Conditioned Media of RAW 264.7 Cells Stimulated with Phellinus linteus Extract Regulates the Epithelial-mesenchymal Transition in Prostate Cancer Cells

상황버섯에 의해 활성화된 RAW 264.7 대식세포주 배양액의 인간 전립선암 세포주의 epithelial-mesenchymal transition 조절

Abstract

Prostate cancer (PCa) is one of the most metastatic tumor. Although hormone therapy or surgical castration is mostly conducted to treat PCa, it has a lot of side effects. Recently, many researchers have been exploring the tumor microenvironment to remedy these circumstances. Immune cells, especially macrophages, are an important composition of the tumor microenvironment. Under normal conditions, macrophages exhibit mild tumoricidal activity against tumors. However, once activated by interferon gamma or lipopolysaccharides, macrophages can kill cancer cells directly or indirectly by secreting cytokines and chemokines. In this study, murine macrophage RAW 264.7 cells were treated with Phellinus linteus extract. To analyze their pro-inflammatory phenotype, we were used several assays such as a real-time polymerase chain reaction, an enzyme-linked immunosorbent and nitric oxide assay. Prostate cancer cells were treated with the RAW 264.7-conditioned media, which was identified as a pro-inflammatory nature, for 48 h, and the expression of epithelial-mesenchymal transition (EMT)-related genes was determined. Not only N-cadherin, Snail, Twist, Slug, and Cadherin 11, which are mechenchymal-related proteins, were decrease, but epithelial marker of E-cadherin was increased. In addition, the mRNA level of vimentin, ccl2, and vegfa were decreased, as the EMT is closely related to the migration and invasion of cancer cells. In conclusion, the RAW 264.7-conditioned media stimulated with P. linteus extract inhibited migration and invasion and regulated the EMT pathway in human prostate cancer cells.

전립선암은 전이성 종양 중의 하나로 치료를 위해 호르몬 요법이나 외과 적 거세 방법이 주로 수행되지만 많은 부작용을 나타내었다. 최근 많은 연구자들이 이러한 상황을 해결하기 위해 종양 미세 환경을 연구하고 있으며 그 중 면역 세포, 특히 대식세포는 종양 미세 환경의 중요한 구성요소이다. 정상적인 조건에서 대식세포는 여러 암세포에 대해 약한 종양 살균 활성을 갖으나 $interferon-{\gamma}$ 또는 lipopolysaccharide에 의해 활성화되면, 염증성 사이토카인 및 케모카인을 분비함으로써 암세포를 직접 또는 간접적으로 사멸 시키게 된다. 본 연구에서는, 마우스 대식세포인 RAW 264.7 세포에 Phellinus linteus 추출물을 처리하여 산화질소의 방출과 pro-inflammatory cytokine들을 real-time PCR과 ELISA 방법으로 분석하였다. RAW 264.7의 조정 배지는 48시간 동안 전립선 암세포처리하여 상피간엽세포전이 관련 유전자의 발현을 측정 하였다. 그 때에 mesenchymal 관련 유전자들인 N-cadherin, snail, twist, slug 및 cadherin 11이 감소했을 뿐만 아니라 epithelial 관련 유전자인 E-cadherin은 증가하였다. 또한 암 전이 및 신생 혈관 형성에 관여하는 vimentin, ccl2 및 vegfa가 감소되었는데, 이는 EMT가 암세포의 이동과 침범에 밀접한 관련이 있기 때문이다. 따라서 Phellinus linteu에 의해 자극된 RAW 264.7 세포의 조정 배지는 인간 전립선 암세포주인 PC-3 세포의 이동과 전이를 억제하고 EMT 경로를 조절한다는 것을 나타낸다.

