Neurotrophin-3 Facilitates Stemness Properties and Associates with Poor Survival in Lung Cancer.

INTRODUCTION: Cancer stem cells (CSCs) shape the tumor microenvironment via neuroendocrine signaling and orchestrate drug resistance and metastasis. Cytokine antibody array demonstrated the upregulation of neurotrophin-3 (NT-3) in lung CSCs. This study aims to dissect the role of NT-3 in lung CSCs during tumor innervation. METHODS: Western blotting, quantitative reverse transcription-PCR, and flow cytometry were used to determine the expression of the NT-3 axis in lung CSCs. NT-3-knockdown and NT-3-overexpressed cells were derived lung CSCs, followed by examining the stemness gene expression, tumorsphere formation, transwell migration and invasion, drug resistance, soft agar colony formation, and in vivo tumorigenicity. Human lung cancer tissue microarray and bioinformatic databases were used to investigate the clinical relevance of NT-3 in lung cancer. RESULTS: NT-3 and its receptor tropomyosin receptor kinase C (TrkC) were augmented in lung tumorspheres. NT-3 silencing (shNT-3) suppressed the migration and anchorage-independent growth of lung cancer cells. Further, shNT-3 abolished the sphere-forming capability, chemo-drug resistance, invasion, and in vivo tumorigenicity of lung tumorspheres with a decreased expression of CSC markers. Conversely, NT-3 overexpression promoted migration and anchorage-independent growth and fueled tumorsphere formation by upregulating the expression of CSC markers. Lung cancer tissue microarray analysis revealed that NT-3 increased in patients with advanced-stage, lymphatic metastasis and positively correlated with Sox2 expression. Bioinformatic databases confirmed a co-expression of NT-3/TrkC-axis and demonstrated that NT-3, NT-3/TrkC, NT-3/Sox2, and NT-3/CD133 worsen the survival of lung cancer patients. CONCLUSION: NT-3 conferred the stemness features in lung cancer during tumor innervation, which suggests that NT-3-targeting is feasible in eradicating lung CSCs.

CXCR3 isoform A promotes head and neck cancer progression by enhancing stem-like property and chemoresistance.

BACKGROUND: The chemokine network orchestrates the cancer stem-like property and consequently participates in cancer progression. CXCR3 contributes cancer progressive property and immunomodulation in the tumor microenvironment. The two major isoforms of CXCR3 are scrutinized and the divergence is showed that CXCR3A promotes cancer cell growth and motility while CXCR3B functions contrarily in many studies. However, rare studies illustrate the role of CXCR3 isoforms in cancer stem-like property and chemoresistance, especially in head and neck cancer (HNC). METHODS: Levels of CXCR3, CXCR3B, and Sox2 were determined in HNC tissue microarray by immunohistochemistry staining to explore potential clinical relevance. Lentivirus-mediated CXCR3-isoform overexpression with MTS assay, clonogenic assay, transwell migration, sphere formation, and chemo-drug susceptibility were implemented to investigate the role of CXCR3-isofoms in HNC. RESULTS: High levels of CXCR3 were significantly associated with advanced stage (p < 0.01), regional lymph node metastasis (p < 0.05), and poor differentiation (p < 0.005) and further correlated with worse survival rate in oral cancer patients (p = 0.036). Higher levels of CXCR3B were found in regional lymphatic invasion of HNC and progressive stage of squamous cell carcinoma. Elevated Sox2 expression was significantly associated with the advanced stage of HNC in the oral cavity, and demonstrated a co-expression pattern with CXCR3B. Furthermore, lentivirus-mediated overexpression of CXCR3A and CXCR3B in SAS human oral cancer cells promoted cell mobility. CXCR3A overexpression enhanced sphere-forming ability and chemoresistance of CSCs by upregulating stemness-related genes. CONCLUSION: This study first provides a novel insight of CXCR3 isoform A in HNC cancer progression via regulating cancer stem-like properties and chemoresistance, suggesting that CXCR3A may be a prognostic marker and novel target for HNC therapy.

Platinum-based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS, but not autophagy.

