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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 190-195
Inhibiting valosin-containing protein suppresses osteosarcoma cell metastasis via AKT/nuclear factor of kappa B signaling pathway in vitro


1 Department of Orthopedics, First Affiliated Hospital of Nanchang University, Nanchang, 330006, P. R. China
2 Department of Pathology, Cancer Hospital of Jiangxi Province, Nanchang, 330006, P. R. China

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Date of Web Publication24-Oct-2013
 

   Abstract 

Background and Aim: The strategies of targeting valosin-containing protein (VCP) may have therapeutic potential for treating cancer metastasis. In this study, we aim to investigate the correlation of VCP protein expression in osteosarcoma (OS) tissues with pulmonary metastasis and its possible molecular mechanism. Materials and Methods: Expression of VCP in 60 OS specimens was detected by immunohistochemistry (IHC) and the relationship with metastasis was analyzed. An artificial micro ribonucleic acid, targeting VCP, was performed to silence the expression of VCP in U2-OS cells. Cell mobility was detected by wound healing and Transwell assays. Western blot and real-time polymerase chain reaction were performed to investigate the expression of VCP in U2-OS cells. Furthermore, the protein of pAKT (phosphorylated serine/threonine protein kinase) and nuclear factor of kappa B protein65 were measured by western blot to evaluate the effect of silencing VCP on AKT/nuclear factor of kappa B (NF-kB) signaling pathway. Results: The results showed that the expression level of VCP protein in cases with pulmonary metastases was significantly higher than that in those without metastasis (P = 0.004). The invasion and migration of U2-OS cells were suppressed by silencing VCP. Furthermore, silencing VCP could down-regulate the phosphorylation of AKT and nuclear transfer of NF-kB. Conclusions: Our findings suggested that inhibition of VCP could suppress OS cells invasion and migration through down-regulating AKT/NF-kB signaling pathway.

Keywords: AKT/nuclear factor of kappa B, immunohistochemistry, metastasis, osteosarcoma, valosin-containing protein

How to cite this article:
Long XH, Zhang ZH, Liu ZL, Huang SH, Luo QF. Inhibiting valosin-containing protein suppresses osteosarcoma cell metastasis via AKT/nuclear factor of kappa B signaling pathway in vitro . Indian J Pathol Microbiol 2013;56:190-5

How to cite this URL:
Long XH, Zhang ZH, Liu ZL, Huang SH, Luo QF. Inhibiting valosin-containing protein suppresses osteosarcoma cell metastasis via AKT/nuclear factor of kappa B signaling pathway in vitro . Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Jun 4];56:190-5. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/190/120358



   Introduction Top


Osteosarcoma (OS) is one of the most common primary malignant bone tumor in childhood and adolescents. It was not until the early 1970's that the introduction of doxorubicin and methotrexate with leucovorin rescue showed promise to improve the survival. When the effective chemotherapy advent, the 5-year survival rate for patients treated with intensive multidrug chemotherapy and aggressive local control has been reported at 55-80%. [1],[2],[3] However, despite the encouraging trend to longer survival, many patients still face a dismal outcome. Numerous articles have reported that the 5-year survival rate of patients with metastatic diseases was <20%. [4],[5],[6] The development of lung metastasis is the main cause of death in patients with OS. Obviously, identification the molecular mechanism of metastasis is significantly important on OS management.

Valosin-containing protein (VCP, also known as p97), which is highly conserved, is a member of the AAA family proteins, involving in a wide variety of cellular functions, such as ubiquitin/proteasome-dependent protein degradation, membrane fusion, cell cycle regulation and endoplasmic reticulum-associated degradation. Recent studies showed that the high levels of VCP expression in cancer cells correlated with the increase of recurrence as well as poor prognosis of patients with cancer of the liver, stomach, prostate, colorectum, esophagus and lung. [7],[8],[9],[10] Recent study revealed that VCP may be contributed to metastasis of pancreas ductal adenocarcinoma. [11] However, it is unclear yet whether VCP is involved in oseosarcoma metastasis and what the molecular mechanism is.

