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WITHDRAWN: The anti-growth and anti-metastasis effects of Schisandrin B on hepatocarcinoma cells in vitro and in vivo
Accepted Manuscript The anti-growth and anti-metastasis effects of Schisandrin B on hepatocarcinoma cells in vitro and in vivo Ruijie Sun, Ruiren Zhai, Changlin Ma, Miao Wei PII:
S0006-291X(17)31134-8
DOI:
10.1016/j.bbrc.2017.06.022
Reference:
YBBRC 37928
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 31 May 2017 Accepted Date: 7 June 2017
Please cite this article as: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
The anti-growth and anti-metastasis effects of Schisandrin B on hepatocarcinoma cells in vitro and in vivo Ruijie Sun,1† Ruiren Zhai,2† Changlin Ma,1 Miao Wei3*
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1. Department of hepatobiliary surgery, Jining First People's Hospital, Jining, Shandong, 272000, China
2. Tumor center Shandong Sunshine Hospital, Weifang, Shandong, 261041, China
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3. Department of health care, Jining First People's Hospital, Jining, Shandong, 272000, China
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#corresponding author: Miao Wei. Department of health care, Jining First People’s Hospital, No. 6 Jiankang Road, Jining, Shandong, 272000, PR China. E-mail: [email protected] †
These authors contributed equally to this work.
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Running title: Sch B inhibits growth and metastasis of HCC
Keywords: Schisandrin B, Anti-growth, Anti-metastasis, Hepatocarcinoma Abbreviations: HCC, Hepatocarcinoma; Sch B, Schisandrin B; HBV, Hepatitis B virus; HCV,
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Hepatitis C virus
ACCEPTED MANUSCRIPT Abstract Hepatocarcinoma (HCC) is the most common liver cancer with high metastasis and recurrence rate which represents extremely poor prognosis. In this study, we aimed to investigate the effects
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of Schisandrin B (Sch B) on HCC cells both in vitro and in vivo and to explore its underlying mechanism. We found that Sch B inhibited the proliferation of HCC cells in a dose-dependent manner as assessed by CCK-8 assay. The flow cytometric assay showed that Sch B significantly
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induced HCC cell apoptosis. Besides, Sch B suppressed HCC cell metastasis as detected by both
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wound-healing assay and transwell migration assay. Mechanistically, the results of western blot indicated that Sch B inhibited cell proliferation by downregulating PCNA, induced cell apoptosis by upregulating Caspase-3 and downregulating Bcl-2, and suppressed cell mobility by downregulating EGF, VEGFA and bFGF. Moreover, Sch B significantly inhibited the HCC
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xenograft model growth in BALB/c nude mice. Taken together, our study indicated that Sch B exhibits potent antitumor activities against HCC and provided a molecular basis for Sch B
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potential applications in the treatment of HCC and other tumor-related diseases.
ACCEPTED MANUSCRIPT Introduction Liver cancer is the second leading cause of cancer-related death worldwide, and the relative 5-year survival rate for liver cancer is 16.6%.[1, 2] Hepatocarcinoma (HCC), which represents the
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predominant form of liver cancer, results in more than 670,000 deaths globally per year.[3, 4] HCC occurs mainly in the liver of patients infected by chronic hepatitis B virus (HBV) or hepatitis C virus (HCV).[5] Other risk factors including alcohol misuse, cigarette smoking and metabolic
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disorders also contribute to HCC incidence.[6] In spite of improvements in HCC therapy over the
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past decade, the prognosis of HCC remains discouraging, mainly due to the high metastasis and recurrence rate of HCC cells.[7] Therefore, there are urgently needed for novel effective strategies and drugs to improve outcomes for patients with HCC.
Schisandrin B (Sch B, C23H28O6) is a dibenzocyclooctadiene lignan isolated from fructus
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schisandrae, a traditional Chinese medicine, and Sch B has been used to treat several human diseases, including hepatitis and myocardial disorders.[8, 9] Previous studies have reported that Sch B possessed multiple functions against cancer. For example, Sch B is a novel inhibitor of
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P-glycoprotein. Overexpression of P-glycoprotein in cancer cells is the most frequent cause for 11]
Sch B also attenuates cancer invasion and metastasis via
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cancer multidrug resistance.[10,
inhibiting epithelial-mesenchymal transition.[12] Besides, Sch B inhibits ATR protein kinase activity in response to DNA damage, suggesting clinical implications in anti-cancer therapy.[13] Although previous studies have reported that Sch B induced human hepatoma SMMC-7721 cell apoptosis accompanying with down-modulation of heat shock protein 70 and up-modulation of caspase-3, the effects of Sch B on HCC cells and the underlying mechanisms of these effects have remained largely unknown.[14]
ACCEPTED MANUSCRIPT In this study, we investigated the anti-growth and anti-metastasis ability of Sch B on human HCC cell line HCCLM3 and the possible molecular mechanisms underlying these actions. In addition, a HCC xenograft nude mice model was performed to evaluate the anti-growth and
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for the potential application of Sch B in HCC.
