# Herbal Compound “Songyou Yin” Renders Hepatocellular Carcinoma Sensitive to Oxaliplatin through Inhibition of Stemness

Authors: Qing-An Jia; Zheng-Gang Ren; Yang Bu; Zhi-Ming Wang; Qiang-Bo Zhang; Lei Liang; Xue-Mei Jiang; Quan-Bao Zhang; Zhao-You Tang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2012)

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## Abstract

We investigated the effect of Chinese herbal compound Song-you Yin on HCC stemness. MHCC97H and Hep3B cell lines were pretreated with SYY for 4 weeks, and their chemosensitivity to oxaliplatin was evaluated. The expression of CSC-related markers, cell invasion and migration, and colony formation were also examined. SYY-treated orthotopic nude mouse models of human HCC were developed to explore the effect of oxaliplatin on tumor growth, metastasis, and survival. The CSC-related molecular changesin vivo were also evaluated. The result showed that MHCC97H and Hep3B cells pretreated with SYY showed significantly increased chemosensitivity to oxaliplatin and the downregulation of CSC-related markers CD90, CD24, and EPCAM. SYY also attenuated cell motility, invasion, and colony formation in MHCC97H and Hep3B cell lines. The reduced tumorigenicity and pulmonary metastasis were observed in SYY-pretreated cell lines. Combination treatment with oxaliplatin and SYY significantly reduced tumor volume and pulmonary metastasis and prolonged survival compared with oxaliplatin treatment alone. Immunohistochemical analysis showed reduced expression of CD90, ABCG2, ALDH, CD44, EPCAM, vimentin, and MMP-9 and increased the expression of E-cadherin, in HCC cells following combination treatment. These data clearly demonstrate that SYY renders hepatocellular carcinoma sensitive to oxaliplatin through the inhibition of stemness.


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## Body

## 1. Introduction

Liver cancer, most commonly hepatocellular carcinoma (HCC), is the fifth most frequently diagnosed cancer in men worldwide, but the second most frequent cause of cancer death [1]. In clinical practice, fewer than 30% of patients with HCC have the chance to be treated with curative options such as liver transplantation, surgical resection, and ablation therapy because HCC is typically confirmed at an advanced stage at diagnosis [2]. As a result, transcatheter hepatic arterial chemoembolization (TACE) and systemic chemotherapy are frequently used [3, 4] although unfortunately the overall response rate to such treatments is poor [5, 6].Recently, biological deterioration of tumor cells after chemotherapy has been reported.In vitro exposure to chemotherapeutic agents enhanced metastatic potential in colorectal, pancreatic, breast, and ovarian carcinoma cells [7–10]. Yamauchi et al. similarly reported the so-called “opposite effect,” an increased metastatic ability in cyclophosphamide-pretreated fibrosarcoma cells [11]. Recent evidence suggests that a certain type of HCC is hierarchically organized by a wide variety of cancer cells including a subset of cells with stem cell features [12–15]. These cancer stem cells (CSCs) are resistant to conventional chemotherapy due to cellular characteristics such as high expression of drug transporters, relative cell cycle quiescence, high levels of DNA repair, and resistance to apoptosis [16, 17]. Costello et al. found that CD34+CD38− CSCs in AML patients exhibited decreased daunorubicin sensitivity compared with CD34+CD38+ cells, which correlated with high expression levels of the drug resistance-related genes LRP and MRP [18]. Similarly, Liu et al. reported that CD133+ glioblastoma cells exhibited less cell death than their CD133− counterparts when treated with multiple chemotherapeutic agents as a result of overexpression of genes that inhibit apoptosis, including FLIP, Bcl-2, and Bcl-XL [19]. The emergence of the CSC theory provides insight into why treatment of tumors with chemotherapy often appears to show an initial response but ultimately results in treatment failure.Our previous study demonstrated that Songyou Yin (SYY, a traditional Chinese medicine containing five herbal compounds) inhibits molecular changes consistent with the epithelial-mesenchymal transition (EMT) in oxaliplatin-treated tumor tissues and cell lines [20]. There is accumulating evidence that the EMT and CSCs form a coalition against cancer therapy [21–24]. Thus, the objective of this study was to investigate whether SYY directly downregulates the proportion of CSCs and inhibits stemness of HCC cells in tumor tissues and HCC cell lines, thus resulting in the sensitization of HCC to oxaliplatin. To date, there is no apparent consensus on the best marker by which to identify CSCs in any particular cancer. Widely used CSC-related markers include CD133, CD90, CD44, CD24, OV6, EPCAM, and staining of side population cells by Hoechst dye [25]. However, defined expression patterns of CSC markers in specific cell lines remain controversial. To select suitable cell lines to explore the effect of SYY on HCC, we first examined the expression of CSC-related markers in a variety of HCC cell lines with high and low metastatic potential. We next investigated changes in CSC proportion and stemness characteristics such as invasion, motility, colony formation, tumorigenesis, and pulmonary metastasis in SYY-treated cell lines and evaluated their changes in chemosensitivity which were also reconfirmed in vivo.


## 2. Materials and Methods

### 2.1. Cell Lines and Animals

The human HCC cell lines with high metastatic potential used in this study were MHCC97H and HCCLM3, which originated from MHCC97 and were established in the authors’ institution [26, 27]. The human HCC cell lines with low metastatic potential were SMMC-7721 (established at second military medical university) and Huh7, Hep3B, and HepG2 (obtained from American Type Culture Collection). MHCC97H and Hep3B cells cultured with 2 mg/mL SYY for 4 weeks were termed MHCC97H-SYY and Hep3B-SYY, respectively. The MHCC97H-RFP (red fluorescent protein) cell line established in the authors’ institute [28] was also cultured with 2 mg/mL SYY (MHCC97H-RFP-SYY). Male BALB/c nu/nu mice (aged 4–6 weeks and weighing approximately 20 g) were obtained from the Chinese Academy of Science and maintained under standard pathogen-free conditions. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Commission.


