Antitumor immunostimulatory activity of polysaccharides from Salvia chinensis Benth

Journal of Ethnopharmacology 168 (2015) 237–247

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Antitumor immunostimulatory activity of polysaccharides from Salvia chinensis Benth Guangwen Shu a, Wenhao Zhao a, Ling Yue b, Hanwen Su c, Meixian Xiang a,n a

College of Pharmacy, South-Central University for Nationalities, Wuhan, PR China Endocrinology department, Wuhan General Hospital of Guangzhou Military Command, Wuhan, PR China c Renmin Hospital of Wuhan University, Wuhan, PR China b

art ic l e i nf o

a b s t r a c t

Article history: Received 30 September 2014 Received in revised form 16 February 2015 Accepted 30 March 2015 Available online 6 April 2015

Ethnopharmacological relevance: Salvia chinensis Benth (S. chinensis) is a traditional herb applied in the treatment of hepatocellular carcinoma (HCC). Polysaccharides abundantly exist in this plant. However, it remains poorly understood if polysaccharides from S. chinensis (PSSC) contribute to its anti-HCC activity. Materials and methods: The in vivo anti-HCC activity of PSSC was evaluated in Kunming mice bearing H22 ascitic hepatoma cells. An array of physiological indexes was measured to evaluate toxicological effects on host animals. Subgroups of immune cells were purified by a magnetic-activated cell sorting system and analyzed by flow cytometry. Reverse transcription real-time PCR and immunoblotting were recruited to determine the effects of PSSC on the cellular signaling of different subgroup of immune cells. Results: PSSC suppressed in vivo proliferation of H22 cells with undetectable toxic effects on tumorbearing mice. PSSC alleviated tumor transplantation-induced CD4þ T cell apoptosis and dysregulation of serum cytokine profiles, which elevated cytotoxic activities of natural killer and CD8þ T cells. PSSC reduced serum levels of prostaglandin E2 (PGE2). Injection of exogenous PGE2 completely abrogated the antitumor immunostimulatory activity of PSSC. Cyclic adenosine monophosphate (cAMP) is the second messager of PGE2. In CD4 þ T cells, PSSC substantially declined intracellular cAMP. This event elevated protein levels of JAK3, enhancing STAT5 phosphorylation and STAT5-depedent expression of antiapoptotic genes. Cyclooxygenase-2 is the key enzyme mediating biosynthesis of PGE2. PSSC suppressed the transcription and translation of cyclooxygenase-2 in tumor associated macrophages. Conclusion: Our data clearly showed antitumor immunostimulatory activity of PSSC against transplanted H22 HCC cells. Suppressing tumor transplantation-induced PGE2 production was implicated in the antitumor immunostimulatory activity of PSSC. These works provides novel insights into the traditional application of S. chinensis against HCC and supported considering PSSC as an adjuvant reagent in clinical HCC treatment. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Salvia chinensis Benth Polysaccharides Antitumor immunostimulatory activity

1. Introduction As one of the most common human malignant diseases, hepatocellular carcinoma (HCC) accounts for approximately 5.6% of all tumors (Rui et al., 2014). This disease is known for its poor prognosis and is the third most common cause of cancer-related mortality (Bosch et al., 2004). Since HCC is generally diagnosed at a later stage, it is only viable to offer surgical removal or liver transplantation to a small proportion of patients, while the majority must rely upon systematic chemotherapy (Llovet et al., 2003). Abbreviations: HCC, Hepatocellular carcinoma; PGE2, prostaglandin E2; S. chinensis, Salvia chinensis Benth; PSSC, polysaccharides from S. chinensis n Corresponding author. Tel./fax: þ 86 27 6784 1196. E-mail address: [email protected] (M. Xiang). http://dx.doi.org/10.1016/j.jep.2015.03.065 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

However, HCC is highly aggressive and resistant to conventional chemotherapeutic reagents (Rampone et al., 2010). In addition, conventional chemotherapeutic reagents frequently exhibit toxic effects on normal cells, resulting in considerable detriment for patient prognosis (Drake and Antonarakis, 2010). The development of novel therapeutic agents against HCC is thus essential. Immunosuppression has been well established as an important contributor to the onset and progression of malignant diseases in both tumor-bearing animals and human cancer patients (Vasievich and Huang, 2011). Epidemiology surveys have unveiled the significance of the hepatic virus infection and chronic inflammatory responses in hepatocarcinogenesis (Berasain et al., 2012; McGivern and Lemon, 2011). Both have the capacity to induce immune system tolerance, giving rise to an effective counterattack against host anti-HCC immune response (Pardee and Butterfield, 2012). On