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anti-metastasis effects of Sch B on HCC cells in vivo. These data provided experimental evidence
ACCEPTED MANUSCRIPT Materials and Methods Cell Culture Human HCC cell line HCCLM3 (with high metastatic potential) was purchased from American
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Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM (high glucose, Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (FBS, Gibco, Rockville, MD) in incubator with 5% CO2 at 37°C.
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Cell Counting kit-8 Assay
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Cell Proliferation assay using the cell counting kit-8 (CCK-8; Dojindo, Japan) was performed according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plates at a density of 5000 cells per well with complete growth medium. When the cells anchored to the plates, various concentrations of Sch B (98% for HPLC, J&K, China) were added and the slides
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were incubated at 37°C with 5% CO2. Each plate had three wells with a non-treated group as control. After 48 h of culture, the growth medium was removed from each well, and then all the wells were filled with 100 ml of fresh medium containing 10 ml CCK-8 solutions. After incubated
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for 2 h at 37˚C, cell proliferation was assessed by absorbance detection at 450 nm with a
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microplate reader (Biotek, Winooski, VT). Cell Apoptosis Analysis
Cell apoptosis was analyzed after various concentrations of Sch B treatment for 48 h staining with Annexin V and PI (BD Bioscience, San Jose, CA) according to the manufacturer’s specifications. After incubated for 15 min at room temperature in the dark, the cells were analyzed by flow cytometry. Annexin V-positive and PI-negative/positive staining cells represent apoptotic cells.
ACCEPTED MANUSCRIPT Wound-Healing Assay After the cells anchored to the plates, the subconfluent cell monolayers were scraped in three parallel lines with a P-200 pipette tip. The detached cells were washed off twice gently, and the
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medium was then replaced with 1% FBS complete medium containing various concentrations of Sch B. To visualize wound healing, images were taken at 0 and 24 h. The percentage of wound closure (original width-width after cell migration/original width) was calculated.
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Transwell Migration Assay
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The 4 × 103 cells were seeded onto the upper part of a transwell chamber (Corning Costar, Rochester, NY) containing a gelatin-coated polycarbonate membrane filter (pore size: 8 mm). Culture medium with 20% FBS was added to the lower chamber to stimulate cell migration. After 24-h pretreatment of various concentrations of Sch B incubation at 37˚C with 5% CO2, the cells
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were stained with crystal violet (Sigma-Aldrich, Germany). Cells on the underside of the filters were observed under a microscope (Olympus IX71, Japan) and counted. Western blot
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RIPA buffer (Sigma-Aldrich, USA) was used to lyse cells with Complete Protease Inhibitor
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Cocktail (Roche, USA). Cell lysates were transferred to 1.5 mL tube and kept at −20°C before use. SDS-PAGE was conducted to separate the cellular proteins. Proteins were separated by 5% stacking gel and 10% running gel. The molecular weight of candidate proteins was referred to the Pre-stained SeeBlue rainbow marker (Invitrogen, Carlsbad, CA, USA) loaded in parallel. The following antibodies were used: anti-PCNA, anti-Caspase-3, anti-Bcl-2, anti-EGF, anti-VEGFA and anti-bFGF (Abcam, Cambridge, UK), anti-GAPDH (Sigma, St. Louis, MO, USA). Blots were detected using a Kodak film developer (Fujifilm, Japan).
ACCEPTED MANUSCRIPT Animal Study A total of 5 × 106 cells/0.2 ml of HCCLM3 cells were injected subcutaneously into each of ten 4-week-old male BALB/c nude mice purchased from Beijing HFK Bioscience Co. Ltd. and kept
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under sterile specific pathogen-free conditions. All experiments were approved by the Animal Care and Ethics Committee of the Jining First People’s Hospital and were in accordance with NIH animal use guidelines. After tumor cells inoculation, the mice were randomly divided into two
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groups of five mice in each group. The mice were given either Sch B (50 mg/kg) or PBS by
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intraperitoneally injecting. Tumor growth was measured every 5 days, and tumor volume was calculated using the formula, volume = 1/2 (length×width2). Twenty-five days after injection, the mice were killed and tumor weights were measured. Immunohistochemistry
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The paraffin-embedded tumor sections were stained for anti-VEGFA (Abcam, Cambridge, UK). VEGFA expression was analyzed using the standard avidin-biotin method. Sections (2 µm) were deparaffinized and pretreated with citrate buffer using a heat-induced epitope retrieval protocol.
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Endogenous peroxidase was blocked with 20% hydrogen peroxide for 15 min at room temperature
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followed by incubation with anti-VEGFA for 30 min. A biotinylated goat anti-mouse immunglobulin G secondary antibody (Dako, Denmark) was then applied to each slide for 30 min. After washing in Tris-hydrochloric acid buffer (TBS), the slides were incubated with peroxidase-conjugated streptavidin complex reagent (Dako, Denmark) and developed with 3,3’-diaminobenzidine for 5 min. The slides were counterstained and dehydrated. Statistical Analysis Experimental values were obtained from at least three independent experiments. Data are
ACCEPTED MANUSCRIPT expressed as means ± SD. Statistical analysis was performed using the Student’s t-test or one-way analysis of variance (GraphPad Prism; GraphPad Software Inc., La Jolla, CA), where appropriate. The Bonferroni post hoc test was used to determine the source of observed differences. P values <
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0.05 were considered significant.