### 2.2. Regents and Antibodies

Oxaliplatin was purchased from Sigma Chemical Co. Monoclonal antibodies used in flow cytometric analysis were mouse anti-human monoclonal antibodies CD90-PE, EPCAM-APC, CD24-FITC, CD133-APC, CD44-PE, IgG-PE isotype, IgG-APC isotype, and IgG-FITC isotype (all purchased from Miltenyi Biotec). Antibodies used for immunofluorescence, immunoblotting and/or, immunohistochemistry were as follows: mouse anti-human monoclonal CD90 (Abcam), rabbit anti-human polyclonal CD133 (Abnova), mouse anti-human monoclonal EPCAM (Millipore), mouse anti-human monoclonal CD44 (Cell Signaling Technologies), rabbit anti-human polyclonal CD24 (Epitomics), mouse anti-human monoclonal MMP-9 (Abcam), mouse anti-human monoclonal E-cadherin (Abcam), mouse anti-human monoclonal Vimentin (Abcam), mouse anti-human monoclonal actin (Beyotime), mouse anti-human monoclonal ABCG2(Millipore), and rabbit anti-human monoclonal ALDH1(ABGENT).


### 2.3. Characterization and Preparation of Herbal Extracts

The Chinese herbal medicine formula SYY, a dietary component authorized by the Chinese State Food and Drug Administration (Grant no. G20070160), includes five Chinese medicinal herbal extracts whose proportions, fingerprint, and protocol of preparation have previously been reported [29]. The SYY used in vitro and in vivo in this study was from the same batch number 20110401 and was produced by Shanghai Fang Xin Pharmaceutical Technology Co., Ltd. Shanghai, China. A 800 mg/mL solution of SYY was sterilized twice by 0.22-μm filtration (Millipore) for further use in vitro.


### 2.4. LDH Cytotoxicity Assay

The cytotoxicity detection kitplus (Roche) is a precise and fast colorimetric assay for the quantitative determination of cytotoxicity by measuring release of lactate dehydrogenase (LDH) activity from damaged cells. MHCC97H cells were cultured to 80% confluence. After trypsin digestion, the cells were counted and pipetted into 96-well plates at a density of 2000 cells/well. Background control (medium only), low controls (spontaneous LDH release), high controls (maximum LDH release), and experimental substance (2 mg/mL and 4 mg/mL SYY) were prepared on the same plate according to the manufacturer’s instructions. The 96-well plates were incubated in a humidified incubator at 37°C in 5% CO2 for 4, 8, 12, 24, 48, and 72 h. Results were expressed as the absorbance of each well at 492 nm (OD492). Cytotoxicity (%) was calculated using the equation: (experimental value − low control)/(high control − low control) × 100%.


### 2.5. Flow Cytometric Analysis

The expression level of CSC-related markers was determined by flow cytometry. Briefly, MHCC97H, HCCLM3, SMMC-7721, Hep3B, Huh7, and HepG2 cells were grown to 80% confluence. After trypsin digestion, the cells were resuspended in medium at a concentration of 1 × 106 cells/mL and incubated with primary antibodies against CD90, CD133, CD24, EPCAM, and CD44 (diluted 1 : 11) at 4°C for 15 min. After washing three times with PBS, the cells were analyzed using a FACSC Flow Cytometer (BD Biosciences). The biomarkers CD90/EPCAM in MHCC97H-SYY and CD24/EPCAM in Hep3B-SYY were also examined for comparison with their parental cell lines.


### 2.6. Immunofluorescence and Western Blot Analysis

The expression of CSC-related markers was also determined by immunofluorescence. Cells were grown on glass cover slips to 40%–50% confluence and then fixed, permeabilized, blocked, and incubated with primary monoclonal antibodies overnight at 4°C. Slides were washed and incubated with anti-mouse or anti-rabbit Cy3-conjugated secondary antibody (Jackson). Cells were counterstained with 4′-6-diamidino-2-phenylindole to visualize cell nuclei and detected by fluorescence microscopy (Olympus).Western blot analysis of protein expression of CD90, CD24, EPCAM, E-cadherin, and vimentin in MHCC97H-SYY, Hep3B-SYY, and their parental cell lines was performed according to the manufacturer’s instructions. The concentration of protein extracted from MHCC97H-SYY, Hep3B-SYY, and parental cell lines was determined using the BCA Protein Assay Kit (Beyotime).


### 2.7. Quantitative Real-Time PCR Analysis

Total RNA was extracted from MHCC97H-SYY, Hep3B-SYY, and their parental cell lines using Trizol Reagent (Invitrogen). Total RNA was reversely transcribed using a Prime Script RT reagents kit (TaKaRa). Messenger RNA expression was determined by real-time PCR using SYBR Premix Ex Taq II (TaKaRa). The primers used for the amplification of human genes were as follows: CD90 forward primer 5′-CAGCATTCTCAGCCACAA C-3′ and reverse primer 5′-TTACCTCCT TCTCCAACCCT-3′; CD24 forward primer 5′-TCGGGTGTGCTATGGATG-3′ and reverse primer 5′-AGA GGG GGTCTGTTGAAG AT-3′; EPCAM forward primer 5′-CAGTGTACTTCAGTTGGTG-3′ and reverse primer 5′-TCAGGTTTTGCTCTTC TC-3′.


### 2.8. Cell Migration and Invasion Assays

Cell migration and invasion of MHCC97H and Hep3B cell lines were assessed by transwell assays (Boyden chambers; Corning). Briefly, 6 × 104 cells in serum-free Dulbecco’s modified Eagle medium (DMEM) were seeded into the upper chamber of each well of 24-well plates containing 8.0-μm pore size membranes. DMEM containing 10% fetal bovine serum (FBS) was added to the lower chamber of each well. After 48 h, cells that had reached the underside of the membrane were stained with Giemsa (Sigma), counted at ×100, and photographed at ×200 magnification. The cell invasion assay was carried out similarly, except that 80 μL matrigel (BD Biosciences) was added to each well 6 h before cells were seeded on the membrane.


### 2.9. Cell Proliferation and Colony Formation Assay

MHCC97H and Hep3B cell lines were pretreated with SYY for 4 weeks and then plated in 96-well plates (3 × 103 cells/well) and exposed to oxaliplatin at increasing concentrations for 24, 48, 72, and 96 h. Cell proliferation assays were carried out with the Cell Counting Kit 8 (CCK8; Dojindo). Results were expressed as the absorbance of each well at 450 nm (OD450).For colony formation assays, 1 × 103 cells were plated in 6-well plates (Corning) and cultured with 1% FBS DMEM with or without SYY (2 mg/mL). Culture medium was replaced every 3 days, and the colonies were fixed with ice-cold 4% paraformaldehyde 14 days after the initiation of treatment. Cells were stained with Giemsa (Sigma) and photographed at ×5 magnification.