These days, cancer can still not be effectively cured because cancer cells readily develop resistance to anticancer drugs. Therefore, an effective combination of drugs with different mechanisms to prevent drug resistance has become a very important issue. Furthermore, the BH3-only protein BNIP3 is involved in both apoptotic and autophagic cell death. In this study, lung cancer cells were treated with a chemotherapy drug alone or in combination to identify the role of BNIP3 and autophagy in combination chemotherapy for treating cancer. Our data revealed that various combinational treatments of two drugs could increase cancer cell death and cisplatin in combination with rapamycin or LBH589, which triggered the cell cycle arrest at the S phase. Cells with autophagosome and pEGFP-LC3 puncta increased when treated with drugs. To confirm the role of autophagy, cancer cells were pre-treated with the autophagy inhibitor 3-methyladenine (3-MA). 3-MA sensitized cancer cells to chemotherapy drug treatments. These results suggest that autophagy may be responsible for cell survival in combination chemotherapy for lung cancer. Moreover, BNIP3 was induced and localized in mitochondria when cells were treated with drugs. The transfection of a dominant negative transmembrane deletion construct of BNIP3 (BNIP3ΔTM) and treatment of a reactive oxygen species (ROS) inhibitor suppressed chemo drug-induced cell death. These results indicate that BNIP3 and ROS may be involved in combination chemo drug-induced cell death. However, chemo drug-induced autophagy may protect cancer cells from drug cytotoxicity. As a result, inhibiting autophagy may improve the effects of combination chemotherapy when treating lung cancer.

A novel intermittent negative air pressure device ameliorates obstructive sleep apnea syndrome in adults.

PURPOSE: Patients with obstructive sleep apnea syndrome (OSAS) have difficulties in compliance with continuous positive airway pressure (CPAP) and the treatment outcome is heterogeneous. We proposed a proof-of-concept study of a novel intermittent negative air pressure (iNAP®) device for physicians to apply on patients who have failed or refused to use CPAP. METHODS: The iNAP® device retains the tongue and the soft palate in a forward position to decrease airway obstruction. A full nightly usage with the device was evaluated with polysomnography. Subgrouping by baseline apnea-hypopnea index (AHI) and body mass index (BMI) with different treatment response criteria was applied to characterize the responder group of this novel device. RESULTS: Thirty-five patients were enrolled: age 41.9 ± 12.2 years (mean ± standard deviation), BMI 26.6 ± 4.3 kg/m2, AHI 41.4 ± 24.3 events/h, and oxygen desaturation index (ODI) 40.9 ± 24.4 events/h at baseline. AHI and ODI were significantly decreased (p < 0.001) by the device. Patients with moderate OSAS, with baseline AHI between 15 to 30 events/h, achieved 64% response rate; and non-obese patients, with BMI below 25 kg/m2, achieved 57% response rate, with response rate defined as 50% reduction in AHI from baseline and treated AHI lower than 20. There were minimal side effects reported. CONCLUSIONS: In a proof-of-concept study, the device attained response to treatment as defined, in more than half of the moderate and non-obese OSAS patients, with minimal side effects.

Galectin-3 promotes CXCR2 to augment the stem-like property of renal cell carcinoma.

Although targeted therapy is usually the first-line treatment for advanced renal cell carcinoma (RCC), some patients can experience drug resistance. Cancer stem cells are tumour-initiating cells that play a vital role in drug resistance, metastasis and cancer relapse, while galectins (Gal) participate in tumour progression and drug resistance. However, the exact role of galectins in RCC stemness is yet unknown. In this study, we grew a subpopulation of RCC cells as tumour spheres with higher levels of stemness-related genes, such as Oct4, Sox2 and Nanog. Among the Gal family, Gal-3 in particular was highly expressed in RCC tumour spheres. To further investigate Gal-3's role in the stemness of RCC, lentivirus-mediated knockdown and overexpression of Gal-3 in RCC cells were used to examine both in vitro and in vivo tumorigenicity. We further assessed Gal-3 expression in RCC tissue microarray using immunohistochemistry. Upon suppressing Gal-3 in parental RCC cells, invasion, colony formation, sphere-forming ability, drug resistance and stemness-related gene expression were all significantly decreased. Furthermore, CXCL6, CXCL7 and CXCR2 were down-regulated in Gal-3-knockdown tumour spheres, while CXCR2 overexpression in Gal-3-knockdown RCC restored the ability of sphere formation. Gal-3 overexpression in RCC promoted both in vitro and in vivo tumorigenicity, and its expression was correlated with CXCR2 expression and tumour progression in clinical tissues. RCC patients with higher co-expressions of Gal-3 and CXCR2 demonstrated a worse survival rate. These results indicate that highly expressed Gal-3 may up-regulate CXCR2 to augment RCC stemness. Gal-3 may be a prognostic and innovative target of combined therapy for treating RCC.