Introduction

Cancer is a malignant tumor formed by abnormal growth of deformed cells when the normal cell undergoes a variety of genetic mutation caused by external stimulators. Cancer is the leading cause of death in the world. It is regarded as one of the most important factors threatening human life worldwide [4]. In Korea, the mortality rate by cancer continues to increase [7]. Prostate cancer is the most common male cancer in the United States and the second leading cause of cancer-related deaths [9]. In Korea, the frequency and mortality rate are increasing due to changes in lifestyle such as westernized eating habits, and aging [12]. The incidence of prostate cancer varies according to genetic factors, race, and age [38, 41]. According to US statistics, prostate cancer is rare in males less than 45 years old, but the incidence is higher after age 50. It is reported that the probability of developing prostate cancer in men over 50 years old is about 30% and that in men over 80 years old is about 80%[45].

Macrophages are known to be involved in host defense and homeostasis by participating in various host responses such as acquired immunity as well as natural immunity. In the inflammatory response, it produces cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 in the early stage of infection. In cancer patients, macrophages infiltrate cancer cells and appear as tumor-associated macrophages (TAMs). TAMs play an important role in the relationship between inflammation and cancer, which is basically an important influence on disease progression. Studies have shown that macrophages are likely to kill tumor cells if properly activated, by interferon-γ (IFN-γ) and lipopolysaccharides (LPS) therapy [1].

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT is a biologic process that approves a polarized epithelial cell, which normally interacts with basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which causes enhanced migratory capacity, invasiveness, elevated resistance to apoptosis and greatly increased production of extracellular matrix components [2]. A number of biomarkers of EMT have been found in recent studies, such as E-cadherin, N-cadherin, vimentin, Snail, Zeb1, and Twist [3]. E-cadherin is located on the cell surface of epithelial tissue and mediates cell-cell adhesion to bind cells in normal epithelial cells. E-cadherin expression is negatively correlated with the occurrence of EMT and tumor invasion [5]. In contrast, vimentin (cytoskeleton marker) and N-cadherin (cell surface markers) are associated with the onset of EMT and progression to invasive carcinomas. Snail, Slug, Zeb1, Zeb2, and Twist can also lower E-cadherin level and consequently trigger EMT [6]. E-cadherin, N-cadherin, Zeb1, Twist, Slug, Snail, and other EMT markers play an important role in the invasion of prostate cancer cells and the regulation of their metastatic potential. Therefore, a therapeutic strategy that intervenes the EMT process or reverses the EMT phenotype may be an alternative to cancer treatment in the future [5].

Conventional anti-cancer drugs cause side effects such as immune function deterioration, hematopoietic disorder, hair loss, and genetic damage. Recently, researches are being conducted to develop anti-cancer agents based on natural products that act specifically on cancer cells with lower side effects and reducing adverse effects [8]. In modern society, the function of food is classified as a primary function of nutrition, a secondary function of taste and palatability, and a tertiary function of biological control which helps to prevent and cure disease [10]. Since our food ingredients are not genotoxic and cytotoxic. Efforts to develop excellent therapeutic agents from everyday natural products have been made worldwide [11]. Phellinus linteus is a white rot fungus that belongs to the Hymenochaetaceae Phellinus. The similar species are P. gilvus, P. isniarius, P. nigricans, P. pini, P. conchaius, P. densus, and P. hatigii. Among them, P. linteus has become a target of mushrooms with novel anti-cancer activity [13]. Many studies have been carried out on the hot water extract of fruit body of P. linteus as it has a blocking effect on gastrointestinal cancer [14]. Also it has an enhancement effect on the immune function when the chemotherapy is treated onto the cancer of the gastrointestinal system including gastric cancer, esophageal cancer, colon cancer, and rectal cancer, as well as liver cancer surgery [14]. It has been reported that the polysaccharide extracted from fruiting body of artificially cultured P. linteus or natural P. linteus has anti-complement acticity, which increased the useful value of P. linteus [15].

In this study, we observed that P. linteus induces RAW 264.7 cells into pro-inflammatory macrophages and the conditioned media regulate the expression of migration, invasion, and EMT related genes in human prostate cancer, PC-3 cells.

