵ cells/mouse). Next day after cell implantation, GLE and GLEA were injected intraperitoneally at a dose of 50 mg/kg mouse. The model group and positive group mice were, respectively, given with 0.9% sodium chloride and lentinan at the same volume on the same schedule. This was repeated every other day for up to 4 wk. At the end of the experiment, the mice were sacrificed, and the tumors were removed. Tumors were weighed and photographed.

Statistical analysis

All experiments were performed in triplicate. Analysis of results was done using t test. All values were expressed as means ± standard deviation (SD). The levels of significance were set at *p < 0.05 and **p < 0.01.

Results

Bioassay-guided separation and compound structure

To examine the biological activities of Ganoderma leucocontextum, we fractionated and monitored its activities by incubating the fractions with cancer cell cultures. Using this approach, we found that the ethanol extract (GLE) of Ganoderma leucocontextum possessed anti-tumor effect. The GLE fraction was then solvent-partitioned with petroleum ether (PE), ethyl acetate (EA) to derive GLPE and GLEA soluble fractions (Figure 1). The effect of ethanol extract and the two soluble fractions were assessed on human breast cancer cells MDA-MB-231 at different concentrations, up to 150 150 µg/mL. The assay showed that the EA fraction significantly induced cancer cell death (Table 1). The results showed that the bioactive compounds of ethanol extract against breast cancer cells were enriched in the GLEA fraction.

Figure 1.

Isolation and purification of GL74 from Ganoderma leucocontextum fruit body.

The dry fruits of Ganoderma leucocontextum were extracted by 95% ethanol, at approximately 80°C. With ODS column chromatography and RP-HPLC, Ganoderma leucocontextum extract was isolated and purified as shown.

Table 1.

Cell viability of ethanol extract and solvent-partitioned fractions. Data represent the mean ± SD of three independent experiments on MDA-MB-231 cells.

Sample	Concentration (μg/mL)	MDA-MB-231 cell viability (%)
GLE	50	52.8 ± 2.04
	100	31.9 ± 1.93
	150	11.4 ± 0.34
GLPE	50	64.0 ± 2.93
	100	57.9 ± 1.61
	150	57.6 ± 1.01
GLEA	25	49.7 ± 7.58
	50	23.7 ± 1.84
	100	0.14 ± 0.15

Column chromatographic separation of the GLEA fraction led to the isolation of 18 fractions (GL1 – 18). All of the isolated fractions were tested for the anti-tumor effects on the breast cancer cells at the same concentrations. Among them, the fractions GL5–GL9 showed potent growth inhibition and induction of cell death of MDA-MB-231 cells with the IC₅₀ values ranging from 14.51 to 51.12 μg/mL, respectively (Table 2).

Table 2.

Cell viability of the different fractions isolated from ethyl acetate fraction. Data represent the mean ± SD of three independent experiments in MDA-MB-231 cells.

sample	Concentration (μg/mL)	MDA-MB-231 cell viability (%)	IC50 (μg/mL)	sample	Concentration (μg/mL)	MDA-MB-231 cell viability (%)	IC50 (μg/mL)
GL1	25	106.8 ± 3.70		GL10	25	85.7 ± 14.2
	50	109.4 ± 11.8			50	70.9 ± 5.03
GL2	25	107.6 ± 6.69		GL11	25	86.9 ± 11.2
	50	97.5 ± 1.55			50	84.2 ± 5.69
GL3	25	111.5 ± 4.62		GL12	25	103.4 ± 0.55
	50	95.1 ± 2.71			50	91.8 ± 6.35
GL4	25	63.7 ± 52.1		GL13	25	107.7 ± 1.32
	50	52.1 ± 6.25			50	94.8 ± 2.80
GL5	25	41.4 ± 2.42	18.51 ± 1.27	GL14	25	104.5 ± 4.47
	50	11.3 ± 2.64			50	98.3 ± 1.0
GL6	25	24.2 ± 2.04	14.51 ± 0.62	GL15	25	98.4 ± 4.20
	50	2.37 ± 0.49			50	95.2 ± 4.4
GL7	25	34.5 ± 5.85	16.79 ± 1.02	GL16	25	107.5 ± 4.85
	50	3.65 ± 1.49			50	94.4 ± 1.33
GL8	25	47.9 ± 3.28	25.31 ± 0.57	GL17	25	108.1 ± 3.83
	50	22.0 ± 1.91			50	121.2 ± 1.95
GL9	25	69.1 ± 1.81	51.12 ± 2.57	GL18	25	94.8 ± 2.10
	50	40.4 ± 2.68			50	100.0 ± 3.21

The bold letters show that the GL5 – GL9 displayed significantly the stronger inhibitory effect on cancer cells MDA-MB-231 in among all fractions.