Triggering receptor expressed on myeloid cells-1 (TREM-1) deficiency augments BAFF production to promote lupus progression.

Sensing of nucleic acids by pattern recognition receptors is the key for the initiation and development of systemic lupus erythematosus (SLE). Triggering receptor expressed on myeloid cells-1 (TREM-1) is a novel innate immune receptor, which can amplify Toll-like receptor (TLR)-induced inflammatory responses. Although patients with lupus exhibit increased serum levels of soluble TREM-1 (sTREM-1), the role of TREM-1 in SLE remains unknown. In current study, we found serum sTREM-1 levels were significantly increased in lupus patients and positively correlated with disease activity. Additionally, diseased B6.lpr mice had elevated TREM-1 in the serum, spleen, and lymph nodes. To investigate the role of TREM-1 in lupus, we established Trem-1-/-.lpr mice. Trem-1-/-.lpr mice exhibited lower survival rates and more severe lupus symptoms, including elevated proteinuria, serum anti-dsDNA antibody levels, renal immune complex depositions and lymphocyte subpopulation expansions in both the spleen and lymph nodes. Besides, Trem-1-/-.lpr mice expressed higher serum B cell-activating factor (BAFF) levels and lymph node dendritic cells (DCs) were the major source of increased BAFF. Activation of membrane-bound TREM-1 could suppress TLR9-induced BAFF expression in bone marrow-derived DCs of B6.lpr mice. Moreover, levels of sTREM-1, which could act as an antagonist of membrane-bound TREM-1, were positively correlated with levels of BAFF in the sera of lupus patients. Our findings suggest a novel modulatory role of TREM-1 in the pathogenesis of SLE. sTREM-1 production is a useful diagnostic marker and a molecular target for combination therapy of lupus.

TNF-α augments CXCR2 and CXCR3 to promote progression of renal cell carcinoma.

Within the tumour microenvironment, a complex network of chemokines and their receptors affects the initiation and progression of tumours. The higher levels of tumour necrosis factor-alpha (TNF-α) are associated with tumour progression and an anti-TNF-α monoclonal antibody has been used successfully to treat patients with renal cell carcinoma (RCC). However, the role of chemokines and their receptors in the TNF-α-promoted progression of RCC remains unclear. In this study, TNF-α was found to enhance the migration, invasion and epithelial-mesenchymal transition (EMT) of RCC cells. To further investigate the molecular mechanism of TNF-α on the progression of RCC, reverse transcription and quantitative PCR was used to screen chemokines and chemokine receptors that were associated with tumorigenesis. The results showed that TNF-α significantly increased the expressions of CXCR2 and CXCR3 and their related ligands in RCC cells. Subsequently, we used a lentiviral shRNA system to knockdown the expression of CXCR2 and/or CXCR3 in RCC cells. CXCR2 and CXCR3 silencing inhibited the induction of Slug and ZEB-1 with TNF-α treatment of RCC cells. In addition, the knockdown of both CXCR2 and CXCR3 resulted in a greater decrease in cell migration, invasion and clonogenic ability compared with either CXCR2 or CXCR3 knockdown alone. Moreover, CXCR2 and CXCR3 silencing significantly reduced the sphere-forming ability of RCC cells. High expression levels of CXCR2 and CXCR3 in cancer tissues correlated with tumour progression of renal cell carcinoma. These findings suggest that TNF-α augments CXCR2 and CXCR3 to promote the progression of renal cell carcinoma leading to a poor prognosis.

Emodin modulates epigenetic modifications and suppresses bladder carcinoma cell growth.