ACCEPTED MANUSCRIPT Results Sch B inhibits the growth of HCC cells To investigate the effect of Sch B on human HCC cells, HCCLM3 cells were treated with Sch B
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at various concentrations for 48 h followed by detection of cell viability. The results showed that Sch B dose-dependently inhibited the proliferation of HCCLM3 cells (P < 0.05, Fig. 1A).
To further elucidate whether the inhibition of cell growth was partially caused by apoptosis, the
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flow cytometric was performed. As shown in Figure 1B, the figure for apoptotic cells were
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significantly increased in a dose-dependent manner after Sch B treatment compared with the untreated group. These results suggest that Sch B inhibits proliferation and promotes apoptosis in HCC cells.
Sch B suppresses the migration and invation of HCC cells
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To investigate the effect of Sch B on the metastatic capability of HCCLM3 cells, the wound-healing assay and transwell chamber assay were carried out. As shown in Figure 2A and C, the wound-healing assay exhibited that the closing rates of scratch wounds were significantly
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decreased by Sch B in a dose-dependent manner compared with the control group (P < 0.05). The
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transwell assay also showed that the number of migratory cells in the Sch B treated groups were obviously decreased in a dose-dependent manner compared with the control group (P < 0.05, Fig. 2B and D). These results implied that Sch B effectively inhibited the motility of HCC cells. Effects of Sch B on expression of PCNA, Caspase-3, Bcl-2, EGF, VEGFA and bFGF To better understand the influence of Sch B on the HCC cell proliferation and apoptosis, the expression of proliferating cell nuclear antigen (PCNA), Caspase-3 and Bcl-2 were evaluated by western blot analysis. Compared with the control group, treatment with Sch B significantly
ACCEPTED MANUSCRIPT decreased the expression of PCNA and Bcl-2 and increased the expression of Caspase-3 in a dose-dependent manner (P < 0.05, Fig. 3A ~ D). Furthermore, the expression of the angiogenic growth factors epidermal growth factor (EGF), vascular endothelial growth factor A (VEGFA) and
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base fibroblast growth factor (bFGF) were examined by western blot assay to investigate the effects of Sch B on HCC cell metastasis. These results showed that the expression levels of all three factors were decreased in a dose-dependent manner compared with the control group (P <
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0.01, Fig. 3E ~ H).
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Sch B inhibits tumor growth in vivo
The anticancer effect of Sch B in vivo was further analyzed in a HCCLM3 tumor xenograft model. As shown in Figure 4A and B, there was a remarkable reduction in tumor weight in mice treated with Sch B at the dose of 50 mg/kg compared with the control mice (P < 0.05). Also, the result of
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immunohistochemistry showed that Sch B significantly decreased the VEGFA expression in tumor tissues compared with the untreated group (Fig. 4C). These results demonstrated that Sch B might
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effectively inhibit HCC cell growth and metastasis in vivo.
ACCEPTED MANUSCRIPT Discussion HCC is one of the leading causes of cancer-related death, becoming a major health problem worldwide. Most patients with HCC experience a recurrence after resection or are diagnosed at
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advanced stages.[7, 15, 16] Despite the introduction of novel therapies has improved patient survival, current common therapeutic strategies for HCC largely rely on surgery, transplantation, use of radiofrequency and transarterial embolization. The overall prognosis of HCC still remains 18]
Thus, continuous investigation of the novel therapeutic strategy for this
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disappointing.[17,
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disease is of great significance. Recently, many bioactive pharmaceutical drugs derived from different natural resources have been identified as effective agent for several diseases.[19-21] For example, salinosporamide A from salinospora tropica potently inhibits the 20S proteasome and exerting anti-cancer activities in experimental models;[22] Besides, taraxasterol, a natural triterpene
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extracted from taraxacum officinale possesses many pharmacological functions, including anti-inflammatory and anti-arthritic activities.[23, 24] Furthermore, several natural products have been reported to have potential in treatment of HCC, such as silymarin, wogonin and
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glycyrrhizin.[25, 26] Thus, exploration of natural products may provide a novel therapeutic strategy
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for the treatment of HCC.