Materials and Methods

Chemicals and Materials

Phellinus linteus was purchased from Kumwhangbio Co. (Jinju, Korea). Diaion HP-20 was purchased from the Mitsubishi Kasei Co. (Tokyo, Japan). Lipopolysaccharide (LPS) and polymyxin B sulfate salt (PB) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies of Ecadherin, vimentin, and snail-1 were purchased from Cell Signaling Technology (Victoria, BC, Canada), CCL2 from Bio X Cell (BxCell, West Lebanon, NH, USA), β-actin from Santa Cruze Biotechnology (Santa Cruz, CA, USA). And goat-anti-rabbit IgG secondary antibody was purchased from Enzo Life Science (Farmingdale, NY, USA). Western blot detection was performed using an enhanced chemiluminescence reagent (WesternBright ECL, Advansta, Menlo Park, CA, USA). N-(1-Naphthyl) ethylenediamine dihydrochloride and sulphanilamide were purchased from Sigma-Aldrich Co.

Extraction of Phellinus linteus

The dried fruit bodies of P. linteus were crushed by a grinder. Grinded fruit bodies of P. linteus were extracted with hot water (PLE) at 100℃ for 20 mins. PLE solution was centrifuged at 13,000 rpm for 10 mins and its supernatant was used for the experiments. Diaion HP-20 is one of the adsorption resins, which binds the unwanted lipophilic compounds and concentrates the target substances from a large amount of aqueous solution. Diaion HP-20 column was used to separate the active portion of the PLE supernatant. The PLE was passed from Diaion HP-20 resin to remove the adsorbed material, yielding two fractions of Phellinus linteus hot water extract/passing (PLEP), which was not adsorbed on resin and Phellinus linteus hot water extract/elution (PLEE), which was adsorbed on resin and eluted with methanol (MeOH) (Table 1). All fractions were dried in Modul 4080C speed vacuum (Hanil Science Industrial Co., Gimpo, Korea) and solvent-free residue was quantitatively re-dissolved in water or MeOH, respectively.

Table 1. Schematic process for purification of bioactive compounds from Phellinus linteus

Cell lines and culture

Mouse macrophage cell line, RAW 264.7 and human prostate cancer cell lines, PC-3 and DU145, were cultured in Dulbecco’s modified Eagle’s minimal medium (DMEM, WelGENE Inc. Daejon, Korea.) supplemented with 10% fetal bovine serum (FBS, WelGENE Inc.), and 100 unit/ml of penicillin, and streptomycin (WelGENE Inc.). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37℃. For the activation of macrophages, RAW 264.7 cells were treated with 100 μg/ml of PLEP or 1 μg/ml of LPS as a positive control for 24 hr. The prostate cancer cells were treated with the conditioned media of RAW 264.7 cells stimulated with or without LPS or PLEP for 48 hr.

Measurement of nitric oxide

Nitrite accumulation was used as an indicator of nitric oxide (NO) production. Quantification of NO was examined in murine macrophage RAW 264.7 cells by the Griess reaction. Griess reagent contains 0.2% naphthylethylenediamine dihydrochloride in DW and 2% sulfanilamide in 5% phosphoric acid. RAW 264.7 cells were dispensed into 96- well plates and stimulated with 10, 30 or 100 μg/ml of PLE, PLEP and PLEE for 24 hr. The supernatants were collected and mixed with 0.5 volumes of Griess reagent and incubated at room temperature for 10 mins. The plates were measured at 540 nm in a VERSA_max microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Cell viability analysis

The cell viability was determined by the colorimetric MTT assay. Prostate cancer PC-3 and DU145 cells were seeded in 48-well plates at a density of 1×10⁴ /well and treated with the conditioned media of RAW 264.7 cells for 24 or 48 hr. 0.5 mg/ml MTT solution was treated and incubated at 37℃ for 3 hr. The supernatant was removed by suction and the dyed precipitates were dissolved in dimethyl sulfoxide. The optical density was measured at 570 nm in a microplate.