### 2.10. Animal Model and Treatment Procedures

Twenty-four nude mice bearing orthotopic xenografts were randomly divided into control group, SYY group, oxaliplatin+SYY group, and oxaliplatin only group. Seven days after orthotopic implantation the mice were treated as follows: SYY group mice were treated intraperitoneally (i.p.) with 0.1 mL 5% glucose solution (GS) once a week and orally with SYY (4 g/kg/d) every day; control group mice were treated with 5% GS and distilled water in the same way as the SYY group; oxaliplatin group mice were treated with oxaliplatin (10 mg/kg) i.p. and 0.1 mL distilled water orally; oxaliplatin+SYY group mice were treated with 0.1 mL oxaliplatin (10 mg/kg) i.p. and 0.1 mL SYY (4 g/kg/d) orally. Tumor weight and lung metastasis were evaluated 4 weeks after the initiation of treatment. Using another 12 nude mice bearing orthotopic xenografts, survival time of an oxaliplatin+SYY group and oxaliplatin only group was determined as the interval between the day of inoculation and the day of death.To evaluate the growth and metastasis potential of SYY-treated HCC cellsin vivo, another 24 male BALB/c nu/nu mice were divided into four groups. Group I mice were subcutaneously injected with 5 × 106 MHCC97H-SYY cells and group II mice were injected with the same number of MHCC97H cells. Tumor weight was measured 4 weeks after injection. Group III mice were injected with 1 × 105 MHCC97H-RFP-SYY cells through the tail vein, and group IV mice were injected with the same number of MHCC97H-RFP cells. Six weeks later, lung metastases were evaluated by fluorescence microscopy and confirmed by microscopic examination of serial sections of every lung tissue block.


### 2.11. Immunohistochemistry

Tumor tissue was fixed, embedded, and sliced into 5μm thick sections. Immunohistochemical staining of CD90, ABCG2, ALDH, CD44, EPCAM, E-cadherin, vimentin, and MMP-9 was carried out using a standard protocol [30].


### 2.12. Statistical Analysis

In vitro LDH cytotoxicity assay data, the proportion of CSC cells, cell migration, invasion, and proliferation assays were compared by Student’s t-test. Tumor weight was compared by analysis of variance (ANOVA), the lung metastasis assay was analyzed using Fisher’s exact test, and survival was compared with Kaplan-Meier method with a log-rank test. Statistical analysis was performed with SPSS 15.0 for Windows (SPSS Inc. Chicago, IL, USA). P<0.05 was considered statistically significant.


## 2.1. Cell Lines and Animals

The human HCC cell lines with high metastatic potential used in this study were MHCC97H and HCCLM3, which originated from MHCC97 and were established in the authors’ institution [26, 27]. The human HCC cell lines with low metastatic potential were SMMC-7721 (established at second military medical university) and Huh7, Hep3B, and HepG2 (obtained from American Type Culture Collection). MHCC97H and Hep3B cells cultured with 2 mg/mL SYY for 4 weeks were termed MHCC97H-SYY and Hep3B-SYY, respectively. The MHCC97H-RFP (red fluorescent protein) cell line established in the authors’ institute [28] was also cultured with 2 mg/mL SYY (MHCC97H-RFP-SYY). Male BALB/c nu/nu mice (aged 4–6 weeks and weighing approximately 20 g) were obtained from the Chinese Academy of Science and maintained under standard pathogen-free conditions. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Commission.


## 2.2. Regents and Antibodies

Oxaliplatin was purchased from Sigma Chemical Co. Monoclonal antibodies used in flow cytometric analysis were mouse anti-human monoclonal antibodies CD90-PE, EPCAM-APC, CD24-FITC, CD133-APC, CD44-PE, IgG-PE isotype, IgG-APC isotype, and IgG-FITC isotype (all purchased from Miltenyi Biotec). Antibodies used for immunofluorescence, immunoblotting and/or, immunohistochemistry were as follows: mouse anti-human monoclonal CD90 (Abcam), rabbit anti-human polyclonal CD133 (Abnova), mouse anti-human monoclonal EPCAM (Millipore), mouse anti-human monoclonal CD44 (Cell Signaling Technologies), rabbit anti-human polyclonal CD24 (Epitomics), mouse anti-human monoclonal MMP-9 (Abcam), mouse anti-human monoclonal E-cadherin (Abcam), mouse anti-human monoclonal Vimentin (Abcam), mouse anti-human monoclonal actin (Beyotime), mouse anti-human monoclonal ABCG2(Millipore), and rabbit anti-human monoclonal ALDH1(ABGENT).


## 2.3. Characterization and Preparation of Herbal Extracts

The Chinese herbal medicine formula SYY, a dietary component authorized by the Chinese State Food and Drug Administration (Grant no. G20070160), includes five Chinese medicinal herbal extracts whose proportions, fingerprint, and protocol of preparation have previously been reported [29]. The SYY used in vitro and in vivo in this study was from the same batch number 20110401 and was produced by Shanghai Fang Xin Pharmaceutical Technology Co., Ltd. Shanghai, China. A 800 mg/mL solution of SYY was sterilized twice by 0.22-μm filtration (Millipore) for further use in vitro.


## 2.4. LDH Cytotoxicity Assay

The cytotoxicity detection kitplus (Roche) is a precise and fast colorimetric assay for the quantitative determination of cytotoxicity by measuring release of lactate dehydrogenase (LDH) activity from damaged cells. MHCC97H cells were cultured to 80% confluence. After trypsin digestion, the cells were counted and pipetted into 96-well plates at a density of 2000 cells/well. Background control (medium only), low controls (spontaneous LDH release), high controls (maximum LDH release), and experimental substance (2 mg/mL and 4 mg/mL SYY) were prepared on the same plate according to the manufacturer’s instructions. The 96-well plates were incubated in a humidified incubator at 37°C in 5% CO2 for 4, 8, 12, 24, 48, and 72 h. Results were expressed as the absorbance of each well at 492 nm (OD492). Cytotoxicity (%) was calculated using the equation: (experimental value − low control)/(high control − low control) × 100%.