Previous studies have reported that Sch B can be used for treatment of several diseases. For example, Sch B has protective function against cerebral functional defects such as dementia not only by antioxidant prevention but also exerting its potent cognitive-enhancing activity through modulation of acetylcholine level.[27] Sch B also reduces hepatic lipid contents in hypercholesterolaemic mice.[28] Besides, Sch B exerts anti-neuroinflammatory activity by inhibiting the Toll-like receptor 4-dependent MyD88/IKK/NF-κB signaling pathway in
ACCEPTED MANUSCRIPT lipopolysaccharide-induced microglia.[29] Other reports showed that Sch B has an antitumor potential in several types of cancer, including gastric cancer, gallbladder cancer and hepatic cancer.[30] Sch B inhibits the proliferation and aberrant mitosis of human gastric cancer SCG-7901
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cells in vitro through down-regulation of cyclin D1 mRNA expression, which causes cell cycle arrest.[31] Sch B also induces apoptosis and cell cycle arrest of gallbladder cancer cells.[32] A research paper by Wu et al. reported that Sch B was able to inhibit the proliferation of human
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hepatoma SMMC-7721 cells and induced apoptosis in vitro.[14] However, there is still largely
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unknown concerning the antitumor activity of Sch B on HCC cells, especially in vivo, and its underlying mechanisms. In the current study, we investigated the anti-growth and anti-metastasis effects of Sch B on HCC cells both in vitro and in vivo, suggesting that Sch B may be a promising candidate for the treatment of HCC.
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Several researches have reported that Sch B can suppress cancer cell growth, such as cholangiocarcinoma.[33] In this study, the results showed that Sch B significantly inhibited the HCC cells proliferation and induced apoptosis with a dose-dependent manner. To further
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understand the mechanism of Sch B on the HCC cell proliferation and apoptosis, the expression of
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PCNA, Caspase-3 and Bcl-2 were measured by western blot assay. PCNA expression is known to be elevated at the late G1 and S phases of proliferating cells.[34-36] Our results showed that Sch B significantly inhibited the expression of PCNA in a dose-dependent manner, indicating that the anti-growth activity of Sch B may be associated with cell cycle arrest. Both Caspase-3 and Bcl-2 play the major role in the transduction of apoptotic signals and the execution of apoptosis in mammalian, which have become recognized as key components of the apoptotic machinery.[37-39] The present data demonstrated that Sch B augmented apoptosis of cultured HCC cells in a
ACCEPTED MANUSCRIPT dose-dependent manner through upregulating Caspase-3 and downregulating Bcl-2 protein expression. Growth factors are always involved in a series of biological events which result in metastatic
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spread of tumors. Sequentially, tumor cells proliferate, loose their anchorage dependence on the extracellular matrix (ECM) and their contacts with neighboring cells, pass through the vessel wall, enter the blood stream, seed the target organ and finally form a new colony.[40-42] EGF, VEGFA
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and bFGF are identified as growth stimulators in a wide variety of events. Accumulating evidence
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is presented that these growth factors have pleiotropic effects on cell motility, chemotaxis, secretion and differentiation which in some cases correlate with metastatic potential.[43, 44] In this study, the wound-healing assay and transwell chamber assay showed that Sch B significantly suppressed HCC cell motility in a dose-dependent manner, while the western blot assay showed
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that Sch B significantly decreased the expression of EFG, VEGFA and bFGF in HCCLM3 cells, indicating that Sch B might suppress the metastasis of HCC cells. Consistent with these results, an obvious inhibition of tumor growth and the expression of VEGFA by Sch B were observed in a
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HCC xenograft mice model, further demonstrating that Sch B was able to inhibit the growth and
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metastasis of HCC cells in vivo. Taken together, our investigation suggests that Sch B effectively inhibits the growth and metastasis of HCC cells both in vitro and in vivo, providing new clues into potential treatment of HCC.
ACCEPTED MANUSCRIPT Conflict of interst The authors declare no conflict of interest.
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ACCEPTED MANUSCRIPT Figure Legends: Figure 1. Effects of Sch B on HCC cell growth. HCCLM3 were treated with various concentrations of Sch B for 48 h. (A) Cell viability was examined using the CCK-8 assay; (B)
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Flow cytometric analysis of apoptosis quantification by dual Annexin V-FITC and propidium iodide. Data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control group.
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Figure 2. Effects of Sch B on HCC cell matastasis in vitro. HCCLM3 cells were treated with Sch
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B (0, 10, 20 and 50 mg/L) for 24 h. (A) Representative micrographs of the wound-healing assay for HCCLM3 cells; (B) Representative micrographs of the transwell migration assay for HCCLM3 cells; (C) Quantification of the wound-healing assay; (D) Quantification of the transwell migration assay. Data are presented as mean ± SD of three independent experiments. *P
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< 0.05, **P < 0.01 and ***P < 0.001 compared with the control group.
Figure 3. Effects of Sch B on the expression of PCNA, Caspase-3, Bcl-2, EGF, VEGFA and Bfgf in HCC cells. HCCLM3 cells were treated with Sch B (0, 10, 20 and 50 mg/L). Data are presented
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as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared
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with the control group.
Figure 4. Effects of Sch B on tumorigenesis in a HCC xenograft BALB/c nude mice model. (A) Images of tumors after removal from the mice; (B) Tumor growth curve. Tumor volumes were measured every five days after treated with Sch B. Data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared with the untreated control group.
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ACCEPTED MANUSCRIPT Highlighs: 1. Schisandrin B significantly inhibited proliferation, induced apoptosis and suppressed metastasis of HCC cells. 2. Mechanistically, Schisandrin B regulated the protein expression associated with
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cell growth and metastasis, such as PCNA, Caspase-3, Bcl-2, EGF, VEGFA and bFGF.