Wound healing assay

This method mimics cell migration during wound healing in vivo. Wound healing assay were carried out as described previously [16]. Briefly, PC-3 cells were seeded at high concentration in a 6-well plate and grown overnight until the density reaches 80%. The monolayer was scratched using pipette tips. And the floating cells were removed and the conditioned media of RAW 264.7 cells was treated along with new media. Migration of PC-3 cells into scratched areas was observed by an optical microscope CKX41 (Olympus, Tokyo, Japan).

Reverse transcriptase (RT)-PCR and quantitative PCR (qPCR) analysis

Using a GeneAll^R RiboEx RNA extraction kit (GeneAll Biotechnology, Seoul, Korea), total RNA from RAW 264.7 cells and PC-3 cells was isolated in accordance with the manufacturer's specifications. The purity and concentration of total RNA were measured by a NanoDrop spectrophotometer ND-8000 (NanoDrop Technologies, Oxfordshire, UK). The Cdna was synthesized using by TOPscript ™ RT DryMIX (Enzynomics, Daejeon, Korea) according to the manufacturer's instructions. qPCR was performed using TOPreal ™ qPCR 2X PreMIX (Enzynomics). All primer sequences for qPCR analysis were described in Table 2. The PCR reactions consisted of pre-incubation process, 3 steps of amplification processes, and melting process. The pre-incubation process was carried out at 95℃ for 600 secs and the 3 steps of amplification processes were performed at 9 5℃ for 10 secs, at 60℃ for 10 secs, at 72℃ for 10 secs and finally a total of 45 cycles. The melting process was carried out at 95℃ for 10 secs, 65℃ for 60 secs, and 95℃ for 30 secs for melting curve. All RT-PCR reactions were performed in triplicate and mRNA expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Table 2. List of human- and mouse-originated primers and antibodies used in this study

Western blotting

PC-3 cells were cultured in a 6-well plate at a density of 2×10⁵ /well and incubated with the conditioned media of RAW 264.7 cells stimulated with 100 μg/ml of PLEP for 48 hr. The cells were detached from the plate surface with trypsin-EDTA. The cells were lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology, Sungnam, Korea). The cell lysates were quantified by Bradford method and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in TBS-T buffer [0.1% Tween 20, 150 mM NaCl, 20 mM Tris (pH 7.4)] at room temperature for 1 hr. The membranes were probed with 5% skim milk containing specific primary anti-body at 4℃ overnight. Finally, the membranes were detected with ECL western blotting detection reagent. Each band was determined by using a fluorescence scanner (LAS 3000, Fuji Film, Tokyo, Japan) and analyzed with Multi Gauge V3.0 software (Fuji Film).

Enzyme-linked immunosorbent (ELISA) assay

ELISA was performed using ELISA Ready-SET-Go! (eBioscience, Seoul, Korea) according to the manufacturer's specifications. In brief, the capture antibodies were coated to 96-well immune-plates and incubated at 4℃ overnight. The plates were washed with 200 μl of PBS containing 0.1% Tween 20 (PBST). Then plates were blocked with 1% BSA for 1 hr at 37℃. Standard and culture supernatants were added into wells and incubated for 3 hr at 37℃. After 3 hr, plates were washed five times with PBST. Then, 100 μl of biotin-conjugated anti-mouse detection antibodies were added into each wells and incubated for 1 hr at room temperature. After 1 hr, the plates were washed five times with PBST. Then avidin-conjugated hourseradish peroxidase were added and incubated for 30 mins. Tetra-methyl-benzidine was used for a substrate. The reaction was stopped by H2SO4 solution.

