ShaoYao decoction ameliorates colitis-associated colorectal cancer by downregulating proinflammatory cytokines and promoting epithelial-mesenchymal transition

Abstracts Background Shaoyao decoction (SYD) is a traditional Chinese medicine prescription formulated by Liu Wan-Su, a master of traditional Chinese medicine in Jin-Yuan Dynasty. SYD is effective in treating ulcerative colitis. Paeonol, a component of SYD, inhibits colorectal cancer (CRC) cell proliferation and induces CRC cell apoptosis. In this study, azoxymethane (AOM)/dextran sodium sulfate (DSS)–induced colitis-associated CRC (caCRC) model and CRC cell lines were used to examine the effects of SYD on CRC in vivo and in vitro. Methods A translational medicine strategy based on phytomics quality control was adopted. Liquid chromatography was employed for the chemical characterization and chemical fingerprinting of SYD. Protein expression and macrophage existence were determined by immunohistochemistry and western blot. Serum cytokines were quantified by Luminex assay. Results AOM/DSS-induced caCRC phenotypically resembled human caCRC. SYD significantly increased the survival rate of the mice, ameliorated the general well-being of the mice, and reduced the incidence and multiplicity of colonic neoplasms. SYD inhibited epithelial–mesenchymal transition (EMT), as indicated by upregulated epithelia cadherin and downregulated neuronal cadherin, fibronectin, vimentin, and transcription factor Snail. SYD reduced the expression levels of serum interleukin 1β, interleukin-6, tumor necrosis factor α, tumor-associated macrophages, and p65. These results showed that SYD can attenuate proinflammatory cytokines and inhibit EMT. Conclusions SYD ameliorates caCRC by suppressing inflammation and inhibiting EMT. SYD might be an alternative therapy for caCRC.


Introduction
Approximately 142,820 (9%) and 50,830 (9%) new cancer cases and deaths of colorectal cancer (CRC) were reported in the US in 2013, respectively [1]. CRC is the third most common cancer in men and the second in women worldwide [2]. Ulcerative colitis, familial adenomatous polyposis, and hereditary nonpolyposis colon cancer syndrome are the three highest risk groups for developing CRC. In a metaanalysis based on 116 studies, the overall prevalence of colorectal cancer in patients with ulcerative colitis was 3.7%. The risk of colorectal cancer increases by 8% in 20 years and by 18% in 30 years [3]. The pathogenesis of colorectal cancer in patients with ulcerative colitis is triggered by inflammation-dependent mechanisms [4]. Furthermore, ameliorating inflammation can control inflammation-mediated reactive oxygen species, microsatellite instability, telomere shortening, and chromosomal instability [4].
Shaoyao decoction (SYD) is a canonical Chinese medicine prescription formulated by Liu Wan-Su in "Plain Questions -anthology on proper therapy for Qi disorder and pathogenesis to save life" in Jin-Yuan dynasty (AD 1186). SYD is commonly used to treat various inflammatory bowel diseases by clearing heat and damp, removing stasis and toxin in the intestine [5]. SYD is effective in ameliorating the major manifestations of ulcerative colitis, such as hematochezia, abdominal pain, diarrhea, and mucosal defense impairment [6,7]. SYD enhances mucous cell proliferation, decreases exfoliated mucosal cell proliferation, and prevents lamina propria edema and neutrophil infiltration [8]. SYD also exerts anti-inflammatory effects that can decrease the expression of intercellular adhesion molecule 1 (ICAM-1) and tumor necrosis factor α (TNF-α) and increase the expression of interleukin (IL)-10 in colon tissues [9]. Paeonol, the primary phenolic component in Radix Paeoniae Alba (Shaoyao), reduces HT29 cell proliferation by downregulating cyclooxygenase-2 and upregulating p27 [10]. Paeonol induces LoVo cell apoptosis by increasing intracellular calcium concentration [11]. However, whether or not SYD can prevent colitisassociated CRC (caCRC) remains elusive.
The epithelial-mesenchymal transition (EMT) serves important functions in the formation of body plan and the differentiation of multiple tissues [12]. EMT is a process in which epithelial cells trans-differentiate and acquire invasive mesenchymal phenotype. Clinical analysis [13] and animal experiments [14] demonstrated that EMT contributes to the loss of intestinal epithelial cells; thus, EMT is recognized as a major contributor to the pathogenesis of inflammatory bowel diseases. Transforming growth factor β (TGF-β), TNF-α, and nuclear factor κB (NF-κB) participate in inflammation development; these factors are also crucial for the initiation of EMT [15]. In the present study, the functions of EMT in azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced caCRC [16] were evaluated to confirm whether or not SYD can inhibit caCRC through EMT regulation.

