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JNK pathway
Molecular Immunology 68 (2015) 671–683
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Toll-like receptor 4 signaling promotes invasion of hepatocellular carcinoma cells through MKK4/JNK pathway Yu-Qing Dong a,b,1 , Chuan-Wei Lu a,1 , Lu Zhang a , Jia Yang a , Waqaar Hameed a , Wei Chen a,∗ a b
Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou 310058, China Department of Clinical Laboratory, the Chinese Medicine Hospital of Hangzhou, Hangzhou 310007, China
a r t i c l e
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Article history: Received 15 March 2015 Received in revised form 21 October 2015 Accepted 22 October 2015 Available online 14 November 2015 Keywords: Toll-like receptor 4 Invasion Hepatocellular carcinoma Jun N-terminal kinase Lipopolysaccharide
a b s t r a c t Toll-like receptor (TLR) 4-mediated signaling has been shown to be important to cell survival, invasion and metastasis in a variety of cancers. The present study aimed to explore the role and downstream pathways of TLR4 signaling in the invasion of hepatocellular carcinoma (HCC) cell lines. We found that LPS, the agonist of TLR4, notably enhanced the invasiveness of HCC cells and the expression of MMP2 and MMP9, as well as the production of IL-6 and TNF␣. LPS treatment dramatically increased the TLR4 expression on HCC cells surface and MKK4/JNK activation, while knockdown of TLR4 inhibited the LPSinduced invasion and the phosphorylation of MKK4 and JNK. Furthermore, silencing of MKK4 or inhibition of JNK activity led to impaired invasiveness of HCCs, low expression level of MMPs and TLR4, as well as limited production of cytokines. However, LPS stimulation only triggered moderate activation of NF-кB. Silencing of NF-кB or NF-кB inhibitor had no obvious effect on the invasive ability of HCCs and TLR4 expression, but suppressed IL-6 and TNF␣ production. These findings suggested that LPS-TLR4 signaling enhanced the invasiveness of HCCs mainly through MKK4/JNK pathway. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chronic inflammation is a major contributor to carcinogenesis and metastasis of tumors (Elinav et al., 2013; Landskron et al., 2014). In patients with chronic liver inflammation, the endotoxin/lipopolysaccharide (LPS) level in the portal and peripheral veins is high due to increased translocation of intestinal bacteria (Dapito et al., 2012; Pidgeon et al., 1999). Elimination of gut-derived bacteria by antibiotics results in significant reduction of hepatocellular carcinoma (HCC) development (Dapito et al., 2012; Iwasaki and Medzhitov, 2004). Toll-like receptor 4 (TLR4), the receptor of LPS, plays a critical role in the development and progression of
Abbreviations: TLR, toll-like receptor; LPS, lipopolysaccharide; HCC, hepatocellular carcinoma; EMT, epithelial-mesenchymal transition; MMP, matrixmetalloproteinase; MAPK, mitogen-activated protein kinase; MKK4, MAP kinase kinase4; JNK, jun N-terminal kinase; ERK, extracellular signal-regulated kinase; IB, inhibitor of NF-kappa B; NF-кB, nuclear factor kappa B; siRNA, small interfer RNA; qRT-PCR, quantitative RT-PCR. ∗ Corresponding author at: Institute of Immunology, Zhejiang University School of Medicine, 388 Yu Hang Tang Rd., Hangzhou 310058, China. Fax: +86 571 88208285. E-mail addresses: [email protected], [email protected] (W. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2015.10.015 0161-5890/© 2015 Elsevier Ltd. All rights reserved.
various human cancers, including colorectal, breast, lung, prostate, pancreatic, head and neck cancer (Ikebe et al., 2009; Kundu et al., 2008; Li et al., 2014a; Ren et al., 2014; Santini et al., 2008; Wang et al., 2013; Yang et al., 2014). In liver cancer, increasing evidence supports the role of TLR4 signaling in the migration and invasion of most HCC cell lines (Dapito et al., 2012; Jing et al., 2012; Liu et al., 2010; Li et al., 2014b). Clinical evidence has also shown that high expression of TLR4 in HCC tissues is strongly associated with poor prognosis due to a high level of metastasis (Jing et al., 2012; Liu et al., 2015). However, the molecular mechanism of TLR4 signaling for the invasion of HCC cells remains unclear. Some evidence supports the idea that NF-KB is a key mediator for TLR4 signaling (Wang et al., 2013; Hartwell et al., 2014; Yu et al., 2013). In contradiction to this opinion, there are suggestions that LPS may promote hepatoma cell invasion through other downstream pathways rather than NF-KB (Liu et al., 2010; Li et al., 2014b). Here, we reported that LPS-TLR4 signaling increased invasive potential of HCC lines, as well as MMPs and inflammatory cytokines production. Importantly, this pro-invasion effect of LPS was mainly mediated by the TLR4/MKK4/JNK signal pathway, but not the NF-KB pathway.
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Fig. 1. Invasion of HCC cell lines and MMPs production in response to LPS. (A) Matrigel invasion assay was performed to observe the changes in HCC cell lines invasion. Photomicrographs of cells that have passed through membrane were taken 48 h after different doses of LPS treatment (200× magnification). The numbers of invading cells through the matrigel were counted in ten fields under the microscope. All data are shown as the means ± SD, *P < 0.05 vs. the control group. Results are representative of three separate experiments. Left panel shows a representative field of HCC cells before and after 1 g/ml LPS treatment. (B) The invasion of HCC cells was assessed by scratch wound-healing assay and was compared between untreated cells (control, upper panel) and cells treated with 1 g/ml LPS (low panel). Cells were monitored for 48 h to evaluate the rate of migration into the scratched area. (C) The mRNA level of MMP2 and MMP9 were analyzed by real-time PCR in three HCC cell lines. Left panel: MMPs mRNA level in HepG2 cells were set as “one fold” and compared the basal mRNA level of these HCC cell lines without LPS treatment. Right panel: HCC cells were treated with
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2. Materials and methods 2.1. Chemicals and reagents LPS (L4391), SP600125 (S5567), PD98059 (P215) and SB203580 (S8307), were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bay 11-7082 (S2913) was obtained from Selleckchem (USA). LPS was dissolved in PBS, whereas SP600125, Bay 11-7082, PD98059 and SB203580 were dissolved in DMSO. PE-TLR4 antibody (124891) was purchased from Biolegend. Antibodies specific to TLR4 (sc10741), p-MKK4 (sc-7990), MKK4 (sc-964), MMP-2 (sc-13595), MMP-9 (sc-21733) and -actin (sc-47778) were obtained from Santa Cruz Biotechnology. Antibodies specific to p-JNK (9251), JNK (9252), p-ERK1/2 (4376), ERK1/2 (9102), p-p38 (9215), p38 (9212), p-IB␣ (2859) and NF-B (8242) were obtained from Cell Signaling Technology. 2.2. Cell culture and transfection Human hepatocellular cell lines HepG2, SMMC7721 and Hep3B were purchased from the American Type Culture Collection (ATCC) (Washington, DC, USA). HepG2 and SMMC7721 cells were cultured in RPMI-1640 (Gibco, USA) medium supplied with 10% fetal bovine serum (FBS, Gibco), 100 units/ml of penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA), then incubated at 37 ◦ C in a humidified 5% CO2 atmosphere. Hep3B cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% FBS. Cells were transfected with siRNAs or/and plasmids with JetPEI (Polyplus) according to the manufacturer’s instructions. 2.3. Small interfering RNA and plasmids TLR4 siRNA (sc-40260), NF-кB (sc-29410) and control siRNA (sc-108060) were purchased from Santa Cruz Biotechnology. The MKK4-specific siRNA (mixture) sequences were 5 -GGACUUGAAAGACCUUGGATT 3 (sense), 5 -UCCAAGGUCUUUCAAGUCCTC3 (antisense) and 5 -GCCGUAUAUGGCACCUGAATT-3 (sense), 5 UUCAGGUGCCAUAUACGGCTT-3 (antisense). The scrambled control RNA sequences were described previously (Wu et al., 2014). Knockdown of TLR4, NF-кB and MKK4 were confirmed by Western Blot. To rule out siRNA off-target, we employed mouse targeting sequences to construct siRNA-resistant plasmid for re-expression. The DNA sequences encoding mouse TLR4, NF-кB and MKK4 were amplified by PCR and ligated into pcDNA3.1 plasmid to generate eukaryotic expression plasmids. The primer sequences were as follow: mouse TLR4 forward primer 5 -CGATTCTAGAACAAAACCAA3 , reverse primer 5 -CTGGAAAGGAAGGTGTCA-3 ; mouse NF-кB p65/RelA forward primer 5 -ACCCTGACCATGGACGATC-3 , reverse primer 5 -CTCCGAAAGCGAGATAAAGA- 3 ; mouse MKK4 forward primer 5 -ACTTCCAACAATGGCGGCTC-3 , reverse primer 5 -TTGCTTCCCCTCAGCCCTTT- 3 .
