|Ahead of print publication
Up-regulation of Wnt5a inhibits proliferation and migration of hepatocellular carcinoma cells
Tianxiao Wang1, Xiaohong Liu2, Jun Wang3
1 School of Medicine and Life Science, University of Jinan-Shandong Academy of Medical Sciences, Jinan, China
2 Department of Pathology, No. 960 Hospital of People' Liberation Army, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, China
3 Department of Oncology, No. 960 Hospital of People' Liberation Army; Department of Cancer Immunotherapy, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, China
No. 25, Shifan Street, Tianqiao District, Jinan
Source of Support: None, Conflict of Interest: None
Objectives: Increasing evidence suggests that Wnt5a plays an important role in tumorigenesis. In particular, its expression is downregulated in hepatocellular carcinoma (HCC). The aim of this study was to explore the effect of Wnt5a overexpression on HCC cells.
Materials and Methods: We transfected the human HCC cell line SMMC-7721 with pcDNA3.1-Wnt5a overexpression vectors or empty pcDNA3.1 vectors. The expression of Wnt5a in transfected SMMC-7721 cells was confirmed by the western blot. Cell proliferation was examined by the colony formation test and cell cycle assay in vitro. The effect of Wnt5a overexpression on cell migration was studied using a scratch assay. In vivo tumorigenesis was assessed using a mouse xenograft model.
Results: Wnt5a overexpression inhibited SMMC-7721 cell proliferation with a significant reduction in S-phase cells and an enrichment of G1-phase cells, a lower colony formation rate, and decreased tumor volumes in the xenograft model compared with those that of control tumors. The in vitro scratch assay revealed that Wnt5a overexpression diminished the capacity of cell migration, which may be mediated by the change in phosphorylated β-catenin and E-cadherin expression.
Conclusion: Wnt5a may act as a tumor suppressor in HCC, partly through the β-catenin/E-cadherin signaling pathway.
Keywords: Hepatocellular carcinoma, migration, proliferation, SMMC-7721, Wnt5a
| > introduction|| |
For decades, hepatocellular carcinoma (HCC) is one of the most frequently occurring tumors worldwide. It is most often developed in cirrhotic livers, and risk factors include chronic infection by hepatitis B and C viruses and nonviral liver diseases., Unfortunately, the cellular mechanisms of hepatocarcinogenesis remain poorly understood. Recent advances have shown that apart from autocrine stimulation by growth factors such as insulin-like growth factor-II and transforming growth factor-α, the dysregulation of at least four growth regulatory pathways is frequently involved in hepatocarcinogenesis., Components of these signaling pathways include retinoblastoma protein, transforming growth factor-β, tumor protein p53, and the wingless-type murine mammary tumor virus integration site family (Wnt). These pathways also interfere with each other at various levels.,,
The Wnt family of genes encodes a large and diverse group of signaling molecules involved in the patterning, proliferation, and differentiation of a variety of organs and cell types., The Wnt ligand binds to its receptor Frizzled and the low-density lipoprotein receptor-related proteins 5 and 6 to activate the canonical Wnt/β-catenin signaling pathway, or functions through β-catenin-independent (noncanonical) pathways, which include the planar cell polarity and Wnt/Ca2+ pathways. Wnt ligands are typically classified into canonical and noncanonical Wnts by the pathways through which they work.,, The Wnt member 5a (Wnt5a) is one of the most highly investigated noncanonical Wnts and has been implicated in almost all aspects of noncanonical Wnt signaling.,, In terms of cancer developmental research, Wnt5a has lived in the shadow of its better-characterized relatives. This is large because of its apparent inability to transform cells or signals through the canonical β-catenin pathway, which is important in cancer.,,, Recent work on a wide variety of human tumors has indicated that Wnt5a plays a critical role in malignant progression, but there is conflicting evidence as to whether it is tumor-promoting or tumor-suppressing.,,,,,
We have previously shown that Wnt5a exhibited a tumor-suppressing effect in HCC, which was probably associated with hepatitis B viral infection.,, The purpose of this study was to further explore the role of Wnt5a in HCC by applying a Wnt5a expression vector to induce Wnt5 overexpression in the human HCC cell line SMMC-7721.
| > Materials And Methods|| |
Cell line and cultures
The human HCC cell lines SMMC-7721 was purchased from the Shanghai Institutes for Biological Sciences (China). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C with 5% CO2.
