Yuehui Li

Toll-like receptor 3 (TLR3) induces apoptosis via death receptors and mitochondria by up-regulating the transactivating p63 isoform α (tap63α)

Toll-like receptor 3 (TLR3), a member of the pathogen recognition receptors, is widely expressed in various cells and has been shown to activate immune signaling pathways by recognizing viral double-stranded RNA. Recently, it was reported that the activation of TLR3 induced apoptosis in some cells, but the detailed molecular mechanism is not fully understood. In this study, we found that in endothelial cells polyinosinic-polycytidylic acid (poly(I-C)) induced dose- and time-dependent cell apoptosis, which was elicited by TLR3 activation, as TLR3 neutralization and down-regulation repressed the apoptosis. Poly(I-C) induced the activation of both caspases 8 and 9, indicating that TLR3 triggered the signaling of both the extrinsic and intrinsic apoptotic pathways. Poly(I-C) up-regulated tumor necrosis factor-related apoptosis-inducing ligand and its receptors, death receptors 4/5, resulting in initiating the extrinsic pathway. Furthermore, poly(I-C) down-regulated anti-apoptotic protein, B cell lymphoma 2 (Bcl-2), and up-regulated Noxa, a key Bcl-2 homology 3-only antagonist of Bcl-2, leading to the priming of the intrinsic pathway. A p53-related protein, the transactivating p63 isoform α (TAp63α), was induced by TLR3 activation and contributed to the activation of both the intrinsic and extrinsic apoptotic pathways. Both the cells deficient in p63 gene expression by RNA interference and cells that overexpressed the N-terminally truncated p63 isoform α (ΔNp63α), a dominant-negative variant of TAp63α, by gene transfection, survived TLR3 activation. Taken together, TAp63α is a crucial regulator downstream of TLR3 to induce cell death via death receptors and mitochondria.

The Expression of Functional Chemokine Receptor CXCR4 Is Associated with the Metastatic Potential of Human Nasopharyngeal Carcinoma

Purpose: Chemokine receptors are implicated in metastasis of several malignant tumors. This study was done to evaluate the contribution of chemokine receptors CXCR4 and CCR7 to metastasis of human nasopharyngeal carcinoma. Experimental Design: Reverse transcription-PCR, immunohistochemistry, and flow cytometry were used to evaluate mRNA and protein expression of CXCR4 and CCR7 in nasopharyngeal carcinoma tumor tissues and cell lines. Chemotaxis assays were used to evaluate the function of CXCR4 in nasopharyngeal carcinoma cells. Antisense CXCR4 was used to inhibit receptor expression and to block metastasis of human nasopharyngeal carcinoma cells in vivo in athymic mice. Results: CXCR4 protein was detected in tumor cells in 31 of 40 primary human nasopharyngeal carcinoma and in 13 of 15 lymph node metastases. CXCR4 transcripts were detected in eight CXCR4 protein–positive primary nasopharyngeal carcinoma tissues and seven nasopharyngeal carcinoma cell lines tested. On the other hand, the transcripts for CCR7 were detected only in four primary nasopharyngeal carcinoma tissues and in none of the nasopharyngeal carcinoma cell lines. In functional experiments, metastatic nasopharyngeal carcinoma cell lines that expressed high levels of CXCR4 were found to migrate in response to the CXCR4 ligand SDF-1α. Transfection of antisense CXCR4 in metastatic nasopharyngeal carcinoma cells inhibited the expression of CXCR4 and SDF-1α-induced cell migration in vitro and reduced the capacity of the tumor cells to form metastasis in the lungs and lymph nodes when injected in athymic mice. Conclusion: The expression of functional CXCR4 but not CCR7 is correlated with the metastatic potential of human nasopharyngeal carcinoma cells. Therefore, CXCR4 may be considered as a potential target for the prevention of nasopharyngeal carcinoma metastasis.

TLR3 activation inhibits nasopharyngeal carcinoma metastasis via downregulation of chemokine receptor CXCR4.