SYD preparation
SYD was composed of nine commonly used herbs: Radix Paeoniae Alba, Radix Angelicae Sinensis, Rhizoma Coptidis, Semen Arecae, Radix Aucklandiae, Radix Et Rhizoma Glycyrrhizae, Radix Et Rhizoma Rhei, Radix Scutellariae, Cortex Cinnamomi. The raw herbs for SYD were purchased from the affiliated Nan Fang Hospital of Southern Medical University. These were mixed in the ratio of 30:15:15:6:6:6:9:15:7.5 (dry weight). Aqueous extracts of SYD were extracted at 80°C by stirring for 1 h using 10 volumes of distilled water (v/m). The extracts were centrifuged at 1,500 g at room temperature. To obtain the semisolid SYD solution, the supernatant was collected and subjected to condensation under reduced pressure at 70°C [17]. The quality of SYD was confirmed by HPLC analysis (Additional file 1: Figures S1 and S2 ,  Table S1). SYD was suspended again in 0.9% saline at a final concentration of 2 g/mL. The solution was stored in aliquots at −20°C.

Animals and experimental procedure
All procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Southern Medical University. All protocols were submitted and validated by Animal Care Ethics Committee of Southern Medical University (No. 2012-055). 6 and 8 weeks male C57BL/6 J mice weighting 15-20 g (specific pathogen-free) were obtained from the Laboratory Animal Center of Southern Medical University. The animals were maintained under controlled conditions (22°C, 12-h/12-h dark/light cycle) in a conventional animal colony. There were 65 mice used in the study, 15 mice in control group, 30 in model group and 20 in SYD group. At the end of the study, there were 15 surviving mice in control group, 7 in model group and 12 in SYD group, respectively.
Procedures of induction of caCRC model by AOM and DSS was shown in Figure 1A [16]. At week 2, mice were injected with AOM (10 mg/kg, i.p.). After 1 week, 3% DSS (International Lab, Chicago, IL, USA) was added to the drinking water for 7 days followed by 14 days of tap water for recovery. This cycle was repeated twice. SYD (7.12 g/kg) or equivalent normal saline was administered by gavage using a tube twice a day. The mice were killed by cervical dislocation at week 16 and the colons (from the ileocecal junction to the anal verge) were removed. After measuring the length and weight, the colons were cut longitudinally along the main axis, washed with phosphate-buffered saline (pH 7.4, 4°C) and macroscopically inspected. The number, size and location of pre-neoplastic and neoplastic lesions (dysplasia and carcinoma) in the colons were recorded based on gross examination. The size of dysplasia was measured by an ocular micrometer. After gross examination, the colons were cut into pieces at about 1 cm intervals. Samples were then fixed in 10% buffered formalin (pH 7.4) or kept in 10 volumes of RNAlater Solution (Ambion, Life technologies, Carlsbad, CA, USA). Remaining samples were flash-frozen in liquid nitrogen and stored at −80°C for further immunohistochemistry, PCR and protein analysis.

Cell viability assays
MTT assay was used to analyze the effects of paeonol and SYD on cell viability in two kinds of colorectal adenocarcinoma cell lines SW480,HCT116. Cells were routinely maintained in RPMI 1640, supplemented with 10% FBS and antibiotics (50 U/ml of penicillin and 50 μg/ml streptomycin) at 37°C in a humidified atmosphere containing 5% CO2. Cell number was measured on a standard microscope by manual counting of cells using a grid with an area of 0.25 mm 2 . 1 × 10 4 Cells were seeded on 96-well plates in a 100 μl volume. The MTT assay was performed by addition of MTT to a final concentration of 0.5 mg/ml of medium. After 4 h, the medium was removed, and 150 μl of DMSO was added to each well. After 10 min of shaking at room temperature, 150 μl of soluble material was placed into a new 96-well plate, and absorbance at 562 nm was read with a background subtraction at 660 nm.

Cell culture and transfection
HCT116 cell line was cultured in RPMI 1640, supplemented with 10% FBS and antibiotics (50 U/ml of penicillin and 50 μg/ml streptomycin) at 37°C in a humidified atmosphere containing 5% CO2. For transfection,cells were seeded in a 6-well plate 24 h before the experiment. The cells were transfected with Snail 1 plasmid using the Attractene reagent (Qiagen, Germany) according to the manufacture's protocol.

Histopathological assessment
For histopathological examination, formalin fixed, paraffinembedded colon tissues were cut into serial sections (5 μm) and stained with haematoxylin and eosin (H&E). Histological alternations such as dysplasia and carcinoma were verified. Assessment of dysplasia and cancer was based on the criteria set forth in the Mouse Models of Intestinal Cancers consensus report: location, presence/absence of prolapse/herniation, size, dysplasia grade, invasion level, presence of desmoplasia and presence of irregular architecture [18]. Carcinoma was defined as high-grade dysplasia of colonic mucosa which invaded beyond the muscularis mucosa and into the submucosa [16].