Wnt5a-expressing plasmids were constructed by subcloning human Wnt5a cDNA into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). To establish stable clones, transfected cells were selected with G418 (Invitrogen-Gibco) or blasticidin (Merck, Darmstadt, Germany). After selection for 2–3 weeks, single colonies were isolated and screened for Wnt5a expression by the western blot.
Cells were lysed with lysis buffer containing a protease inhibitor mixture (Roche Applied Science, Mannheim, Germany) on ice for 30 min. Proteins were separated with 10% sodium dodecyl sulphate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were probed with specific antibodies and reactive proteins were detected using enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sources and concentrations of the antibodies used were as follows: polyclonal anti-Wnt5a (LS-C47384, Lifespan, 1:1000), anti-phosphorylated (P-)β-catenin (sc-57535, Santa Cruz Biotechnology, 1:1000), and anti-E-cadherin (H-108, Santa Cruz Biotechnology, 1:1000). Experiments were performed three times.
Cell cycle analysis
SMMC-7721 cells were collected with trypsin/ethylenediaminetetraacetic acid (EDTA), washed with fluorescence-activated cell sorting buffer composed of phosphate-buffered saline (PBS), 2 mM EDTA, and 0.5% bovine serum albumin, and stained with propidium iodide at 0.05 mg/mL. The cells were washed and fixed with 1% paraformaldehyde, and the fluorescence of 10,000 cells was quantified using a FACSCalibur with CellQuest software (BD Biosciences, Pharmingen).
Colony formation assay
Control and Wnt5a-transfected SMMC-7721 cells were trypsinized, counted, and seeded into six-well plates at a density of 500 cells per well in RPMI 1640 serum-free medium. After 15 days, the cells were washed with PBS, fixed in 10% methanol for 15 min, and subjected to Giemsa staining for 10 min. Visualized colonies were then photographed and scored. Each experiment was repeated at least three times.
Cell migration assay
Transfected SMMC-7721 cells were seeded onto fibronectin-coated six-well plates in RPMI 1640 medium-containing 0.5% FBS. Cells were starved for 24 h prior to scratch assays, then they were scratched using a sterile pipette tip, washed twice, and incubated in serum-free medium. The extent of scratch closure was quantified by measuring the area of the scratch before and 24 h after migration and the results are expressed as the percentage of wound closure.
Xenograft studies in nude mice
Four-week-old male nude BALB/c mice were housed in a sterile environment. The animals were maintained under specific-pathogen-free conditions in the Animal Laboratory Unit of the General Hospital of Jinan Military Command. All animals used in this study were handled in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Bioethics Committee of the General Hospital of Jinan Military Command. SMMC-7721 cells (5 × 106), which were transfected with the pcDNA3.1-Wnt5a or control vector, were injected into the anterior subcutaneous tissue of the hind leg of the experimental mice (n = 3 per group). After 25 days, the mice were sacrificed, and all tumors were excised, photographed, and measured. Tumor volume (V) was calculated using the formula V = (A × B 2)/2, where A is the largest diameter and B is the smallest diameter of the tumor.
All statistical analyses were carried out using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). If not otherwise stated, the data are represented as the mean of at least three independent experiments ± standard deviation. Statistically significances between the control and experimental group were calculated by the independent t-test and a value of P < 0.05 was considered statistically significant.
| > Results|| |
Effect of Wnt5a overexpression on the cell cycle of SMMC-7721 hepatocellular carcinoma cells
The SMMC-7721 HCC cell line was selected for this study based on its low baseline Wnt5a protein expression levels. SMMC-7721 cells were transfected with plasmid vectors capable of constitutively driving the expression of Wnt5a (SMMC-7721/Wnt5a) or an empty vector (SMMC-7721/pcDNA3.1) serving as a control. The expression of Wnt5a was measured by the western blot to assess whether transfection with the pcDNA3.1-Wnt5a expression vector was successful [Figure 1]a.