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the other hand, it has been shown that immunotherapy is beneficial in the inhibition of HCC cell proliferation and repression of its metastasis and recurrence (Avella et al., 2012). These observations suggest that the stimulation of immune responses in the host can provide alternative strategies for the treatment of HCC. CD4 þ T cells are fundamental components of the adaptive immune response. Activation, maturation, and active participation of CD4 þ T cells are highly significant in an effective antitumor immune response (Dobrzanski, 2013). Generally, CD4 þ T cells can be divided into two subtypes according to their secreted cytokine profiles: T helper 1 (Th1) cells that secrete IFN-γ/IL-2 and T helper 2 (Th2) cells that secrete IL-4/IL-5 (Du et al., 2009). The cytokines secreted by Th1 cells have great potential for stimulating cytotoxicity of CD8 þ T and natural killer (NK) cells against tumor cells, providing an effective antitumor immune response. In contrast, Th2 cytokines suppress the cytotoxic activities of CD8 þ and NK cells, resulting in the onset and progression of tumors (Lakshmi Narendra et al., 2013). It has been suggested that CD4 þ T celldependent antitumor immune responses could be impaired through various mechanisms. For instance, increased apoptotic CD4 þ T cell could be detected in both cancer patients and tumorbearing animals (Bhattacharyya et al., 2007). Moreover, an alteration from the cytokine profiles typical for Th1 cells to those for Th2 cells is another pathway mediating the repressed cellular immune response (Grimm et al., 2010). Prostaglandin E2 (PGE2) is a metabolic of arachidonic acid and a critical contributor to the antitumor immunosuppression associated with malignant diseases such as HCC (Lee et al., 2009; Li et al., 2012). The removal of PEG2 receptors has been shown to enhance antitumor immune response and reduce carcinogenesis (Yang et al., 2003). Cyclooxygenase (COX) is the key enzyme that catalyzes the conversion of arachidonic acid to PGE2. COX isoforms can be categorized into two types: COX-1 and COX-2. COX-1 is constitutively expressed in most tissues. Although basal levels of intracellular COX-2 are low, its expression can be induced by a series of stimulus (Tiwari et al., 2014). In most cases, inducible COX-2 is responsible for the high PGE2 levels frequently detected in inflammatory-related pathology processes (Niu et al., 2014; Olajide et al., 2014). PGE2 is known to bind to its receptor and elevate cellular levels of cyclic adenosine monophosphate (cAMP), working as the secondary messenger and activating the downstream signaling cascade of PGE2 (Mosenden et al., 2011). PGE2 is able to directly induce CD4þ T cell apoptosis and exacerbate phenotypes of Th2 while inhibiting those of Th1 (Ghosh et al., 2012; Li et al., 2013), resulting in the suppression of antitumor immune responses. These observations highlight the PGE2 pathway as a potential target to ameliorate tumor-related immunosuppression. Recently, traditional medicines have received increasing attention from the scientific community due to their significant potential as therapeutic agents. Salvia chinensis Benth (S. chinensis, Family Lamiaceae) is a traditional Chinese medicine commonly found in South Central China. It has been documented in an early material medica “Bencao Gangmu” (Ming Dynasty, about 600 years ago) that the whole plant of this species is able to ameliorate blood stasis and eliminate swells. Since in the theories of the traditional Chinese medicine, tumors or malignant tissues were considered as stasis and swells, this herb are traditionally used in the treatment of malignant diseases, including HCC. Consistent with the traditional usage of S. chinensis, its anti-tumor activity was supported by an array of modern scientific studies (Liu et al., 2012; Yang et al., 2011; Shoemaker et al., 2005). Our previous work identified the major chemical composition of total flavonoids from S. chinensis and revealed that they are able to induce HCC cell apoptosis by repressing cellular NF-κB signaling (Xiang et al., 2013). In addition to flavonoids, polysaccharides constitute another type of major secondary metabolic product in S. chinensis.

Previous studies have highlighted the immunomodulatory activity of natural polysaccharides which is beneficial for treating malignant diseases (Xu et al., 2009; Zhao et al., 2012a). However, the extent to which polysaccharides from S. chinensis (PSSC) can stimulate anti-HCC immune response in vivo remains uncertain. In this work, the monosaccharide composition of PSSC was examined and the efficacy of PSSC for antitumor immunostimulatory activity against transplanted H22 HCC cells was evaluated. Possible molecular mechanisms underlying the anti-tumor immunomodulatory activity of PSSC and the toxicological effects of PSSC on tumor-bearing animals were also assessed.

2. Materials and methods 2.1. Phytochemical investigations Fresh S. chinensis was collected in Wuhan, Hubei Province, PR China and identified by Prof. Dingrong Wan (South-Central University for Nationalities, Wuhan, PR China). Voucher specimens (No. SC-2012028) were deposited at the Herbarium of Medical Plants, College of Pharmacy, South-Central University for Nationalities. The plant materials were cleaned, dried at room temperature and milled into powders. Phytochemical investigations were performed as described previously (Li et al., 2014). Briefly, the powders (1.0 kg) were extracted by distilled water (1 L, 100 1C) under reflux for 3 times for 1.5 h each time. The extracts were combined, filtered, and concentrated under reduced pressure to 500 mL. The insoluble fractions were removed through centrifugation of the sample at 4500g for 20 min. Then, the supernatants were precipitated with 4 volumes of 95% ethanol at 4 1C overnight. The sediments were fully dissolved in distilled water and subjected to intensive dialysis for 3 days at 4 1C (cut off molecular weight determined at 7500 Da). The protein contaminants in the retentate were removed through extraction with Sevage reagent (CH3Cl: BuOH ¼4: 1, v/v). This procedure was repeated six times. After separating the organic phase by centrifugation, the water phase was precipitated with 3 volumes of 95% ethanol and washed with 100% ethanol, acetone, and ether, yielding brownish crude PSSC. The crude PSSC was then dissolved in distilled water and loaded into a DEAE-Sepharose CL-6B column. The column was eluted with distilled water, 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, 1.0 M NaCl, and 0.2 M NaOH, respectively. Eluted fluids were monitored using the phenol-sulfuric acid method. The major fraction was the 0.2 M NaOH fraction. This fraction was collected, neutralized by 0.2 M HCl, dialyzed, and further purified by size-exclusion chromatography on a Sephadex G-100 column. The column was eluted with 0.1 M NaCl at a flow rate of 0.5 mL/min. The main polysaccharide fraction was then collected, concentrated, dialyzed, and lyophilized, providing a white refined PSSC (17.2 g). The homogeneity and molecular mass of refined PSSC were determined by an Agilent high performance liquid chromatography (HPLC) system (Agilent Technologies, USA) equipped with a TSK-GEL G3000 PWXL column. The column was eluted with Na2SO4 (0.1 M). Detection was performed with a RID-10A refractive index detector. Linear regression was calibrated with T-series dextran standards at different molecular weights (Mw 2000 kDa, 670 kDa, 410 kDa, 270 kDa, 150 kDa, 80 kDa, 50 kDa, 12 kDa, and 5 kDa). The PSSC showed a single, sharp and symmetric peak in HPLC. The UV absorption of PSSC at both 260 and 280 nm was undetectable, indicating an absence of nucleic acids or proteins in PSSC. The average molecular weight of PSSC was determined as 327 kDa. Monosaccharide composition of PSSC was determined as described previously (He et al., 2013). Briefly, PSSC was hydrolyzed into monosaccharides in 2 M trifluoroacetic acid at 100 1C for 8 h.