Annexin-Ⅴ/propidium iodide (PI) staining

Analysis of apoptotic cells was measured by an FITC Annexin-Ⅴ/PI detection kit. PC-3 cells were treated with the conditioned media of RAW 264.7 cells stimulated with PLEP for 48 hr and harvested cells were washed with 1x PBS. Then collected cells were resuspended in 1x binding buffer (100 μl) and stained with Annexin-Ⅴ/PI staining sol- ution at room temperature for 15 mins. Finally, the stained cells were analyzed by a flow cytometry (FACS Calibur, Becton Dickinson Co. CA, USA) and quantitative analysis of apoptotic cells was performed by the Cell Quest software (Becton Dickinson Co.)

Statistical analysis

All experiments were performed at least three times. Unless otherwise stated, the data are expressed as means ± SD. Differences between the three groups were performed using by ANOVA, followed by Tukey-Kramer multiple comparison tests. Results were statistically significant at *p<0.05.

Results

Various fraction of Phellinus linteus induces NO production in RAW 264.7 cells

PLE is the extract derived from natural product by hot water extraction. Therefore, it may contain both compounds to increase and decrease NO production. Diaion HP-20 column was used to separate some compounds, which adsorbs substances with molecular weight of 1,500 or more and hydrophobic character. By fractionation with Diaion HP-20, the passing fraction, PLEP, increased NO production and, on the contrary, the elution fraction with methanol, PLEE, reduced NO production induced by LPS (Fig. 1A, Fig. 1B). NO production in PLEP was higher than that in PLE, because of the molecules that reduce NO production were collected in the elution fraction. Therefore, PLEP was used for the subsequent experiments. In addition, endotoxin inhibitor polymyxin B (PB) was used to confirm whether the PLE and PLEP exhibits LPS-like mode action or is contained with LPS. PB was pre-treated at a concentration of 5 μg/ml in RAW 264.7 cells for 1 hr before treatment with samples (Fig. 1C). As a result, NO production was not changed after treatment with PB, while NO induction by LPS was reduced up to base line. This result indicates that PLE and PLEP increase NO production through different pathways from LPS.

Fig. 1. Effects of PLE and its fractions on nitric oxide production in RAW 264.7 cells. (A) Induction of NO productions; (B) Inhibition of NO production; (C) Effect of polymyxin B. CON, untreated negative control; LPS, 1 μg/ml of lipopolysaccharide; PLE, P. linteus hot water extract; PLEP, P. linteus hot water extract/passing; PLEE, P. linteus hot water extract/MeOH elution. Data are presented as mean ± SD (n=3 in each group). **p<0.01, ***p<0.001 vs. CON group.

PLEP activates macrophage RAW 264.7 cells into pro-inflammatory phenotype

Previously we found that PLEP promotes the production of NO from RAW 264.7 cells. Production of NO is one of the important evidence that macrophages have nature of pro-inflammatory. Macrophages exposed to activating agents such as LPS release a various of inflammatory cytokines and other substances including interleukin-1β (IL-1β), IL-6, IL-8, and TNF-α, all of which directly induce tumoricidal activity of macrophages [17]. Therefore, the effect of PLEP was investigated on the release of inflammatory cytokines from RAW 264.7 cells stimulated with 10-100 μg/ml of PLEP by monitoring the gene expression of pro-inflammatory markers. The mRNA expression of iNOS, which converts arginine to NO, was increased in pattern similar to results of the NO production. The gene expression of Il-1β, Il-6 and COX2 also showed the increase pattern (Fig. 2A). Cyclooxygenase (COX), known as prostaglandin-endoperoxide synthase (PTGS), is an enzyme that is responsible for the formation of instrumental biological mediators called prostanoids, including prostaglandins, prostacyclin and thromboxane [18]. In particularly, COX2, Il-1β, and TNF-α expressions were higher after treatment of 100 μg/ml of PLEP than that of 1 μg/ml of LPS as a positive control. ELISA was performed to observe the pro-inflammatory cytokines production from RAW 264.7 cells. As a result, the levels of IL-6 and TNF-α were similar to mRNA expression which was observed in Fig. 2A. The production of IL-6 and TNF-α in the supernatant of RAW 264.7 cells stimulated with PLEP was increased in a dose-dependent manner (Fig. 2B). It seemed that PLEP not only regulates the mRNA expression levels but also actually controls to secrete cytokines. Based on these results, it was suggested that PLEP can lead RAW 264.7 cells to tumoricidal phenotype by producing NO and pro-inflammatory cytokines. Also biomarkers of M2 type macrophages, Il-4, and Arginase-1 were examined by RT-PCR. However, the expression levels were too low to be detected (data not shown).