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and cells were washed twice with complete media to remove detached cells, followed by the acquisition of the original image of the wound under a microscope. The LPS group was added with 1 g/ml LPS. Dishes were then put back in an incubator and photographs were taken at 48 h after wounding. The difference between the widths of the wounds is taken as the migration distance. 2.5. Transwell invasion assay Cell migration in vitro was determined using 6.5 mm Transwell chambers with 8 mm pores (Corning, NY, USA). Transwell filters were coated with matrigel on the upper surface of the polycarbonic membrane and the matrigel solidified after 1 h of incubation at 37 ◦ C. Cells (5 × 104 cells) were harvested in 100 l of serum free RPMI-1640 medium and added to the upper compartment of the chamber. For LPS group, 1 g/ml LPS was added to both upper and lower part of the chambers. Cells were allowed to migrate for 48 h. Uninvaded cells in the upper chamber were removed with cotton swabs and invaded cells were fixed with 100% methanol for 5 min, followed with staining with DAPI for 15 min. The number of cells was counted from five randomly selected visual fields, using an inverted microscope at 200× magnification. 2.6. MTT assay Cell proliferation was assessed by the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. HepG2, SMMC7721 and Hep3B cells were seeded in a 96-well plate at a density of 1 × 104 cells/well. After incubated with or without 1 g/ml LPS for 12 h, 24 h and 48 h, MTT (20 l) was added into wells and incubated for an additional 4 h. Cells were pelleted and lysed in 150 L of DMSO, and the absorbance of each well was determined at 490 nm. 2.7. Quantitative real-time PCR Total RNA was extracted using Trizol according to the manufacturer’s protocol. Real-time quantitative PCR, using SYBR Green detection chemistry (Takara), was performed on a 7500 real-time PCR system (Applied Biosystems) as we described previously (Lai et al., 2013). The following primers were used: murine TLR4, 5 -AAGCCGAAAGGTGATTGTTG3 (forward) and 5 -CTGAGCAGGGTCTTCTCCAC-3 (reverse); murine MMP-2, 5 -TGATCTTGACCAGAATACCATCGA-3 (forward) and 5 -GGCTTGCGAGGGAAGAAGTT-3 (reverse); MMP-9, 5 -TGGGGGGCAACTCGGC-3 (forward) murine and 5 -GGAATGATCTAAGCCCAG-3 (reverse); murine 5 -GCACCACACCTTCTACAATGA-3 (forward) and actin, 5 -TGTCACGCACGATTTCCC-3 (reverse). All reactions were performed in triplicate. For data analysis, the relative levels of the target gene mRNA transcripts to the -actin were calculated by 2−Ct and statistical significance was determined by the one way ANOVA using SPSS (version 10.0).
2.4. Scratch wound healing assay 2.8. Western blot analysis HepG2, SMMC7721 and Hep3B cells were seeded at a density of 2 × 106 cells/well in a 6-well plate and cultured overnight. A wound was created by scratching with a 250-l pipette tip,
After treatment, the cells were washed twice with cold PBS and lysed with cell lysis buffer (Cell Signaling Technology) sup-
1 g/ml LPS for indicated time. MMPs mRNA level in untreated cells was set as “one fold” for each cell lines. Data are from three independent experiments (means ± SD). Statistical analysis was done by comparison with untreated cells. *P < 0.05, **P < 0.01. (D) The protein level of MMP2 and MMP9 in HCC cells treated with 1 g/ml LPS for 48 h was detected by Western Blot. The relative levels of MMP2 and MMP9 were analyzed by densimetric analysis using ImageJ software. Data are expressed as the means ± SD from three separate experiments. Statistical analysis was done by comparison with untreated cells. *P < 0.05, **P < 0.01 vs. the untreated group.
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plemented with PMSF (Beyotime Institute of Biotechnology). To detect the nuclear translocation of NF-кB, we isolated nuclear fraction from LPS-treated cells using NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA) according to the manufacturer’s protocols. Protein concentrations were determined using
a BCA protein assay kit (Pierce). The lysates were loaded onto SDS-PAGE gels and transferred onto nitrocellulose membranes and then incubated with antibodies as described previously (Wu et al., 2014). Protein signals were detected by ECL reagent (Thermo Fisher).
Fig. 2. LPS enhanced TLR4 expression and downstream pathways activation. (A) The mRNA level of TLR4 were analyzed by real-time PCR in three HCC cell lines. Left panel: TLR4 mRNA level in HepG2 cells was set as “one fold” and compared the basal mRNA level of these HCC cell lines without LPS treatment. Right panel: HCC cells were treated with 1 g/ml LPS for indicated time. TLR4 mRNA level in untreated cells was set as “one fold” for each cell lines. Each bar represented triplicate analyses of mean ± SD. **P < 0.01 vs. the untreated group. (B) TLR4 expression on the surface of these HCC cells was determined by FACS analysis using mean fluorescence intensity as a readout. HCC cells were treated with LPS for 36 h, then fixed and stained cells with PE-TLR4 Ab. (C) Three HCC cells were treated with 1 g/ml LPS for indicated time, and the phosphorylation of IкB were examined by Western Blot. Cytoplasmic and nuclear fractions were extracted from HCC cells treated with LPS and the translocation of NF-кB was detected. Histone H3 as a loading control for nuclear NF-кB. Quantitative analysis of the relative levels of IкB and NF-кB from three separate experiments was shown as the means ± SD. *P < 0.05 vs. the untreated cells. (D) Western blot and quantitative analysis of the relative levels of MKK4 and JNK in HCC cells after LPS treatment. *P < 0.05, **P < 0.01 vs. the untreated cells.
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Fig. 2. (Continued).
2.9. Flow cytometry The expression of TLR4 protein was measured by flow cytometry. HCC cells (5 × 105 ) were treated with 1 g/ml LPS for 36 h. Cells were collected and washed three times with PBS followed by incubation with Rat serum for 10 min to block nonspecific binding of TLR4 antibody. Subsequently, cells were washed for three times and incubated with 3 l human PE-TLR4 antibody in 50 l PBS for 30 min. Cells were washed again and analyzed by flow cytometry. The control was performed by using isotype-IgG antibody. For apoptosis analysis, HCC cells were collected and stained with Annexin V and 7-AAD, then analyzed by flow cytometry. For quantification, Annexin V-positive cell fraction was considered as apoptotic cell fraction. 2.10. ELISA After stimulation with LPS at the indicated time points, the cell supernatants were collected and analyzed using ELISA kits of IL-6 and TNF␣ (R&D company) according to the manufacturer’s protocols. 2.11. Statistical analysis Data are expressed as means ± standard deviation (SD). Statistical significance was determined by two-tailed Student’s t test and
one way ANOVA using SPSS (version 10.0). Differences resulting in P values less than 0.05 were considered statistically significant. 3. Results 3.1. LPS enhanced the invasion of HCC cell lines and MMPs production To evaluate the impact of LPS on cellular invasion, we choose three HCC cell lines with different invasive abilities, including HepG2, SMMC7721 and Hep3B cells. Different doses of LPS (0.01, 0.1, 1 and 10 g/ml) were used to stimulate these cell lines and matrigel invasion assay was performed. We found that treatment of 1 and 10 g/ml LPS significantly enhanced the invasive ability of three HCC cells compared with the LPS-untreated cells (Fig. 1A). However, 10 g/ml LPS stimulation significantly increased the apoptosis of HCC cells, so we used 1 g/ml LPS as the proper dose in our system (Fig. S1). As shown in Fig. 1A, HepG2 was the most invasive cell line among these three cell lines, and the invasiveness of Hep3B cells was very low. However, some study showed LPS had no effect on invasion of HepG2 cells (Jing et al., 2012; Wang et al., 2013). Similarly, the results of scratch wound-healing assay showed that HepG2 cells treated with 1 g/ml LPS were capable of closing the wound completely within 48 h, whereas cells without LPS treatment showed significantly slower rates of wound-healing (Fig. 1B). LPS treatment also triggered higher wound-healing abil-
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ity in SMMC7721 and Hep3B cells compared with that in untreated cells (Fig. 1B). These data indicated LPS significantly promoted the invasive abilities of these HCC cell lines. To determine whether other factors may affect the number of HCC cells in transwell chambers, we detected the proliferation and apoptosis of HCC cells after LPS stimulation. Although the survival and proliferation of HepG2 and SMMC7721 after LPS treatment were slightly increased, the change was no significant (Fig. S2). Annexin V staining results also revealed that 1 g/ml LPS treatment did not affect the apoptosis of these HCC cells (Fig. S1). We further assessed the expression level of important proteins related to invasion, MMP2 and MMP9. In these HCC cell lines, HepG2 cells had higher basal mRNA level of MMP2 and MMP9 compared with SMMC7721 and Hep3B cell, which could explain the higher invasion ability of HepG2 without LPS treatment (Fig. 1C). The real-time PCR results showed that MMP2 and MMP9 mRNA expression in these HCC cells were significantly upregulated at 24 h after LPS stimulation, except the expression of MMP9 in Hep3B cells (Fig. 1C). Furthermore, the increased protein level of MMP2 and MMP9 in these cells was confirmed by Western blot (Fig. 1D).
Inflammatory cytokines, IL-6 and TNF␣, secreted by HCC cells were also detected by ELISA. After LPS treatment, the production of IL-6 and TNF␣ in culture supernatants of HepG2 and SMMC7721 were significantly increased, whereas TNF␣ in supernatants of Hep3B had no obvious change (Fig. S3). 3.2. LPS promoted TLR4 expression and downstream pathways activation Because LPS is the main agonist of TLR4 on tumor cells surface, we want to determine whether LPS stimulation will affect the expression of TLR4 on surface of HCC cells. For gene transcription, SMMC7721 cells showed higher TLR4 mRNA level than HepG2 and Hep3B cells (Fig. 2A). LPS triggered a significant increase of TLR4 mRNA expression in all these HCCs (Fig. 2A). TLR4 transcription level in HepG2 cells dramatically increased and up to 18 folds after LPS stimulation, whereas TLR4 transcription level in SMMC7721 cells increased to about 9 folds. The upregulation of TLR4 protein level on the surface of HCC cells was further confirmed by flow cytometry (Fig. 2B).