Toll-like receptor 3 (TLR3), a receptor for viral double strain RNA (dsRNA), mediates anti-viral immune response by activating the innate immunity and cross-priming CD8+ T cells. TLR3 agonists can directly trigger apoptosis in human cancer cells, and have been used as adjuvants to treat cancer patients with the aim of inducing an IFN-dependent immune response. In this study, we examined the effect of TLR3 activation on the metastasis of nasopharyngeal carcinoma (NPC). We found that NPC cells expressed TLR3 gene transcript and protein. TLR3 activation down-regulated the expression of chemokine receptor CXCR4 in a dose-dependent manner, and inhibited cell migration in response to CXCR4 ligand stromal cell-derived factor-1α (SDF-1α) in chemotaxis assays. Furthermore, TLR3 activation significantly reduced the capacity of NPC cells to form metastasis in draining lymph nodes when injected in athymic mice. The anti-metastasis activity of endogenous human TLR3 expression in cancer cells reveals a novel aspect of the multiple-faced TLR biology, which may open new clinical prospects for using TLR3 agonists to control cancer metastasis in selected cancers.

The role of MALAT1 correlates with HPV in cervical cancer.

Cervical cancer, the second most common type of cancer in women worldwide, is responsible for >275,100 mortalities each year and is associated with high-risk human papilloma virus (HR-HPV). HPVs have two important oncogenes, E6 and E7, which have crucial roles in malignant transformation in cervical cancer. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA originally identified in non-small cell lung cancer. Previous studies have revealed that MALAT1 is expressed in numerous tissue types, and is significant in maintaining the normal function of the body. However, it also appeared to be notably upregulated in numerous carcinoma types compared with adjacent non-cancerous tissues. In the present study, it was identified that MALAT1 expression was upregulated in cervical cancer cell lines compared with normal cervical squamous cell samples. Further study into the effect of MALAT1 on cellular phenotype revealed that MALAT1 was able to promote cell migration and proliferation. Of note, it was revealed that the expression of MALAT1 was decreased with the knockdown of HPV16 E6/E7 in CaSki cells. Furthermore, the investigations in clinical samples also revealed that MALAT1 was expressed in HPV-positive cervical squamous cells, but not in HPV-negative normal cervical squamous cells. These results indicate that HPV correlates with MALAT1 deregulation in cervical cancer.

The expression of functional toll‐like receptor 4 is associated with proliferation and maintenance of stem cell phenotype in endothelial progenitor cells (EPCs)

Endothelial dysfunction is involved in various cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) contribute to re‐endothelialization and neo‐vascularization, and the increase of EPCs in peripherial circulation benefits the prognosis of cardiovascular disease. However, little is known about the biological stimuli that initiate the proliferation and the maintenance of stem cell phenotype of EPCs. Here we reported that human umbilical vein blood derived EPCs expressed gene transcripts coding for Toll‐like receptor (TLR) 1–6, TLR8–10, TLR4 co‐receptor CD14, and myeloid differentiation factor 88 (MyD88), a TLR adaptor molecule. Protein expression of TLR2, 4, CD14, and MyD88 was also detected by FACS or Western blot. The activation of TLR4 by LPS modulated the expression of TLRs, induced the phosphorylation of NF‐κB, P38, and ERK42/44, and up‐regulated the gene expression of cytokines IL‐8, IFN‐α, IFN‐β, and TNF‐α, suggesting EPCs expressed functional TLR4. Unexpectedly LPS treatment failed to induce apoptosis in EPCs, but instead promoted cell proliferation of EPCs. Furthermore, the treatment of EPCs with LPS up‐regulated stem cell markers AC133 and CD34 in both mRNA and protein levels, and down‐regulated the protein expression of differential marker eNOS. These results suggested that TLR4 functions to maintain the stem cell phenotype of EPCs and enlarge its population, which reveals a novel aspect of the multiple‐faced TLR biology, and may open new prospects for using TLR4 agonists to promote the production of EPCs for clinical use. J. Cell. Biochem. 111: 179–186, 2010.

GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit

AIMnTo investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC).nnnMETHODSnSemiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Changs liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed.nnnRESULTSnWe identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ.nnnCONCLUSIONnGABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC.

PAX6, a novel target of microRNA-7, promotes cellular proliferation and invasion in human colorectal cancer cells.