|Figure 1: Effect of Wnt5a overexpression on the cell cycle progression, colony-forming potential, and motility of SMMC-7721 cells. (a) Expression of Wnt5a in SMMC-7721 cells. The overexpression of Wnt5a in SMMC-7721/Wnt5a cells indicated successful transfection. β-actin detection confirmed equal loading. (Wnt5: SMMC-7721/Wnt5a; Control: SMMC-7721/pcDNA3.1). (b) Flow cytometric analysis of cell cycle progression. (c) Effect of Wnt5a on clonogenicity of SMMC-7721 cells. SMMC-7721/Wnt5a cells displayed lower clonogenicity than that of SMMC-7721/pcDNA3.1 cells after 25 days of culture. (d) The motility of SMMC-7721/pcDNA3.1 and SMMC-7721/Wnt5a cells was determined by the wound migration assay. The spreading of SMMC-7721/Wnt5a cells along the edges of the wound was significantly decreased compared with those that of SMMC-7721/pcDNA3.1 cells after 48 h|
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To examine the mechanism underlying the effect of Wnt5a on SMMC-7721 cell proliferation, we evaluated the cell cycle in control and Wnt5a-overexpressing cells by flow cytometry. As shown in [Figure 2], the proportion of SMMC-7721/Wnt5a cells in the G1 phase was significantly increased relative to that of SMMC-7721/pcDNA3.1 cells (P = 0.003). Conversely, the proportion of SMMC-7721/Wnt5a cells in the S phase was significantly decreased relative to that of SMMC-7721/pcDNA3.1 cells (P = 0.001). This result showed that cell cycle progression from the G0/G1 to S phase was suppressed in SMMC-7721/Wnt5a cells, compared with those that of the control group, and a blockade in the G1 phase was observed in cells transfected with Wnt5a expression vectors [Figure 1]b.
|Figure 2: Xenograft studies in nude mice and effect of key mediator expression. (a) Nude mice injected with SMMC-7721/Wnt5a cells showed decreased tumor volumes compared with those that of mice injected with SMMC-7721/pcDNA3.1 cells. (b) The expression of P-β-catenin and E-cadherin was upregulated in SMMC-7721/Wnt5a cells|
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Effect of Wnt5a overexpression on the colony-forming potential of SMMC-7721 cells
To investigate whether Wnt5a expression affected the colony formation ability of SMMC-7721 cells, the same number of SMMC-7721/Wnt5a and SMMC-7721/pcDNA3.1 cells were seeded at a low density in six-well plates (1000 cells per well). After 15 days, the existing colonies were visualized and counted microscopically [Figure 1]c. The number and size of colonies formed by SMMC-7721/Wnt5a cells were lower than those of SMMC-7721/pcDNA3.1 colonies. Statistical analysis revealed that the colony formation rate of SMMC-7721/Wnt5a cells was 5.12% ± 1.67%, which was significantly lower than that of 12.33% ± 1.44% observed for SMMC-7721/pcDNA3.1 cells (T = −25.112; P < 0.01).
Effect of Wnt5a expression on the motility of SMMC-7721 cells
We examined whether Wnt5a could regulate cell motility by performing anin vitro wound healing assay [Figure 1]d. We observed that 48 h after a scratch was made in the SMMC-7721 cell monolayer, control cells had migrated into the scratch zone and the boundary area became unclear. In contrast, the scratch area was still visible in cultures-containing SMMC-7721/Wn5a cells. These results confirmed that Wnt5a overexpression resulted in a decrease in the migration capacity of SMMC-7721 cells.
Xenograft studies in nude mice
To test the tumor-suppressing efficiency of Wnt5a overexpression in vivo, we established a xenograft model in nude mice through subcutaneous transplantation of SMMC-7721/Wnt5a and SMMC-7721/pcDNA3.1 cells. As shown in [Figure 2]a, the tumor volume was decreased in nude mice injected with SMMC-7721/Wnt5a cells compared with that in mice injected with control cells (P = 0.003).