Fig. 3. TLR4 silencing blocked LPS-induced invasion and downstream pathways activation in HCC cells. (A) HepG2 and SMMC7721 cells with high TLR4 protein level were transfected with control (Ctrl) siRNA or TLR4 siRNA at a final concentration of 40 nM. 36 h after transfection, efficient knockdown of TLR4 was confirmed by Western blot assay and densimetric analysis. Data was expressed as the means ± SD from three separate experiments. *P < 0.05 vs. the control siRNA transfection group. (B) Invasiveness of TLR4-silencing cells determined by Transwell assay. 24 h after TLR4 siRNA transfection, cells were treated with 1 g/ml LPS stimulation for another 48 h. The numbers of migration cells were counted in ten fields. Data from three independent experiments are shown as the means ± SD, *P < 0.05, **P < 0.01 vs. LPS-untreated cells, # P < 0.05, ## P < 0.01 vs. the control siRNA group treated with LPS. (C and D) The effect of TLR4 silencing on mRNA level (C) and protein level (D) of MMP2 and MMP9 before and after LPS treatment was assessed in HCC cells by RT-PCR and Western Blot. 24 h after siRNA transfection, cells were treated with LPS for indicated time(C) or for 48 h (D). Data from three separate experiments was shown as the means ± SD, *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05, ## P < 0.01 vs. the control siRNA group treated with LPS. (E) The effect of TLR4 silencing on the phosphorylation of IкB, MKK4 and JNK was determined by Western Blot and densimetric analysis. 36 h after siRNA transfection, cells were treated with LPS for 2 h (MKK4, JNK) or 12 h (IкB) and then collected for Western blot assay. Data from three separate experiments was shown as the means ± SD, *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05 vs. the control siRNA group treated with LPS.
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Fig. 3. (Continued).
To investigate the potential downstream pathways of LPS-TLR4 signaling in HCC cells, we detect the activation of NF-кB and MAPK signal pathways after LPS stimulation. It had been reported that LPS-TLR4 signaling could activate the NF-кB pathway in various HCCs (Jing et al., 2012; Wang et al., 2013; Yu et al., 2013). We found that LPS could significantly, but not dramatically increase the phosphorylation of IкB and nuclear translocation of NF-кB in these HCC cells at 2 h after treatment, and the activation of IкB and NF-кB maintained a plateau until 24 h (Fig. 2C). At the same time, the phosphorylation of JNK was dramatically increased in these HCC cells after LPS stimulation, whereas the total JNK remained unchanged (Fig. 2D). Furthermore, the upstream activating kinase of JNK, MKK4, showed higher phosphorylation level in all HCCs after LPS treatment (Fig. 2D). But LPS did not affect the activation of other two MAPK members, ERK and p38, in HCC cells (Fig. S4). These data indicated that MKK4/JNK and NF-кB pathways might involve in LPS-TLR4 signaling in HCCs. 3.3. LPS-induced invasion of HCCs could be inhibited by TLR4 silencing To determine the role of TLR4 in LPS-induced migration and invasion, we knocked down TLR4 by siRNA in HepG2 and SMMC7721 cells with high invasive abilities. Western Blot results confirmed the low protein level of TLR4 in these HCC cell lines after
TLR4 silencing (Fig. 3A). Knockdown of TLR4 in HCC cells dramatically decreased the number of cells invading the basal membrane after LPS treatment compared with cells transfected with control siRNA (Fig. 3B). Without LPS treatment, silencing of TLR4 had no obvious effect on the invasion of HCC cells, may due to the low basal level of TLR4 (Fig. 3B). To rule out the possibility of siRNA off target effect, we want to construct a siRNA-resistant plasmid to restore TLR4 expression in TLR4-silencing cells. Because we used pooled siRNAs of TLR4 from Santa, the third codon mutation for amino acids in the siRNA targeting sequence was not suitable for human TLR4 in our system, so we employed the mouse TLR4 sequence for re-expression. The same strategy was used for re-expression of NF-кB and MKK4 in the following experiments. We found that reexpression of mouse TLR4 in TLR4-silencing cells could partially rescued the decrease of cell migration (Figs. S5 and S6). Accordantly, reduced MMP2 and MMP9 mRNA level and protein level were observed in HCC cells after TLR4 silencing in LPS-treated HCC cells, whereas TLR4 knockdown did not change the expression level of MMP2 and MMP9 in LPS-untreated cells (Fig. 3C,D). For the signal pathway, LPS-induced phosphorylation of MKK4 and JNK was significantly decreased after TLR4 knockdown, but the phosphorylation of IкB was moderately reduced in these two cells (Fig. 3E). In LPS untreated cells, the phosphorylation of MKK4, JNK and IкB was no altered (Fig. 3E). These data suggested that TLR4 sig-
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Fig. 4. Knockdown of NF-кB did not alter LPS-triggered invasion of HCCs. (A) The silencing of NF-кB in HepG2 and SMMC7721 cells was determined by Western Blot and densimetric analysis. Data was expressed as the means ± SD from three separate experiments. *P < 0.05 vs. the control siRNA transfected cells. (B) The effect of NF-кB or MKK4 silencing on the migration of HCC cells after LPS stimulation was determined by the Transwell assay. Values are expressed as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. LPS-untreated cells, # P < 0.05 vs. the control siRNA group treated with LPS. (C and D) The effect of NF-кB or MKK4 silencing on mRNA level (C) and protein level (D) of MMP2 and MMP9 before and after LPS treatment was assessed in HCC cells by RT-PCR and Western Blot. *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05, ## P < 0.01 vs. the control siRNA group treated with LPS. (E) The
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Fig. 4. (Continued).
naling was critical for LPS triggered-invasion and MMPs production in HCC cells. 3.4. NF-кB activation was not critical for LPS-triggered invasion of HCCs To further identify the downstream signal pathways contributed to LPS-induced invasion, we inhibited the NF-кB and MAPK pathways using kinase inhibitors or siRNAs respectively in HCC cells. It had been reported that NF-кB was an important transcription factor controlling MMP2 and MMP9 expression. However, silencing of NF-кB expression resulted in slightly but not significantly reduction of the cell migration in HepG2 and SMMC7721 cells treated with LPS (Fig. 4A,B). Without LPS treatment, the migration of these
HCC cells did not change after NF-кB silencing (Fig. 4B). Accordantly, knockdown of NF-кB had no obvious effect on the mRNA level and protein level of MMP2 and MMP9 (Fig. 4C,D). In addition, we observed the effect of NF-кB silencing on the expression of TLR4 and the production on inflammatory cytokines. The protein level of TLR4 did not altered in HCC cells treated with NF-кB siRNA compared with that in cells treated with control siRNA both before and after LPS treatment (Fig. 4E). Interestingly, the production of IL-6 and TNF␣ were significantly decreased after NF-кB knockdown (Fig. S7). To exclude the siRNA off target, we re-expressed mouse NF-кB in NF-кB silencing cells and found re-expression of mouse NF-кB could significantly alleviate the decrease of IL-6 and TNF␣ production (Figs 4 F and S8). These data indicated that NF-кB
regulation of TLR4 expression by NF-кB or MKK4 silencing. Cells were transfected with control siRNA, NF-кB or MKK4 siRNA. After 24 h, cells were treated LPS for another 36 h and then were collected. The expression level of TLR4 was assessed by Western blot and densimetric analysis. *P < 0.05 vs. the untreated
group, # P < 0.05 vs. the control siRNA group treated with LPS. (F) Re-expression of NF-кB in NF-кB silencing cells. Cells were transfected with control siRNA, NF-кB siRNA
for 24 h and then transfected with an empty vector (mock), siRNA-resistant NF-кB plasmid. After 48 h, cells were collected for Western Blot analysis. Data showed the quantitative analysis of the relative levels of NF-кB from three independent experiments. *P < 0.05 vs. the control siRNA group, # P < 0.05 vs. the mock group treated with LPS.
(G) The effect of NF-кB overexpression on invasiveness of HCCs. Cells transfected with control siRNA, NF-кB siRNA for 24 h and then transfected with an empty vector or
NF-кB plasmid for another 24 h, followed with the treatment of LPS for 48 h. The cell migration was determined by Transwell assay. *P < 0.05 vs. LPS untreated cells. # P < 0.05 vs. the mock group treated with LPS.
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Fig. 5. MKK4/JNK pathway was required for LPS-induced invasion in HCC cells. (A) The silencing of MKK4 was determined by Western Blot and densimetric analysis. Data was expressed as the means ± SD from three separate experiments. *P < 0.05 vs. the control siRNA transfection group. (B) The effects of MKK4 silencing and SP600125 on the phosphorylation of JNK were determined by Western blot and densimetric analysis. For the inhibitor treatment, cells were pretreated with SP600125 (20 M) for 30 min, then incubated with LPS for another 2 h. Data are expressed as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05 vs. the control siRNA group, P < 0.05 vs. the DMSO group. (C) The effect of SP600125 on the migration of HCC cells after LPS stimulation was determined by the Transwell assay. After pretreatment with SP600125 for 30 min, cells were further treated with LPS in presence or absence of SP600125 for 48 h. *P < 0.05 vs. the untreated group, # P < 0.05 vs. the DMSO group treated with LPS. (D and E) The effect of SP600125 on mRNA level (D) and protein level (E) of MMP2 and MMP9 before and after LPS treatment was assessed in HCC cells by RT-PCR and Western Blot. *P < 0.05, **P < 0.01 vs. the LPS untreated cells. # P < 0.05, ## P < 0.01 vs. the DMSO group treated with LPS. (F) The effect of SP600125 on the TLR4 expression. Data from three separate experiments was shown as the means ± SD, *P < 0.05 vs. the LPS untreated cells. # P < 0.05 vs. the DMSO group treated with LPS.