Ethanol extract and ethyl acetate fraction of Ganoderma leucocontextum inhibited breast cancer growth in vitro and in vivo

We evaluated the effects of GLE and GLEA on the activities of human breast carcinoma cell lines, including MDA-MB-231, MCF-7, MDA-MB-468, SK-BR-3, and mouse breast cancer cell line 4T1. We found that GLE induced MDA-MB-231 cell death in a concentration-dependent manner (Figure 2a). Similar antitumor effects were observed in MCF-7, MDA-MB-468, SK-BR-3 and 4T1 cells with GLE treatment (Figure 2a). GLEA, which was purified from ethanol extract, displayed significantly stronger inhibitory effects on the breast cancer cell lines than GLE at the same concentrations (Figure 2b). At the same concentrations, GLE and GLEA had little effect on the viability and morphology of MCF-10A mammary epithelial (Figure 2a,b), suggesting specific effect of GLE and GLEA on breast cancer cells.

Figure 2.

Ganoderma leucocontextum ethanol extract (GLE), ethyl acetate fraction (GLEA) and GL74 inhibited proliferation and colony formation.

(a). Breast cancer cells MDA-MB-231, MCF-7, MDA-MB-468, SK-BR-3 and 4T1 were treated with GLE for 48 h. A normal breast epithelial cell line MCF-10A - was used as a control. (b). Breast cancer cells MDA-MB-231, MCF-7, MDA-MB-468, SK-BR-3 and 4T1 were treated with GLEA for 48 h. A normal breast epithelial cell line MCF-10A - was used as a control. (c-d). The photos of colony sizes and colony number formed by 4T1 cells were examined after treatment with GLE (c) and GLEA (d) for 2 wk.

We further performed colony formation assay using GLE and GLEA on mouse breast cancer cell 4T1. The experimental results showed clearly that GLE inhibited colony formation in the concentration-dependent manner: the treated group formed less and smaller colonies relative to the control group (Figure 2c). GLEA treatment had stronger inhibitory effects on colony formation size and number than GLE in the concentration-dependent manner (Figure 2d).

Based on colony formation results, we further tested the anticancer of GLE and GLEA in vivo. Strain Balb/c mice were injected with 4T1 cells. GLE and GLEA were injected into the mice, respectively, every other day. After Day 30, all mice were sacrificed. GLE and GLEA were found to possess inhibitory effect on tumor growth in vivo. The data displayed that GLE decreased tumor weight and size in mice with the tumor inhibition rate being 33.6% (Figure 3). GLEA, which was isolated from GLE, showed stronger antitumor activity in the tumor-bearing mice than GLE at the same dose with the tumor inhibitory rate of 50.2% (Figure 3). Our results further confirmed the antitumor activity of Ganoderma leucocontextum.

Figure 3.

GLE and GLEA inhibited tumor growth in mice.

Mice were injected intraperitoneally with 4T1 cells followed by delivery of GLE, GLEA and buffer vehicle. GLE and GLEA inhibited tumor growth. GLEA was purified from GLE and showed a stronger effect on inhibiting tumor growth than GLE did.

Structural analysis of compound GL74

The fraction GL7 was eluted with 70% methanol that markedly induced breast cancer cell death. Using this fraction, we further purified by RP-HPLC using 71% methanol to elute and obtained a compound GL74. GL74 was a colorless powder, and its molecular formula was determined to be C₃₀H₄₆O₃ by HRESIMS data (m/z 455.3(M+) (Supplementary Figure S1). The ¹³C and ¹H NMR (Supplementary Figure S2, S3) data were consistent with previously reported data on 26,27-dihydroxy-5α-lanosta-7,9(11), 24-trien-3-one (Ganoderiol F) [27]. Thus, GL74 was identified as 26,27-dihydroxy-5α-lanosta-7,9(11), 24-trien-3-one (Ganoderiol F), and its structure is shown in Figure 4.