Now, it has been confirmed that matrix metalloproteinases (MMPs) are obbligato in degradation of basement membrane and epimatrix, among which MMP-2 and MMP-9 are mostly correlated with tumor invasion and metastasis. MMP-2 and MMP-9 over-express in OS and promote OS cells migration and invasion by degrading some components of basement membrane and epimatrix. A large number of studies indicated that activation and transposition of nuclear factor of kappa B (NF-κB) gene, an upstream gene of MMPs, are closely related to tumor invasion and migration. [12],[13] And now it has been confirmed that phosphorylation and activation of AKT is an important regulative factor in gene transcription, which NF-κB depend upon and is essential for inhibitor of Kappa B (IκB) degradation and NF-κB activation mediated by inhibitor of Kappa B kinase complexes (IKKs). However, whether VCP functions in AKT/NF-κB signaling pathway in OS metastasis has not been reported.

This study was to investigate the role VCP act in pulmonary metastasis of OS and the molecular mechanism involved in. A potential strategy for OS therapy was wished to be found.


   Materials and Methods Top


Patient specimens

A total of 60 cases of OS samples were obtained from patients who underwent surgery in our hospital from 2005 to 2011. The pulmonary metastasis survey was performed with plain films and chest computed tomography scans at first diagnosis. All patients do not have a history of prior therapies with anticancer drugs or radiotherapy. The samples were fixed with 10% formalin and embedded in paraffin and then, were cut into 2 μm sections. In all cases, consent was taken from the Institute Ethics Committee.

Immunohistochemistry (IHC) analysis

Immunoperoxidase procedure (S-P procedure) and hematoxylin and eosin (H and E) staining were performed on paraffin-embedded sections. Antigen retrieval was performed with heating the sections in 10 mmol/L citrate buffer (pH 6.0) for 20 min. A rabbit anti-VCP polyclonal antibody (Santa Cruz, USA) was used as the primary antibody at a final dilution of 1:800. For negative controls (NC), sections were incubated with phosphate buffered saline (PBS) instead of the primary antibodies. The sections were chemiluminescence stained and counterstained using hematoxylin. Positive staining in endothelial cells in the non-cancerous areas in each specimen was used as internal positive control (PC). Stained sections were evaluated in a blind manner without prior knowledge of the clinical pathological features of patients. Staining intensity in the cytoplasm of the tumor cells was shown in comparison to that of endothelial cells and categorized as follows: Weaker (level 1), equal (level 2) to or stronger (level 3) than that of endothelial cells. When staining intensity of the tumor cells was different among areas in the same specimen, the predominant pattern was chosen. Cases with negative endothelial cell staining were regarded as having poor antigen preservation and were excluded from further analysis.

Cell culture and transfection

The human OS cell line U2-OS was cultured in HAM'S/F-12 medium, containing 10% fetal bovine serum (FBS), 1% antibiotics (penicillin and streptomycin), at 37°C in a humidified air atmosphere containing 5% CO 2 . U2-OS cells were seeded in six-well plates at 30% confluence on the day before transfection. Transfection with recombinant plasmid (a micro ribonucleic acid [miRNA] vector), which targeting VCP gene or negative plasmid was performed using lipofectamine 2,000 reagent (Invitrogen, USA). Transfection complexes were prepared according to the manufacturer's instructions (Invitrogen, USA).

Real-time polymerase chain reaction (RT-PCR)

RT-PCR was used to detect the level of VCP messenger RNA (mRNA). The cellular total RNA was extracted with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The concentration of total RNA was determined by spectrophotometry at 260 nm and the purity was determined by the 260/280 ratio with a BioPhotometer (Eppendorf, Hamburg, Germany). RT-PCR was routinely performed with the Two Steps kit (Promega, USA) according to the manual instruction to obtain complementary deoxyribonucleic acid , which was then used as the template for amplification. The quantity of each target was normalized against the quantity of β-actin. Primers were designed with Primer Premier 5.0 software, using Basic Local Alignment search tool to search human genome database to ensure that the sequences target mRNA specifically. VCP forward primer 5'-GCCTTGAATGAAGTAGGGTAT-3' and VCP reverse primer 5'-GTTGGGTCTG TTGGTTGC-3', 466 bp. βcactin forward primer 5'-GTGGACATCCGCAAAGAC-3' and reverse primer 5'-GAAAGGGTGTAACGCAACT-3', 303 bp. After amplification, deoxyribonucleic acid (DNA) electrophoresis was performed on standard 1% agarose gels. And DNA was labeled with ethidium bromide and images were acquired using Canon DIGITAL IXUS 900Ti. The expression level of VCP mRNA in cells transfected by recombinant plasmid (VCP-Knockdown group, KD) was compared with normal U2-OS cells (not transfected by any plasmid, PC group, PC) and those cells transfected by negative plasmid (NC group, NC). All experiments were repeated by 6 times.