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Fig. 5. (Continued).
might be more important for cytokines production but not for cell migration. Because lots of studies showed the critical role of NF-кB in LPS-induced invasion of tumor cell, we only observed the limited activation of NF-кB after LPS treatment and that knockdown of NF-кB had no obvious effect on cell invasion. Therefore, we overexpressed NF-кB both in HepG2 and SMMC7721 cells and detected the LPS-induced cell invasion (Fig. 4F). The invasive ability of these two cells with NF-кB overexpression were significantly increased after LPS treatment compared with that of control plasmid-transfected cells (Fig. 4G). We also observed that re-expression of NF-кB also obviously enhanced the cell invasion in NF-кB-silencing cells compared NF-кB-silencing cells with the mock-transfected (Fig. 4G). These data implied that high expression level of NF-кB might contribute to cell migration.
3.5. LPS-induced invasion was mainly mediated by MKK4/JNK pathway in HCCs To determine the role of MKK4/JNK signal pathway in HCC cells invasion, we employed MKK4 siRNA and the JNK specific inhibitor, SP600125. The silencing of MKK4 expression by siRNA was confirmed by Western blot (Fig. 5A). MKK4 siRNA transfection and SP600125 treatment dramatically suppressed the phosphorylation of JNK in HCC cells after LPS treatment (Fig. 5B). HepG2 and SMMC7721 cells pre-treated with SP600125 before LPS exposure showed lower invasive abilities compared with cells only treated with LPS (Fig. 5C). Silencing of MKK4 expression in HepG2 and SMMC7721 cells also compromised the increase of cell invasion induced by LPS (Fig. 4B). Without LPS treatment, MKK4 silencing and SP600125 treatment had no effect on the migration of
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HCCs (Figs. 4 B and 5 C). Consistently, knockdown of MKK4 and SP600125 treatment significantly reduced LPS-induced MMP2 and MMP9 expression both in mRNA level and protein level (Figs. 4 C,D, 5 D,E). We also found that re-expressed mouse MKK4 in MKK4 silencing cells could partially rescue HCCs invasiveness which suppressed by MKK4 silencing, excluding the siRNA off target (Figs. S9 and S10). Furthermore, we explored whether blocking of MKK4/JNK activity had effect on TLR4 expression as well as IL-6 and TNF␣ production. Both MKK4 knockdown and SP600125 treatment could significantly inhibit the TLR4 expression and these inflammatory cytokines production after LPS treatment (Figs. 4 E, 5 F, S7 and S11). In addition, we found the specific inhibitors for ERK and p38, PD98059 and SB203580, did not affect LPS-triggered invasion in HepG2 and SMMC 7721 (Fig. S12). Altogether, these data indicated that LPS-TLR4 signaling for invasion of HCC cells might be mainly dependent on MKK4/JNK pathway, but not NF-кB pathway.
4. Discussion TLR4 signaling is critical for the survival, proliferation and invasion of tumor cells. In the present study, we focused on the mechanism of LPS/TLR4 signaling-triggered invasion in HCC cell lines and assessed the potential role of NF-кB and JNK signal pathways. We found that LPS treatment could significantly upregulate the expression of TLR4 on the surface of HCC cells and the activation of both MKK4/JNK and NF-кB pathways. Blocking of TLR4 or MKK4/JNK activity remarkably suppressed the invasive activity of HCC cells, as well as MMPs expression and cytokine production. These data suggests that LPS-TLR4 signaling promotes invasion of HCC cells through the MKK4/JNK pathway. TLR4 was not only detected on the surface of most HCC cell lines, including SMMC7721, Hep3B and PLC/PRF/5, but also in HCC tissues from HCC patients (Hsiao et al., 2013; Li et al., 2014b; Jing et al., 2012; Wang et al., 2013). However, there are contradictory opinions for the expression of TLR4 in HepG2 cells. Some studies confirmed the expression of TLR4 in HepG2 cells (Hsiao et al., 2013; Li et al., 2014b; Kanda et al., 2013; Mamedova et al., 2013). But on the contrary, two research groups have reported that TLR4 is almost absent in HepG2 cells, therefore LPS had little effect on the invasion and metastasis of HepG2 cells (Jing et al., 2012; Wang et al., 2013). Our data showed HepG2 cells expressed moderate levels of TLR4 compare with SMMC7721 cells, but LPS treatment induced more dramatic increase of TLR4 mRNA level in HepG2 cells than that in SMMC7721. Therefore, LPS treatment could dramatically enhance the invasive activity of HepG2 cells. For the cell signaling responsible for TLR4 expression, blocking MKK4/JNK activity resulted in significantly decrease of TLR4 expression triggered by LPS treatment, whereas silencing of NF-кB did not alter TLR4 expression, suggesting the critical role of MKK4/JNK in up-regulation of TLR4 expression triggered by LPS. The invasive potential of SMMC7721, HepG2 and Hep3B is inconsistent in different laboratories due to different cell sources and different experiment conditions. Jing et al. (2012) showed SMMC7721 was the most invasive HCC cells and HepG2 was the least. We found that HepG2 cells showed higher invasion activity than SMMC7721 cells both before LPS treatment and after LPS treatment. Numerous clinical and experimental studies have demonstrated an increase in particular MMPs, especially MMP2 and MMP-9 associated with cancer progression (Deakin and Chaplain, 2013; Hong et al., 2014; Wang et al., 2014b; Yang et al., 2014). Our data showed that the mRNA level and protein level of MMP2 and MMP9 were higher in HepG2 cells compared with that in SMMC7721 cells both before LPS treatment and after LPS treatment. However, the expression level of TLR4 is not consistent with
the expression level of MMPs and invasive activity in these two cell lines. There may exist other signal pathways except TLR4 signaling to induce MMPs expression in HCC cells. Here, we focus on the downstream pathways activated by TLR4 signaling in the process of invasion of HCC cells. In the TLR4 signaling pathway, NF-кB activation was the most studied and was closely related to epithelial-mesenchymal transition (EMT), invasion and proliferation in various HCC cell lines (Hartwell et al., 2014; Inoue et al., 2007; Jing et al., 2012; Song et al., 2014; Yeh et al., 2012; Yu et al., 2013). However, Liu et al. (2010) found NF-кB inhibition had no effect on LPS-induced adhesion of HepG2 cells, suggesting that other signaling pathways may be responsible for LPS-induced invasion. In the present study, LPS only moderately increased the nuclear translocation of NF-кB, but obviously promoted the phosphorylation of MKK4 and JNK in these HCC cells. Furthermore, knockdown of MKK4 or blocking JNK activity significantly suppressed the invasiveness of these cells and the production of MMPs. However, knockdown of NF-кB did not affect the invasive ability and MMPs production in these cells. Overexpression of NF-кB could enhance the sensitivity of cell migration responded to LPS-TLR4 signaling. It is difficult to determine the role of NF-кB in the migration of these HCCs in our system, which may need further investigation. In addition, both NF-кB silencing and MKK4/JNK blocking could inhibit the production of IL-6 and TNF␣ induced by LPS treatment. It indicated the powerful potential of NF-кB in regulation of inflammatory cytokines production. Therefore, our data suggested that LPS promoted the invasive activity of these HCC cells mainly through MKK4/JNK pathway. Except NF-кB, the JNK pathway also controls the expression of MMP2 and MMP9 in other cell types (Gweon and Kim, 2014; Lin et al., 2012; Wang et al., 2014a). Moreover, the study from Li et al. (2014b) supported our results. They found that TLR4/JNK signaling was required for LPS-induced EMT in HepG2 cells, but they did not observe the activation of NF-кB and other MAPKs at the same time. In conclusion, our study has shown that increased invasiveness of hepatocellular carcinoma cells triggered by LPS-TLR4 signaling is mainly mediated by the TLR4/MKK4/JNK pathway. Our findings suggest that MKK4/JNK may be the potential targets for the treatment of hepatocellular cancer. Ackownledgements This work was supported by the National Natural Science Foundation of China Grant (No. 31170841 and 31370879), and Qianjiang Talent project of Zhejiang Province (No. 2013R10040). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molimm.2015. 10.015. Reference: Dapito, D.H., Mencin, A., Gwak, G.Y., Pradere, J.P., Jang, M.K., Mederacke, I., Caviglia, J.M., Khiabanian, H., Adeyemi, A., Bataller, R., Lefkowitch, J.H., Bower, M., Friedman, R., Sartor, R.B., Rabadan, R., Schwabe, R.F., 2012. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell 21, 504–516. Deakin, N.E., Chaplain, M.A., 2013. Mathematical modeling of cancer invasion: the role of membrane-bound matrix metalloproteinases. Front. Oncol. 3, 70. Elinav, E., Nowarski, R., Thaiss, C.A., Hu, B., Jin, C., Flavell, R.A., 2013. Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms. Nat. Rev. Cancer 13, 759–771. Gweon, E.J., Kim, S.J., 2014. Resveratrol attenuates matrix metalloproteinase-9 and -2-regulated differentiation of HTB94 chondrosarcoma cells through the p38 kinase and JNK pathways. Oncol. Rep. 32, 71–78. Hartwell, H.J., Petrosky, K.Y., Fox, J.G., Horseman, N.D., Rogers, A.B., 2014. Prolactin prevents hepatocellular carcinoma by restricting innate immune activation of c-Myc in mice. Proc. Natl. Acad. Sci. U. S. A. 111, 11455–11460.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Toll-like receptor 4 signaling promotes invasion of hepatocellular carcinoma cells through MKK4/JNK pathway Yu-Qing Dong a,b,1 , Chuan-Wei Lu a,1 , Lu Zhang a , Jia Yang a , Waqaar Hameed a , Wei Chen a,∗ a b
Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou 310058, China Department of Clinical Laboratory, the Chinese Medicine Hospital of Hangzhou, Hangzhou 310007, China
a r t i c l e
i n f o
Article history: Received 15 March 2015 Received in revised form 21 October 2015 Accepted 22 October 2015 Available online 14 November 2015 Keywords: Toll-like receptor 4 Invasion Hepatocellular carcinoma Jun N-terminal kinase Lipopolysaccharide
a b s t r a c t Toll-like receptor (TLR) 4-mediated signaling has been shown to be important to cell survival, invasion and metastasis in a variety of cancers. The present study aimed to explore the role and downstream pathways of TLR4 signaling in the invasion of hepatocellular carcinoma (HCC) cell lines. We found that LPS, the agonist of TLR4, notably enhanced the invasiveness of HCC cells and the expression of MMP2 and MMP9, as well as the production of IL-6 and TNF␣. LPS treatment dramatically increased the TLR4 expression on HCC cells surface and MKK4/JNK activation, while knockdown of TLR4 inhibited the LPSinduced invasion and the phosphorylation of MKK4 and JNK. Furthermore, silencing of MKK4 or inhibition of JNK activity led to impaired invasiveness of HCCs, low expression level of MMPs and TLR4, as well as limited production of cytokines. However, LPS stimulation only triggered moderate activation of NF-кB. Silencing of NF-кB or NF-кB inhibitor had no obvious effect on the invasive ability of HCCs and TLR4 expression, but suppressed IL-6 and TNF␣ production. These findings suggested that LPS-TLR4 signaling enhanced the invasiveness of HCCs mainly through MKK4/JNK pathway. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chronic inflammation is a major contributor to carcinogenesis and metastasis of tumors (Elinav et al., 2013; Landskron et al., 2014). In patients with chronic liver inflammation, the endotoxin/lipopolysaccharide (LPS) level in the portal and peripheral veins is high due to increased translocation of intestinal bacteria (Dapito et al., 2012; Pidgeon et al., 1999). Elimination of gut-derived bacteria by antibiotics results in significant reduction of hepatocellular carcinoma (HCC) development (Dapito et al., 2012; Iwasaki and Medzhitov, 2004). Toll-like receptor 4 (TLR4), the receptor of LPS, plays a critical role in the development and progression of
Abbreviations: TLR, toll-like receptor; LPS, lipopolysaccharide; HCC, hepatocellular carcinoma; EMT, epithelial-mesenchymal transition; MMP, matrixmetalloproteinase; MAPK, mitogen-activated protein kinase; MKK4, MAP kinase kinase4; JNK, jun N-terminal kinase; ERK, extracellular signal-regulated kinase; IB, inhibitor of NF-kappa B; NF-кB, nuclear factor kappa B; siRNA, small interfer RNA; qRT-PCR, quantitative RT-PCR. ∗ Corresponding author at: Institute of Immunology, Zhejiang University School of Medicine, 388 Yu Hang Tang Rd., Hangzhou 310058, China. Fax: +86 571 88208285. E-mail addresses: [email protected], [email protected] (W. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.molimm.2015.10.015 0161-5890/© 2015 Elsevier Ltd. All rights reserved.
various human cancers, including colorectal, breast, lung, prostate, pancreatic, head and neck cancer (Ikebe et al., 2009; Kundu et al., 2008; Li et al., 2014a; Ren et al., 2014; Santini et al., 2008; Wang et al., 2013; Yang et al., 2014). In liver cancer, increasing evidence supports the role of TLR4 signaling in the migration and invasion of most HCC cell lines (Dapito et al., 2012; Jing et al., 2012; Liu et al., 2010; Li et al., 2014b). Clinical evidence has also shown that high expression of TLR4 in HCC tissues is strongly associated with poor prognosis due to a high level of metastasis (Jing et al., 2012; Liu et al., 2015). However, the molecular mechanism of TLR4 signaling for the invasion of HCC cells remains unclear. Some evidence supports the idea that NF-KB is a key mediator for TLR4 signaling (Wang et al., 2013; Hartwell et al., 2014; Yu et al., 2013). In contradiction to this opinion, there are suggestions that LPS may promote hepatoma cell invasion through other downstream pathways rather than NF-KB (Liu et al., 2010; Li et al., 2014b). Here, we reported that LPS-TLR4 signaling increased invasive potential of HCC lines, as well as MMPs and inflammatory cytokines production. Importantly, this pro-invasion effect of LPS was mainly mediated by the TLR4/MKK4/JNK signal pathway, but not the NF-KB pathway.
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Fig. 1. Invasion of HCC cell lines and MMPs production in response to LPS. (A) Matrigel invasion assay was performed to observe the changes in HCC cell lines invasion. Photomicrographs of cells that have passed through membrane were taken 48 h after different doses of LPS treatment (200× magnification). The numbers of invading cells through the matrigel were counted in ten fields under the microscope. All data are shown as the means ± SD, *P < 0.05 vs. the control group. Results are representative of three separate experiments. Left panel shows a representative field of HCC cells before and after 1 g/ml LPS treatment. (B) The invasion of HCC cells was assessed by scratch wound-healing assay and was compared between untreated cells (control, upper panel) and cells treated with 1 g/ml LPS (low panel). Cells were monitored for 48 h to evaluate the rate of migration into the scratched area. (C) The mRNA level of MMP2 and MMP9 were analyzed by real-time PCR in three HCC cell lines. Left panel: MMPs mRNA level in HepG2 cells were set as “one fold” and compared the basal mRNA level of these HCC cell lines without LPS treatment. Right panel: HCC cells were treated with
Y.-Q. Dong et al. / Molecular Immunology 68 (2015) 671–683
2. Materials and methods 2.1. Chemicals and reagents LPS (L4391), SP600125 (S5567), PD98059 (P215) and SB203580 (S8307), were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bay 11-7082 (S2913) was obtained from Selleckchem (USA). LPS was dissolved in PBS, whereas SP600125, Bay 11-7082, PD98059 and SB203580 were dissolved in DMSO. PE-TLR4 antibody (124891) was purchased from Biolegend. Antibodies specific to TLR4 (sc10741), p-MKK4 (sc-7990), MKK4 (sc-964), MMP-2 (sc-13595), MMP-9 (sc-21733) and -actin (sc-47778) were obtained from Santa Cruz Biotechnology. Antibodies specific to p-JNK (9251), JNK (9252), p-ERK1/2 (4376), ERK1/2 (9102), p-p38 (9215), p38 (9212), p-IB␣ (2859) and NF-B (8242) were obtained from Cell Signaling Technology. 2.2. Cell culture and transfection Human hepatocellular cell lines HepG2, SMMC7721 and Hep3B were purchased from the American Type Culture Collection (ATCC) (Washington, DC, USA). HepG2 and SMMC7721 cells were cultured in RPMI-1640 (Gibco, USA) medium supplied with 10% fetal bovine serum (FBS, Gibco), 100 units/ml of penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA), then incubated at 37 ◦ C in a humidified 5% CO2 atmosphere. Hep3B cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% FBS. Cells were transfected with siRNAs or/and plasmids with JetPEI (Polyplus) according to the manufacturer’s instructions. 2.3. Small interfering RNA and plasmids TLR4 siRNA (sc-40260), NF-кB (sc-29410) and control siRNA (sc-108060) were purchased from Santa Cruz Biotechnology. The MKK4-specific siRNA (mixture) sequences were 5 -GGACUUGAAAGACCUUGGATT 3 (sense), 5 -UCCAAGGUCUUUCAAGUCCTC3 (antisense) and 5 -GCCGUAUAUGGCACCUGAATT-3 (sense), 5 UUCAGGUGCCAUAUACGGCTT-3 (antisense). The scrambled control RNA sequences were described previously (Wu et al., 2014). Knockdown of TLR4, NF-кB and MKK4 were confirmed by Western Blot. To rule out siRNA off-target, we employed mouse targeting sequences to construct siRNA-resistant plasmid for re-expression. The DNA sequences encoding mouse TLR4, NF-кB and MKK4 were amplified by PCR and ligated into pcDNA3.1 plasmid to generate eukaryotic expression plasmids. The primer sequences were as follow: mouse TLR4 forward primer 5 -CGATTCTAGAACAAAACCAA3 , reverse primer 5 -CTGGAAAGGAAGGTGTCA-3 ; mouse NF-кB p65/RelA forward primer 5 -ACCCTGACCATGGACGATC-3 , reverse primer 5 -CTCCGAAAGCGAGATAAAGA- 3 ; mouse MKK4 forward primer 5 -ACTTCCAACAATGGCGGCTC-3 , reverse primer 5 -TTGCTTCCCCTCAGCCCTTT- 3 .