Cytokine measurement by multiplex immunoassay
The levels of interleukin-1β (IL-1β), IL-6 and TNF-α in the serum were measured using mice cytokine multiplex kit (Millipore, Billerica, MA, USA). Quantification of cytokines was performed using the Luminex system (Austin, TX, USA) [19].

Statistical analysis
Each experiment was repeated at least three times. Data were presented as mean ± SD. All data were analyzed using SPSS statistical package (version 13.0, SPSS Inc, Chicago, IL, USA). Survival rate was analyzed with Kaplan-Meier survival analysis. Data between two groups were compared with 2-independent samples tests. Ranked data were analyzed by nonparametric test (K Independent Samples Test). Mean values of data from more than 3 groups were analyzed by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered as statistically significant.
Result SYD increased the survival rate of mice treated with AOM/DSS Body weight loss and bloody stools were observed in mice acutely exposed to DSS. These symptoms were relieved during the recovery period ( Figure 1B). The colon weight was increased and the length was shortened significantly in the mice that received both AOM and DSS compared with the control mice ( Table 1). The significant increases in colon weight and colon length ratio were caused by apparent mucosal thickening. SYD substantially ameliorated the body weight fluctuation and mucosal thickening. The Kaplan-Meier survival curves showed that SYD significantly increased the survival rate of the mice ( Figure 1C).

SYD reduced the incidence and multiplicity of colonic neoplasms and oncogenic protein expression
The colonic neoplasm formation rate was 100% in the mice treated with AOM/DSS. Neoplasms were principally distributed in the middle and distal colon (Figure 2A). The total multiplicity of colorectal neoplasms decreased by 42.8% after SYD treatment (P < 0.01, Table 2). Moreover, SYD significantly retarded the development of large neoplasms (diameter > 3 mm) by 59.2% ( Figure 2B, Table 2). The tumour weight and tumour volume were decreased by SYD ( Figure 2C). The neoplasms in the colon were mostly tubular adenoma or adenocarcinoma according to the criteria of Mouse Models of Intestinal Cancers ( Figure 2D). The neoplasms was mostly high-differentiation with invasion in model group.SYD treatment could ameliorate level of differentiation and invasion of neoplasms (P = 0.041, Table 3).
The expression levels of proliferating cell nuclear antigen (PCNA) and β-catenin increased in dysplastic and neoplastic lesions ( Figure 3A and Figure 3B). Intense nuclear staining for p53 (CM5 clone: detects both mutant and wild-type forms) was detected in the neoplastic epithelium and in nondysplastic crypts, implicating the involvement of p53 mutation in our AOM/DSS-induced caCRC model [18]. The expression levels of these neoplastic markers suggested that AOM/DSS-induced caCRC phenotypically resembled human caCRC even though the expression of COX-2 was not high in adenocarcinoma ( Figures 3A and 3B, Table 4).

SYD and Paeonol inhibited CRC cell proliferation
SYD significantly decreased cell viability in a time-and dose-dependent manner; the dose ranged from 0 mg/mL to 8 mg/mL. The viabilities of SW480 and HCT116 cells decreased to 50.2266% and 53.6704% after 24 h of treatment with 2 mg/mL SYD ( Figure 4A). Paeonol, an ingredient of Radix Paeoniae Alba, significantly decreased cell viability in a time-and dose-dependent manner; the dose ranged from 0 mM/L to 0.8 mM/L. The viabilities of SW480 and HCT116 cells decreased to 48.0659% and 51.3992% after 48 h of treatment with 0.4 mM /L Paeonol ( Figure 4B).

SYD and Paeonol decreased oncogenic proteins in CRC cells
SYD and Paeonol decreased the expression levels of PCNA, β-catenin, p53, and COX-2 in CRC cells in a dose-dependent manner. ( Figure 5A and Figure 5B, Table 5 and Table 6).  Table 4). Western blot analysis further confirmed the effects of SYD on the expression of E-cad, N-cad, and vimentin. Snail was upregulated in the model group, and it was downregulated by SYD ( Figure 6B, Table 4).   Overexpression of Snail, a transcription factor crucial in EMT, induced the upregulation of N-cad and vimentin. SYD and Paeonol decreased the expression of N-cad induced by Snail overexpression. (Figure 5C, Table 5 and Table 6) SYD decreased serum cytokines, macrophage proliferation, and NF-κB expression

SYD inhibited Snail-induced EMT
The number of macrophages and the expression of NF-κB were increased significantly in the model group compared with those in the control group; SYD counteracted these Table 3 Analysis of differentiation and invasion   Table 4. effects ( Figure 7A). Serum IL-1β, IL-6, and TNF-α were upregulated in the model group and were significantly inhibited by SYD ( Figure 7B).