BackgroundPaired box 6 (PAX6), a highly conserved transcriptional factor, has been implicated in tumorigenesis.AimWe aimed to explore the roles and molecular mechanisms of PAX6 and microRNA (miR-7) in colorectal cancer cells.MethodsTissue microarray immunohistochemistry and Western blot were applied to examine the PAX6 expression. Real-time RT-PCR and Western blot were performed to determine the expression of miR-7 and PAX6. Luciferase reporter assay was used to determine whether PAX6 was a target of miR-7. Effects of miR-7 and PAX6 on colorectal cell proliferation, cell cycle progression, colony formation and invasion were then investigated. Western blot was used to determine the activities of the ERK and PI3K signal pathways, as well as the protein expression of MMP2 and MMP9.ResultsThe protein levels of PAX6 were gradually increased, while the expression of miR-7 was gradually reduced with malignancy of colorectal cancer. PAX6 was further identified as a target of miR-7, and its protein expression was negatively regulated by miR-7 in human colorectal cancer cells. Overexpression of PAX6 in Caco-2 and SW480 cells enhanced cellular proliferation, cell cycle progression, colony formation, and invasion, while miR-7 upregulation repressed these biological processes. Furthermore, the activities of ERK and PI3K signal pathways, as well as the protein levels of MMP2 and MMP9, were upregulated in PAX6-overexpressed Caco-2 and SW480 cells but deregulated in miR-7-overexpressed Caco-2 and SW480 cells.ConclusionsOur study suggests that as a novel target of miR-7, PAX6 may serve as a promising therapeutic target for colorectal cancer.

Autoantibodies as potential biomarkers for nasopharyngeal carcinoma

Autoantibody signatures, as new biomarkers, may improve the early detection of nasopharyngeal carcinoma (NPC). We constructed a T7 phage cDNA library from mixed NPC tissues, and we isolated 31 tumor‐associated proteins using biopan enrichment techniques with sera from NPC patients and from healthy population. DNA sequence analysis showed that among 31 phage‐displayed proteins, 22 have sequence identity with known or putative tumor‐associated proteins. The results of immunochemical reactivity of patients sera with phage‐expressed proteins showed enrichment in the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in the sera of patients but not in the sera of healthy donors. The autoantibody against phage‐expressed protein MAGE, HSP70, Fibronectin, and CD44 measured by ELISA had greater predictive value than that against EBNA‐1, respectively. The antibody levels against MAGE in sera positively correlated with the clinical stages of NPC, and the antibody levels against other three proteins partly correlated with the clinical stages of NPC. Our studies suggested that the autoantibodies against tumor‐associated antigens in the sera of NPC patients could be used as a screening test for NPC. Studies of the corresponding proteins may have significances in tumor biology, novel drug development, and immunotherapy.

Stanniocalcin1 (STC1) Inhibits Cell Proliferation and Invasion of Cervical Cancer Cells

STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-κB p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-κB p65 activation in cervical cancer.

Identification of genes associated with tumor development in CaSki cells in the cosmic space

It is important to understand the mechanisms of tumor development for curing cervical cancer. However, the molecular basis determining the different characteristics of tumor remains unclear. Space environment as a special study model can expand the study field of tumor development. To approach this, after human cervical carcinoma CaSki cells were flown on “Shen Zhou IV” space shuttle mission, the cell morphology and proliferation was investigated after flying to ground. We found that the growth of 48A9 CaSki cell (flight group) became slow compared with ground groups. Observation of cells by light microscopy revealed differences in cell morphology between ground controls and flight groups, and the flight group exhibited morphologic differences, characterized by rounder, smoother, decreased, smaller and low-adhension cells. Transmission electron microscope images showed the structure of the ultrastructural characteristics of 48A9 CaSki cells were clearly distinct from those of the ground CaSki cells in aspects of mitochondrion, cytoplasm, nucleus and ribosomes. MTT and soft agar assay showed that 48A9 CaSki cells grew slowly compared to ground control. Furthermore, suppression subtractive hybridization combining with reverse Northern blot was used to identify differently expression genes between flight and ground groups. These differentially expressed genes included cytoskeleton, cell differentiation, cell apoptosis, signal transduction, DNA repair, protein synthesis, substance metabolism, and antigen presentation. The identification of differently expressed genes which is likely to increase our understanding of the molecular processes underlying tumor development will provide new insight into tumor development mechanisms, and may facilitate the development of new anticancer strategies.