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and cells were washed twice with complete media to remove detached cells, followed by the acquisition of the original image of the wound under a microscope. The LPS group was added with 1 g/ml LPS. Dishes were then put back in an incubator and photographs were taken at 48 h after wounding. The difference between the widths of the wounds is taken as the migration distance. 2.5. Transwell invasion assay Cell migration in vitro was determined using 6.5 mm Transwell chambers with 8 mm pores (Corning, NY, USA). Transwell filters were coated with matrigel on the upper surface of the polycarbonic membrane and the matrigel solidified after 1 h of incubation at 37 ◦ C. Cells (5 × 104 cells) were harvested in 100 l of serum free RPMI-1640 medium and added to the upper compartment of the chamber. For LPS group, 1 g/ml LPS was added to both upper and lower part of the chambers. Cells were allowed to migrate for 48 h. Uninvaded cells in the upper chamber were removed with cotton swabs and invaded cells were fixed with 100% methanol for 5 min, followed with staining with DAPI for 15 min. The number of cells was counted from five randomly selected visual fields, using an inverted microscope at 200× magnification. 2.6. MTT assay Cell proliferation was assessed by the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. HepG2, SMMC7721 and Hep3B cells were seeded in a 96-well plate at a density of 1 × 104 cells/well. After incubated with or without 1 g/ml LPS for 12 h, 24 h and 48 h, MTT (20 l) was added into wells and incubated for an additional 4 h. Cells were pelleted and lysed in 150 L of DMSO, and the absorbance of each well was determined at 490 nm. 2.7. Quantitative real-time PCR Total RNA was extracted using Trizol according to the manufacturer’s protocol. Real-time quantitative PCR, using SYBR Green detection chemistry (Takara), was performed on a 7500 real-time PCR system (Applied Biosystems) as we described previously (Lai et al., 2013). The following primers were used: murine TLR4, 5 -AAGCCGAAAGGTGATTGTTG3 (forward) and 5 -CTGAGCAGGGTCTTCTCCAC-3 (reverse); murine MMP-2, 5 -TGATCTTGACCAGAATACCATCGA-3 (forward) and 5 -GGCTTGCGAGGGAAGAAGTT-3 (reverse); MMP-9, 5 -TGGGGGGCAACTCGGC-3 (forward) murine and 5 -GGAATGATCTAAGCCCAG-3 (reverse); murine 5 -GCACCACACCTTCTACAATGA-3 (forward) and actin, 5 -TGTCACGCACGATTTCCC-3 (reverse). All reactions were performed in triplicate. For data analysis, the relative levels of the target gene mRNA transcripts to the -actin were calculated by 2−Ct and statistical significance was determined by the one way ANOVA using SPSS (version 10.0).
2.4. Scratch wound healing assay 2.8. Western blot analysis HepG2, SMMC7721 and Hep3B cells were seeded at a density of 2 × 106 cells/well in a 6-well plate and cultured overnight. A wound was created by scratching with a 250-l pipette tip,
After treatment, the cells were washed twice with cold PBS and lysed with cell lysis buffer (Cell Signaling Technology) sup-
1 g/ml LPS for indicated time. MMPs mRNA level in untreated cells was set as “one fold” for each cell lines. Data are from three independent experiments (means ± SD). Statistical analysis was done by comparison with untreated cells. *P < 0.05, **P < 0.01. (D) The protein level of MMP2 and MMP9 in HCC cells treated with 1 g/ml LPS for 48 h was detected by Western Blot. The relative levels of MMP2 and MMP9 were analyzed by densimetric analysis using ImageJ software. Data are expressed as the means ± SD from three separate experiments. Statistical analysis was done by comparison with untreated cells. *P < 0.05, **P < 0.01 vs. the untreated group.
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plemented with PMSF (Beyotime Institute of Biotechnology). To detect the nuclear translocation of NF-кB, we isolated nuclear fraction from LPS-treated cells using NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA) according to the manufacturer’s protocols. Protein concentrations were determined using
a BCA protein assay kit (Pierce). The lysates were loaded onto SDS-PAGE gels and transferred onto nitrocellulose membranes and then incubated with antibodies as described previously (Wu et al., 2014). Protein signals were detected by ECL reagent (Thermo Fisher).
Fig. 2. LPS enhanced TLR4 expression and downstream pathways activation. (A) The mRNA level of TLR4 were analyzed by real-time PCR in three HCC cell lines. Left panel: TLR4 mRNA level in HepG2 cells was set as “one fold” and compared the basal mRNA level of these HCC cell lines without LPS treatment. Right panel: HCC cells were treated with 1 g/ml LPS for indicated time. TLR4 mRNA level in untreated cells was set as “one fold” for each cell lines. Each bar represented triplicate analyses of mean ± SD. **P < 0.01 vs. the untreated group. (B) TLR4 expression on the surface of these HCC cells was determined by FACS analysis using mean fluorescence intensity as a readout. HCC cells were treated with LPS for 36 h, then fixed and stained cells with PE-TLR4 Ab. (C) Three HCC cells were treated with 1 g/ml LPS for indicated time, and the phosphorylation of IкB were examined by Western Blot. Cytoplasmic and nuclear fractions were extracted from HCC cells treated with LPS and the translocation of NF-кB was detected. Histone H3 as a loading control for nuclear NF-кB. Quantitative analysis of the relative levels of IкB and NF-кB from three separate experiments was shown as the means ± SD. *P < 0.05 vs. the untreated cells. (D) Western blot and quantitative analysis of the relative levels of MKK4 and JNK in HCC cells after LPS treatment. *P < 0.05, **P < 0.01 vs. the untreated cells.
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Fig. 2. (Continued).
2.9. Flow cytometry The expression of TLR4 protein was measured by flow cytometry. HCC cells (5 × 105 ) were treated with 1 g/ml LPS for 36 h. Cells were collected and washed three times with PBS followed by incubation with Rat serum for 10 min to block nonspecific binding of TLR4 antibody. Subsequently, cells were washed for three times and incubated with 3 l human PE-TLR4 antibody in 50 l PBS for 30 min. Cells were washed again and analyzed by flow cytometry. The control was performed by using isotype-IgG antibody. For apoptosis analysis, HCC cells were collected and stained with Annexin V and 7-AAD, then analyzed by flow cytometry. For quantification, Annexin V-positive cell fraction was considered as apoptotic cell fraction. 2.10. ELISA After stimulation with LPS at the indicated time points, the cell supernatants were collected and analyzed using ELISA kits of IL-6 and TNF␣ (R&D company) according to the manufacturer’s protocols. 2.11. Statistical analysis Data are expressed as means ± standard deviation (SD). Statistical significance was determined by two-tailed Student’s t test and
one way ANOVA using SPSS (version 10.0). Differences resulting in P values less than 0.05 were considered statistically significant. 3. Results 3.1. LPS enhanced the invasion of HCC cell lines and MMPs production To evaluate the impact of LPS on cellular invasion, we choose three HCC cell lines with different invasive abilities, including HepG2, SMMC7721 and Hep3B cells. Different doses of LPS (0.01, 0.1, 1 and 10 g/ml) were used to stimulate these cell lines and matrigel invasion assay was performed. We found that treatment of 1 and 10 g/ml LPS significantly enhanced the invasive ability of three HCC cells compared with the LPS-untreated cells (Fig. 1A). However, 10 g/ml LPS stimulation significantly increased the apoptosis of HCC cells, so we used 1 g/ml LPS as the proper dose in our system (Fig. S1). As shown in Fig. 1A, HepG2 was the most invasive cell line among these three cell lines, and the invasiveness of Hep3B cells was very low. However, some study showed LPS had no effect on invasion of HepG2 cells (Jing et al., 2012; Wang et al., 2013). Similarly, the results of scratch wound-healing assay showed that HepG2 cells treated with 1 g/ml LPS were capable of closing the wound completely within 48 h, whereas cells without LPS treatment showed significantly slower rates of wound-healing (Fig. 1B). LPS treatment also triggered higher wound-healing abil-
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ity in SMMC7721 and Hep3B cells compared with that in untreated cells (Fig. 1B). These data indicated LPS significantly promoted the invasive abilities of these HCC cell lines. To determine whether other factors may affect the number of HCC cells in transwell chambers, we detected the proliferation and apoptosis of HCC cells after LPS stimulation. Although the survival and proliferation of HepG2 and SMMC7721 after LPS treatment were slightly increased, the change was no significant (Fig. S2). Annexin V staining results also revealed that 1 g/ml LPS treatment did not affect the apoptosis of these HCC cells (Fig. S1). We further assessed the expression level of important proteins related to invasion, MMP2 and MMP9. In these HCC cell lines, HepG2 cells had higher basal mRNA level of MMP2 and MMP9 compared with SMMC7721 and Hep3B cell, which could explain the higher invasion ability of HepG2 without LPS treatment (Fig. 1C). The real-time PCR results showed that MMP2 and MMP9 mRNA expression in these HCC cells were significantly upregulated at 24 h after LPS stimulation, except the expression of MMP9 in Hep3B cells (Fig. 1C). Furthermore, the increased protein level of MMP2 and MMP9 in these cells was confirmed by Western blot (Fig. 1D).
Inflammatory cytokines, IL-6 and TNF␣, secreted by HCC cells were also detected by ELISA. After LPS treatment, the production of IL-6 and TNF␣ in culture supernatants of HepG2 and SMMC7721 were significantly increased, whereas TNF␣ in supernatants of Hep3B had no obvious change (Fig. S3). 3.2. LPS promoted TLR4 expression and downstream pathways activation Because LPS is the main agonist of TLR4 on tumor cells surface, we want to determine whether LPS stimulation will affect the expression of TLR4 on surface of HCC cells. For gene transcription, SMMC7721 cells showed higher TLR4 mRNA level than HepG2 and Hep3B cells (Fig. 2A). LPS triggered a significant increase of TLR4 mRNA expression in all these HCCs (Fig. 2A). TLR4 transcription level in HepG2 cells dramatically increased and up to 18 folds after LPS stimulation, whereas TLR4 transcription level in SMMC7721 cells increased to about 9 folds. The upregulation of TLR4 protein level on the surface of HCC cells was further confirmed by flow cytometry (Fig. 2B).