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Table 1 Monosaccharide compositions (%) of PSSC. Monosaccharide

Composition

Monosaccharide

Composition

Arabinose Fructose Galactose Glucose Mannose

19.7 3.3 16.6 3.1 2.9

Rhamnose Ribose Xylose Galacturonic acid Glucuronic acid

21.3 2.6 3.4 24.8 2.3

Then, the monosaccharides were labeled with 1-phenyl-3-methyl5-pyrazolone (PMP). PMP-labeled monosaccharides were analyzed using a Shimadzu LC-2010A HPLC system (Shimadzu, Japan). As shown in Table 1, major monosaccharide units in PSSC were arabinose, galactose, rhamnose, and galacturonic acid. Other monosaccharides, including fructose, glucose, mannose, ribose, xylose, and glucuronic acid, also existed in PSSC. One of major monosaccharide units in PSSC was galacturonic acid containing ionizable carboxylic acid group which is in favor of elevating the solubility of PSSC in water. 2.2. Cells, animals and animal experimentations Male Kunming mice (18–22 g) were obtained from the Experimental Animal Center, Hubei Institute of Health and Epidemic Prevention. The mice were maintained in the standard specific pathogen-free circumstance. All of the animal experimental protocols were approved by the Animal Care and Use Committee of South-Central University for Nationalities. Murine H22 ascitic hepatoma cells were maintained by transplanting them into the peritoneal cavities of Kunming mice on a weekly basis. The in vivo HCC cell growth model was established by subcutaneously transplanting 1  106 of H22 cells into the right flanks of the mice. The mice were randomly divided into groups and treated as described in the text. Each group contained ten mice. Drug treatment began 24 h after transplantation. The negative control group received 0.9% normal saline (0.9% NaCl). Cisplatin, a conventional approved drug in HCC treatment, was used as the positive control. All of the drugs, including PSSC, cisplatin, and PGE2 (purchased from Cayman Chemical), were administrated through intraperitoneal injection each day for 10 consecutive days. At day 11, all of the animals were sacrificed. The transplanted tumors were dissected out and immediately weighed. The tumor inhibitory rate (TIR) was calculated by the formula: Tumor inhibitory rate (%)¼(WControl WTreated)/WControl  100%. Where, WTreated and WControl constituted the average tumor weight of the treated and vehicle control mice, respectively. In an array of previous studies, the highest experimental dosages of plant polysaccharides on mice were around 100 mg/ kg (Li et al., 2014; Hua et al., 2014; Zhang et al., 2014). The highest dosage of PSSC was thus selected as 100 mg/kg here. The dosage of cisplatin was chosen according to our previous report (Zhao et al., 2012b). In our pilot experiment, mice without tumor transplantation were intraperitoneally injected with different dosages of PGE2. After 12 h, average serum level of PGE2 from the mice receiving 0.03 mg/kg exogenous PGE2 was the closest to that of the mice with tumor transplantation but treated with saline (Supplementary Fig. 1). Therefore, the highest dosage of PGE2 was selected as 0.03 mg/kg. 2.3. Blood and serum physiochemical parameters determination Blood samples were collected from the experimental mice before being sacrificed under diethyl ether anesthesia. The concentrations of red blood cells (RBC), hemoglobin (HGB), white blood cells (WBC), and platelets (PLD) were determined by an

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automatic blood cell counting apparatus (Mindray, Shenzhen, PR China). Serum samples were separated from the blood via centrifugation at 6500g for 8 min. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CRE) and uric acid (UA) in serum were measured by an automatic biochemical analyzer (Sysmex, Japan). 2.4. Flow cytometry analysis (FACS), purification of subsets of immune cells, and cell apoptosis detection CD4 þ T cell percentages were determined after the single-cell suspensions of spleens and lymph nodes were incubated with rat anti-mouse FITC-CD4 (1: 500 dilution according to the instructions of the manufacturer) from BD Biosciences (San Jose, California, USA) in 500 μL of serum free Dulbecco's Modified Eagle Medium (DMEM). The unbound antibodies were removed by washing the cells with serum free DMEM for three times. The samples were subsequently analyzed through flow cytometry (Becton Dickinson FACS Calibur, Franklin Lakes, NJ, USA). CD4 þ T, CD8 þ T and NK cells in the signal-cell suspensions were purified by a magneticactivated cell sorting (MACS) system using rat anti-mouse CD4, CD8, CD49b and goat anti-rat IgG microbeads (BD Biosciences). To isolate tumor associated macrophages, the transplanted tumors were dissected out, immediately chopped into small pieces, and incubated in the digestion mixture as described previously (Fujiwara et al., 2011) for 45 min at 37 1C. Cells were washed with PBS and re-suspended in DMEM containing 10% fetal calf serum (FCS, Invitrogene, Carlsbad, CA, USA). Cells detached from culture flasks were collected and sorted using CD11b magnetic beads purchased from Miltenyi Biotechnology (Auburn, CA, USA). The purity of each subset was over 95% as determined by FACS. The ratio of the apoptotic nucleus in the purified CD4 þ T cell population was determined using FACS as described in previous methods (Zhang et al., 2011). Briefly, purified CD4 þ T cells were fixed with cold 70% ethanol (stored at  20 1C) for at least 2 h on ice. The cells were then resuspended and washed twice with PBS with 20 mM of EDTA. Following the removal of intercellular RNA through incubation with RNaseA (1 mg/mL) at 37 1C for 1 h, the cells were stained with 30 μg/mL propidium iodine and then analyzed by FACS. Ratios of apoptotic cells were represented by their Sub-G1 DNA content percentages. 2.5. Caspase-3 activity assay Caspase-3 activity assay kits were obtained from Beyotime Biotechnology (Nantong, Jiangsu, PR China) and used to measure the activities of caspase-3 in purified CD4 þ T cells. 2.6. Enzyme-linked immunosorbent assay (ELISA) The concentrations of IFN-γ, IL-2, IL-4, and IL-10 in mouse serum were quantified using the corresponding ELISA kits as purchased from Jiancheng Biotechnology (Nanjing, Jiangsu, PR China). PGE2 levels in mouse serum and cyclic adenosine monophosphate (cAMP) levels in CD4 þ T cells were also determined using the corresponding ELISA kits as purchased from Cayman Chemical. 2.7. Cytotoxic activity determination CD8 þ T and NK cells were effector cells. Concentrations of purified NK and CD8 þ T cells were adjusted to 107/mL in DMEM containing 10% FCS. H22 cells were used as target cells and their concentration were adjusted to 105/mL in DMEM supplied with 10% FCS. To measure the cytotoxic activities of effector cells, 100 μL of effector cells were mixed with 100 μL of target cells thoroughly

