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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 1  |  Page : 119-123

Lipopolysaccharide promoted the growth rate of MG-63 cells via the extracellular regulated kinase pathway


Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei, PR China

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Hao Peng
Department of Orthopedics, Renmin Hospital of Wuhan University, 99 Zhangzhidong Road, Wuhan, Hubei 430060
PR China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_673_17

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 > Abstract 

Objective: The objective of this study is to investigate the effects of lipopolysaccharide (LPS) on the growth rate of MG-63 cells.
Materials and Methods: MG-63 cells were exposed to various concentrations of LPS, and the growth rate was determined with the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, variations in the phosphorylation state of extracellular regulated kinase (ERK1/2), Jun N-terminal kinase, and p38 were determined by western blotting.
Results: There was a correlation between the increased phosphorylation of ERK1/2 and the growth of LPS-treated MG-63 cells. The increased phosphorylation was mediated by the LPS receptor toll-like receptor-4.
Conclusions: LPS promoted the growth of MG-63 cells through the ERK pathway.

Keywords: Extracellular regulated kinase, lipopolysaccharide, mitogen-activated protein kinase, MG-63, osteosarcoma


How to cite this article:
Niu X, Peng H. Lipopolysaccharide promoted the growth rate of MG-63 cells via the extracellular regulated kinase pathway. J Can Res Ther 2018;14:119-23

How to cite this URL:
Niu X, Peng H. Lipopolysaccharide promoted the growth rate of MG-63 cells via the extracellular regulated kinase pathway. J Can Res Ther [serial online] 2018 [cited 2021 Jun 24];14:119-23. Available from: https://www.cancerjournal.net/text.asp?2018/14/1/119/226750


 > Introduction Top


The survival rate of patients with osteosarcoma has been significantly improved with the advent of many effective chemotherapeutic drugs since the late 1970s.[1],[2] Meanwhile, limb salvage surgery has gradually become the mainstay treatment for osteosarcoma because of its functional and physiological benefits over traditional amputation procedures.[3] Historically, there were several attempts to improve contiguous joint function and decrease prosthesis-related complications by means of intercalary resection. It aimed to preserve a part or all of the joint surface.[4],[5],[6] Furthermore, intercalary resection may result in superior joint function and longer survival because normal articular cartilage and ligamentous stability was preserved.[7] However, many orthopedic oncologists were reluctant to perform joint-saving procedures for patients with tumor breaking through the epiphyseal growth plate because of unreliable margin. There would be a high risk of local recurrence and the catastrophic oncologic outcomes in the contaminated margin.[8]

Lipopolysaccharide (LPS) or endotoxin was a major component of the cell wall in Gram-negative bacteria.[9] Under normal physiological conditions, LPS could not be detectable in the circulatory system. However, a detectable amount (~1.0 ng/ml) was usually present in the portal venous circulation. To understand its regulatory mechanisms, LPS has been widely involved in several experimental models.[10],[11] For example, LPS was a potent stimulator of inflammation, which was associated with numerous aspects of chronic inflammation, such as altering cytokine levels. It was resulted from the stimulation of inflammatory cells in the tumor microenvironment.[12] LPS exposure could also lead to carcinogenesis, induce tumor cell proliferation and survival, facilitate invasion and metastasis, and promote angiogenesis.[12]

In general, osteosarcoma was observed in knee joints, which were usually infected with bacteria, and LPS was found to be involved.[13],[14] In this study, the effects of LPS on the MG-63 osteosarcoma cell line were investigated. In addition, the involvement of the mitogen-activated protein kinase (MAPK) signaling cascade in the LPS-mediated effects was also explored.


 > Materials and Methods Top


Unless otherwise specified, all chemicals and reagents in this study were purchased from the Sigma Chemical Company (St. Louis, MO, USA). LPS was purchased from Chemicon International (Temecula, CA, USA). Antibodies to IgG, GAPDH, extracellular regulated kinase (ERK1/2), phospho-ERK1/2, Jun N-terminal kinase (JNK), phospho-JNK, p38, and phospho-p38 were purchased from Abcam (Cambridge, MA, USA). The MG-63 cell line (ScienCell, Carlsbad, CA, USA) was cultured at 37°C in humidified air with 5% CO2.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

