Kuang-Hui Sun

Monoclonal anti‐double‐stranded DNA autoantibody stimulates the expression and release of IL‐1β, IL‐6, IL‐8, IL‐10 and TNF‐α from normal human mononuclear cells involving in the lupus pathogenesis

In our previous reports, we found polyclonal anti‐double‐stranded DNA antibodies (anti‐dsDNA) purified from patients with active systemic lupus erythematosus (SLE) exerted inhibitory effect on [3H]thymidine incorporation of human mononuclear cells (MNC). However, the other immunological effects of anti‐dsDNA on the functions of MNC have not yet been reported. In this study, two monoclonal antibodies, 12B3 and 9D7, with different anti‐dsDNA activity were evaluated for their effects on the expression and release of different cytokines from human MNC. We confirmed absence of endotoxin in the two monoclonal antibody preparations and the used medium as detected by Limulus amoebocyte lysate test. The mRNA expression and release of different cytokines including interleukin (IL)‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) were measured. We found the two monoclonal anti‐dsDNA not only dose‐responsively suppressed the phytohaemagglutinin (PHA)‐induced thymidine uptake of human MNC but stimulated the mRNA expression of IL‐1β, IL‐6 and IL‐8 in normal human MNC detected by reverse transcription–polymerase chain reaction (RT–PCR). Enzyme‐linked immunosorbent assay (ELISA) measurement of cytokines in MNC culture supernatants revealed that anti‐dsDNA enhanced IL‐1β, IL‐8, TNF‐α and IL‐10 release from resting MNC. These effects of anti‐dsDNA antibodies were not affected by polymyxin B, a potent binder and neutralizer of lipopolysaccharide (LPS). These in vitro studies suggest that anti‐dsDNA possess a dual effect on normal human MNC: (a) to enhance the release of proinflammatory cytokines (IL‐1β, IL‐8 and TNF‐α) from MNC to augment inflammatory reaction; and (b) to polarize the immune reaction towards the T helper 2 (Th2) (increased IL‐10 production) pathway. This unique effect of anti‐dsDNA may play a role in lupus pathogenesis by augmenting inflammatory reactions and autoantibody production which are commonly found in patients with active SLE.

Tumor-derived tumor necrosis factor-alpha promotes progression and epithelial-mesenchymal transition in renal cell carcinoma cells

Pro‐inflammatory cytokines and chemokines are involved in promoting tumorigenesis by facilitating tumor proliferation and metastasis. The serum levels of interleukin (IL)‐6, IL‐1β, and tumor necrosis factor‐alpha (TNF‐α) are significantly elevated in patients with renal cell carcinoma (RCC). However, the mechanisms of how these cytokines participate in the progression of RCC remains unknown. In the present study, we investigated the effects of tumor‐derived cytokines on invasion and the epithelial‐mesenchymal transition (EMT) of RCC cells. We found that expression of IL‐1β, IL‐6, TNF‐α, hypoxia‐inducible factor‐alpha (HIF‐1α), and matrix metalloproteinase‐2 (MMP2) were significantly elevated in high malignancy A498 cells compared to low malignancy 786‐O cells. The invasion ability of A498 was three‐fold higher than that of 786‐O cells. The invasiveness of 786‐O cells was markedly enhanced by adding conditioned medium derived from A498 cells. This phenomenon was significantly inhibited by immunodepletion of TNF‐α followed by MMP2, IL‐6, or IL‐1β from A498 conditioned medium. Synergistic inhibition was also noted after simultaneous immunodepletion of TNF‐α, IL‐1β, and IL‐6. RCC cell lines with higher malignancy produced more TNF‐α, which was correlated with their stronger invasive ability. The invasiveness of 786‐O cells was significantly promoted by TNF‐α in a dose‐dependent manner. Moreover, TNF‐α induced the EMT of 786‐O cells by repressing E‐cadherin, promoting vimentin expression, and activating MMP9 activity. Our findings demonstrate that pro‐inflammatory cytokines, especially TNF‐α, can enhance invasion and the EMT of renal cancer cells, which provides a therapeutic target to prevent and treat advanced RCC. (Cancer Sci 2008; 99: 905–913)