3. Schisandrin B significantly inhibited the HCC xenograft model growth in BALB/c
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nude mice.
S0006-291X(17)31134-8
DOI:
10.1016/j.bbrc.2017.06.022
Reference:
YBBRC 37928
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 31 May 2017 Accepted Date: 7 June 2017
Please cite this article as: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The anti-growth and anti-metastasis effects of Schisandrin B on hepatocarcinoma cells in vitro and in vivo Ruijie Sun,1† Ruiren Zhai,2† Changlin Ma,1 Miao Wei3*
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1. Department of hepatobiliary surgery, Jining First People's Hospital, Jining, Shandong, 272000, China
2. Tumor center Shandong Sunshine Hospital, Weifang, Shandong, 261041, China
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3. Department of health care, Jining First People's Hospital, Jining, Shandong, 272000, China
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#corresponding author: Miao Wei. Department of health care, Jining First People’s Hospital, No. 6 Jiankang Road, Jining, Shandong, 272000, PR China. E-mail: [email protected] †
These authors contributed equally to this work.
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Running title: Sch B inhibits growth and metastasis of HCC
Keywords: Schisandrin B, Anti-growth, Anti-metastasis, Hepatocarcinoma Abbreviations: HCC, Hepatocarcinoma; Sch B, Schisandrin B; HBV, Hepatitis B virus; HCV,
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Hepatitis C virus
ACCEPTED MANUSCRIPT Abstract Hepatocarcinoma (HCC) is the most common liver cancer with high metastasis and recurrence rate which represents extremely poor prognosis. In this study, we aimed to investigate the effects
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of Schisandrin B (Sch B) on HCC cells both in vitro and in vivo and to explore its underlying mechanism. We found that Sch B inhibited the proliferation of HCC cells in a dose-dependent manner as assessed by CCK-8 assay. The flow cytometric assay showed that Sch B significantly
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induced HCC cell apoptosis. Besides, Sch B suppressed HCC cell metastasis as detected by both
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wound-healing assay and transwell migration assay. Mechanistically, the results of western blot indicated that Sch B inhibited cell proliferation by downregulating PCNA, induced cell apoptosis by upregulating Caspase-3 and downregulating Bcl-2, and suppressed cell mobility by downregulating EGF, VEGFA and bFGF. Moreover, Sch B significantly inhibited the HCC
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xenograft model growth in BALB/c nude mice. Taken together, our study indicated that Sch B exhibits potent antitumor activities against HCC and provided a molecular basis for Sch B
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potential applications in the treatment of HCC and other tumor-related diseases.
ACCEPTED MANUSCRIPT Introduction Liver cancer is the second leading cause of cancer-related death worldwide, and the relative 5-year survival rate for liver cancer is 16.6%.[1, 2] Hepatocarcinoma (HCC), which represents the
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predominant form of liver cancer, results in more than 670,000 deaths globally per year.[3, 4] HCC occurs mainly in the liver of patients infected by chronic hepatitis B virus (HBV) or hepatitis C virus (HCV).[5] Other risk factors including alcohol misuse, cigarette smoking and metabolic
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disorders also contribute to HCC incidence.[6] In spite of improvements in HCC therapy over the
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past decade, the prognosis of HCC remains discouraging, mainly due to the high metastasis and recurrence rate of HCC cells.[7] Therefore, there are urgently needed for novel effective strategies and drugs to improve outcomes for patients with HCC.
Schisandrin B (Sch B, C23H28O6) is a dibenzocyclooctadiene lignan isolated from fructus
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schisandrae, a traditional Chinese medicine, and Sch B has been used to treat several human diseases, including hepatitis and myocardial disorders.[8, 9] Previous studies have reported that Sch B possessed multiple functions against cancer. For example, Sch B is a novel inhibitor of
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P-glycoprotein. Overexpression of P-glycoprotein in cancer cells is the most frequent cause for 11]
Sch B also attenuates cancer invasion and metastasis via
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cancer multidrug resistance.[10,
inhibiting epithelial-mesenchymal transition.[12] Besides, Sch B inhibits ATR protein kinase activity in response to DNA damage, suggesting clinical implications in anti-cancer therapy.[13] Although previous studies have reported that Sch B induced human hepatoma SMMC-7721 cell apoptosis accompanying with down-modulation of heat shock protein 70 and up-modulation of caspase-3, the effects of Sch B on HCC cells and the underlying mechanisms of these effects have remained largely unknown.[14]
ACCEPTED MANUSCRIPT In this study, we investigated the anti-growth and anti-metastasis ability of Sch B on human HCC cell line HCCLM3 and the possible molecular mechanisms underlying these actions. In addition, a HCC xenograft nude mice model was performed to evaluate the anti-growth and
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for the potential application of Sch B in HCC.
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anti-metastasis effects of Sch B on HCC cells in vivo. These data provided experimental evidence
ACCEPTED MANUSCRIPT Materials and Methods Cell Culture Human HCC cell line HCCLM3 (with high metastatic potential) was purchased from American
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Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM (high glucose, Gibco, Rockville, MD) supplemented with 10% fetal bovine serum (FBS, Gibco, Rockville, MD) in incubator with 5% CO2 at 37°C.