The deregulation of epigenetics was involved in early and subsequent carcinogenic events. Reversing cancer epigenetics to restore a normal epigenetic condition could be a rational approach for cancer treatment and specialized prevention. In the present study, we found that the expression levels of two epigenetic markers, histone H3K27 trimethylation (H3K27me3), was low but histone H3S10 phosphorylation (pH3Ser10) was high in human bladder cancer tissues, which showed opposite expression patterns in their normal counterparts. Thus, we investigated whether a natural product, emodin, has the ability to reverse these two epigenetic modifications and inhibit bladder cancer cell growth. Emodin significantly inhibited the cell growth of four bladder cancer cell lines in a dose- and time-dependent manner. Emodin treatment did not induce specific cell cycle arrest, but it altered epigenetic modifications. Emodin treatment resulted in the suppression of pH3Ser10 and increased H3K27me3, contributing to gene silencing in bladder cancer cells. Microarray analysis demonstrated that oncogenic genes including fatty acid binding protein 4 (FABP4) and fibroblast growth factor binding protein 1 (HBP17), RGS4, tissue inhibitor of metalloproteinase 3 (TIMP3), WNT5b, URB, and collagen, type VIII, alpha 1 (COL8A1) responsible for proliferation, survival, inflammation, and carcinogenesis were significantly repressed by emodin. The ChIP assays also showed that emodin increased H3K27me3 but decreased pH3Ser10 modifications on the promoters of repressed genes, which indicate that emodin reverses the cancer epigenetics towards normal epigenetic situations. In conclusion, our work demonstrates the significant anti-neoplastic activity of emodin on bladder cancer cells and elucidates the novel mechanisms of emodin-mediated epigenetic modulation of target genes. Our study warrants further investigation of emodin as an effective therapeutic or preventive agent for bladder cancer.

Galectin-1 upregulates CXCR4 to promote tumor progression and poor outcome in kidney cancer.

Galectin-1, a ß-galactoside-binding lectin, is involved in many physiologic and pathologic processes, including cell adhesion, differentiation, angiogenesis, and tumor progression. However, the role of galectin-1 in kidney cancer remains elusive. This study evaluated the role of galectin-1 in the progression and clinical prognosis of renal cell carcinoma. We found significant overexpression of galectin-1 in both kidney cancer cell lines and metastatic tissue specimens from patients with renal cell carcinoma. Knockdown of galectin-1 gene expression in renal cancer cell lines reduced cell invasion, clonogenic ability, and epithelial-mesenchymal transition in vitro; reduced tumor outgrowth in vivo; and inhibited the angiogenesis-inducing activity of these cells in vitro and in vivo. Galectin-1 knockdown decreased CXCR4 expression levels in kidney cancer cells, and restoration of CXCR4 expression in galectin-1-silenced cells rescued cell motility and clonogenic ability. Additional studies suggested that galectin-1 induced CXCR4 expression through activation of nuclear factor-κB (NF-κB). Analysis of patient specimens confirmed the clinical significance and positive correlation between galectin-1 and CXCR4 expression levels and revealed concomitant overexpression of galectin-1 and CXCR4 associated adversely with overall and disease-free survival. Our findings suggest that galectin-1 promotes tumor progression through upregulation of CXCR4 via NF-κB. The coordinated upregulation of galectin-1 and CXCR4 may be a novel prognostic factor for survival in patients with renal cell carcinoma and the galectin-1-CXCR4 axis may serve as a therapeutic target in this disease.

Suspension bead array of the single-stranded multiplex polymerase chain reaction amplicons for enhanced identification and quantification of multiple pathogens.

Rapid identification of single and multiple infectious agents is vital in clinical settings and during biothreat attack. This study assesses the assay of single-stranded multiplex polymerase chain reaction (PCR) amplicons by suspension bead array (SSMP-SBA) for multiple pathogens identification in a single-tube reaction. A 15-plex assay for identification of 11 highly infectious pathogens was developed to evaluate the performance of SSMP-SBA. Pathogen-specific amplicons were obtained by sequential amplification of genomic DNAs using gene-specific primers tagged with artificial unique sequences and unique primers of which the reverse primer was modified by biotin and phosphorothioate. The SSMP products generated by T7 exonuclease-mediated DNA hydrolysis were hybridized to 15 sets of beads coupled with gene-specific and control oligonucleotide probes for pathogen identification and quantification by flow cytometry. This method was validated via assessment of 57 reference strains and one clinical bacterial isolate. All 11 pathogens can be detected by the 15-plex SSMP-SBA assay, and this design significantly enhanced the signal-to-noise ratio and improved the assay performance. This assay achieves similar sensitivity to our in-house real-time PCR system with the limit of detection equivalent to 5-100 genome copies and a linear dynamic range crossing three to five logs. In the validation assay, a 100% accuracy rate was achieved when the pathogens were among the target species. Notably, the species of pathogens were accurately identified from the samples with multiple infections. SSMP-SBA presents superior performance with multiplexing capability in a single-tube reaction and provides a new approach for detection and species identification of multiple pathogen infections.