Galectin-1 promotes lung cancer progression and chemoresistance by upregulating p38 MAPK, ERK and cyclooxygenase-2

Purpose: This study is aimed at investigating the role and novel molecular mechanisms of galectin-1 in lung cancer progression. Experimental Design: The role of galectin-1 in lung cancer progression was evaluated both in vitro and in vivo by short hairpin RNA (shRNA)-mediated knockdown of galectin-1 in lung adenocarcinoma cell lines. To explore novel molecular mechanisms underlying galectin-1–mediated tumor progression, we analyzed gene expression profiles and signaling pathways using reverse transcription PCR and Western blotting. A tissue microarray containing samples from patients with lung cancer was used to examine the expression of galectin-1 in lung cancer. Results: We found overexpression of galectin-1 in non–small cell lung cancer (NSCLC) cell lines. Suppression of endogenous galectin-1 in lung adenocarcinoma resulted in reduction of the cell migration, invasion, and anchorage-independent growth in vitro and tumor growth in mice. In particular, COX-2 was downregulated in galectin-1–knockdown cells. The decreased tumor invasion and anchorage-independent growth abilities were rescued after reexpression of COX-2 in galectin-1–knockdown cells. Furthermore, we found that TGF-β1 promoted COX-2 expression through galectin-1 interaction with Ras and subsequent activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal–regulated kinase (ERK), and NF-κB pathway. Galectin-1 knockdown sensitized lung cancer cells to platinum-based chemotherapy (cisplatin). In addition, galectin-1 and COX-2 expression was correlated with the progression of lung adenocarcinoma, and high clinical relevance of both proteins was evidenced (n = 47). Conclusions: p38 MAPK, ERK, and COX-2 activation are novel mediators for the galectin-1–promoted tumor progression and chemoresistance in lung cancer. Galectin-1 may be an innovative target for combined modality therapy for lung cancer. Clin Cancer Res; 18(15); 4037–47. ©2012 AACR.

IL-27 Directly Restrains Lung Tumorigenicity by Suppressing Cyclooxygenase-2-Mediated Activities

Gene transfer of IL-27 to tumor cells has been proven to inhibit tumor growth in vivo by antiproliferation, antiangiogenesis, and stimulation of immunoprotection. To investigate the nonimmune mechanism of IL-27 that suppresses lung cancer growth, we have established a single-chain IL-27-transduced murine Lewis lung carcinoma (LLC-1) cell line (LLC-1/scIL-27) to evaluate its tumorigenic potential in vivo. Mice inoculated with LLC/scIL-27 displayed retardation of tumor growth. Production of IL-12, IFN-γ, and cytotoxic T cell activity against LLC-1 was manifest in LLC/scIL-27-injected mice. Of note, LLC-1/scIL-27 exhibited decreased expression of cyclooxygenase-2 (COX-2) and PGE2. On the cellular level, the LLC/scIL-27 transfectants had reduced malignancy, including down-regulation of vimentin expression and reduction of cellular migration and invasion. The suppression of tumorigenesis by IL-27 on lung cancer cells was further confirmed by the treatment with rIL-27 on the murine LLC-1 and human non-small cell lung carcinoma (NSCLC) cell lines. PGE2-induced vimentin expression, movement, and invasiveness were also suppressed by the treatment with rIL-27. Our data show that IL-27 not only suppresses expression of COX-2 and PGE2 but also decreases the levels of vimentin and the abilities of cellular migration and invasion. Furthermore, inoculation of LLC/scIL-27 into immunodeficient NOD/SCID mice also exhibited reduced tumor growth. Our data indicate that IL-27-induced nonimmune responses can contribute to significant antitumor effects. Taken together, the results suggest that IL-27 may serve as an effective agent for lung cancer therapy in the future.