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Cell Counting kit-8 Assay
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Cell Proliferation assay using the cell counting kit-8 (CCK-8; Dojindo, Japan) was performed according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plates at a density of 5000 cells per well with complete growth medium. When the cells anchored to the plates, various concentrations of Sch B (98% for HPLC, J&K, China) were added and the slides
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were incubated at 37°C with 5% CO2. Each plate had three wells with a non-treated group as control. After 48 h of culture, the growth medium was removed from each well, and then all the wells were filled with 100 ml of fresh medium containing 10 ml CCK-8 solutions. After incubated
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for 2 h at 37˚C, cell proliferation was assessed by absorbance detection at 450 nm with a
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microplate reader (Biotek, Winooski, VT). Cell Apoptosis Analysis
Cell apoptosis was analyzed after various concentrations of Sch B treatment for 48 h staining with Annexin V and PI (BD Bioscience, San Jose, CA) according to the manufacturer’s specifications. After incubated for 15 min at room temperature in the dark, the cells were analyzed by flow cytometry. Annexin V-positive and PI-negative/positive staining cells represent apoptotic cells.
ACCEPTED MANUSCRIPT Wound-Healing Assay After the cells anchored to the plates, the subconfluent cell monolayers were scraped in three parallel lines with a P-200 pipette tip. The detached cells were washed off twice gently, and the
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medium was then replaced with 1% FBS complete medium containing various concentrations of Sch B. To visualize wound healing, images were taken at 0 and 24 h. The percentage of wound closure (original width-width after cell migration/original width) was calculated.
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Transwell Migration Assay
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The 4 × 103 cells were seeded onto the upper part of a transwell chamber (Corning Costar, Rochester, NY) containing a gelatin-coated polycarbonate membrane filter (pore size: 8 mm). Culture medium with 20% FBS was added to the lower chamber to stimulate cell migration. After 24-h pretreatment of various concentrations of Sch B incubation at 37˚C with 5% CO2, the cells
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were stained with crystal violet (Sigma-Aldrich, Germany). Cells on the underside of the filters were observed under a microscope (Olympus IX71, Japan) and counted. Western blot
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RIPA buffer (Sigma-Aldrich, USA) was used to lyse cells with Complete Protease Inhibitor
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Cocktail (Roche, USA). Cell lysates were transferred to 1.5 mL tube and kept at −20°C before use. SDS-PAGE was conducted to separate the cellular proteins. Proteins were separated by 5% stacking gel and 10% running gel. The molecular weight of candidate proteins was referred to the Pre-stained SeeBlue rainbow marker (Invitrogen, Carlsbad, CA, USA) loaded in parallel. The following antibodies were used: anti-PCNA, anti-Caspase-3, anti-Bcl-2, anti-EGF, anti-VEGFA and anti-bFGF (Abcam, Cambridge, UK), anti-GAPDH (Sigma, St. Louis, MO, USA). Blots were detected using a Kodak film developer (Fujifilm, Japan).
ACCEPTED MANUSCRIPT Animal Study A total of 5 × 106 cells/0.2 ml of HCCLM3 cells were injected subcutaneously into each of ten 4-week-old male BALB/c nude mice purchased from Beijing HFK Bioscience Co. Ltd. and kept
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under sterile specific pathogen-free conditions. All experiments were approved by the Animal Care and Ethics Committee of the Jining First People’s Hospital and were in accordance with NIH animal use guidelines. After tumor cells inoculation, the mice were randomly divided into two
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groups of five mice in each group. The mice were given either Sch B (50 mg/kg) or PBS by
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intraperitoneally injecting. Tumor growth was measured every 5 days, and tumor volume was calculated using the formula, volume = 1/2 (length×width2). Twenty-five days after injection, the mice were killed and tumor weights were measured. Immunohistochemistry
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The paraffin-embedded tumor sections were stained for anti-VEGFA (Abcam, Cambridge, UK). VEGFA expression was analyzed using the standard avidin-biotin method. Sections (2 µm) were deparaffinized and pretreated with citrate buffer using a heat-induced epitope retrieval protocol.
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Endogenous peroxidase was blocked with 20% hydrogen peroxide for 15 min at room temperature
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followed by incubation with anti-VEGFA for 30 min. A biotinylated goat anti-mouse immunglobulin G secondary antibody (Dako, Denmark) was then applied to each slide for 30 min. After washing in Tris-hydrochloric acid buffer (TBS), the slides were incubated with peroxidase-conjugated streptavidin complex reagent (Dako, Denmark) and developed with 3,3’-diaminobenzidine for 5 min. The slides were counterstained and dehydrated. Statistical Analysis Experimental values were obtained from at least three independent experiments. Data are
ACCEPTED MANUSCRIPT expressed as means ± SD. Statistical analysis was performed using the Student’s t-test or one-way analysis of variance (GraphPad Prism; GraphPad Software Inc., La Jolla, CA), where appropriate. The Bonferroni post hoc test was used to determine the source of observed differences. P values <
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0.05 were considered significant.