Pre-existing Fas ligand (FasL) in cancer cells elicits tumor-specific protective immunity, but delayed induction of FasL expression after inoculation facilitates tumor formation.

Overexpression of Fas ligand (FasL) in cancer cells elicits potential antitumor effects via recruitment of neutrophils. Conversely, FasL-expressing tumors may counterattack tumor-infiltrating lymphocytes by delivering apoptotic death signals via Fas/FasL interactions, which may lead to tumor escape. In order to distinguish the role of FasL in antitumor activity and tumor progression, Lewis lung carcinoma cells (LLC-1) were used to establish the cell line LLC-FasL, in which FasL expression was repressed by doxycycline (Dox) treatment and induced in the absence of Dox. LLC-FasL cells promote tumor regression when expressing FasL, whereas tumor outgrowth is observed by depletion of FasL expression. To investigate whether initial expression of FasL during tumor formation is critical for FasL-mediated tumor regression, Dox-treated LLC-FasL cells were inoculated into Dox-treated mice, but Dox treatment was stopped 5 days after inoculation. When low cell numbers were inoculated, we observed 80% survival and no tumor formation, whereas no mice survived inoculation with high cell numbers, despite the delayed induction of FasL by Dox withdrawal. The inoculation of a high density of cells may establish a favorable tumor microenvironment before the expression of FasL. Our findings demonstrate that FasL may elicit antitumor activity when it is initially present on injected cancer cells and thus can act prior to tumor microenvironment formation. Furthermore, a well-established tumor microenvironment abrogates FasL-mediated antitumor activity.

TGFß1 induces CXCL1 to promote stemness features in lung cancer.

Chemokines critically orchestrate the tumorigenesis, metastasis, and stemness features of cancer cells that lead to poor outcomes. High plasma levels of transforming growth factor-ß1 (TGFß1) correlate with poor prognostic features in advanced lung cancer patients, thus suggesting the importance of TGFß1 in the lung tumor microenvironment. However, the role of chemokines in TGFß1-induced tumor stemness features remains unclear. Here, we clarify the previously undocumented role of CXCL1 in TGFß1-induced lung cancer stemness features. CXCL1 and its receptor CXCR2 were significantly upregulated in TGFß1-induced lung cancer stem cells (CSCs). CXCL1 silencing (shCXCL1) suppressed stemness gene expression, tumorsphere formation, colony formation, drug resistance, and in vivo tumorigenicity in TGFß1-induced lung tumorspheres. Immunohistochemistry staining showed that patients with stage II/III lung cancer had higher expression levels of CXCL1. The levels of CXCL1 were positively associated with lymph node metastasis and correlated with the expression of the CSC transcription factor Oct-4. Furthermore, online database analysis revealed that CXCL1 expression was negatively correlated with lung cancer survival in patients. Patients with high TGFß1/CXCL1/CD44 co-expression had a worse survival rate. We suggest that CXCL1 serves as a crucial factor in TGFß1-induced stemness features of lung cancer.

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.

PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFß1-promoted stem-like properties in head and neck cancer.