TNF-α Induces Epithelial–Mesenchymal Transition of Renal Cell Carcinoma Cells via a GSK3β-Dependent Mechanism

TNF-α is a cytokine with antitumorigenic property. In contrast, low dose, chronic TNF-α production by tumor cells or stromal cells may promote tumor growth and metastasis. Serum levels of TNF-α are significantly elevated in renal cell carcinoma (RCC) patients. Here, we showed that TNF-α induced epithelial–mesenchymal transition (EMT) and promoted tumorigenicity of RCC by repressing E-cadherin, upregulating vimentin, activating MMP9, and invasion activities. In addition, TNF-α treatment inhibited glycogen synthase kinase 3β (GSK-3β) activity through serine-9 phosphorylation mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in RCC cells. Inhibition of PI3K/AKT by LY294002 reactivated GSK-3β and suppressed the TNF-α–induced EMT of RCC cells. Inactivation of GSK-3β by LiCl significantly increased MMP9 activity and EMT of RCC cells. Activation of GSK-3β by transduction of constitutively active GSK-3β into RCC cells suppressed TNF-α–mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient GSK-3β, in contrast, potentiated EMT, anchorage-independent growth and drastically enhanced tumorigenicity in vivo. Most importantly, a 15-fold inactivation of GSK-3β activity, 3-fold decrease of E-cadherin, and 2-fold increase of vimentin were observed in human RCC tumor tissues. These results indicated that inactivation of GSK-3β plays a pivotal role in the TNF-α–mediated tumorigenesis of RCC. Mol Cancer Res; 10(8); 1109–19. ©2012 AACR.

Anti‐SSB/La is one of the antineutrophil autoantibodies responsible for neutropenia and functional impairment of polymorphonuclear neutrophils in patients with systemic lupus erythematosus

Decreased number and impaired functions of polymorphonuclear neutrophils (PMN) due to the presence of anti‐PMN autoantibodies in the serum render patients with systemic lupus erythematosus (SLE) susceptible to bacterial infections. However, the cognate antigens and pathological mechanisms of anti‐PMN autoantibodies in SLE are rarely reported in the literature. In this study, we found approximately 20% of SLE sera contained anti‐PMN autoantibodies detected by human PMN‐coated cellular ELISA. A membrane protein with molecular weight of 50 kDa was identified as the cognate antigen of anti‐PMN in Western blot after membrane‐biotinylation and streptavidin column elution. The 50 kDa molecule was proved to be SSB/La after immunoscreening, molecular cloning and nucleotide sequencing of the gene from the human leucocyte cDNA library. Human anti‐SSB/La autoantibodies purified from active SLE sera passing through the recombinant SSB/La conjugated Sepharose 4B affinity column could bind and penetrate into normal human PMN. Functional analysis revealed that the anti‐SSB/La autoantibodies exerted a number of potent effects on human PMN, including suppressed phagocytosis, accelerated apoptosis and enhanced IL‐8 production. These in vitro results suggest that anti‐SSB/La is one of the anti‐PMN autoantibodies capable of penetrating into PMN and responsible for neutropenia and functional impairment of PMN in patients with SLE.

ABNORMAL SPLENIC AND THYMIC IL-4 AND TNF-ALPHA EXPRESSION IN MRL-LPR/LPR MICE

The MRL‐lpr/lpr and MRL‐++ mice were studied for the expression of cytokines in the spleen, lymph node., thymus, kidney and brain through the reverse transcription‐polymerase chain reaction (RT‐PCR). The frequencies of IL‐4 and TNF‐a expression in the thymus and spleen were significantly higher in MRL‐lpr/lpr mice than in MRL‐++ mice from the age of 17 to 32 weeks. More importantly, IL‐4 transcript was demonstrated in the early rather than in the terminal stage of the lupus disease. At the 20th week, MRL‐lpr/lpr mice with active disease exhibited higher concentrations of IL‐1α, IL‐6 and TNF‐a in serum than MRL‐++ mice. Interestingly, in MRL‐lpr/lpr but not MRL‐++ mice, the IL‐6 concentration in culture supernatants of the thymic cells was significantly higher than that of the splenic or lymph node cells. On the other hand, IL‐6 and IL‐l/? were expressed in the brain and kidney of MRL‐lpr/lpr mice but not of MRL‐++ mice. Cultured MRL‐lpr/lpr mesangial cells could also express IL‐6 but to a lesser extent. These results suggest that the abnormal splenic and thymic IL‐4 and TNF‐α expression may predispose the development of autoimmune reactions. The expression of IL‐1ß and IL‐6 in the brain and kidney may be implicated in the damage of these two organs in MRL‐lpr/lpr mice.