Figure 4.

Identification of Ganoderiol F.

(a). Comparison of GL74 13C spectroscopic data with the most close molecule in the literature [27]. b. The structure of Ganoderiol F.

Ganoderiol f inhibited cancer cell proliferation, cell migration, colony formation, and modulated cell cycle progression

Ganoderiol F was obtained from many species of Ganoderma spp., and has been reported to possess a variety of biological activities including anti-inflammatory, anti-cancer effects [28,29]. We examined the roles of Ganoderiol F in inhibiting cancer cell proliferation in a number of breast cancer cell lines including MDA-MB-231, MDA-MB-468, SK-BR-3, MCF-7, and 4T1. The data showed that the survival rates of all cancer cell lines were decreased in a concentration- and time-dependent manner when the cells were treated with Ganoderiol F (Figure 5a,b). Importantly, at the equal concentrations, Ganoderiol F had little effect on the viability and morphology of MCF-10A mammary epithelial (Figure 5a). In addition, Ganoderiol F displayed a significant effect on MDA-MB-231 cell survival when the cells were treated for 24 h on the gradient concentrations (Figure 5c).

Figure 5.

The purified Ganoderiol F inhibited cell proliferation, decreased cell survival and colony formation.

(a). Cancer cell cultures (MDA-MB-231, SK-BR-3, MDA-MB-468, MCF-7, and 4T1) and MCF-10A were treated with Ganoderiol F (0–88 μM) for 48 h. Cell viability was determined. Each experiment was repeated three times. Data represent the mean ± SD of three independent experiments. Ganoderiol F significantly decreased cancer cell viability relative to the non-cancer cells MCF-10A.(c). MDA-MB-231 cells were treated with Ganoderiol F (22, 44 μM) for up to 72 h followed by cell viability determination. Data represent the mean ± SD of three independent experiments. (c). GL74 decreased cancer cell survival. MDA-MB-231 cells were cultured overnight. After cell attachment, cells were cultured in serum-free DMEM, to which GL74 was added at different concentrations as shown. The cultures were maintained for 48 h. Cell viability was analyzed by trypan blue staining and cell counting assay as described in Materials and Methods. Data represent the mean ± SD of three experiments. (d). Colony formation in 4T1 cells was assayed with the purified Ganoderiol F for 14 d. Left, Number of colonies. Right, Photos of colony sizes. Data represent the mean ± SD of three experiments.

To verify that Ganoderiol F decreased the viability of breast cancer cells, we further tested the effect of Ganoderiol F on colony formation of 4T1 cells. The experimental results showed that Ganoderiol F inhibited colony formation in both the sizes and number of the colonies in a dose-depended manner (Figure 5d).

We compared the effects of GLE, GLEA, and Ganoderiol F on cancer cell migration. In the scratch migration assay, the assay showed that at the concentration of 50 μg/mL, both GLE and GLEA displayed an inhibitory effect on MDA-MB-231 cell migration, whereas Ganoderiol F exerted a significant inhibitory effect on cancer cell migration at the concentration of 15 μg/mL (33 μM) (Figure 6a, Fig S4).

Figure 6.

GLE, GLEA and GL74 inhibited cell migration and cell cycle progression.

(a). Cell migration was tested by scratch assays. MDA-MB-231 cells were scraped and treated with or without GLE, GLEA and GL74. Cell migration patterns were recorded by light microscopy at 0 and 24 h. Migrated distance was measured and quantified. (b). Histogram of cell cycles in MDA-MB-231.(c). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were detected in MDA-MB-231 treated with GL74 relative to the control.