ACCEPTED MANUSCRIPT Results Sch B inhibits the growth of HCC cells To investigate the effect of Sch B on human HCC cells, HCCLM3 cells were treated with Sch B
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at various concentrations for 48 h followed by detection of cell viability. The results showed that Sch B dose-dependently inhibited the proliferation of HCCLM3 cells (P < 0.05, Fig. 1A).
To further elucidate whether the inhibition of cell growth was partially caused by apoptosis, the
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flow cytometric was performed. As shown in Figure 1B, the figure for apoptotic cells were
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significantly increased in a dose-dependent manner after Sch B treatment compared with the untreated group. These results suggest that Sch B inhibits proliferation and promotes apoptosis in HCC cells.
Sch B suppresses the migration and invation of HCC cells
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To investigate the effect of Sch B on the metastatic capability of HCCLM3 cells, the wound-healing assay and transwell chamber assay were carried out. As shown in Figure 2A and C, the wound-healing assay exhibited that the closing rates of scratch wounds were significantly
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decreased by Sch B in a dose-dependent manner compared with the control group (P < 0.05). The
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transwell assay also showed that the number of migratory cells in the Sch B treated groups were obviously decreased in a dose-dependent manner compared with the control group (P < 0.05, Fig. 2B and D). These results implied that Sch B effectively inhibited the motility of HCC cells. Effects of Sch B on expression of PCNA, Caspase-3, Bcl-2, EGF, VEGFA and bFGF To better understand the influence of Sch B on the HCC cell proliferation and apoptosis, the expression of proliferating cell nuclear antigen (PCNA), Caspase-3 and Bcl-2 were evaluated by western blot analysis. Compared with the control group, treatment with Sch B significantly
ACCEPTED MANUSCRIPT decreased the expression of PCNA and Bcl-2 and increased the expression of Caspase-3 in a dose-dependent manner (P < 0.05, Fig. 3A ~ D). Furthermore, the expression of the angiogenic growth factors epidermal growth factor (EGF), vascular endothelial growth factor A (VEGFA) and
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base fibroblast growth factor (bFGF) were examined by western blot assay to investigate the effects of Sch B on HCC cell metastasis. These results showed that the expression levels of all three factors were decreased in a dose-dependent manner compared with the control group (P <
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0.01, Fig. 3E ~ H).
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Sch B inhibits tumor growth in vivo
The anticancer effect of Sch B in vivo was further analyzed in a HCCLM3 tumor xenograft model. As shown in Figure 4A and B, there was a remarkable reduction in tumor weight in mice treated with Sch B at the dose of 50 mg/kg compared with the control mice (P < 0.05). Also, the result of
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immunohistochemistry showed that Sch B significantly decreased the VEGFA expression in tumor tissues compared with the untreated group (Fig. 4C). These results demonstrated that Sch B might
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effectively inhibit HCC cell growth and metastasis in vivo.
ACCEPTED MANUSCRIPT Discussion HCC is one of the leading causes of cancer-related death, becoming a major health problem worldwide. Most patients with HCC experience a recurrence after resection or are diagnosed at
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advanced stages.[7, 15, 16] Despite the introduction of novel therapies has improved patient survival, current common therapeutic strategies for HCC largely rely on surgery, transplantation, use of radiofrequency and transarterial embolization. The overall prognosis of HCC still remains 18]
Thus, continuous investigation of the novel therapeutic strategy for this
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disappointing.[17,
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disease is of great significance. Recently, many bioactive pharmaceutical drugs derived from different natural resources have been identified as effective agent for several diseases.[19-21] For example, salinosporamide A from salinospora tropica potently inhibits the 20S proteasome and exerting anti-cancer activities in experimental models;[22] Besides, taraxasterol, a natural triterpene
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extracted from taraxacum officinale possesses many pharmacological functions, including anti-inflammatory and anti-arthritic activities.[23, 24] Furthermore, several natural products have been reported to have potential in treatment of HCC, such as silymarin, wogonin and
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glycyrrhizin.[25, 26] Thus, exploration of natural products may provide a novel therapeutic strategy
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for the treatment of HCC.
Previous studies have reported that Sch B can be used for treatment of several diseases. For example, Sch B has protective function against cerebral functional defects such as dementia not only by antioxidant prevention but also exerting its potent cognitive-enhancing activity through modulation of acetylcholine level.[27] Sch B also reduces hepatic lipid contents in hypercholesterolaemic mice.[28] Besides, Sch B exerts anti-neuroinflammatory activity by inhibiting the Toll-like receptor 4-dependent MyD88/IKK/NF-κB signaling pathway in
ACCEPTED MANUSCRIPT lipopolysaccharide-induced microglia.[29] Other reports showed that Sch B has an antitumor potential in several types of cancer, including gastric cancer, gallbladder cancer and hepatic cancer.[30] Sch B inhibits the proliferation and aberrant mitosis of human gastric cancer SCG-7901
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cells in vitro through down-regulation of cyclin D1 mRNA expression, which causes cell cycle arrest.[31] Sch B also induces apoptosis and cell cycle arrest of gallbladder cancer cells.[32] A research paper by Wu et al. reported that Sch B was able to inhibit the proliferation of human
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hepatoma SMMC-7721 cells and induced apoptosis in vitro.[14] However, there is still largely
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unknown concerning the antitumor activity of Sch B on HCC cells, especially in vivo, and its underlying mechanisms. In the current study, we investigated the anti-growth and anti-metastasis effects of Sch B on HCC cells both in vitro and in vivo, suggesting that Sch B may be a promising candidate for the treatment of HCC.