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in 96-well round-bottom plates for a total volume of 200 μL per well (effector to target ratio is 50: 1). As a negative control, the target cells (100 μL) were incubated with 100 μL of DMEM supplied with 10% FCS without any effector cells. After 5-h incubation at 37 1C in 5% CO2 atmosphere, the cell-free supernatants were harvested and the cytotoxic activity of CD8 þ T and NK cells were quantified based on measuring the activity of lactate dehydrogenase released from damaged target cells using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, San Luis Obispo, California, USA). 2.8. RNA isolation, reverse transcription and quantitative real-time PCR Reverse-transcription and quantitative real-time PCR analysis were performed as described in previous methods (Tong et al., 2011). Briefly, total RNA was extracted from the purified CD4 þ T cells using the Trizol reagent (Invitrogen, California, USA). The integrity of the RNA was verified by agarose gel electrophoresis and ethidium bromide staining. After quantifying RNA concentration by measuring its optical density at 260 nm, 5 mg of the total RNA was reverse transcribed at 42 1C for 30 min by ReverTra Ace-α (Toyobo, Osaka, Japan) to provide cDNA. The transcription levels of Bcl-xL, Mcl-1, and COX-2 were analyzed through real-time PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. The primer pair sets were listed as follows (50 to 30 ). Bcl-xL: forward: GCT GGG ACA CTT TTG TGG AT; reverse: CTA GGC CCA ACC CTG TGA TA. Mcl-1 forward: AAA GGC GGC TGC ATA AGT C; reverse: TGG CGG TAT AGG TCG TCC TC. COX-2: forward: TTC CAA TCC ATG TCA AAA CCG T; reverse: TGC ACA TTG TAA GTA GGT GGA C. GAPDH: forward: GCA GTG GCA AAG TGG AGA TT; reverse: GGA GAC AAC CTG GTC CTC AG. 2.9. Immunoblotting The indicated subgroups of immune cells were lysed in a buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 5 μg/mL aprotinin, 2 μg/mL leupeptin, 50 mM NaF, 1 mM PMSF, and 1 mM Na3VO4. Concentrations of proteins were quantified using a commercial kit from Bio-Rad (Richmond, CA, USA). Protein samples (25 μg) were resolved on SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Amersham Biosciences). Membranes were blocked with TBST containing 5% bovine albumin (Sigma) for 1 h at room temperature and incubated with indicated antibodies (1 μg/mL) for 2 h. After cleansing in TBST for 30 min, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies for 45 min. Membranes were finally washed extensively with TBST and protein bands were examined by enhanced chemiluminescence (ECL Western Blotting Kit, Amersham). Antibodies against JAK3, STAT5, phosphorylated STAT5 (Tyr 694), Bcl-xL, Mcl1, COX-2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.10. Chromatin immunoprecipitation (ChIP) ChIP was performed as described in previous work (Shu et al., 2011) with polyclonal rabbit anti-STAT5 antibodies. Precipitated DNA (10% of total) was subsequently analyzed through real-time PCR using a primer specific to the promoter regions of the Bcl-xL coding gene in the mouse. The levels of input DNA were used as the internal control. The primer sequences used for detecting the precipitated DNA were as follows (50 to 30 ): mouse Bcl-xL promoter: forward: GTC GCC GGA GAT AGA TTT GA; reverse: AAG GTC TGA GTC CGG GTT CT.

2.11. Histopathological examinations At the end of our study, transplanted H22 tumors were dissected out, washed with ice-cold saline, fixed in buffered neutral 10% formalin and embedded in paraffin. Then, tumor samples were cut into 5-μm-thick sections and subjected to hematoxylin-eosin (HE) staining. Photographs were taken at 100  magnifications.

2.12. Statistical analysis Data shown in this paper are the representatives or statistics (mean value 7standard deviation) of the results from all the mice of each group. The data were subjected to the two-tailed Student's t test or one-way analysis of variance (ANOVA), followed by the Dunnett's t test for a comparison between the treated and saline control groups. A p value of o0.05 was considered to be statistically significant, and a p value of o0.01 was considered to be statistically very significant.

3. Results 3.1. PSSC suppressed H22 HCC cell growth in vivo As shown in Table 2 and Fig. 1A, the average tumor weight in the saline-receiving group was 1.46 70.2 g. In the PSSC (10, 30, 100 mg/kg) groups, the average tumor weights were determined as 1.17 70.2, 0.87 70.19, and 0.63 70.17 g, respectively. As compared with the negative control group, the average tumor weights from 30 and 100 mg/kg PSSC groups showed statistical significance. Although the cisplatin treatment attained a very high tumor inhibition rate (79.4%), the average body weight of the tumorbearing mice in this group was also dramatically decreased. In contrast, the PSSC treatment exhibited few effects on the body weights of the tumor-bearing mice. To compensate the decreased body weight with cisplatin, we expressed the tumor weight relative to the animal bodyweight. Cisplatin reduced the ratio of tumor weight to bodyweight by 72%. Treating tumor-bearing mice with 30 and 100 mg/kg PSSC gave rise to decreases in this ratio by 41 and 58%, respectively (Fig. 1B). Moreover, our histopathological examinations showed that in transplanted tumor tissues, PSSC resulted in cell vacuolization, nuclear shrinkage, decreased number of tumor cells in a unit area and attenuated signaling of cytoplasm staining (Fig. 1C). These data further confirmed the HCC-repressing activity of PSSC.