Western blot

Total protein from the cells was extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer containing 60 μg/ml Phenylmethanesulfonyl fluoride (PMSF). Protein concentration was determined by Bradford assay. Equal amounts of protein were electrophoresed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a pure nitrocellulose blotting membrane (0.22 um). Membranes were blocked with 5% difco skim milk for 1 h at room temperature, then blocked with primary antibodies (polyclonal rabbit anti-VCP immunoglobulin [IgG], 1:200; rabbit anti-pAKT IgG, 1:1000; rabbit anti-nuclear factor of kappa B protein65 [NF-κBp65] IgG; 1:1000; goat anti-AKT IgG, 1:1000; goat anti-MMP9 IgG, 1:1000; monoclonal mouse anti-GAPDH, 1:5000, Santa Cruz, USA) for one night at 4°C. Membranes were washed before incubated with appropriate peroxidase-conjugated secondary antibodies (anti-rabbit, anti-goat and anti-mouse, 1:2000, ZSGB, China). The immune complexes were detected with pro-light HRP kit (TIAN GEN, China). The protein expression level in cells transfected by recombinant plasmid (a miRNA vector) was compared with those in cells transfected by negative plasmid and normal U2-OS cells. All experiments were repeated by 6 times.

Migration assay

The cell migration was assessed with "wound healing assay" determining the ability of the cells to move into a cellular space in a two-dimensional in vitro. In brief, cells were grown to confluence in 6-well tissue culture plastic dishes to a density of approximately 5 × 10 6 cells/well. The cells were denuded by dragging a rubber policeman (Fisher Scientific, Hampton, NH, USA) through the center of the plate. Cultures were rinsed with PBS and replaced with fresh quiescent medium alone or containing 10% FBS, following which the cells were incubated at 37°C for 24 h. Photographs were taken at 0 and 24 h and the migrated distance was measured by Image J National Center for Biotechnology Information (NCBI). The cells migration rate was obtained by counting three fields per area and represented as the average of six independent experiments performed over multiple days. The migration rate of cell transfected by recombinant plasmid (a miRNA vector) which targeting VCP gene, was compared with those cells transfected by negative plasmid and normal U2-OS cells. All experiments were repeated 6 times.

Transwell invasion assay

Invasion of U2-OS cells was measured using the BD BioCoat TM BD Matrigel TM Invasion Chamber (BD Bioscience, NJ, U S A) according to the manufacturer's protocol. The medium in the lower chamber contained 5% fetal calf serum as a source of chemoattractants. Cells were suspended in serum-free medium and added to the upper chambers at the same time. Cells that passed through the Matrigel-coated membrane were stained with Diff-Quik (Sysmex, Kobe, Japan) and photographed (×400). Photographs were taken at 24 h and cell counting was measured by Image J (NCBI). The values for the invasion were obtained by counting three fields per memberane and represented as the average of six independent experiments performed over multiple days. The number of invasion cell tranfected by recombinant plasmid (a miRNA vector), which targeting VCP gene, was compared with those cell transfected by negative plasmid and normal U2-OS cells. All experiments were repeated 6 times.

Statistical analysis

All measurement data were expressed as the χ– ± standard deviation The difference of cells invasion and migration between inhibition VCP and control group were evaluated by one-way t-test. The count data was analyzed using non-parametric Two-Independent-Samples test. Value of P < 0.05 was considered statistically significant. All analyses were performed using the Statistical Package for Social Sciences (SPSS) version 13.0 (SPSS, Chicago, IL).