## 2.5. Flow Cytometric Analysis

The expression level of CSC-related markers was determined by flow cytometry. Briefly, MHCC97H, HCCLM3, SMMC-7721, Hep3B, Huh7, and HepG2 cells were grown to 80% confluence. After trypsin digestion, the cells were resuspended in medium at a concentration of 1 × 106 cells/mL and incubated with primary antibodies against CD90, CD133, CD24, EPCAM, and CD44 (diluted 1 : 11) at 4°C for 15 min. After washing three times with PBS, the cells were analyzed using a FACSC Flow Cytometer (BD Biosciences). The biomarkers CD90/EPCAM in MHCC97H-SYY and CD24/EPCAM in Hep3B-SYY were also examined for comparison with their parental cell lines.


## 2.6. Immunofluorescence and Western Blot Analysis

The expression of CSC-related markers was also determined by immunofluorescence. Cells were grown on glass cover slips to 40%–50% confluence and then fixed, permeabilized, blocked, and incubated with primary monoclonal antibodies overnight at 4°C. Slides were washed and incubated with anti-mouse or anti-rabbit Cy3-conjugated secondary antibody (Jackson). Cells were counterstained with 4′-6-diamidino-2-phenylindole to visualize cell nuclei and detected by fluorescence microscopy (Olympus).Western blot analysis of protein expression of CD90, CD24, EPCAM, E-cadherin, and vimentin in MHCC97H-SYY, Hep3B-SYY, and their parental cell lines was performed according to the manufacturer’s instructions. The concentration of protein extracted from MHCC97H-SYY, Hep3B-SYY, and parental cell lines was determined using the BCA Protein Assay Kit (Beyotime).


## 2.7. Quantitative Real-Time PCR Analysis

Total RNA was extracted from MHCC97H-SYY, Hep3B-SYY, and their parental cell lines using Trizol Reagent (Invitrogen). Total RNA was reversely transcribed using a Prime Script RT reagents kit (TaKaRa). Messenger RNA expression was determined by real-time PCR using SYBR Premix Ex Taq II (TaKaRa). The primers used for the amplification of human genes were as follows: CD90 forward primer 5′-CAGCATTCTCAGCCACAA C-3′ and reverse primer 5′-TTACCTCCT TCTCCAACCCT-3′; CD24 forward primer 5′-TCGGGTGTGCTATGGATG-3′ and reverse primer 5′-AGA GGG GGTCTGTTGAAG AT-3′; EPCAM forward primer 5′-CAGTGTACTTCAGTTGGTG-3′ and reverse primer 5′-TCAGGTTTTGCTCTTC TC-3′.


## 2.8. Cell Migration and Invasion Assays

Cell migration and invasion of MHCC97H and Hep3B cell lines were assessed by transwell assays (Boyden chambers; Corning). Briefly, 6 × 104 cells in serum-free Dulbecco’s modified Eagle medium (DMEM) were seeded into the upper chamber of each well of 24-well plates containing 8.0-μm pore size membranes. DMEM containing 10% fetal bovine serum (FBS) was added to the lower chamber of each well. After 48 h, cells that had reached the underside of the membrane were stained with Giemsa (Sigma), counted at ×100, and photographed at ×200 magnification. The cell invasion assay was carried out similarly, except that 80 μL matrigel (BD Biosciences) was added to each well 6 h before cells were seeded on the membrane.


## 2.9. Cell Proliferation and Colony Formation Assay

MHCC97H and Hep3B cell lines were pretreated with SYY for 4 weeks and then plated in 96-well plates (3 × 103 cells/well) and exposed to oxaliplatin at increasing concentrations for 24, 48, 72, and 96 h. Cell proliferation assays were carried out with the Cell Counting Kit 8 (CCK8; Dojindo). Results were expressed as the absorbance of each well at 450 nm (OD450).For colony formation assays, 1 × 103 cells were plated in 6-well plates (Corning) and cultured with 1% FBS DMEM with or without SYY (2 mg/mL). Culture medium was replaced every 3 days, and the colonies were fixed with ice-cold 4% paraformaldehyde 14 days after the initiation of treatment. Cells were stained with Giemsa (Sigma) and photographed at ×5 magnification.


## 2.10. Animal Model and Treatment Procedures

Twenty-four nude mice bearing orthotopic xenografts were randomly divided into control group, SYY group, oxaliplatin+SYY group, and oxaliplatin only group. Seven days after orthotopic implantation the mice were treated as follows: SYY group mice were treated intraperitoneally (i.p.) with 0.1 mL 5% glucose solution (GS) once a week and orally with SYY (4 g/kg/d) every day; control group mice were treated with 5% GS and distilled water in the same way as the SYY group; oxaliplatin group mice were treated with oxaliplatin (10 mg/kg) i.p. and 0.1 mL distilled water orally; oxaliplatin+SYY group mice were treated with 0.1 mL oxaliplatin (10 mg/kg) i.p. and 0.1 mL SYY (4 g/kg/d) orally. Tumor weight and lung metastasis were evaluated 4 weeks after the initiation of treatment. Using another 12 nude mice bearing orthotopic xenografts, survival time of an oxaliplatin+SYY group and oxaliplatin only group was determined as the interval between the day of inoculation and the day of death.To evaluate the growth and metastasis potential of SYY-treated HCC cellsin vivo, another 24 male BALB/c nu/nu mice were divided into four groups. Group I mice were subcutaneously injected with 5 × 106 MHCC97H-SYY cells and group II mice were injected with the same number of MHCC97H cells. Tumor weight was measured 4 weeks after injection. Group III mice were injected with 1 × 105 MHCC97H-RFP-SYY cells through the tail vein, and group IV mice were injected with the same number of MHCC97H-RFP cells. Six weeks later, lung metastases were evaluated by fluorescence microscopy and confirmed by microscopic examination of serial sections of every lung tissue block.


## 2.11. Immunohistochemistry

Tumor tissue was fixed, embedded, and sliced into 5μm thick sections. Immunohistochemical staining of CD90, ABCG2, ALDH, CD44, EPCAM, E-cadherin, vimentin, and MMP-9 was carried out using a standard protocol [30].


## 2.12. Statistical Analysis

In vitro LDH cytotoxicity assay data, the proportion of CSC cells, cell migration, invasion, and proliferation assays were compared by Student’s t-test. Tumor weight was compared by analysis of variance (ANOVA), the lung metastasis assay was analyzed using Fisher’s exact test, and survival was compared with Kaplan-Meier method with a log-rank test. Statistical analysis was performed with SPSS 15.0 for Windows (SPSS Inc. Chicago, IL, USA). P<0.05 was considered statistically significant.