Table 2 The inhibitory effect of PSSC on the growth of transplanted H22 tumor cells in mice. Group

Non-tumor saline cisplatin PSSC PSSC PSSC a b

Dose Mice weight (g) (mg/ Start kg)

End

– – 5 10 30 100

26.4 70.9 26.9 70.6 19.6 71.1 b 27.4 70.9 27.5 70.8 27.8 71.1

21.0 7 0.6 21.7 7 0.5 21.3 7 0.8 21.4 7 0.8 21.5 7 0.5 21.2 7 0.8

a

Number of mice Start/ end

Tumor weight (g)

TIR (%)

10/10 10/10 10/8 10/10 10/10 10/10

– 1.46 7 0.20 0.30 70.05 b 1.177 0.20 0.87 70.19 b 0.63 70.17 b

– – 79.4 19.9 40.4 56.8

Tumor inhibitory rate. p o 0.01, compared to tumor-bearing mice treated with saline.

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Fig. 1. PSSC suppressed H22 HCC tumor growth in vivo. (A) Images of the tumor-bearing mice from the experimental groups. (B) At the end of PSSC treatment, transplanted tumors were dissected out and immediately weighted. Tumor weight was expressed relative to the animal bodyweight to compensate the decreased bodyweight with cisplatin. The pictorial illustration of the changes in tumor dimensions upon PSSC treatment is also shown. (C) Effects of PSSC on the histopathology data of transplanted H22 tumor tissues.

Table 3 Effect of PSSC on blood WBC, RBC, HGB and PLT of tumor-bearing mice. Group

Nontumor Saline Cisplatin PSSC PSSC PSSC a

Dose (mg/ kg)

– – 5 10 30 100

Concentration of blood cells

Group

WBC (109/L)

RBC (1012/ L)

HGB (g/L)

PLT (109/ L)

8.717 1.0

7.91 71.1

1097 7.6

6667 84

8.05 71.0 8.28 71.2 8.12 70.94 8.59 70.98 8.98 71.3

1127 9.3 987 12.3 1057 9.0 1027 7.7 1167 10.7

6757 86 681 7 77 636 7 75 650 7 86 688 7 85

10.05 7 1.2 5.85 7 0.87 10.26 7 1.0 10.177 0.90 11.497 1.1

a

Table 4 Effect of PSSC on serum hepatic function indexes of tumor-bearing mice.

p o 0.01, compared to tumor-bearing mice treated with saline.

3.2. The toxicological impacts of PSSC on tumor-bearing mice As shown in Table 3, the concentrations of WBC, HGB, and PLT were comparable among all of the experimental groups. Cisplatin injection was shown to dramatically decline the amount of WBC in the blood (from 8.71  109/L to 5.85  109/L on average), indicating a strong toxic impact on the immune system of the host. In contrast, this effect was undetected in the PSSC-receiving groups. The toxic effects on normal hepatocytes were determined by the serum hepatic function markers in all of the experimental groups. The results showed that after cisplatin treatment, serum activities of ALT and AST increased by l.6 and 1.3 folds, respectively, while

Non-tumor Saline Cisplatin PSSC PSSC PSSC a

Dose (mg/kg)

– – 5 10 30 100

Hepatic function indexes (U/L) ALT

AST

92.2 7 5.8 88.7 7 6.4 148.17 7.6 a 90.17 5.4 95.3 7 6.1 89.4 7 6.7

224 718 209 721 297 726 225 719 213 720 221 722

a

p o 0.01, compared to tumor-bearing mice treated with saline.

these two parameters remained largely unchanged in all of the PSSC groups (Table 4). The effects of PSSC on the kidneys of mice were examined through serum renal function markers, including BUN, UA, and CRE. As shown in Table 5, cisplatin resulted in more than 2-fold increases in the levels of serum BUN and CRE. However, the effects of PSSC on these parameters were insubstantial. These data verified very low toxic effects on tumorbearing mice from PSSC as compared to cisplatin. 3.3. PSSC alleviated tumor transplantation-induced CD4 þ T cell apoptosis and associated antitumor immune suppression As PSSC exhibited few cytotoxic effects against HCC cells in vitro (Supplementary Fig. 2), it was supposed that the stimulation of an

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antitumor immune response in the host contributed to the in vivo antitumor activity of PSSC. As shown in Fig. 2A, the ratio of CD4 þ T cells declined significantly (by 52% in spleens and 38% in lymph nodes) after tumor transplantation. PSSC resulted in substantial elevations of CD4 þ T cell percentages in both spleens (by 0.54and 1.3-fold in 30 and 100 mg/kg group, respectively) and lymph nodes (by 0.39- and 0.67-fold in 30 and 100 mg/kg group, respectively). FACS revealed that in spleens of tumor-bearing mice, the percentage of apoptotic CD4 þ T cells was reduced to 12.5, 8.3, and 3.8% in 10, 30, and 100 mg/kg group; apoptotic CD4 þ T cells in lymph nodes decreased to 14.5 and 9.1% in 30 and 100 mg/kg group, respectively (Fig. 2B). Consistently, caspase-3 activity assay

Table 5 Effect of PSSC on renal function indexes of tumor-bearing mice. Group

Non-tumor Saline Cisplatin PSSC PSSC PSSC a

Dose (mg/kg)

– – 5 10 30 100

Renal function indexes (U/L) BUN

UA

CRE

7.84 7 1.0 6.95 7 0.87 17.17 1.59 a 7.55 7 0.98 8.107 0.91 7.197 0.88

197 712 205 710 211 715 206 711 188 717 201 713

59.9 7 7.6 61.3 7 8.2 144.17 9.3a 55.7 7 8.3 58.6 7 8.8 57.8 7 9.1

p o 0.01, compared to tumor-bearing mice treated with saline.