BACKGROUND: Resistance to chemotherapeutic drugs is a key factor for cancer recurrence and metastases in head and neck cancer (HNC). Cancer stem cells (CSCs) in tumors have self-renewal, differentiation, and higher drug resistance capabilities, resulting in a poor prognosis for patients. In glucose metabolism, pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase and impedes pyruvate from being metabolized into acetyl-CoA and entering the tricarboxylic acid cycle to generate energy. Studies have reported that PDK1 and PDK2 inhibition suppresses the growth, motility, and drug resistance of cancer cells. Furthermore, while TGFß1 levels are persistently elevated in HNC patients with poor prognosis, the role of PDK isoforms in the TGFß1-promoted progression and stem-like properties of HNC is unclear. METHODS: Levels of PDK1 and PDK2 were evaluated in HNC tissue microarrays by immunohistochemistry to explore potential clinical relevance. PDK1 and PDK2 were knocked down by the lentivirus shRNA system to investigate their role in TGFß1-promoted tumor progression in vitro. RESULTS: We found that PDK2 levels were increased in the later stage of HNC tissues compared to constant PDK1 expression. After PDK1 and PDK2 knockdown, we discovered increased ATP production and decreased lactate production in TGFß1-treated and untreated HNC cells. However, only PDK2 silencing significantly inhibited the clonogenic ability of HNC cells. We subsequently found that TGFß1-promoted migration and invasion capabilities were decreased in PDK1 and PDK2 knockdown cells. The tumor spheroid-forming capability, motility, CSC genes, and multidrug-resistant genes were downregulated in PDK1 and PDK2 silencing CSCs. PDK1 and PDK2 inhibition reversed cisplatin and gemcitabine resistance of CSCs, but not paclitaxel resistance. CONCLUSION: The results demonstrated that the PDK1- and PDK2-mediated Warburg effect contributes to the TGFß1-enhanced stemness properties of HNC. Therefore, PDK1 and PDK2 may serve as molecular targets for the combination therapy of HNC.

Fucoidan from Sargassum hemiphyllum inhibits the stemness of cancer stem cells and epithelial-mesenchymal transitions in bladder cancer cells.

A variety of anticancer activities have been established for fucoidan from brown algae, whereas whether cancer stem cells (CSCs) are inhibited by sulfated polysaccharides is unexplored. In this study, fucoidan extracted from Sargassum hemiphyllum was showed heat stable and might tolerate 140 °C treatment. Fucoidan did not exhibit cytotoxicity in 5637 and T24 bladder cancer cells. After fucoidan treatment, the stress fibers were aggregated into thick and abundant underneath the plasma membrane and getting around the cells, and the structure of F-actin showed a remarkable change in the filopodial protrusion in T24 and 5637 cells. Using culture inserts, transwell assays and time lapse recordings showed that fucoidan inhibited cell migration. In the epithelial-mesenchymal transition (EMT), fucoidan downregulated the expression of vimentin, a mesenchymal marker, and upregulated the expression of E-cadherin, an epithelial marker. Additionally, the transcription levels of Snail, Slug, Twist1, Twist2, MMP2 and MMP9 were significantly decreased by fucoidan, indicating EMT suppression. CSCs are implicated in tumor initiation, metastatic spread, drug resistance and tumor recurrence. Our results showed that fucoidan inhibited stemness gene expression and sphere formation in bladder CSCs. For the first time, our findings demonstrated that fucoidan inhibits CSC formation and provides evidence as potential anticancer therapy.

Immunotherapy for lung cancers.

Lung cancer is the leading cause of cancer-related deaths worldwide. Although treatment methods in surgery, irradiation, and chemotherapy have improved, prognosis remains unsatisfactory and developing new therapeutic strategies is still an urgent demand. Immunotherapy is a novel therapeutic approach wherein activated immune cells can specifically kill tumor cells by recognition of tumor-associated antigens without damage to normal cells. Several lung cancer vaccines have demonstrated prolonged survival time in phase II and phase III trials, and several clinical trials are under investigation. However, many clinical trials involving cancer vaccination with defined tumor antigens work in only a small number of patients. Cancer immunotherapy is not completely effective in eradicating tumor cells because tumor cells escape from host immune scrutiny. Understanding of the mechanism of immune evasion regulated by tumor cells is required for the development of more effective immunotherapeutic approaches against lung cancer. This paper discusses the identification of tumor antigens in lung cancer, tumor immune escape mechanisms, and clinical vaccine trials in lung cancer.

Reversing interleukin-2 inhibition mediated by anti-double-stranded DNA autoantibody ameliorates glomerulonephritis in MRL-lpr/lpr mice.