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Several researches have reported that Sch B can suppress cancer cell growth, such as cholangiocarcinoma.[33] In this study, the results showed that Sch B significantly inhibited the HCC cells proliferation and induced apoptosis with a dose-dependent manner. To further
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understand the mechanism of Sch B on the HCC cell proliferation and apoptosis, the expression of
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PCNA, Caspase-3 and Bcl-2 were measured by western blot assay. PCNA expression is known to be elevated at the late G1 and S phases of proliferating cells.[34-36] Our results showed that Sch B significantly inhibited the expression of PCNA in a dose-dependent manner, indicating that the anti-growth activity of Sch B may be associated with cell cycle arrest. Both Caspase-3 and Bcl-2 play the major role in the transduction of apoptotic signals and the execution of apoptosis in mammalian, which have become recognized as key components of the apoptotic machinery.[37-39] The present data demonstrated that Sch B augmented apoptosis of cultured HCC cells in a
ACCEPTED MANUSCRIPT dose-dependent manner through upregulating Caspase-3 and downregulating Bcl-2 protein expression. Growth factors are always involved in a series of biological events which result in metastatic
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spread of tumors. Sequentially, tumor cells proliferate, loose their anchorage dependence on the extracellular matrix (ECM) and their contacts with neighboring cells, pass through the vessel wall, enter the blood stream, seed the target organ and finally form a new colony.[40-42] EGF, VEGFA
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and bFGF are identified as growth stimulators in a wide variety of events. Accumulating evidence
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is presented that these growth factors have pleiotropic effects on cell motility, chemotaxis, secretion and differentiation which in some cases correlate with metastatic potential.[43, 44] In this study, the wound-healing assay and transwell chamber assay showed that Sch B significantly suppressed HCC cell motility in a dose-dependent manner, while the western blot assay showed
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that Sch B significantly decreased the expression of EFG, VEGFA and bFGF in HCCLM3 cells, indicating that Sch B might suppress the metastasis of HCC cells. Consistent with these results, an obvious inhibition of tumor growth and the expression of VEGFA by Sch B were observed in a
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HCC xenograft mice model, further demonstrating that Sch B was able to inhibit the growth and
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metastasis of HCC cells in vivo. Taken together, our investigation suggests that Sch B effectively inhibits the growth and metastasis of HCC cells both in vitro and in vivo, providing new clues into potential treatment of HCC.
ACCEPTED MANUSCRIPT Conflict of interst The authors declare no conflict of interest.
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ACCEPTED MANUSCRIPT Figure Legends: Figure 1. Effects of Sch B on HCC cell growth. HCCLM3 were treated with various concentrations of Sch B for 48 h. (A) Cell viability was examined using the CCK-8 assay; (B)
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Flow cytometric analysis of apoptosis quantification by dual Annexin V-FITC and propidium iodide. Data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control group.
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Figure 2. Effects of Sch B on HCC cell matastasis in vitro. HCCLM3 cells were treated with Sch
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B (0, 10, 20 and 50 mg/L) for 24 h. (A) Representative micrographs of the wound-healing assay for HCCLM3 cells; (B) Representative micrographs of the transwell migration assay for HCCLM3 cells; (C) Quantification of the wound-healing assay; (D) Quantification of the transwell migration assay. Data are presented as mean ± SD of three independent experiments. *P
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< 0.05, **P < 0.01 and ***P < 0.001 compared with the control group.
Figure 3. Effects of Sch B on the expression of PCNA, Caspase-3, Bcl-2, EGF, VEGFA and Bfgf in HCC cells. HCCLM3 cells were treated with Sch B (0, 10, 20 and 50 mg/L). Data are presented
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as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared
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with the control group.
Figure 4. Effects of Sch B on tumorigenesis in a HCC xenograft BALB/c nude mice model. (A) Images of tumors after removal from the mice; (B) Tumor growth curve. Tumor volumes were measured every five days after treated with Sch B. Data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared with the untreated control group.
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT Highlighs: 1. Schisandrin B significantly inhibited proliferation, induced apoptosis and suppressed metastasis of HCC cells. 2. Mechanistically, Schisandrin B regulated the protein expression associated with
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cell growth and metastasis, such as PCNA, Caspase-3, Bcl-2, EGF, VEGFA and bFGF.
3. Schisandrin B significantly inhibited the HCC xenograft model growth in BALB/c
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nude mice.