We determined whether the Ganoderiol F-induced cancer cell apoptosis occurred through cell cycle arrest. After treatment of MDA-MB-231 cells with Ganoderiol F for 48 h, FCM data showed that cells increased in G0/G1 phase (17.5%) but decreased in S phase (15.0%), and decreased in G2/M phase (2%) at the concentration of 44 μM (Figure 6b). Thus, Ganoderiol F induced significant cell cycle arrest in G1 phase accompanied by a reduction in the S and G2/M phases in MDA-MB-231 cells (p < 0.05 at 22 μM, p < 0.01 at 44 μM), compared with the control (Figure 6c).

Ganoderiol F regulated cell cycle proteins and cell apoptotic proteins

The transition from G1 to S phase is controlled by a series of cyclins and cyclin-dependent kinases (CDKs). We further examined whether Ganoderiol F inhibited cancer cell proliferation and induced cancer cell death through regulating the expression of cyclins and cyclin-dependent kinases. After treatment of MDA-MB-231 cells with Ganoderiol F for 48 h, Western bolt analysis showed that the expression of CDK4, CDK2, Cyclin D1, Cyclin D3 and Cyclin E1 significantly decreased in a dose-dependent manner, whereas the expression of CDK6 was not affected significantly (Figure 7a).

Figure 7.

Effect of GL74 on cell cycle-associated proteins.

(a). Protein expression of CDK4, CDK6, CDK2, CyclinD1, Cyclin D3, Cyclin E, c-Mcy, Foxo3, Bcl-2, Bcl-xl and Bcl-w in MDA-MB-231 cells after GL74 treatment at different concentrations (22–44 µM) for 48 h. Cell lysate was prepared and analyzed by western blotting. The data showed that GL74 reduced expression of CDK4, CDK6, CDK2, CyclinD1, Cyclin D3, Cyclin E, c-Mcy, Bcl-2, Bcl-xl and Bcl-w, but increased expression of Foxo3a.(b). Pathway associated with cell cycle progression.

It has been reported that c-Myc can active Cyclin D1-CDK4/CDK6 complex and Cyclin E-CDK complex during the G0 to S transition [30]. Western blotting showed that the protein levels of c-Myc in MDA-MB-231 cells treated with Ganoderiol F decreased compared with the control (Figure 7a). Foxo3 is known as a tumor suppressor. We detected an increase in Foxo3 protein levels in the cancer cells treated with Ganoderiol F (Figure 7a). Thus, Ganoderiol F affected the expression of c-Myc and Foxo3 and modulated cell cycle progression (Figure 7b).

In addition, we also examined the expression of other proteins that are associated with cancer cell apoptosis including Bcl-2 and Bcl-xl. MDA-MB-231 treated with Ganoderiol F greatly decreased expression of Bcl-2 and Bcl-xl (Figure 7a), confirming the induction of Ganoderiol F in cancer cell apoptosis.

Discussion

Ganoderma leucocontextum, called “Bai-Rou-Ling-Zhi (白肉灵芝)” with white context, has been regarded as folk medicine in the Southwestern China for the prevention and treatment of various diseases. In the last five years, it has been reported that its bioactive compounds have various effects, including anti-diabetic, antitumor, neuroprotective, pancreatic lipase inhibition [31–33]. In this study, we found that ethanol extract and active fraction (GLEA) inhibited cancer growth in vitro and in vivo. We further identified Ganoderiol F as a CDK4/CDK6 inhibitor for the inhibition of cell proliferation and induction of apoptosis in human breast cancer cells using bioactivity-guided fractionation and chemical analysis. Our compound purified from the ethanol extract possessed significant antitumor activities in vitro and in vivo. These results provided strong evidence for the molecular basis of the antitumor activity of Ganoderma leucocontextum. Our finding thus afforded a potential use of Ganoderma leucocontextum as a functional food supplementary.

Triterpenoids isolated from Ganoderma spp., tetracyclic lanostane-type with a high degree of oxidation, are well-known as the typical secondary metabolism compounds in this genus, which have been identified with anticancer activity [10,34]. To date, more than 300 compounds have been identified, including ganoderiols, ganoderic acids, ganoderenic acids, ganolucidic acids, lucidenic acids, lycialdehydes, and lanosterols, which have been studied as potential anticancer agents [35–41]. It is anticipated that more compounds are to be identified in the future.