   Results Top


Correlation of VCP expression levels in OS tissues with pulmonary metastasis

Hematoxylin-eosin staining for OS tissues with or without pulmonary metastasis all showed that OS is cell-rich and has significant cellular atypia, ansionucleosi, prominent nucleoli, abundant cytoplasm and a small amount of bone-like matrix [Figure 1]a and b. IHC staining for VCP in OS tissues without pulmonary metastasis showed that the staining intensity in the cytoplasm of the tumor cells are weaker (level 1) than that of endothelial cells [Figure 1]c while these are stronger (level 3) than endothelial cells in OS tissues with lung metastases [Figure 1]d. The expression level of VCP in OS samples with pulmonary metastasis is higher than that in those without pulmonary metastatic disease (P = 0.004) [Table 1].
Figure 1: Valosin-containing protein (VCP) expression in osteosarcoma (OS) with and without pulmonary metastasis (×400). Representative images of hematoxylin-eosin staining for OS tissues without (a) and
with (b) pulmonary metastasis and immunohistochemistry staining for VCP in OS tissues without (c) and with (d) pulmonary metastasis


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Table 1: The relationship of VCP protein expression levels in OS tissues to pulmonary metastasis

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VCP was down regulated by the recombinant plasmid which targeting VCP gene

An artificial miRNA, targeting VCP gene, was performed to silence VCP in vitro. VCP mRNA and protein were measured by RT-PCR and western blot. The results showed that VCP mRNA and protein were down-regulated by the recombinant plasmid [Figure 2]a and b.
Figure 2: The inhibitory effect of recombinant plasmid on valosin-containing protein (VCP) messenger ribonucleic acid (a) and protein (b) expression. Columns, mean (n = 6); bars, standard deviation; *P < 0.05 versus negative control (NC) and positive control (PC) group. (NC, NC group; PC, PC group; KD, VCP-Knockdown group)

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Silencing VCP inhibits migration of U2-OS cells in vitro

To investigate the effect of silencing VCP on U2-OS cell migration, the U2-OS cells were transfected with recombinant plasmid targeting VCP gene. Wound healing assay was performed to measure the migration of U2-OS cells. The results of Wound healing assay revealed that the migration rate (44 ± 13.3%) in cell transfected by the recombinant plasmid were significantly lower than those of cells transfected with negative plasmid (74 ± 16.5%) and normal U2-OS cells (74 ± 23.4%) (P < 0.05) [Figure 3]a.
Figure 3: The effect of inhibiting valosin-containing protein on osteosarcoma cell migration and invasion in vitro. A representative image of six experiments of wound healing (a) and Transwell invasion assay (b) is shown for each group. Columns, mean (n = 6); bars, standard deviation; *P < 0.05 versus negative control and positive control group

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Silencing VCP inhibits invasion of U2-OS cells in vitro

To investigate the effect of silencing VCP on U2-OS cell invasion, the U2-OS cells were transfected with recombinant plasmid targeting VCP gene. The results of Transwell invasion assay revealed that the number of transmembrane cells (19 ± 4.0 piece) in cell transfected by the recombinant plasmid were lower significant than those of cells transfected with negative plasmid (45 ± 8.9 piece) and normal U2-OS cells (55 ± 5.6 piece) (P < 0.05) [Figure 3]b.

The effect of silencing VCP on the phosphorylation of AKT

To investigate the effect of inhibition VCP on the phosphorylation of AKT, the expression level of pAKT protein in U2-OS cell was measured using western blot. The results showed that the pAKT protein expression level in cell transfected by recombinant plasmid was significant lower than in those cells transfected with negative plasmid and normal U2-OS cells [Figure 4].
Figure 4: Inhibiting valosin-containing protein down-regulate phosphorylati on of AKT and the expression of matrix metalloproteinases-9 and nuclear factor of kappa B protein65 in U2-osteosarcoma cell. (Columns, mean [n = 6]; bars, standard deviation. *P < 0.05, **P < 0.05, #P < 0.05, vs. positive control and negative control group)

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The inhibition of VCP suppresses nuclear transfer of NF-κB

To investigate the effect of inhibition VCP on nuclear transfer of NF-κB, the protein expression level of NF-κBp65 and MMP9 was detected. It found that NF-κBp65 and MMP9 protein were decreased significantly in cells transfected by recombinant plasmid for 24 h when compared with that in cells transfected by negative plasmid and normal U2-OS cells [Figure 4].


   Discussion Top


OS is the most common pediatric primary bone tumor. Its early metastasis resulted in low cure rate and high mortality. OS metastasis involved in local invasion, access to the circulation, seeding and eventual proliferation within a favorable distant target organ. Several genes including cyclooxygenase-2, COP9 constitutive photomorphogenic homolog subunit 3, fatty acid synthase, MMP2 and MMP9 have been recently reported to be involved in the course of OS metastasis. [14],[15],[16] However, the process and mechanism of metastasis still remains incompletely characterized. Clearly, identification of molecular mechanisms of OS cell metastasis is very important for management of patients with OS.