Fig. 2. The production of mRNA and cytokine in RAW 264.7 cells stimulated with PLEP. (A) mRNA expression; (B) Cytokine production. Data are presented as mean ± SD (n=3 in each group). *p<0.05, **p<0.01, ***p<0.001 vs. CON group.

Conditioned media of RAW 264.7 cells does not affect the cell viability of two different types of prostate cancer cells

The most generally used cell lines for prostate xenograft models are LNCaP, PC-3, and DU145 [19, 20]. The PC-3 cell line was originally derived from a bone metastasis of human prostatic adenocarcinoma origin [21]. Intravenous injection of PC-3 cell line has led to the establishment of lymph node metastases [22]. The PC-3 cells have a high metastatic potential and are negative for AR expression [20]. The DU145 cell line, which has weaker metastatic potential compared with the PC-3 cell line, was derived from a brain metastasis of human prostatic adenocarcinoma origin [23]. Two types of prostate cancer cells were used to observe the anti-cancer activity of conditioned media of RAW 264.7 cells stimulated with PLEP. To investigate the anti-cancer activities of PLEP itself and conditioned media of RAW 264.7 cells treated with PLEP, MTT assay was performed (Fig. 3A). As a result, PLEP itself and conditioned media of RAW 264.7 cells with LPS or PLEP did not affect the cell viability of prostate cancer cell lines. In addition, apoptosis analysis exhibited that conditional media of RAW 264.7 cells with stimulated with PLEP also did not affect the phenotype (Fig. 3B).

Fig. 3. Conditioned media of RAW 264.7 cells stimulated with PLEP does not affect apoptosis in prostate cancer cells. (A) Cell viability of prostate cancer cells; (B) Rate of apoptotic cell death of PC-3 cells. Various volumes of conditioned media of RAW 264.7 cells were treated into PC-3 cells and incubated for 48 hr. CON, un-treated negative control; CM, conditioned media of RAW 264.7 cells; LPS-CM, conditioned media of RAW 264.7 cells stimulated with LPS; PLEP-CM, conditioned media of RAW 264.7 cells stimulated with PLEP. Data are presented as mean ± SD (n=3 in each group). *p<0.05, ***p<0.001 vs. CON group.

Conditioned media of RAW 264.7 cells represses the epithelial-mesenchymal transition (EMT)