MG-63 cells were cultured in 96-well plates (1 × 103 cells/well) in media supplemented with leptin. Control cells were switched from RPMI-1640 medium to Dulbecco's Modified Eagle's Medium containing 0.1% dimethyl sulfoxide (DMSO). The cells were treated with LPS (0, 10, 20, and 40 ng/mL) once daily for 9 days, then 20 μL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (final concentration, 0.5%) was added to each well. After incubating for 4 h at 37°C in the dark, 150 μL of DMSO was added to each well and incubated for 10 min to dissolve the formazan crystals. The absorbance in each well was measured with a model EXL800 microplate reader (Cole-Parmer, Vernon Hills, IL, USA) at 490 nm. This experiment was repeated for three independent times. The viability of the LPS-treated cells was expressed as a percentage of the growth population plus/minus the standard error of the mean relative to that of the untransfected control cells. The cell growth rate was calculated as follows: Growth % = (mean experimental absorbance-mean control absorbance/mean control absorbance) ×100.

Immunofluorescent staining

MG-63 cells were fixed in 3.7% of paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min, and blocked with 1% bovine serum albumin and 10% goat serum prepared in PBS overnight at 4°C. The samples were incubated with primary antibodies diluted in PBS. Then the binding primary antibody was detected with an Alexa Fluor 488 goat anti-rabbit IgG (H + L) secondary antibody. Fluorescent images were captured with a Nikon A1 confocal microscope (Tokyo, Japan), and immunofluorescent staining was performed in triplicate.

Western blotting

MG-63 cell lysates were electrophoresed with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for immunoblotting. The membranes were blocked and then incubated with primary antibodies overnight at 4°C. The membranes were subsequently washed 3 times with PBS, incubated with species-compatible peroxidase-conjugated secondary antibodies. Immunoreactive bands were developed with ECL reagents (Pierce, Rockford, IL, USA).

Statistical analysis

Statistically significant differences in gene expression levels between treatment groups were determined with one-way analysis of variance, followed by the Newman–Keuls post hoc test. GraphPad Prism version 5 software (GraphPad Software, La Jolla, CA, USA, www.graphpad.com/company/) was applied for statistical analysis. Replicates were included in the statistical model. Differences were considered statistically significant at the 95% confidence level (P< 0.05). Data were presented as mean ± standard deviation.


 > Results Top


Determination of the optimal lipopolysaccharide concentration that maximally stimulated MG-63 cell growth

MG-63 cells were exposed to various concentrations of LPS (0, 10, 20, and 40 ng/mL) for 0, 24, 48, and 72 h to determine the effects of LPS on cell growth. The cell growth was optimal with LPS of 20 ng/mL at 48 h [Figure 1].
Figure 1: Growth percentage after treating MG-63 cells with increasing concentrations (0, 10, 20, and 40 ng/mL) of lipopolysaccharide for 0, 24, 48, and 72 h

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Lipopolysaccharide promoted MG-63 cell growth through the extracellular regulated kinase 1/2 pathway

MG-63 cells were treated with LPS at 20 ng/mL, and the phosphorylation status of MAPKs (ERK1/2, JNK, and p38) was examined at 0, 2, 4, and 8 h after treatment and the signaling pathway through which LPS acted was identified. The phosphorylated ERK1/2 level was higher than that of JNK and p38 at 4 h [Figure 2]a and [Figure 2]b. This result suggested that ERK1/2 may play a key role in the stimulation ability of LPS on MG-63 cell growth. To verify this, U0126 (a specific inhibitor of ERK1/2 phosphorylation) was applied to suppress ERK1/2 phosphorylation. U0126 was found to effectively inhibit the phosphorylated ERK1/2 level [Figure 3]a and [Figure 3]b. In the presence of U0126, MG-63 cell growth was not promoted with LPS (20 ng/mL) [Figure 3]c.
Figure 2: Effects of lipopolysaccharide at a concentration of 20 mg/mL on the activation of mitogen-activated protein kinases in MG-63 cells. (a) Relative mRNA expression levels of total and phosphorylated extracellular regulated kinase, Jun N-terminal kinase, and p38. (b) Relative protein expression levels of total and phosphorylated extracellular regulated kinase, Jun N-terminal kinase, and p38 by Western blotting. Results were presented as the mean ± standard deviation (n = 5). *Significantly different from the control (P < 0.05)