OBJECTIVE: Our previous study demonstrated that anti-double-stranded DNA (anti-dsDNA) antibodies involved in lupus nephritis down-regulate the production of interleukin-2 (IL-2) in T cells, which in turn, contributes to the defective production of cytotoxic cells and to activation-induced cell death in vitro. To reveal novel molecular targets for lupus therapy, the molecular mechanisms of IL-2 down-regulation by anti-dsDNA were studied. METHODS: Anti-dsDNA monoclonal antibody (mAb) 9D7 was used to study the molecular mechanisms of IL-2 production in vitro. Treatment with arginine-rich peptide, a penetration inhibitor, was used to verify the effect of internalization of anti-dsDNA on the production of IL-2. The signaling pathway for IL-2 expression induced by anti-dsDNA was analyzed by using kinase inhibitors. The therapeutic effects of these inhibitors were evaluated in MRL-lpr/lpr mice. RESULTS: Inhibition of IL-2 production in activated Jurkat cells and human T cells pretreated with mAb 9D7 was reversed by treatment with the arginine-rich peptide. Levels of pAkt and phosphorylated glycogen synthase kinase 3 (pGSK-3) were reduced in activated Jurkat cells that had been pretreated with mAb 9D7. The inhibition of IL-2 production by mAb 9D7 was counteracted by pretreating the cells with LiCl (a GSK-3 inhibitor). However, IL-2 reduction was not recovered in the cells pretreated with ERK and JNK inhibitors. Furthermore, MRL-lpr/lpr mice injected with LiCl or with arginine-rich peptide restored the IL-2 production and reduced the manifestations of lupus. CONCLUSION: These findings suggest that penetration of T cells by anti-dsDNA may inhibit IL-2 production by activating GSK-3. Moreover, blocking GSK-3 activation as well as inhibiting anti-dsDNA penetration is a potential therapeutic approach for lupus.

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-gamma, 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 PGE(2). 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. PGE(2)-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 PGE(2) 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.

Characterization of a salt-tolerant xylanase from Thermoanaerobacterium saccharolyticum NTOU1.

A xylanase gene was PCR-cloned from Thermoanaerobacterium saccharolyticum and expressed in Escherichia coli. The xylanase (XynA) consisted of a signal peptide, glycoside hydrolase family 10 domains, carbohydrate-binding modules, and surface layer homology domains. It was optimally active at 70-73°C and at pH 5-7. It had enhanced activity with NaCl with optimal activity at 0.4 M but was tolerant up to 2 M NaCl. The thermostable and salt-tolerant properties of this xylanase suggest that it may be useful for industrial applications.

Nucleotide-binding domain of phosphoglycerate kinase 1 reduces tumor growth by suppressing COX-2 expression.

Phosphoglycerate kinase 1 (PGK-1) is a multifunctional protein that is involved in the glycolytic pathway and the generation of the angiogenesis inhibitor angiostatin. In a previous study, we showed that the overexpression of full-length PGK-1 in Lewis lung carcinoma (LLC-1) can reduce tumor growth in vivo by downregulation of COX-2 expression. Phosphoglycerate kinase 1 has two functional domains: a catalytic domain (CD); and a nucleotide-binding domain (NBD). To identify the functional domain of PGK-1 responsible for its antitumor effects, we evaluated the tumorigenicity of LLC-1 cells overexpressing full-length PGK-1 (LLC-1/PGK), CD (LLC-1/CD), and NBD (LLC-1/NBD). Although no difference in tumor cell growth was observed in vitro, the tumor invasiveness was reduced in the LLC-1/PGK, LLC-1/CD, and LLC-1/NBD cells compared to parental LLC-1 cells in vivo. In addition, in vivo tumor growth retardation by LLC-1/CD and LLC-1/NBD cells was observed, similar to that by LLC-1/PGK cells. However, the reduced stability of COX-2 mRNA and downregulation of the COX-2 protein and its metabolite, prostaglandin E2, was only found in LLC-1/PGK and LLC-1/NBD cells. Low levels of COX-2 were also observed in the tumor mass formed by the modified cells when injected into mice. The results indicate that COX-2 suppression by PGK-1 is independent of its catalytic activity. COX-2 targeting by PGK-1 can be attributed to its NBD and is probably a result of the destabilization of COX-2 gene transcripts brought about by the mRNA-binding property of PGK-1.