To identify whether PLEP also plays a role similar to LPS, EMT-related genes such as E-cadherin, N-cadherin, snail, twist, slug, and cadherin 11 were observed by using RT- PCR (Fig. 4A). The EMT is important developmental program that is activated during cancer metastasis and invasion. Cellular contacts greatly limit the ability of epithelial cells to move or migrate. It has been proposed that tumor cells lose this restriction during the last steps of the process of tumorigenesis, concomitantly with the loss of epithelial characteristics. One of the proteins that is down-regulated at this stage of tumorigenesis is E-cadherin, a molecule integral in the establishment of homotypic adhesion junctions [24-26]. The strong evidence that down-regulation of E-cadherin during the process of epithelial differentiation occurs at the transcriptional level [29-31]. Expression of E-cadherin was in creased in a dose-dependent manner after treatment with conditioned media of RAW 264.7 cells stimulated with PLEP or LPS which increased to 1.81-fold in PC-3 cells treated with conditioned media of RAW 264.7 cells stimulated with PLEP and 1.71-fold with LPS. On the other hand, the level of mRNA expression of N-cadherin was decreased. When the conditioned media of RAW 264.7 cells stimulated with PLEP showed 0.59-fold decrease compare to control. Loss of E-cadherin or switch of E-cadherin to N-cadherin leads to destruction of cell-cell adhesion, which drives adenoma to become carcinoma [32]. Mesenchymal markers, Snail and Twist, are able to down-regulated the level of E-cadherin and drive EMT to occur [34]. Expression of snail was decreased to 0.68-fold with conditioned media stimulated with PLEP and 0.55-fold with LPS. Expression of twist was decreased to 0.39-fold with conditioned media stimulated with PLEP and 0.42-fold with LPS. Expression of Slug was decreased to 0.47-fold with conditioned media stimulated with PLEP and 0.41-fold with LPS. Expression of cadherin 11 was decreased to 0.65-fold with conditioned media stimulated with PLEP and 0.59-fold with LPS. In the same manner, the expression of EMT-related proteins showed similar results with mRNA expression. The protein expression of E-cadherin was increased in a dose-dependent manner in PC-3 cells after treatment of conditioned media of RAW 264.7 cells and on the other hand, snail-1 protein was decreased (Fig. 4B). Thus, conditioned media of RAW 264.7 cells activated by PLEP inhibit EMT by a mechanism that reduces N-cadherin and increases E-cadherin in PC-3 cells.

Fig. 4. Conditioned media of RAW 264.7 cells activated with PLEP suppress EMT process in PC-3 cells. (A) mRNA expression; (B) Protein expression. The various volumes (5, 10, and 20%) of the conditioned media were treated in PC-3 cells for 48 hr. The mRNA expression levels were determined by real-time PCR and normalized to GAPDH. Data are presented as mean ± SD (n=3 in each group). *p<0.05, **p<0.01, ***p<0.001 vs. CON group.

Conditioned media of RAW 264.7 cells inhibits migration, invasion, and angiogenesis ability of PC-3 cells

Cell migration and invasion are important steps in many physiological events such as implantation of embryo, embryogenesis, morphogenesis, neurogenesis, angiogenesis, wound healing, and inflammation [35, 39]. Cell migration and invasion are also involved in the pathophysiology of many diseases, such as cancer. Furthermore, the capacity to produce metastases in very different cancers is the main features of malignant tumors, which is one of the main causes of death for cancer [42]. There is a report that decreased expression of migration-related genes, vimentin and CCL2, inhibited the migration ability [43]. As a result of mRNA expression of vimentin and ccl2 was decreased to 0.40 and 0.54-fold with conditioned media with PLPE and 0.37 and 0.36-fold with LPS, respectively (Fig. 5A). On the other hand, the protein expression of vimentin and CCL2 protein was further reduced after treatment with the conditioned media of RAW 264.7 cells stimulated with PLEP (Fig. 5B). Wound healing assay was performed to confirm the migration efficiency of PC-3 cells. PC-3 cells were scratched with 200 μl tips and treated with conditioned media of RAW 264.7 cells for 24 hr. Images of the wound were taken at x100 magnifications using an optical microscope. Cell migration ability was more inhibited in PC-3 treated with conditioned media of RAW 264.7 activated with PLEP than LPS (Fig. 5C). These results indicate that the conditioned media of RAW 264.7 cells stimulated with PLEP inhibits the migration of PC-3 cells through regulation of vimentin and CCL2. Additionally, the expression of angiogenesis-related gene, vegfa, was down-regulated to 0.43-fold with conditioned media with PLEP and 0.41-fold with LPS (Fig. 5A). Angiogenesis is physiological process through which new blood vessels shape from pre-existing vessels [44]. Angiogenesis is an advanced field of cancer research and has been proposed as a new treatment. These results implied that conditioned media of RAW 264.7 cells stimulated with PLEP inhibits the capability of invasion and angiogenesis through reducing the mRNA expression of vegfa.