Cold-induced ependymin expression in zebrafish and carp brain: implications for cold acclimation.

Cold acclimation has been suggested to be mediated by alternations in the gene expression pattern in the cold‐adapted fish. To investigate the mechanism of cold acclimation in fish brain at the molecular level, relevant subsets of differentially expressed genes of interest were identified and cloned by the PCR‐based subtraction suppression hybridization. Characterization of the selected cold‐induced cDNA clones revealed one encoding ependymin. This gene was shown to be brain‐specific. The expression of ependymin was induced by a temperature shift from 25°C to 6°C in Cyprinus carpio or 12°C in Danio rerio. Activation of ependymin was detected 2 h after cold exposure and peaked at more than 10‐fold at 12 h. This peak level remains unchanged until the temperature returns to 25°C. Although the amount of soluble ependymin protein in brain was not changed by cold treatment, its level in the fibrous insoluble polymers increased 2‐fold after exposure to low temperature. These findings indicate that the increase in ependymin expression is an early event that may play an important role in the cold acclimation of fish.

Autocrine CCL2 promotes cell migration and invasion via PKC activation and tyrosine phosphorylation of paxillin in bladder cancer cells

The amount of monocyte chemoattractant protein-1 (MCP-1/CCL2) produced by a transitional cell carcinoma is directly correlated with high recurrence and poor prognosis in bladder cancer. However, the mechanisms underlying the effects of CCL2 on tumor progression remain unexplored. To investigate the role played by CCL2, we examined cell migration in various bladder cancer cell lines. We found that high-grade cancer cells expressing high levels of CCL2 showed more migration activity than low-grade bladder cancer cells expressing low levels of the chemokine. Although the activation of CCL2/CCR2 signals did not appreciably affect cell growth, it mediated cell migration and invasion via the activation of protein kinase C and phosphorylation of tyrosine in paxillin. Blocking CCL2 and CCR2 with small hairpin RNA (shCCL2) or a specific inhibitor reduced CCL2/CCR2-mediated cell migration. The antagonist of CCR2 promoted the survival of mice bearing MBT2 bladder cancer cells, and CCL2-depleted cells showed low tumorigenicity compared with shGFP cells. In addition to observing high-levels of CCL2 in high-grade human bladder cancer cells, we showed that the CCL2/CCR2 signaling pathway mediated migratory and invasive activity, whereas blocking the pathway decreased migration and invasion. In conclusion, high levels of CCL2 expressed in bladder cancer mediates tumor invasion and is involved with advanced tumorigenesis. Our findings suggest that this CCL2/CCR2 pathway is a potential candidate for the attenuation of bladder cancer metastases.

Anti-dsDNA antibody up-regulates interleukin 6, but not cyclooxygenase, gene expression in glomerular mesangial cells: a marker of immune-mediated renal damage?

Abstract:Objective and Design: To determine whether anti-double stranded DNA antibody (anti-dsDNA) can affect the synthesis of eicosanoids and cytokines in rat glomerular mesangial cells (RMC).¶Materials or Subjects: Glomerular mesangial cells were isolated and subcultured from Sprague-Dawley rats. Monoclonal anti-dsDNA (12B3 clone) was derived from autoimmune MRL-lpr/lpr mouse by hybridoma technology.¶Methods: The mRNA expression of cyclo-oxygenase type 1 (COX-1), type 2 (COX-2), Th1 (IL-2 and IFN-γ)/Th2 (IL-4 and IL-10) and proinflammatory (IL-6 and TNF-α) and anti-inflammatory (TGF-β) cytokines of RMC ± anti-dsDNA was detected by RT-PCR. The PGE2 production by RMC ± anti-dsDNA was measured by ELISA. The statistical significance was assessed by non-parametric Wilcoxon signed rank test.¶Results: We found RMC spontaneously expressed COX-1, but not COX-2. The incubation of RMC with anti-dsDNA (50ng/ml) did not affect COX expression and PGE2 production by RMC. RMC also spontaneously expressed IL-6, TNF-α and TGF-β mRNA. However, only IL-6 was up-regulated by anti-dsDNA.¶Conclusions: Increased IL-6 expression in RMC may become a marker of anti-dsDNA-mediated immune damage of mesangial cells.¶