## 3. Results

### 3.1. Expression of CSC-Related Markers in HCC Cell Lines

The expression of CSC-related markers in the cell lines HCCLM3, MHCC97H, HepG2, SMMC7721, Hep3B, and Huh7 was examined by flow cytometry and immunofluorescence (Additional file 1 (see Supplementary Material available online at doi:10.1155/2012/908601)). The proportion of CSCs in each cell line is presented in Figure1. The expression of CD90/EpCAM in MHCC97H was 2.13±0.37% and 1.35±0.24%. The expression of CD24/EpCAM in Hep3B was 99.48±3.87% and 64.30±3.09%, all of which were consistent with reports in the literature. Moreover, these CSC-related markers were previously used to study CSCs [12–14]. Therefore, MHCC97H and Hep3B were selected for further study.Figure 1
The expression of the CSC-related markers in HCCLM3, MHCC97H, HepG2, SMMC7721, Hep3B and Huh7 HCC cell cultures.


### 3.2. SYY Exhibited No Significant Cytotoxicity in HCC Cell Lines but Increased Their Sensitivity to Oxaliplatin

MHCC97H cells that were treated with SYY (2 mg/mL and 4 mg/mL) for 4, 8, 12, 24, 48, and 72 h demonstrated no significant increase in release of lactate dehydrogenase (LDH) compared with the control group. When LDH release of the control group was set at 100%, the relative emission of LDH at 4, 8, 12, 24, 48, and 72 h was98.91±3.09%, 97.84±2.17%, 96.50±4.72%, 98.75±3.00%, 101.58±3.86%, and 101.75±4.81% in cells treated with 2 mg/mL SYY and 98.84±3.19%, 99.00±2.43%, 99.58±2.72%, 101.58±3.86%, 101.75±4.81%, and 100.41±3.47% in cells treated with 4 mg/mL SYY. There was no statistically significant difference between the control and experimental groups (Figure 2(a)), indicating that SYY did not exhibit acute cytotoxicity to MHCC97H cells.SYY exhibited no significant cytotoxicity in HCC cell lines but increased their sensitivity to oxaliplatin: (a) MHCC97H cells treated with 2 mg/mL and 4 mg/mL SYY demonstrated no increase in LDH release indicating no acute cytotoxicity and (b) MHCC97H and Hep3B cell lines treated with 2 mg/mL SYY for 4 weeks showed increased chemosensitivity to oxaliplatin compared with parental cell lines.
(a)
(b)The IC50 of oxaliplatin was lower in SYY-treated MHCC97H cells (MHCC97H-SYY) and Hep3B cells (Hep3B-SYY) compared with the corresponding parental cell lines MHCC97H and Hep3B. The CCK8 assay showed that the IC50 of oxaliplatin in Hep3B-SYY was 0.41±0.16 μmol/L, compared with 1.38±0.28 μmol/L for Hep3B (P=0.0376). The IC50 of oxaliplatin against MHCC97H-SYY was also lower than that for MHCC97H (12.15±1.64 μmol/L versus 28.93±2.18 μmol/L; P=0.0011; Figure 2(b)).


### 3.3. SYY Reduced Expression of CSC-Related Markers and Inhibited the Stemness of HCC Cell Lines

We further explored the underlying mechanism of the chemosensitization to oxaliplatin by SYY and found that the expression of the CSC-related marker CD90 in MHCC97H and MHCC97H-SYY cells was1.83%±0.42% and 0.61%±0.19% (P<0.01), respectively, and the expression of EpCAM was 1.37%±0.35% and 0.50%±0.19% (P<0.01), respectively. Similarly, the expression of CD24 in Hep3B and Hep3B-SYY was 62.74%±4.45% and 7.75%±2.00% (P<0.01), respectively, and the expression of EPCAM was 99.28%±0.50% and 92.71%±2.30% (P<0.01), respectively (Figure 3(a), Additional file 2). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses gave similar results, confirming the downregulation of CSC-related markers in SYY-treated cells. The upregulation of E-cadherin and the downregulation of vimentin were also observed in SYY-treated cell lines (Figure 3(b)).Hepatoma cell lines MHCC97H and Hep3B treated with SYY (2 mg/mL) for 4 weeks exhibited decreased stemness. (a) Tumor cells that were treated with SYY for 4 weeks showed reduced expression of CSC-related markers compared with their parental cell lines by flow cytometry. (b) RT-PCR and Western blot analyses confirmed the decreased expression of CSC-related markers in SYY-treated cell lines. (c) The transwell assay demonstrated that MHCC97H-SYY and Hep3B-SYY cells migrated through the basement membrane less efficiently than the parental cell lines MHCC97H and Hep3B. (d) Colony formation assay demonstrated that MHCC97H-SYY and Hep3B-SYY had a significantly lower proliferation rate and colony-forming ability than untreated parental cells. (e) MHCC97H-SYY cell lines also showed diminished subcutaneous tumor growth capacity and pulmonary metastasis compared with parental cells in nude mouse models.
(a)
(b)
(c)
(d)
(e)The transwell assay for cell migration and invasiveness demonstrated that MHCC97H-SYY and Hep3B-SYY cells passed through the basement membrane less efficiently than the parental cell lines MHCC97H and Hep3B (P<0.01). The number of cells crossing the basement membrane was lower in MHCC97H-SYY than in MHCC97H in both the cell migration assay (149.50±18.13 versus 44.36±8.15; P=0.0005) and invasion assay (127.83±18.01 versus 38.66±5.57; P=0.002). Similarly, the passage of Hep3B-SYY cells through the basement membrane was lower than that of Hep3B for cell migration (95.66.50±14.12 versus 48.02±25.51; P=0.0083) and invasion (88.33±7.74 versus 41.51±5.57; P=0.0001) (Figure 3(c), Additional file 3). In colony formation assays, MHCC97H-SYY and Hep3B-SYY exhibited a significantly lower proliferation rate and colony-forming ability than their parental cells. The number of colonies formed was lower in MHCC97H-SYY than in the parental cells (19.83±3.43 versus 4.67±1.63; P<0.001) and in Hep3B-SYY compared with Hep3B (3.85±1.43 versus 3.13±1.03; P=0.0395; Figure 3(d), Additional file 4).MHCC97H-SYY cell lines also showed diminished subcutaneous tumor growth capacity compared with parental cells in nude mouse models. Four weeks after implantation, the tumor weight was2.46±0.59 g and 1.89±0.86 g in MHCC97H and MHCC97H-SYY groups, respectively, (P=0.0038). Six weeks after tail vein injection, the mean number of lung metastatic nodules in the MHCC97H-RFP group and MHCC97H-RFP-SYY group was 13.66±6.35 and 4.83±4.87, respectively, (P=0.048; Figure 3(e), Additional file 5).