unveiled that in 30 and 100 mg/kg PSSC group, activity of caspase3 in CD4 þ T cells was reduced by 60 and 81% in spleens, and 57 and 80% in lymph nodes (Fig. 2C). H22 cell transplantation remarkably decreased the cytotoxic activities of both NK and CD8 þ T cells. PSSC treatment dramatically elevated their cytotoxic activities. As shown in Fig. 2D, treating tumor-bearing mice with 10, 30, and 100 mg/kg PSSC led to increases in cytotoxic activities of NK cells against H22 HCC cells of 0.50-, 1.0- and 1.8-fold, and increases in those of CD8 þ T cells of 0.44, 0.73 and 1.4-fold, respectively. The effects of PSSC on the profile of serum cytokines were also evaluated. As shown in the upper five rows of Table 6, transplanting H22 cells decreased serum concentrations of IFN-γ (from 21.8 to 9.2 pg/mL) and IL-2 (from 25 to 12 pg/mL) and elevated those of IL-4 (from 80 to 146 pg/mL) and IL-10 (from 13.7 to 38.6 pg/mL). After treating tumor-bearing mice with PSSC at the dosages of 10, 30, and 100 mg/kg, serum levels of IFN-γ increased to 14.7, 17.6 and 22.1 pg/ mL; those of IL-2 increased to 16, 19 and 26 pg/mL, respectively. Serum levels of IL-4 displayed 19, 37, and 49% reductions, and those of IL-10 showed 18, 47 and 61% reductions, respectively. 3.4. PSSC Stimulates antitumor immune response by repressing PGE2 production PGE2 is an important factor that mediates antitumor immune tolerance. The effects of PSSC on serum concentrations of PGE2 were thus evaluated. It was revealed that tumor transplantation

Fig. 2. PSSC stimulated an anti-tumor immune response in tumor-bearing mice. (A) After the mice were treated as indicated, ratios of CD4þ T cells in their spleens and lymph nodes were measured by FACS. (B and C) CD4þ T cells were then purified from spleens and lymph nodes. Apoptotic CD4 þ T cell were quantified by FACS (B) and caspase-3 activity assay (C). (D) Next, NK and CD8 þ T cells were purified from the spleens of the animals. Their cytotoxic activities against H22 cells mouse were determined. n p o 0.05, nnp o0.01, as compared to the saline-receiving group.

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Table 6 Effect of PSSC on serum levels of IFN-γ, IL-2, IL-4, and IL-10. Group

Non-tumor Saline PSSC PSSC PSSC PSSC þPGE2 PSSC þPGE2 a

Dose (mg/kg) Serum concentration of cytokines (pg/mL)

– – 10 30 100 100 þ0.01 100 þ0.03

IFN-γ

IL-2

IL-4

IL-10

21.8 7 3.1 9.2 7 1.9 a 14.7 7 2.7 b 17.6 7 2.5 c 22.17 2.9 c 14.3 7 2.4 d 9.6 7 2.1 d

257 2.0 807 8.1 13.7 71.9 127 3.2 a 1467 17 a 38.6 74.2 a b 167 2.6 1187 15 31.6 73.7 b 197 1.9 b 927 12 c 20.3 72.6 c 267 2.8 c 747 9.5 c 14.9 72.1 c 187 2.1 e 957 10 e 25.2 72.8 117 1.7 d 1377 13 d 34.7 73.1 d

p o 0.01, compared to non-tumor mice. p o0.05, compared to tumor-bearing mice treated with saline. po 0.01, compared to tumor-bearing mice treated with saline. d po 0.01, compared to tumor-bearing mice treated with 100 mg/kg PSSC. e p o 0.05, compared to tumor-bearing mice treated with 100 mg/kg PSSC. b c

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gave rise to a sharp increase in the serum levels of PGE2 (from 30.7 to 125.5 pg/mL), which was dose-dependently alleviated by PSSC (Fig. 3A, decreased to 90.4, 52.9 and 32.7 pg/mL in 10, 30, and 100 mg/kg PSSC group, respectively). To understand the role of suppressed PGE2 production in the antitumor immunostimulatory activity of PSSC, the tumor-bearing mice were concurrently treated with PSSC and different dosages of exogenous PGE2. It was found that exogenous PGE2 dose-dependently reduced the tumorinhibitory impacts of PSSC (Fig. 3B, average tumor weight increased to 1.06 and 1.48 g after co-treating the mice with 0.01 and 0.03 mg/kg PGE2, respectively). PGE2 also impaired the protective effects of PSSC on CD4 þ T cells against tumor transplantation-induced apoptosis. As shown in Fig. 3C, after cotreating the mice with 0.01 and 0.03 mg/kg PGE2, the percentage of CD4 þ T cells in spleens decreased to 20.1 and 10.1%, and those in lymph nodes decreased to 28.9 and 18.1%. The apoptotic ratio of CD4 þ T cells in spleens increased to 10.5 and 17.1% and 17.0 and

Fig. 3. The effects of PGE2 on the anti-tumor immunostimulatory activity of PSSC. (A) After the animals were treated as indicated, the serum levels of PGE2 were quantified by ELISA. (B) After the indicated treatment, transplanted tumors were dissected out and immediately weighted (left). Images of tumor-bearing mice are also shown (right). (C) After the indicated treatment, spleens and lymph nodes were dissected out. Their single-cell suspensions were prepared. Ratios of CD4þ T cells in the single-cell suspensions were measured by FACS (D and E). Next, CD4 þ T cells were purified from the spleens and lymph nodes. Apoptotic CD4 þ T cell were quantified by both FACS (D) and a caspase-3 activity assay (E). (F) NK and CD8 þ T cells were then purified from the spleens of the animals. Their cytotoxic activities against H22 cells were quantified. n p o 0.05, nnp o 0.01, as compared to saline treatment (A) or mice treated with PSSC alone (B–F).