Discussion
Variations in climatic conditions, geographic locations, and harvesting, processing, and extraction methods could challenge the quality control of herbal extracts from Chinese compound prescriptions [20]. In the present study, several specific chemical markers and batch-tobatch comparison for quality controls were utilized. This strategy (i.e., formulomics) was based on liquid chromatography for chemical characterization and chemical fingerprinting of SYD [21,22]. The strict extract quality control of SYD is important to translate the TCM formula to clinical drugs [21]. Basing on the capacity of SYD to downregulate the expression of proinflammatory cytokines, such as ICAM-1 and TNF-α, we hypothesized that SYD ameliorates caCRC by suppressing inflammation [23,24]. Repeated DSS administration causes chronic inflammation, and AOM induces the formation of O6-methylguanine upon metallic activation [25]. Therefore, DSS in combination with AOM resulted in 100% incidence of colonic neoplasms in mice in the present experiment. DSS administration caused mass death in the model group; the effect of this drug was alleviated by SYD treatment. Tumor numbers were reduced and CRC cell proliferation was inhibited by SYD.  These results suggested that SYD elicited antitumor effects by inhibiting CRC cell proliferation. Increased PCNA and β-catenin expression suggested that DSS/AOM-induced neoplasm resembled human caCRC. The increase in β-catenin due to the constitutive Wnt activation for AOM induces mutations in the exon 3 of Ctnnb [25]. Wild-type p53 is a rapidly degraded protein with a low cellular level [26]; thus, the accumulation of p53 in the model group also characterizes caCRC for the early loss of p53 function [18]. Downregulation of PCNA, β-catenin, and p53 by SYD further confirmed the efficacy of SYD in DSS/AOM-induced caCRC.
EMT disrupts cell-cell adherence, causes apico-basal polarity loss, and triggers matrix remodeling to promote CRC pathogenesis and metastasis [27]. EMT endows cells Western blot of β-catenin, Cox2, p53 and PCNA expression in SW480 and HCT116 exposed to 0-4 mg/mL SYD. The expression levels of these proteins were reduced in a dose-dependent manner. Semiquantitive analysis of these proteins is showed in Table 5. (B) Western blot of β-catenin, Cox2, p53 and PCNA expression in SW480 and HCT116 exposed to 0-0.4 mM/L Paeonol. The expression levels of these proteins were reduced in a dose-dependent manner. Semiquantitive analysis of these proteins is showed in Table 6. (C) Western blot of E-cad, N-cad and vimentin expression in SW480 with Snail overexpression exposed to 0-4 mg/mL SYD and 0-0.4 mM/L Paeonol. The expression levels of N-cad and vimentin were reduced in a dose-dependent manner, while E-cad was increased in a dose-dependent manner. Semiquantitive analysis of these proteins is showed in Tables 5 and 6.  with migratory and invasive capacity, induces stem cell properties, prevents apoptosis and senescence, and contributes to immunosuppression [12]. In the present study, SYD ameliorated DSS/AOM-induced EMT, as indicated by upregulated E-cad and downregulated N-cad, fibronectin, and vimentin. In a previous report, Snail overexpression results in dramatic changes in signaling pathways that are involved in EMT and contributes to the acquisition of stem/progenitor-like character [28]. Snail also represses epithelial gene expression and thereby promotes EMT and tumor invasion by activating the beta-catenin pathway [29]. SYD and paeonol significantly decreased Snailinduced expression of N-Cad and vimentin. This result suggested that SYD executed its protective activity by downregulating Snail-induced EMT [28,29]. The major function of SYD was to resolve colorectal inflammation through heat clearing and detoxification. Paeonol decreases lipopolysaccharide-induced TNF-α,  Table 4.
Mutant p53 accumulated in inflamed colon and in cancerous glands concomitantly with NF-κB activation and sustained DNA damage. Mice were susceptible to chronic inflammation. However, it also greatly accelerated the inflammation-associated colon cancer under this situation [37]. TNF-α is a critical factor produced by macrophages to accelerate EMT [38]. This factor increases Snail protein expression and Snail nuclear translocation to initiate cancer invasion and metastasis through EMT activation [27]. EMT can be induced by activating the NF-κB pathway and enhancing Snail [39]. AOM exacerbated DSS-induced colitis to caCRC by triggering p53 mutation, β-catenin accumulation, and EMT induction upon tumor-associated macrophage (TAM) infiltration, and NF-κB promoted IL-1β, IL-6, and TNF-α release. Therefore, SYD might ameliorate caCRC by suppressing NF-κB activation and cytokine-induced EMT.