Fig. 3. TLR4 silencing blocked LPS-induced invasion and downstream pathways activation in HCC cells. (A) HepG2 and SMMC7721 cells with high TLR4 protein level were transfected with control (Ctrl) siRNA or TLR4 siRNA at a final concentration of 40 nM. 36 h after transfection, efficient knockdown of TLR4 was confirmed by Western blot assay and densimetric analysis. Data was expressed as the means ± SD from three separate experiments. *P < 0.05 vs. the control siRNA transfection group. (B) Invasiveness of TLR4-silencing cells determined by Transwell assay. 24 h after TLR4 siRNA transfection, cells were treated with 1 g/ml LPS stimulation for another 48 h. The numbers of migration cells were counted in ten fields. Data from three independent experiments are shown as the means ± SD, *P < 0.05, **P < 0.01 vs. LPS-untreated cells, # P < 0.05, ## P < 0.01 vs. the control siRNA group treated with LPS. (C and D) The effect of TLR4 silencing on mRNA level (C) and protein level (D) of MMP2 and MMP9 before and after LPS treatment was assessed in HCC cells by RT-PCR and Western Blot. 24 h after siRNA transfection, cells were treated with LPS for indicated time(C) or for 48 h (D). Data from three separate experiments was shown as the means ± SD, *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05, ## P < 0.01 vs. the control siRNA group treated with LPS. (E) The effect of TLR4 silencing on the phosphorylation of IкB, MKK4 and JNK was determined by Western Blot and densimetric analysis. 36 h after siRNA transfection, cells were treated with LPS for 2 h (MKK4, JNK) or 12 h (IкB) and then collected for Western blot assay. Data from three separate experiments was shown as the means ± SD, *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05 vs. the control siRNA group treated with LPS.
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Fig. 3. (Continued).
To investigate the potential downstream pathways of LPS-TLR4 signaling in HCC cells, we detect the activation of NF-кB and MAPK signal pathways after LPS stimulation. It had been reported that LPS-TLR4 signaling could activate the NF-кB pathway in various HCCs (Jing et al., 2012; Wang et al., 2013; Yu et al., 2013). We found that LPS could significantly, but not dramatically increase the phosphorylation of IкB and nuclear translocation of NF-кB in these HCC cells at 2 h after treatment, and the activation of IкB and NF-кB maintained a plateau until 24 h (Fig. 2C). At the same time, the phosphorylation of JNK was dramatically increased in these HCC cells after LPS stimulation, whereas the total JNK remained unchanged (Fig. 2D). Furthermore, the upstream activating kinase of JNK, MKK4, showed higher phosphorylation level in all HCCs after LPS treatment (Fig. 2D). But LPS did not affect the activation of other two MAPK members, ERK and p38, in HCC cells (Fig. S4). These data indicated that MKK4/JNK and NF-кB pathways might involve in LPS-TLR4 signaling in HCCs. 3.3. LPS-induced invasion of HCCs could be inhibited by TLR4 silencing To determine the role of TLR4 in LPS-induced migration and invasion, we knocked down TLR4 by siRNA in HepG2 and SMMC7721 cells with high invasive abilities. Western Blot results confirmed the low protein level of TLR4 in these HCC cell lines after
TLR4 silencing (Fig. 3A). Knockdown of TLR4 in HCC cells dramatically decreased the number of cells invading the basal membrane after LPS treatment compared with cells transfected with control siRNA (Fig. 3B). Without LPS treatment, silencing of TLR4 had no obvious effect on the invasion of HCC cells, may due to the low basal level of TLR4 (Fig. 3B). To rule out the possibility of siRNA off target effect, we want to construct a siRNA-resistant plasmid to restore TLR4 expression in TLR4-silencing cells. Because we used pooled siRNAs of TLR4 from Santa, the third codon mutation for amino acids in the siRNA targeting sequence was not suitable for human TLR4 in our system, so we employed the mouse TLR4 sequence for re-expression. The same strategy was used for re-expression of NF-кB and MKK4 in the following experiments. We found that reexpression of mouse TLR4 in TLR4-silencing cells could partially rescued the decrease of cell migration (Figs. S5 and S6). Accordantly, reduced MMP2 and MMP9 mRNA level and protein level were observed in HCC cells after TLR4 silencing in LPS-treated HCC cells, whereas TLR4 knockdown did not change the expression level of MMP2 and MMP9 in LPS-untreated cells (Fig. 3C,D). For the signal pathway, LPS-induced phosphorylation of MKK4 and JNK was significantly decreased after TLR4 knockdown, but the phosphorylation of IкB was moderately reduced in these two cells (Fig. 3E). In LPS untreated cells, the phosphorylation of MKK4, JNK and IкB was no altered (Fig. 3E). These data suggested that TLR4 sig-
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Fig. 4. Knockdown of NF-кB did not alter LPS-triggered invasion of HCCs. (A) The silencing of NF-кB in HepG2 and SMMC7721 cells was determined by Western Blot and densimetric analysis. Data was expressed as the means ± SD from three separate experiments. *P < 0.05 vs. the control siRNA transfected cells. (B) The effect of NF-кB or MKK4 silencing on the migration of HCC cells after LPS stimulation was determined by the Transwell assay. Values are expressed as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. LPS-untreated cells, # P < 0.05 vs. the control siRNA group treated with LPS. (C and D) The effect of NF-кB or MKK4 silencing on mRNA level (C) and protein level (D) of MMP2 and MMP9 before and after LPS treatment was assessed in HCC cells by RT-PCR and Western Blot. *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05, ## P < 0.01 vs. the control siRNA group treated with LPS. (E) The
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Fig. 4. (Continued).
naling was critical for LPS triggered-invasion and MMPs production in HCC cells. 3.4. NF-кB activation was not critical for LPS-triggered invasion of HCCs To further identify the downstream signal pathways contributed to LPS-induced invasion, we inhibited the NF-кB and MAPK pathways using kinase inhibitors or siRNAs respectively in HCC cells. It had been reported that NF-кB was an important transcription factor controlling MMP2 and MMP9 expression. However, silencing of NF-кB expression resulted in slightly but not significantly reduction of the cell migration in HepG2 and SMMC7721 cells treated with LPS (Fig. 4A,B). Without LPS treatment, the migration of these
HCC cells did not change after NF-кB silencing (Fig. 4B). Accordantly, knockdown of NF-кB had no obvious effect on the mRNA level and protein level of MMP2 and MMP9 (Fig. 4C,D). In addition, we observed the effect of NF-кB silencing on the expression of TLR4 and the production on inflammatory cytokines. The protein level of TLR4 did not altered in HCC cells treated with NF-кB siRNA compared with that in cells treated with control siRNA both before and after LPS treatment (Fig. 4E). Interestingly, the production of IL-6 and TNF␣ were significantly decreased after NF-кB knockdown (Fig. S7). To exclude the siRNA off target, we re-expressed mouse NF-кB in NF-кB silencing cells and found re-expression of mouse NF-кB could significantly alleviate the decrease of IL-6 and TNF␣ production (Figs 4 F and S8). These data indicated that NF-кB
regulation of TLR4 expression by NF-кB or MKK4 silencing. Cells were transfected with control siRNA, NF-кB or MKK4 siRNA. After 24 h, cells were treated LPS for another 36 h and then were collected. The expression level of TLR4 was assessed by Western blot and densimetric analysis. *P < 0.05 vs. the untreated
group, # P < 0.05 vs. the control siRNA group treated with LPS. (F) Re-expression of NF-кB in NF-кB silencing cells. Cells were transfected with control siRNA, NF-кB siRNA
for 24 h and then transfected with an empty vector (mock), siRNA-resistant NF-кB plasmid. After 48 h, cells were collected for Western Blot analysis. Data showed the quantitative analysis of the relative levels of NF-кB from three independent experiments. *P < 0.05 vs. the control siRNA group, # P < 0.05 vs. the mock group treated with LPS.
(G) The effect of NF-кB overexpression on invasiveness of HCCs. Cells transfected with control siRNA, NF-кB siRNA for 24 h and then transfected with an empty vector or
NF-кB plasmid for another 24 h, followed with the treatment of LPS for 48 h. The cell migration was determined by Transwell assay. *P < 0.05 vs. LPS untreated cells. # P < 0.05 vs. the mock group treated with LPS.
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Fig. 5. MKK4/JNK pathway was required for LPS-induced invasion in HCC cells. (A) The silencing of MKK4 was determined by Western Blot and densimetric analysis. Data was expressed as the means ± SD from three separate experiments. *P < 0.05 vs. the control siRNA transfection group. (B) The effects of MKK4 silencing and SP600125 on the phosphorylation of JNK were determined by Western blot and densimetric analysis. For the inhibitor treatment, cells were pretreated with SP600125 (20 M) for 30 min, then incubated with LPS for another 2 h. Data are expressed as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. the untreated group, # P < 0.05 vs. the control siRNA group, P < 0.05 vs. the DMSO group. (C) The effect of SP600125 on the migration of HCC cells after LPS stimulation was determined by the Transwell assay. After pretreatment with SP600125 for 30 min, cells were further treated with LPS in presence or absence of SP600125 for 48 h. *P < 0.05 vs. the untreated group, # P < 0.05 vs. the DMSO group treated with LPS. (D and E) The effect of SP600125 on mRNA level (D) and protein level (E) of MMP2 and MMP9 before and after LPS treatment was assessed in HCC cells by RT-PCR and Western Blot. *P < 0.05, **P < 0.01 vs. the LPS untreated cells. # P < 0.05, ## P < 0.01 vs. the DMSO group treated with LPS. (F) The effect of SP600125 on the TLR4 expression. Data from three separate experiments was shown as the means ± SD, *P < 0.05 vs. the LPS untreated cells. # P < 0.05 vs. the DMSO group treated with LPS.