VCP is a member of the AAA family proteins, which plays various important roles in cells by using its ATPase activity. In the early studies, VCP is known to be involved in the ubiquitin/proteasome degradation pathway, which works in both the up-regulation of cell proliferation and the down-regulation of cell death in human cancer cells. Recent studies revealed that VCP may be contributed to cancer cell metastasis. [11] In this study, the expression levels of VCP protein in OS tissues was detected by IHC and the correlation of VCP expression levels with pulmonary metastasis was analyzed. The results revealed that the expression levels of VCP in OS tissues with pulmonary metastasis was higher than in those tissues without pulmonary metastasis (P = 0.004). It indicated that inhibiting VCP may be a treatment strategy for treating OS metastasis.

To confirm that VCP involve in OS metastasis, we performed wound healing invasion assay and Transwell migration assay in vitro to investigate the effect of inhibiting VCP expression on U2-OS cells mobility. The recombinant plasmid of miRNA, targeting VCP, was constructed for inhibiting VCP in OS cell. The inhibitory effect was evaluated by RT-PCR and western and the results showed that VCP mRNA and protein expression were significant inhibited by the recombinant plasmid. The wound healing assay and Transwell assay showed that the migrated rate and invasion cell of U2-OS cells transfected with recombinant plasmid was significant lower than those cells transfected with negative recombinant plasmid. The result is similar to Asai et al. reported. [17] The results suggested that inhibition VCP could suppress invasion and migration of OS cells in vitro.

What is the molecular mechanism of silence VCP inhibit OS cell migration and invasion? Chiho Mori-Konya et al. [18] report that VCP is highly modified to regulate its molecular function via acetylation and phosphorylation. Recent study showed that inhibiting VCP (p97) suppress OS cell migration and invasion through down-regulation NF-kappaB. [17] AKT is essential for NF-κB activation by stimulation of the IKK complex, which phosphorylates IκB. [19] Franck Vandermoere showed that VCP as an essential target of AKT signaling. [20] Confocal microscopy indicated a co-localization between AKT and VCP upon AKT stimulation. Interestingly, in this study, we found that a lower expression level of pAKT protein in those cells transfected by recombinant plasmid, targeting VCP gene, than that in cells transfected by negative plasmid and normal U2-OS cells were observed. It suggested that inhibiting VCP could down-regulate phosphorylation of AKT. NF-κB is composed of DNA-binding subunits (p50 and p52) and subunits with transcriptional activity (p65 and RelB or c-Rel), which dimerize in various combinations. The primary form of NF-κB is a heterodimer of the p50 and p65 subunits and is localized mainly in the cytoplasm in an inactive form bound to an inhibitory protein termed IκB. [21],[22],[23] It has been shown previously that NF-κB up-regulates MMP-9. [12],[24] For the development of metastases, the cancerous cells must degrade the components of the extracellular matrix (ECM). A significant role in cancerous progression was quickly allotted to the MMPs, because of their capacity to degrade ECM thus supporting tumor invasion. Among all MMPs, MMP-2 and MMP-9 are recognized to be particularly involved in the degradation of ECM components. [25],[26] In present study, western blot was also performed to evaluate the expression level of NF-κBp65, MMP-9 protein. The results showed that NF-κBp65, MMP-9 have a lower expression level in cells inhibited VCP than in those cell transfected with negative plasmid and normal U2-OS cells. It indicated that VCP inhibition reduced the nuclear translocation of NF-κB, attenuated the activation on MMP-9 expression. The result is similar to previous reported. [17]

In summary, our results demonstrate that silencing VCP could suppress invasion and migration abilities in OS cells through down-regulating AKT/NF-κB pathway in vitro. However, previous studies showed that tumor microenviroment might influence tumor progression, invasion and cell migration. So, furthermore experiments in vivo are necessary to clear that targeting VCP and AKT/NF-κB pathway may be a potential therapeutic strategy for treating OS metastases.

 
   References Top

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Correspondence Address:
Zhi Li Liu
Department of Orthopedics, First Affiliated Hospital of Nanchang University, Yong Wai Zheng Street 17, Nanchang, Jiangxi, 330006, P. R. China

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.120358

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