Western blot of Wnt5a-overexpressing SMMC-7721 cells
The protein expression levels of Wnt5a, P-β-catenin, and E-cadherin were assessed in control and Wnt5a-overexpressing SMMC-7721 cells by the western blot [Figure 2]b. The protein expression levels of P-β-catenin and E-cadherin were also increased in SMMC-7721/Wnt5a cells compare with those in the control cells. All western blots were performed three times.
| > Discussion|| |
Our results demonstrated that the overexpression of Wnt5a in SMMC-7721 cells decreased cell proliferation, and the phenomenon was associated with the blockade of cell cycle progression at the G1 phase. Consistent with this finding, the colony-forming potential of Wnt5a-overexpressing SMMC-7721 cells was diminished as compared to that of control cells. In agreement with ourin vitro data, xenografting Wnt5a-overexpressing SMMC-7721 cells into nude mice yielded tumors with smaller volumes than that from control cells, which confirmed our hypothesis that Wnt5a acts as a tumor-suppressing gene in HCC.
Recent studies have indicated that the up-regulation of Wnt5a was associated with tumor invasiveness and metastasis in metastatic melanoma, gastric cancer, and nonsmall-cell lung cancer.,, Wnt5a was expressed predominantly in the metastatic but not primary lesions of metastatic melanoma. It has also been reported that Wnt5a knockdown in human colon cancer cells reduced directional migration, deregulated focal adhesion site formation, and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Ourin vitro scratch assay showed that SMMC-7721/Wnt5a cells had a decreased wound-healing capacity, indicating that Wnt5a overexpression decreased the motility of SMMC-7721 cells. These observations suggested that the complex Wnt5a-regulated signaling pathways and the functional role of Wnt5a depend on cellular and stimulus factors during the development of HCC.
As a highly evolutionary conserved noncanonical Wnt ligand, Wnt5A encoded by the human WNT5A gene has been shown to contribute to tumor suppression as well as oncogenic signaling. However, the role Wnt5A plays is related to the availability of key receptors and intercellular interactions among different cancer type. For example, several downstream signaling pathways involving tumor cell migration and invasion can be tightly controlled by Wnt5A in breast cancer and HCC. Therefore, restoring WNT5A signaling is viewed as an innovative therapeutic option., β-catenin is recognized as the key mediator of the canonical Wnt signal pathway and also binds to E-cadherin. Together, they contribute to the process of cell adhesion and migration in many tumor types., The loss of E-cadherin expression and disassembly of the E-cadherin/catenin complex on the cell surface induce the transition from a stationary to a motile phenotype and enable tumor cells to disseminate and metastasize. Previous studies have shown that in pancreatic cancer cells, Wnt5A/c-Jun N-terminal kinase signaling promoted the mRNA expression of vimentin but decreased that of E-cadherin, suggesting its regulatory effects on epithelial-mesenchymal transition (EMT). Kanzawa et al. reported that Wnt5a regulated the induction of EMT and the maintenance of cancer stem cell properties in MKN-7 cells. The present study showed that SMMC-7721/Wnt5a cells exhibited elevated levels of P-β-catenin and E-cadherin, which were associated with the decreased migration of these cells. We hypothesize that the inhibition of abnormal cell adhesion and migration processes by Wnt5a may be mediated by the β-catenin/E-cadherin pathway.
| > Conclusion|| |
In summary, our experiments demonstrated that Wnt5a affects the biological behavior of SMMC-7721 HCC cells. Specifically, increased Wnt5a expression decreased the proliferative and migratory capacity of this HCC cell line. We presumed that Wnt5a might be a potential therapeutic target for the inhibition of HCC progression.
Financial support and sponsorship
This study was funded in part by the Shandong Provincial Nature Science Foundation grant number ZR2010HQ038 and ZR2010HM05.
Conflicts of interest
There are no conflicts of interest.
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