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23.2% in lymph nodes, respectively (Fig. 3D). Consistently, in spleen CD4 þ T cells, caspase-3 activity was elevated by 0.95and 3.4-fold; in lymph node CD4 þ T cells, caspase-3 activity was elevated by 1.2- and 3.6-fold (Fig. 3E). PSSC-induced elevation of the cytotoxic activities of NK and CD8 þ T cells against H22 HCC cells were also significantly attenuated by PGE2. After co-treating tumor-bearing mice with 0.01 and 0.03 mg/kg PGE2, cytotoxic activities of NK cells against H22 HCC cells showed 33 and 66% reductions; those of CD8 þ T cells showed 26 and 50% reductions, respectively (Fig. 3F). PSSC-medicated increases of Th1 cytokines and declines of Th2 cytokines were consistently undermined by PGE2. As shown in the lower 3 rows of Table 6, after co-treating tumor-bearing mice with PGE2 at the dosages of 0.01 and 0.03 mg/kg, serum levels of IFN-γ dropped to 14.3 and 9.6 pg/mL; those of IL-2 dropped to 18 and 11 pg/mL. In contrast, serum levels of IL-4 were elevated to 95 and 137 pg/mL and those of IL-10 were elevated to 25.2 and 34.7 pg/ mL, respectively. These data suggested that reducing PGE2 production is critical in mediating the antitumor immunostimulatory activity of PSSC. 3.5. PSSC activates JAK3 expression and JAK3/STAT5 signaling in CD4 þ T cells The effects of transplanting H22 cells and PSSC administration on cAMP/JAK3 pathway in CD4 þ T cells were evaluated. As shown in Fig. 4A, H22 tumor transplantation resulted in a 4.8-fold increase in the level of intracellular cAMP in CD4 þ T cells, which was reduced by 51 and 75% after treating the mice with 30 and 100 mg/kg PSSC, respectively. Protein levels of JAK3 and phosphorylated STAT5 were also reduced after tumor transplantation, which was dose-dependently ameliorated by PSSC (Fig. 4B). Bcl-xL and Mcl-1 are two conventional STAT5 responsive genes inhibiting cell apoptosis (Nam et al., 2012). Their protein levels in CD4 þ T cells dramatically declined following the transplantation of H22 cells but rose again after PSSC treatment (Fig. 4B). Consistently, as shown in Fig. 4C, mRNA level of Bcl-xL was elevated by 0.89- and 1.6-folds in 30 and 100 mg/kg PSSC group, respectively, and those of Mcl-1 was elevated by 0.59- and 1.3-fold. ChIP assay showed that transplanting H22 cells mitigated the association between STAT5 protein and Bcl-xL promoter, which was rescued by PSSC (Fig. 4D, increased by 0.5-, 1.4- and 2.2-fold in 10, 30, and 100 mg/ kg group as compared to the tumor-bearing mice treated with normal saline, respectively). 3.6. PSSC suppresses COX-2 transcription in tumor associated macrophages (TAMs) COX-2 is the key enzyme that mediates the biosynthesis of PGE2 from arachidonic acid. In tumor-bearing mice treated with saline, the transcription levels of COX-2 in the homogenized tumor tissues (HTT) showed only a marginal elevation as compared to those in the mouse spleens, lymph nodes, and livers. However, COX-2 mRNA levels in TAMs were significantly higher (more than 15 fold as compared to those in HTT, Fig. 5A), implying a possibility that TAMs were the major PGE2 source contributing to the steep increase in serum PGE2 after tumor transplantation. To explore possible molecular mechanisms underlying PSSC-mediated inhibition of serum PGE2, the effects of PSSC on COX-2 expression in TAMs were assessed. After treating the mice with 10, 30, and 100 mg/kg PSSC, mRNA level of COX-2 in TAMs displayed reductions of 45, 68 and 86%, respectively (Fig. 5B). In line with this observation, COX-2 proteins in TAMs were also substantially downregulated by PSSC (Fig. 5C). These findings suggested that inhibiting COX-2 expression in TAMs is implicated in PSSC-induced reduction of serum PGE2 in tumor-bearing mice.

Fig. 4. PSSC upregulated JAK3 expression and JAK3/STAT5 signaling cascade in the CD4þ T cells. (A) After the animals were treated as indicated, the relative cAMP levels in the spleen CD4þ T cells were determined. (B) Protein levels of JAK3, phosphorylated STAT5, Bcl-xL, Mcl-1, and STAT5 were detected by immunoblotting using protein levels of β-actin as the loading control. (C) The transcription levels of Bcl-xL and Mcl-1 in the spleen CD4 þ T cells from tumor-bearing mice were quantified by reverse transcription real-time PCR. (D) CD4 þ T cells were purified from the mouse spleens and subjected to a ChIP assay using the antibodies against STAT5. The precipitated DNA was analyzed through real-time PCR using primers specific to the Bcl-xL promoter. np o 0.05, nnp o0.01, as compared to saline treatment.

4. Discussion S. chinensis is a traditional herb widely utilized in the treatment of malignant diseases such as HCC. Polysaccharides were contained abundantly in this plant. However, extents to which PSSC can stimulate anti-HCC immune response in vivo remains uncertain. In this study, intraperitoneal injections of PSSC dramatically repressed the in vivo growth of transplanted H22 HCC cells. Moreover, the tumor inhibitory rates of PSSC were positively associated with PSSC dosages. These data clearly established the in vivo HCC-suppressing capacity of PSSC and indicated that PSSC is an important contributor to the antitumor activity of S. chinensis.

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Fig. 5. PSSC inhibited COX-2 expression in TAMs. (A) Indicated tissues of tumorbearing mice treated with only saline were dissected out immediately after the sacrifice of the animals. The transcription levels of COX-2 in these tissues were quantified by transcription real-time PCR. The COX-2 mRNA levels in different tissues were normalized to those of homogenized tumor tissues (HTT). (B and C) Next, TAMs were prepared. The mRNA and protein levels of COX-2 in TAMs were then detected using reverse transcription and real-time PCR (B) and immunoblotting (C). *po 0.05, **p o 0.01, as compared to saline treatment.