Fig. 5. Conditioned media of RAW 264.7 cells represses migration, invasion and angiogenesis ability in PC-3 cells. (A) mRNA expression; (B) Protein expression; (C) Wound healing. Data are presented as mean ± SD (n=3 in each group). *p<0.05, **p<0.01, ***p<0.001 vs.CON group.

Discussion

Prostate cancer is a first leading cause of cancer incidence in the male population, especially, in the United State. That is also extremely increasing as a fifth incidence rate in Korean male. One of the causes of high mortality in prostate cancer is the spread of tumor cells to many tissues, including lymph nodes and bones [46]. More than 70% of patients were diagnosed with bone metastases. Many studies are underway to develop anti-cancer drugs and preventive therapies. However, synthetic drugs already developed are known to have side effects or toxicity, depending on their use and frequency of use. Now ingredients that were ingested in everyday life are considered to be safe for human body because of no cytotoxicity and genotoxicity unlike conventional anti-cancer drugs. Many studies have been researched on the hot water extract of fruit body of Phellinus linteus as it has been known of blocking effect on digestive system cancer. It also has an effect on the enhancement of immune function when the chemotherapy is combined for the cancer of the gastrointestinal system including gastric cancer, esophageal cancer, colon cancer, rectal cancer, and liver cancer [14].

It has been reported that macrophages are likely to kill tumor cells if properly activated by stimulator, such as interferon (IFN) -γ, and lipopolysaccharides (LPS) [10]. In this study, it was proved that the P. linteus hot water extract (PLE) produces nitric oxide (NO) from macrophages RAW 264.7 cells (Fig. 1A). Since PLE is a natural product-derived extract, it contains biological compounds showing both activities to increase NO production and decrease NO (Fig. 1B). To solve this problem, a simple purification was carried out using Diaion HP-20. As a result, molecules (PLEP) capable of activating NO production, and molecules (PLEE) inhibiting NO production could be separated (Fig. 1B). PLEP that activates NO production was confirmed by using real-time PCR and ELISA (Fig. 2). To determine the anti-cancer effect of conditioned media of RAW 264.7 cells activated by PLEP, we performed MTT and Annexin V-PI double staining assay. As a result, anti-cancer activity of conditioned media of RAW 264.7 cells stimulated with PLEP was not observed (Fig. 3).

EMT is a very important step in metastasis, during which non-motile, polarized epithelial cells separate their cell junctions and change into individual, motile mesenchymal cells [27, 28, 33]. Conditioned media of RAW 264.7 cells stimulated with PLEP was suppressed EMT by regulating EMT biomarkers in human prostate cancer PC-3 cells. It was also investigated the effect of conditioned media on migration and invasion by RT-PCR, western blotting, and wound healing analysis. As a result, vimentin, CCL2, and VEGFA, were down-regulated and the wound healing was also inhibited by the conditioned media of RAW 264.7 cells stimulated with PLEP (Fig. 5). However, the mRNA level of vimentin and ccl2 was not matched with protein levels. This inconsistency of mRNA and protein correlation has been observed with several reasons [36, 37, 40]. For one example, in the NIH3T3 cells, some genes, which is related with housekeeping genes, have stable mRNAs and proteins, thus mRNAs and proteins tended to have good correlation. However, specific genes related with transcription factors, genes with cell cycling and coding genes with signaling are unstable and protein is also same as well [36]. Therefore, this inconsistence of mRNA and protein levels from vimentin and CCL2 can occur.

In conclusion, it was suggested that PLEP induces pro-in flammatory effect of RAW 264.7 cells and its conditioned media has an effect on migration and invasion by inhibiting EMT of PC-3 prostate cancer cells. Although the conditioned media of RAW 264.7 macrophages does not directly kill cancer cells, its anti-cancer effect can be expected through inhibiting migration and invasion of cancer cells. Finally, this new direction will be helpful for cancer therapy after in vivo animal experiments for more accurate treatment.