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Fig. 5. (Continued).
might be more important for cytokines production but not for cell migration. Because lots of studies showed the critical role of NF-кB in LPS-induced invasion of tumor cell, we only observed the limited activation of NF-кB after LPS treatment and that knockdown of NF-кB had no obvious effect on cell invasion. Therefore, we overexpressed NF-кB both in HepG2 and SMMC7721 cells and detected the LPS-induced cell invasion (Fig. 4F). The invasive ability of these two cells with NF-кB overexpression were significantly increased after LPS treatment compared with that of control plasmid-transfected cells (Fig. 4G). We also observed that re-expression of NF-кB also obviously enhanced the cell invasion in NF-кB-silencing cells compared NF-кB-silencing cells with the mock-transfected (Fig. 4G). These data implied that high expression level of NF-кB might contribute to cell migration.
3.5. LPS-induced invasion was mainly mediated by MKK4/JNK pathway in HCCs To determine the role of MKK4/JNK signal pathway in HCC cells invasion, we employed MKK4 siRNA and the JNK specific inhibitor, SP600125. The silencing of MKK4 expression by siRNA was confirmed by Western blot (Fig. 5A). MKK4 siRNA transfection and SP600125 treatment dramatically suppressed the phosphorylation of JNK in HCC cells after LPS treatment (Fig. 5B). HepG2 and SMMC7721 cells pre-treated with SP600125 before LPS exposure showed lower invasive abilities compared with cells only treated with LPS (Fig. 5C). Silencing of MKK4 expression in HepG2 and SMMC7721 cells also compromised the increase of cell invasion induced by LPS (Fig. 4B). Without LPS treatment, MKK4 silencing and SP600125 treatment had no effect on the migration of
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HCCs (Figs. 4 B and 5 C). Consistently, knockdown of MKK4 and SP600125 treatment significantly reduced LPS-induced MMP2 and MMP9 expression both in mRNA level and protein level (Figs. 4 C,D, 5 D,E). We also found that re-expressed mouse MKK4 in MKK4 silencing cells could partially rescue HCCs invasiveness which suppressed by MKK4 silencing, excluding the siRNA off target (Figs. S9 and S10). Furthermore, we explored whether blocking of MKK4/JNK activity had effect on TLR4 expression as well as IL-6 and TNF␣ production. Both MKK4 knockdown and SP600125 treatment could significantly inhibit the TLR4 expression and these inflammatory cytokines production after LPS treatment (Figs. 4 E, 5 F, S7 and S11). In addition, we found the specific inhibitors for ERK and p38, PD98059 and SB203580, did not affect LPS-triggered invasion in HepG2 and SMMC 7721 (Fig. S12). Altogether, these data indicated that LPS-TLR4 signaling for invasion of HCC cells might be mainly dependent on MKK4/JNK pathway, but not NF-кB pathway.
4. Discussion TLR4 signaling is critical for the survival, proliferation and invasion of tumor cells. In the present study, we focused on the mechanism of LPS/TLR4 signaling-triggered invasion in HCC cell lines and assessed the potential role of NF-кB and JNK signal pathways. We found that LPS treatment could significantly upregulate the expression of TLR4 on the surface of HCC cells and the activation of both MKK4/JNK and NF-кB pathways. Blocking of TLR4 or MKK4/JNK activity remarkably suppressed the invasive activity of HCC cells, as well as MMPs expression and cytokine production. These data suggests that LPS-TLR4 signaling promotes invasion of HCC cells through the MKK4/JNK pathway. TLR4 was not only detected on the surface of most HCC cell lines, including SMMC7721, Hep3B and PLC/PRF/5, but also in HCC tissues from HCC patients (Hsiao et al., 2013; Li et al., 2014b; Jing et al., 2012; Wang et al., 2013). However, there are contradictory opinions for the expression of TLR4 in HepG2 cells. Some studies confirmed the expression of TLR4 in HepG2 cells (Hsiao et al., 2013; Li et al., 2014b; Kanda et al., 2013; Mamedova et al., 2013). But on the contrary, two research groups have reported that TLR4 is almost absent in HepG2 cells, therefore LPS had little effect on the invasion and metastasis of HepG2 cells (Jing et al., 2012; Wang et al., 2013). Our data showed HepG2 cells expressed moderate levels of TLR4 compare with SMMC7721 cells, but LPS treatment induced more dramatic increase of TLR4 mRNA level in HepG2 cells than that in SMMC7721. Therefore, LPS treatment could dramatically enhance the invasive activity of HepG2 cells. For the cell signaling responsible for TLR4 expression, blocking MKK4/JNK activity resulted in significantly decrease of TLR4 expression triggered by LPS treatment, whereas silencing of NF-кB did not alter TLR4 expression, suggesting the critical role of MKK4/JNK in up-regulation of TLR4 expression triggered by LPS. The invasive potential of SMMC7721, HepG2 and Hep3B is inconsistent in different laboratories due to different cell sources and different experiment conditions. Jing et al. (2012) showed SMMC7721 was the most invasive HCC cells and HepG2 was the least. We found that HepG2 cells showed higher invasion activity than SMMC7721 cells both before LPS treatment and after LPS treatment. Numerous clinical and experimental studies have demonstrated an increase in particular MMPs, especially MMP2 and MMP-9 associated with cancer progression (Deakin and Chaplain, 2013; Hong et al., 2014; Wang et al., 2014b; Yang et al., 2014). Our data showed that the mRNA level and protein level of MMP2 and MMP9 were higher in HepG2 cells compared with that in SMMC7721 cells both before LPS treatment and after LPS treatment. However, the expression level of TLR4 is not consistent with
the expression level of MMPs and invasive activity in these two cell lines. There may exist other signal pathways except TLR4 signaling to induce MMPs expression in HCC cells. Here, we focus on the downstream pathways activated by TLR4 signaling in the process of invasion of HCC cells. In the TLR4 signaling pathway, NF-кB activation was the most studied and was closely related to epithelial-mesenchymal transition (EMT), invasion and proliferation in various HCC cell lines (Hartwell et al., 2014; Inoue et al., 2007; Jing et al., 2012; Song et al., 2014; Yeh et al., 2012; Yu et al., 2013). However, Liu et al. (2010) found NF-кB inhibition had no effect on LPS-induced adhesion of HepG2 cells, suggesting that other signaling pathways may be responsible for LPS-induced invasion. In the present study, LPS only moderately increased the nuclear translocation of NF-кB, but obviously promoted the phosphorylation of MKK4 and JNK in these HCC cells. Furthermore, knockdown of MKK4 or blocking JNK activity significantly suppressed the invasiveness of these cells and the production of MMPs. However, knockdown of NF-кB did not affect the invasive ability and MMPs production in these cells. Overexpression of NF-кB could enhance the sensitivity of cell migration responded to LPS-TLR4 signaling. It is difficult to determine the role of NF-кB in the migration of these HCCs in our system, which may need further investigation. In addition, both NF-кB silencing and MKK4/JNK blocking could inhibit the production of IL-6 and TNF␣ induced by LPS treatment. It indicated the powerful potential of NF-кB in regulation of inflammatory cytokines production. Therefore, our data suggested that LPS promoted the invasive activity of these HCC cells mainly through MKK4/JNK pathway. Except NF-кB, the JNK pathway also controls the expression of MMP2 and MMP9 in other cell types (Gweon and Kim, 2014; Lin et al., 2012; Wang et al., 2014a). Moreover, the study from Li et al. (2014b) supported our results. They found that TLR4/JNK signaling was required for LPS-induced EMT in HepG2 cells, but they did not observe the activation of NF-кB and other MAPKs at the same time. In conclusion, our study has shown that increased invasiveness of hepatocellular carcinoma cells triggered by LPS-TLR4 signaling is mainly mediated by the TLR4/MKK4/JNK pathway. Our findings suggest that MKK4/JNK may be the potential targets for the treatment of hepatocellular cancer. Ackownledgements This work was supported by the National Natural Science Foundation of China Grant (No. 31170841 and 31370879), and Qianjiang Talent project of Zhejiang Province (No. 2013R10040). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molimm.2015. 10.015. Reference: Dapito, D.H., Mencin, A., Gwak, G.Y., Pradere, J.P., Jang, M.K., Mederacke, I., Caviglia, J.M., Khiabanian, H., Adeyemi, A., Bataller, R., Lefkowitch, J.H., Bower, M., Friedman, R., Sartor, R.B., Rabadan, R., Schwabe, R.F., 2012. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell 21, 504–516. Deakin, N.E., Chaplain, M.A., 2013. Mathematical modeling of cancer invasion: the role of membrane-bound matrix metalloproteinases. Front. Oncol. 3, 70. Elinav, E., Nowarski, R., Thaiss, C.A., Hu, B., Jin, C., Flavell, R.A., 2013. Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms. Nat. Rev. Cancer 13, 759–771. Gweon, E.J., Kim, S.J., 2014. Resveratrol attenuates matrix metalloproteinase-9 and -2-regulated differentiation of HTB94 chondrosarcoma cells through the p38 kinase and JNK pathways. Oncol. Rep. 32, 71–78. Hartwell, H.J., Petrosky, K.Y., Fox, J.G., Horseman, N.D., Rogers, A.B., 2014. Prolactin prevents hepatocellular carcinoma by restricting innate immune activation of c-Myc in mice. Proc. Natl. Acad. Sci. U. S. A. 111, 11455–11460.
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