### 3.4. Oxaliplatin and SYY Combination Treatment Resulted in Enhanced Inhibition of Tumor Growth and Reduced Pulmonary Metastasis

First, we evaluated the effect of SYY alone on tumor growth and pulmonary metastasis in nude mice using MHCC97H-RFP cells. Treatment with a low dose of SYY (2 g/kg) did not significantly inhibit tumor growth (2.31±1.15 g versus 2.40±1.21 g; P>0.05) or the number of pulmonary metastasis nodules (15.00±8.41 versus 25.83±5.25P=0.0526) compared with the control group. Similarly, a medium dose of SYY (4 g/kg) did not decrease tumor growth (2.22±0.31 g versus 2.40±1.21 g; P>0.05) although there was a significant decrease in the number of pulmonary metastasis nodules compared with the control group (7.00±6.71 versus 25.83±5.25; P=0.0009). However, treatment with the high dose of SYY (8 g/kg) significantly inhibited tumor growth (1.32±0.64 g versus 2.40±1.21 g; P=0.0466) and the number of pulmonary metastasis nodules (0.67±1.63 versus 25.83±5.25; P<0.0001) compared with the control group (Figure 4(a)).Oxaliplatin and SYY cotreatment resulted in a smaller tumor volume and a lower percentage of pulmonary metastasis than oxaliplatin treatment alone. (a) Treatment with low-dose SYY (2 g/kg) did not significantly inhibit tumor growth or decrease the incidence of pulmonary metastasis. Treatment with 4 g/kg SYY decreased the number of pulmonary metastasis nodules but did not inhibit tumor growth. In contrast, high-dose SYY (8 g/kg) treatment significantly inhibited tumor growth and pulmonary metastasis. (b) Cotreatment with oxaliplatin and SYY (4 g/kg) reduced tumor volume, decreased the percentage of pulmonary metastasis, and prolonged survival compared with oxaliplatin treatment alone.
(a)
(b)SYY (4 g/kg) enhanced the antitumor effect of oxaliplatin. The tumor weight was lower in the SYY+OXA group than in the OXA alone group (0.83±0.20 g versus 1.92±0.79 g; P=0.0069), and a lower number of pulmonary metastasis nodules were observed compared with oxaliplatin treatment alone (22.34±9.06 versus 44.50±7.34; P=0.0143). Combination treatment with oxaliplatin and SYY also prolonged survival compared with oxaliplatin treatment alone (58.17±9.22 days versus 46.33±5.96 days; P=0.0005; Figure 4(b)).


### 3.5. Combination Treatment with Oxaliplatin and SYY Showed Decreased the Expression of CSC-Related Markers and Reversed EMT

To investigate the role of SYY in modulating the proportion of CSCsin vivo, we examined tumor tissues by immunohistochemistry and observed a decreased proportion of ABCG2-, CD44-, ALDH1-, EpCAM-, and CD90-positive CSCs after cotreatment with SYY and oxaliplatin compared with oxaliplatin treatment alone. A significant decrease in the expression of matrix metalloproteinase 9 (MMP-9), which is involved in the breakdown of extracellular matrix during tumor metastasis, was also observed after cotreatment with oxaliplatin and SYY. In addition, the typical membranous E-cadherin expression was significantly upregulated in tumors treated with SYY and oxaliplatin (Figure 5).Oxaliplatin and SYY cotreatment reduced the proportion of CSCs, decreased the expression of MMP-9, and reversed EMT with increased expression of E-cadherin compared with oxaliplatin treatment alone.


## 3.1. Expression of CSC-Related Markers in HCC Cell Lines

The expression of CSC-related markers in the cell lines HCCLM3, MHCC97H, HepG2, SMMC7721, Hep3B, and Huh7 was examined by flow cytometry and immunofluorescence (Additional file 1 (see Supplementary Material available online at doi:10.1155/2012/908601)). The proportion of CSCs in each cell line is presented in Figure1. The expression of CD90/EpCAM in MHCC97H was 2.13±0.37% and 1.35±0.24%. The expression of CD24/EpCAM in Hep3B was 99.48±3.87% and 64.30±3.09%, all of which were consistent with reports in the literature. Moreover, these CSC-related markers were previously used to study CSCs [12–14]. Therefore, MHCC97H and Hep3B were selected for further study.Figure 1
The expression of the CSC-related markers in HCCLM3, MHCC97H, HepG2, SMMC7721, Hep3B and Huh7 HCC cell cultures.


## 3.2. SYY Exhibited No Significant Cytotoxicity in HCC Cell Lines but Increased Their Sensitivity to Oxaliplatin

MHCC97H cells that were treated with SYY (2 mg/mL and 4 mg/mL) for 4, 8, 12, 24, 48, and 72 h demonstrated no significant increase in release of lactate dehydrogenase (LDH) compared with the control group. When LDH release of the control group was set at 100%, the relative emission of LDH at 4, 8, 12, 24, 48, and 72 h was98.91±3.09%, 97.84±2.17%, 96.50±4.72%, 98.75±3.00%, 101.58±3.86%, and 101.75±4.81% in cells treated with 2 mg/mL SYY and 98.84±3.19%, 99.00±2.43%, 99.58±2.72%, 101.58±3.86%, 101.75±4.81%, and 100.41±3.47% in cells treated with 4 mg/mL SYY. There was no statistically significant difference between the control and experimental groups (Figure 2(a)), indicating that SYY did not exhibit acute cytotoxicity to MHCC97H cells.SYY exhibited no significant cytotoxicity in HCC cell lines but increased their sensitivity to oxaliplatin: (a) MHCC97H cells treated with 2 mg/mL and 4 mg/mL SYY demonstrated no increase in LDH release indicating no acute cytotoxicity and (b) MHCC97H and Hep3B cell lines treated with 2 mg/mL SYY for 4 weeks showed increased chemosensitivity to oxaliplatin compared with parental cell lines.
(a)
(b)The IC50 of oxaliplatin was lower in SYY-treated MHCC97H cells (MHCC97H-SYY) and Hep3B cells (Hep3B-SYY) compared with the corresponding parental cell lines MHCC97H and Hep3B. The CCK8 assay showed that the IC50 of oxaliplatin in Hep3B-SYY was 0.41±0.16 μmol/L, compared with 1.38±0.28 μmol/L for Hep3B (P=0.0376). The IC50 of oxaliplatin against MHCC97H-SYY was also lower than that for MHCC97H (12.15±1.64 μmol/L versus 28.93±2.18 μmol/L; P=0.0011; Figure 2(b)).