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Figure 3: Effects of mitogen-activated protein kinase after extracellular regulated kinase 1/2 was inhibited with U0126. (a) Relative protein expression levels of total and phosphorylated extracellular regulated kinase, Jun N-terminal kinase, and p38. (b) Relative protein expression levels of total and phosphorylated extracellular regulated kinase, Jun N-terminal kinase, and p38 by Western blotting. Results are presented as the mean ± standard deviation (n = 5). (c) Growth percentage of MG-63 cells. *Significantly different from the control (P < 0.05)

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The lipopolysaccharide receptor toll-like receptor-4 in silenced MG-63 cells

The LPS receptor, toll-like receptor (TLR-4), was detected with immunocytochemistry [Figure 4]a and [Figure 4]b. The shRNA was efficiently transfected into MG-63 cells [Figure 4]c. This receptor was silenced to confirm that LPS functioned through TLR-4. The phosphorylated ERK1/2 level was lower in silenced cells than that of in control cells without significant difference [Figure 5]a and [Figure 5]b. The growth rate of MG-63 cells at 0, 24, 48, and 72 h was lower in the shRNA group than that of in the control group [Figure 5]c.
Figure 4: The expression of toll-like receptor-4 in cultured synovial MG-63 cells. (a) Toll-like receptor-4 expression in MG-63 cells. (b) Toll-like receptor-4 expression in MG-63 cells of the negative group (the primary antibody was replaced by phosphate-buffered saline and the secondary antibody was added). (c) Toll-like receptor-4 expression in MG-63 cells after toll-like receptor-4 silencing by RNA interference

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Figure 5: Effects of toll-like receptor-4 silencing by RNA interference on mitogen-activated protein kinase activation in MG-63 cells. (a) Relative mRNA expression levels of total and phosphorylated extracellular regulated kinase 1/2, Jun N-terminal kinase, and p38. (b) Relative protein expression levels of total and phosphorylated extracellular regulated kinase, Jun N-terminal kinase, and p38 by Western blotting. Results were presented as the mean ± standard deviation (n = 5). (c) Growth percentage of MG-63 cells after 0, 24, 48, and 72 h (20 ng/mL lipopolysaccharide vs. toll-like receptor-4 siRNA + 20 ng/mL lipopolysaccharide)

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 > Discussion Top


Osteosarcoma, one of the most common type of primary bone cancers, was a malignant neoplasm notorious for its high aggressiveness and early metastatic potential.[15],[16],[17] However, little has been known about the signaling pathways that are crucial for its progression, as well as the molecular mechanisms of osteosarcoma. Histologically, osteosarcoma can be categorized into several different types, including osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, and giant cell-rich osteosarcoma.[18] In this study, the MG-63 osteosarcoma cell line was employed as the model. LPS has been the main component of the outer cell wall of Gram-negative bacteria, which was the most biologically active compound.[19] For example, LPS exposure could lead to carcinogenesis, as well as inducing tumor cell proliferation and survival.[20] In this study, the result showed that the rate of MG-63 cell growth could be enhanced with LPS.

This study showed that LPS increased the rate of MG-63 cell growth through the ERK1/2 pathway. The optimal concentration of LPS was 20 ng/mL. This study also showed that the effects of LPS were made through its receptor, TLR-4, which was expressed at high levels in MG-63 cells. Experiments in which the TLR-4 shRNA blocked LPS-mediated growth provided further evidence for the roles of this receptor in cellular processes.

To investigate the mechanism on the promotion effects from LPS on MG-63 cell growth, the signaling pathways downstream of TLR-4 was investigated. Extracellular signals were converted into specific cellular responses by MAPK signaling pathways, through a cascade of phosphorylation events.[21] Thus, precise regulation of these signaling pathways would be essential for normal cell functions, and their deregulation lead to various diseases, including tumorigenesis. Our results indicated that MG-63 cell growth would be promoted by LPS through its receptor TLR-4, through the ERK1/2 signaling pathway. Moreover, several studies also showed that the TLR-4 was promoted through the ERK1/2 signaling pathway, for example, Coriolus versicolor mushroom polysaccharides made effects on TLR-4 to activate the ERK1/2 signaling pathways.[22]


 > Conclusions Top


In summary, our results provided evidence that the LPS signaling pathway promoted MG-63 cell growth mainly through an ERK1/2-dependent pathway. Thus, our findings suggested that the LPS–TLR-4 axis might be useful in inhibiting the proliferation of MG-63 cells. Further studies are necessary before any potential clinical applications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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