Ganoderiol F had been recognized as a bioactive compound isolated from Ganoderma spp. [42,43]. In our study, we provided strong evidence showing that Ganoderiol F possesses cell proliferation, cell migration, and colony formation in the inhibition of breast cancer cell growth and induction of cell death. Our results further our current understanding of the bioactive compound Ganoderiol F. It is therefore a potential for this compound to be developed as an agent for cancer treatment.

Many breast cancer patients are treated by targeting ERs and estrogen-associated pathways, if the cancer patients are estrogen and progesterone hormone receptor positive [1]. However, there are 30% of breast cancer patients who do not respond or later become resistant to ER therapies. It has been reported that dysregulation of the cyclin D1-CDK4/CDK6 axis appears to be an early step in the breast cancer pathogenesis, and cyclin D is overexpressed in ductal carcinoma in situ and maintained in metastatic lesions [1,44]. Thus, alternative approaches are needed for the treatment of this group of patients. Ganoderiol F may be a potential agent for such treatment.

Furthermore, we explored the molecular mechanism underlying the effect of Ganoderiol F on cancer cell death. Our data showed that cyclin D1 and cyclin D3 were decreased after Ganoderiol F treatment in breast cancer cells MDA-MB-231. It has been known that Cyclin D activates CDK4 and CDK6, and increases CDK2 activity [45]. We further found that the protein levels of CDK4, CDK6 and CDK2 decreased in MDA-MB-231 cells treated with Ganoderiol F. Cyclin E binds and activates CDK2 to facilitate the cells through the G1-S transition and DNA replication [46]. Our results showed that MDA-MB-231 cells treated with Ganoderiol F showed decreased Cyclin E levels. c-Myc has been shown to regulate CDK4 and CDK6 activities and affect cell cycle progression [47,48]. We detected decreased c-Myc levels in MDA-MB-231 cells after Ganoderiol F treatment. Meanwhile, we found that Foxo3 was up-regulated after Ganoderiol F treatment in MDA-MB-231 cells. Consistently, the anti-apoptotic molecules Bcl-2 and Bcl-w were down-regulated after Ganoderiol F treatment.

These data provided evidence that Ganoderiol F reduced and inhibited expression of cyclin D-CDK4/CDK6 and cyclin E-CDK2 to arrest the cell cycle progression in G1 phase, thereby blocking cell proliferation, and up-regulated pro-apoptotic Foxo3 and down-regulated anti-apoptotic Bcl-2 and Bcl-w leading to induction of cell apoptosis, after Ganoderiol F treatment.

It should be mentioned that there were other ingredients and molecules that possessed anti-cancer activity. For example, in the 18 fractions, we found at least 6 of them possessed anti-cancer activity, but we focused on the fraction with the highest activity in this study. Other fractions could contain novel molecules with potent anti-cancer activity. This awaits further investigation.

In summary, we investigated the anti-cancer activity of Ganoderma leucocontextum. Small molecules of anti-cancer activity were obtained in the ethanol extract, which were monitored by their anticancer activity and purified in the ethyl acetate fraction. We further showed that the ethanol extract and ethyl acetate fraction inhibited tumor growth in vitro and in vivo. The major active component Ganoderiol F was purified from GL7. We found that Ganoderiol F treatment decreased c-Myc expression in MDA-MB-231 cells. This led to decrease cyclin D expression, and reduce the activation of cyclin E-CDK2, and up-regulate pro-apoptotic Foxo3 and down-regulate anti-apoptotic Bcl-2 and Bcl-w. Ultimately, cell cycle progression was arrested that led to induced cells death. These results uncovered the molecular mechanism by which Ganoderiol F served as a CDK4/CDK6 inhibitor to exert its anti-cancer activity. Future work could lead to structural modification of Ganoderiol F to improve its effective therapeutic application in clinical settings.

Funding Statement

This work was supported by National Natural Science Foundation of China (No. 31700020), National Natural Science Foundation of Guangdong, China [No. 2017A030310088], Science and Technology Project of Guangzhou, China [No. 201707020022, 201807010106], GDAS, Project of Science and Technology Development [2019GDASYL-0302002].

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary Material

Supplemental data for this article can be accessed here.