## 3.3. SYY Reduced Expression of CSC-Related Markers and Inhibited the Stemness of HCC Cell Lines

We further explored the underlying mechanism of the chemosensitization to oxaliplatin by SYY and found that the expression of the CSC-related marker CD90 in MHCC97H and MHCC97H-SYY cells was1.83%±0.42% and 0.61%±0.19% (P<0.01), respectively, and the expression of EpCAM was 1.37%±0.35% and 0.50%±0.19% (P<0.01), respectively. Similarly, the expression of CD24 in Hep3B and Hep3B-SYY was 62.74%±4.45% and 7.75%±2.00% (P<0.01), respectively, and the expression of EPCAM was 99.28%±0.50% and 92.71%±2.30% (P<0.01), respectively (Figure 3(a), Additional file 2). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses gave similar results, confirming the downregulation of CSC-related markers in SYY-treated cells. The upregulation of E-cadherin and the downregulation of vimentin were also observed in SYY-treated cell lines (Figure 3(b)).Hepatoma cell lines MHCC97H and Hep3B treated with SYY (2 mg/mL) for 4 weeks exhibited decreased stemness. (a) Tumor cells that were treated with SYY for 4 weeks showed reduced expression of CSC-related markers compared with their parental cell lines by flow cytometry. (b) RT-PCR and Western blot analyses confirmed the decreased expression of CSC-related markers in SYY-treated cell lines. (c) The transwell assay demonstrated that MHCC97H-SYY and Hep3B-SYY cells migrated through the basement membrane less efficiently than the parental cell lines MHCC97H and Hep3B. (d) Colony formation assay demonstrated that MHCC97H-SYY and Hep3B-SYY had a significantly lower proliferation rate and colony-forming ability than untreated parental cells. (e) MHCC97H-SYY cell lines also showed diminished subcutaneous tumor growth capacity and pulmonary metastasis compared with parental cells in nude mouse models.
(a)
(b)
(c)
(d)
(e)The transwell assay for cell migration and invasiveness demonstrated that MHCC97H-SYY and Hep3B-SYY cells passed through the basement membrane less efficiently than the parental cell lines MHCC97H and Hep3B (P<0.01). The number of cells crossing the basement membrane was lower in MHCC97H-SYY than in MHCC97H in both the cell migration assay (149.50±18.13 versus 44.36±8.15; P=0.0005) and invasion assay (127.83±18.01 versus 38.66±5.57; P=0.002). Similarly, the passage of Hep3B-SYY cells through the basement membrane was lower than that of Hep3B for cell migration (95.66.50±14.12 versus 48.02±25.51; P=0.0083) and invasion (88.33±7.74 versus 41.51±5.57; P=0.0001) (Figure 3(c), Additional file 3). In colony formation assays, MHCC97H-SYY and Hep3B-SYY exhibited a significantly lower proliferation rate and colony-forming ability than their parental cells. The number of colonies formed was lower in MHCC97H-SYY than in the parental cells (19.83±3.43 versus 4.67±1.63; P<0.001) and in Hep3B-SYY compared with Hep3B (3.85±1.43 versus 3.13±1.03; P=0.0395; Figure 3(d), Additional file 4).MHCC97H-SYY cell lines also showed diminished subcutaneous tumor growth capacity compared with parental cells in nude mouse models. Four weeks after implantation, the tumor weight was2.46±0.59 g and 1.89±0.86 g in MHCC97H and MHCC97H-SYY groups, respectively, (P=0.0038). Six weeks after tail vein injection, the mean number of lung metastatic nodules in the MHCC97H-RFP group and MHCC97H-RFP-SYY group was 13.66±6.35 and 4.83±4.87, respectively, (P=0.048; Figure 3(e), Additional file 5).


## 3.4. Oxaliplatin and SYY Combination Treatment Resulted in Enhanced Inhibition of Tumor Growth and Reduced Pulmonary Metastasis

First, we evaluated the effect of SYY alone on tumor growth and pulmonary metastasis in nude mice using MHCC97H-RFP cells. Treatment with a low dose of SYY (2 g/kg) did not significantly inhibit tumor growth (2.31±1.15 g versus 2.40±1.21 g; P>0.05) or the number of pulmonary metastasis nodules (15.00±8.41 versus 25.83±5.25P=0.0526) compared with the control group. Similarly, a medium dose of SYY (4 g/kg) did not decrease tumor growth (2.22±0.31 g versus 2.40±1.21 g; P>0.05) although there was a significant decrease in the number of pulmonary metastasis nodules compared with the control group (7.00±6.71 versus 25.83±5.25; P=0.0009). However, treatment with the high dose of SYY (8 g/kg) significantly inhibited tumor growth (1.32±0.64 g versus 2.40±1.21 g; P=0.0466) and the number of pulmonary metastasis nodules (0.67±1.63 versus 25.83±5.25; P<0.0001) compared with the control group (Figure 4(a)).Oxaliplatin and SYY cotreatment resulted in a smaller tumor volume and a lower percentage of pulmonary metastasis than oxaliplatin treatment alone. (a) Treatment with low-dose SYY (2 g/kg) did not significantly inhibit tumor growth or decrease the incidence of pulmonary metastasis. Treatment with 4 g/kg SYY decreased the number of pulmonary metastasis nodules but did not inhibit tumor growth. In contrast, high-dose SYY (8 g/kg) treatment significantly inhibited tumor growth and pulmonary metastasis. (b) Cotreatment with oxaliplatin and SYY (4 g/kg) reduced tumor volume, decreased the percentage of pulmonary metastasis, and prolonged survival compared with oxaliplatin treatment alone.
(a)
(b)SYY (4 g/kg) enhanced the antitumor effect of oxaliplatin. The tumor weight was lower in the SYY+OXA group than in the OXA alone group (0.83±0.20 g versus 1.92±0.79 g; P=0.0069), and a lower number of pulmonary metastasis nodules were observed compared with oxaliplatin treatment alone (22.34±9.06 versus 44.50±7.34; P=0.0143). Combination treatment with oxaliplatin and SYY also prolonged survival compared with oxaliplatin treatment alone (58.17±9.22 days versus 46.33±5.96 days; P=0.0005; Figure 4(b)).