Cisplatin is a conventional chemotherapeutic drug approved for HCC treatment. In this work, cisplatin showed stronger tumorsuppressing activity than PSSC. However, it also led to various severe toxic effects on the tumor-bearing animals. At the end of the 10-day treatment, 2 out of 10 mice died in the cisplatin group. In addition, the body weights of the remaining mice substantially decreased. In contrast, mice deaths did not occur in the PSSCreceiving groups and no significant decline was observed in the body weights of the mice. Furthermore, it has previously been reported that cisplatin has the potential to exhibit severe toxic effects on the liver and kidney of patients (Cayir et al., 2011). Similarly, our work observed that cisplatin injection resulted in considerable increases in the serum levels of ALT, AST, BUN and CRE, indicating its substantial hepatotoxicity and nephrotoxicity. In contrast, the PSSC treatment provided no adverse effects for these parameters, indicating that PSSC was safe for the liver and kidneys of the host. These data collectively suggested that toxic effects of PSSC on tumor-bearing animals were undetectable. In mammals, CD4 þ T cells play a significant role in the moderation of antitumor immune defense. Normally, the amount

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of CD4 þ T cells is maintained by homeostasis with highly orchestrated molecular mechanisms that control their proliferation and apoptosis (Marrack and Kappler, 2004). To evade the antitumor immune response of the host, malignant cells are capable of inducing CD4 þ T cell apoptosis (Biswas et al., 2006; Finke et al., 1999). Our previous findings revealed that transplanting H22 HCC cells elevated CD4 þ T cell apoptosis and resulted in a substantial decrease in the percentage of CD4 þ T cells in tumorbearing mice (Shu et al., 2013). The effects of PSSC on CD4 þ T cell homeostasis were thus evaluated. We found that PSSC alleviated tumor transplantation-induced CD4 þ T cell apoptosis. Moreover, the transplantation of H22 cells led to decreases in the serum levels of Th1 cytokines, including IFN-γ and IL-2. Meanwhile, the serum levels of IL-4 and IL-10, two representative Th2 cytokines, were elevated. PSSC revived the dysregulated cytokine profile of the tumor-bearing mice. CD4 þ T cells were capable of stimulating cytotoxic activity in NK and CD8 þ cytotoxic T cells against cancer cells. Therefore, the effects of PSSC on the cytotoxic activities of NK and CD8 þ T cells against H22 cells were tested. Our findings showed that PSSC is able to augment the cytotoxic activity of both the NK and CD8 þ T cells against H22 cells. These data collectively demonstrated that PSSC has the capacity of alleviating systematic immune suppression induced by tumor transplantation, which is closely associated with in vivo anti-tumor activity of PSSC. PGE2 has been implicated as a potential inhibitor of T cell function in the context of malignant diseases and a major contributor to crippled anti-tumor immune function (Pockaj et al., 2004; Sharma et al., 2005). To understand whether PGE2 pathway was involved in CD4 þ T cell apoptosis induced by H22 transplantation, serum levels of PGE2 in response to tumor transplantation and PSSC treatment were determined. Our data showed that serum levels of PGE2 were dramatically increased after the transplantation of H22 HCC cells. PSSC was shown to decrease the serum concentration of PGE2 to its basal level. These data indicated that overproduced PGE2 is implicated in tumorinduced CD4 þ T cell apoptosis in the host and inhibiting PGE2 biosynthesis plays critical roles in the anti-HCC immunostimulatory activity of PSSC. This point is supported by our finding that concurrent treatment of tumor-bearing mice with exogenous PGE2 abrogated immunostimulatory effects of PSSC. Previous studies reported that PGE2 inhibited T cell proliferation by increasing intracellular cAMP (Kolenko et al., 1999). This event in turn reduces the expression of JAK3 which catalyzes the tyrosine phosphorylation of STAT5 and activates the anti-apoptotic signal cascade downstream of STAT5 (Chattopadhyay et al., 2009; Rani et al., 2011). Our findings unveiled that in CD4 þ T cells, PSSC decreased intracellular cAMP and elevated JAK3/STAT5 antiapoptotic signaling which are downstream of PGE2. These data collectively unveiled that transplanting H22 cells leads to an excess of serum PGE2, which elevates intracellular cAMP and impaired JAK3/STAT5 anti-apoptotic pathway in CD4 þ T cells. Suppressing PGE2 production which reactivated JAK3/STAT5 signaling is implicated in the protective properties of PSSC on CD4 þ T cells against tumor transplantation-induced apoptosis. TAMs are an important component of the HCC microenvironment, participating in both the mediation of HCC immune tolerance and the promotion of HCC progression (Shirabe et al., 2012). PSSC treatment was shown to result in substantial decreases in both the mRNA and protein levels of COX-2 in TAMs. The possibility that PSSC also inhibits COX-2 expression in other tissues cannot be disregarded. Nevertheless, the transcription levels of COX-2 in TAMs were much higher than those in the other examined tissues, including homogenized transplanted tumors. These data implied that TAMs are the major source of overproduced PGE2, significantly contributing to tumor immunosuppression in the mice transplanted with H22 cells. It is thus

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reasonable to hypothesize that a reduction of COX-2 expression in TAMs is influential in the declined PGE2 production mediated by PSSC. The abnormal arachidonic acid metabolic pathway accounting for tumor immunosuppression has also been observed in TAMs in a renal cancer microenvironment (Daurkin et al., 2011). These findings suggest that therapeutic approaches directed at arachidonic acid metabolism in TAMs provide a novel strategy through which tumor immunosuppression can be counteracted.

5. Conclusion In this work, we unveiled that PSSC was able to stimulate an anti-tumor immune response in mice transplanted with H22 HCC cells with low toxic effects on the hosts. Suppressing tumor transplantation-induced PGE2 production was the key event mediating the anti-tumor immunostimulatory activity of PSSC. PSSC activates JAK3/STAT5 anti-apoptotic pathway in CD4 þ T cells. Moreover, the downregulation of COX-2 expression in TAMs was implicated in the declined PGE2 production induced by PSSC in tumor-bearing mice. These data provide novel insights into the traditional application of the herb S. chinensis against HCC, supporting for the use of PSSC as a potential adjuvant reagent in the stimulation of an anticancer immune response in clinical HCC treatment.

Declaration of interest The authors declare no conflict of interest.

Acknowledgments This work is supported by grants from the Natural Science Foundation of China (31301147 and 31200264), Innovation and Entrepreneurship Training Program Funded by South Central University for Nationalities (GCX15008) and "Chenguang Planning" from the Natural Science Foundation of Wuhan City (2013070104010029 and 2014070404010210).

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