## 3.5. Combination Treatment with Oxaliplatin and SYY Showed Decreased the Expression of CSC-Related Markers and Reversed EMT

To investigate the role of SYY in modulating the proportion of CSCsin vivo, we examined tumor tissues by immunohistochemistry and observed a decreased proportion of ABCG2-, CD44-, ALDH1-, EpCAM-, and CD90-positive CSCs after cotreatment with SYY and oxaliplatin compared with oxaliplatin treatment alone. A significant decrease in the expression of matrix metalloproteinase 9 (MMP-9), which is involved in the breakdown of extracellular matrix during tumor metastasis, was also observed after cotreatment with oxaliplatin and SYY. In addition, the typical membranous E-cadherin expression was significantly upregulated in tumors treated with SYY and oxaliplatin (Figure 5).Oxaliplatin and SYY cotreatment reduced the proportion of CSCs, decreased the expression of MMP-9, and reversed EMT with increased expression of E-cadherin compared with oxaliplatin treatment alone.


## 4. Discussion

For HCC patients with advanced stage disease, TACE and systemic chemotherapy are usually the treatment of choice. However, these therapies often transiently shrink tumors by targeting the tumor bulk but fail to kill CSCs, leading to eventual treatment failure and tumor relapse. In the present study, we found that the Chinese herbal medicine SYY renders HCC cells sensitive to oxaliplatin. CSCs possess certain characteristics that make them difficult to kill: they have the property of self-protection through increased activity of multiple drug resistance transporters such as ABCB1 and/or ABCG2 [31–33]; they divide much more slowly than other cells, allowing them to evade traditional chemotherapies that hit rapidly multiplying cells [34], and they overexpress antiapoptotic proteins such as BCL-2 and survivin and have a high capacity for DNA repair, leading to resistance to apoptosis and anticancer drugs [35, 36]. The discovery of CSCs not only explained why treatment with chemotherapy often seems to be initially successful but ultimately fails to eradicate the tumor, but also had a profound impact on our current perception of cancer diagnosis, management, and treatment options [37].The most critical issue in the field of CSCs is to develop phenotypic assays that can be used to reliably identify CSCs [38]. Currently, the most widely used method to identify these cells is through their expression of markers such as CD133, CD90, CD24, CD44, OV6, and EpCAM and through staining of side population cells by Hoechst dye [25]. However the identification of specific CSC markers in a defined cell line remains controversial. In the current study, the expression of CD90/EpCAM in MHCC97H cells and CD24/EpCAM in Hep3B cells was consistent with reports in the literature and their previous use as CSC-related markers to study hepatoma CSCs [12–14]. MHCC97H and Hep3B cells that were pretreated with SYY (2 mg/mL) for 4 weeks showed increased sensitivity to chemotherapy compared with their parental cell lines. One of the typical feature of CSCs is resistance to chemotherapy [39]. In the present study, we found that the expression of CSC-related markers in MHCC97H-SYY (CD90/EpCAM) and Hep3B-SYY (CD24/EpCAM) cells was significantly lower than in their parental cell lines.Traits such as invasiveness, motility, colony-forming ability, tumorigenicity, and lung metastasis have been attributed to the existence of CSCs and are considered characteristics associated with the stemness of cancer [40]. Therefore, we explored changes in the stemness characteristics of SYY-treated cell lines and tissues that reflect changes in CSCs. SYY-treated MHCC97H-SYY and Hep3B-SYY cells showed lower motility, invasiveness, and colony-forming ability in vitro. We injected the tail vein of BALB/c nu/nu mice with MHCC97H-RFP-SYY cells and found a reduced incidence of lung metastasis compared with mice injected with MHCC97H. In the nude mice models, MHCC97H-SYY also showed diminished subcutaneous tumor growth capacity compared with the parental cells. Cotreatment of nude mice with oxaliplatin and SYY resulted in smaller tumor volume and a lower percentage of pulmonary metastasis than oxaliplatin treatment alone. Consistent with these findings, we found that oxaliplatin and SYY cotreatment decreased the expression of CSC-related markers and reversed EMT compared with oxaliplatin treatment alone. Together, these findings indicate that SYY suppresses the stemness of HCC by decreasing expression of CSC-related markers in tumor tissues and cell lines.SYY is a Chinese herbal medicine formula consisting of five herbs. Some components of SYY have demonstrated value in the treatment of malignancies [41, 42]. In our previous study, we reported that SYY effectively inhibited tumor growth and metastasis, and increased survival in a HCC nude mouse model bearing a MHCC97H xenograft [29]. Xiong et al. reported that SYY inhibits molecular changes consistent with EMT in oxaliplatin-treated tumor tissues and cell lines [20]. We suggest that the downregulation of CSC-related markers in the current study was responsible for the increased chemosensitivity of SYY-treated tissues and cell lines and the repression of stemness. There are two ways to decrease the proportion of CSCs: induction of CSC differentiation or direct elimination of CSCs. The efficacy of retinoic acid-induced differentiation to target the stem-like tumor cells in glioma has recently been demonstrated [43]. Elimination therapy is antigen-based therapy that targets different aspects of CSCs, and can either take the form of vaccines or monoclonal antibody therapy or be based on the spontaneous response of the immune system to cancer [44]. At present it is not clear whether SYY targets CSC through the induction of CSC differentiation or direct elimination of CSCs. This question will be investigated in further studies using isolated CSCs.


## 5. Conclusions

SYY can render hepatocellular carcinoma sensitive to oxaliplatin through the inhibition of stemness.



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*Source: 908601-2012-12-20.xml*