GPC3 Promotes Lung Squamous Cell Carcinoma Progression and HLA-A2-Restricted GPC3 Antigenic Peptide-Modified Dendritic Cell-Induced Cytotoxic T Lymphocytes to Kill Lung Squamous Cell Carcinoma Cells.

Lung squamous cell carcinoma (LUSC) is associated with poor clinical prognosis and lacks available targeted agents. GPC3 is upregulated in LUSC. Our study aimed to explore the roles of GPC3 in LUSC and the antitumor effects of HLA-A2-restricted GPC3 antigenic peptide-sensitized dendritic cell (DC)-induced cytotoxic T lymphocytes (CTLs) on LUSC. LUSC cells with GPC3 knockdown and overexpression were built using lentivirus packaging, and cell viability, clone formation, apoptosis, cycle, migration, and invasion were determined. Western blotting was used to detect the expression of cell cycle-related proteins and PI3K-AKT pathway-associated proteins. Subsequently, HLA-A2-restricted GPC3 antigenic peptides were predicted and synthesized by bioinformatic databases, and DCs were induced and cultured in vitro. Finally, HLA-A2-restricted GPC3 antigenic peptide-modified DCs were co-cultured with T cells to generate specific CTLs, and the killing effects of different CTLs on LUSC cells were studied. A series of cell function experiments showed that GPC3 overexpression promoted the proliferation, migration, and invasion of LUSC cells, inhibited their apoptosis, increased the number of cells in S phase, and reduced the cells in G2/M phase. GPC3 knockdown downregulated cyclin A, c-Myc, and PI3K, upregulated E2F1, and decreased the pAKT/AKT level. Three HLA-A2-restricted GPC3 antigenic peptides were synthesized, with GPC3522-530 FLAELAYDL and GPC3102-110 FLIIQNAAV antigenic peptide-modified DCs inducing CTL production, and exhibiting strong targeted killing ability in LUSC cells at 80 : 1 multiplicity of infection. GPC3 may advance the onset and progression of LUSC, and GPC3522-530 FLAELAYDL and GPC3102-110 FLIIQNAAV antigenic peptide-loaded DC-induced CTLs have a superior killing ability against LUSC cells.

Mapping variation of extracellular matrix in human keloid scar by label-free multiphoton imaging and machine learning.

Significance: Rapid diagnosis and analysis of human keloid scar tissues in an automated manner are essential for understanding pathogenesis and formulating treatment solutions. Aim: Our aim is to resolve the features of the extracellular matrix in human keloid scar tissues automatically for accurate diagnosis with the aid of machine learning. Approach: Multiphoton microscopy was utilized to acquire images of collagen and elastin fibers. Morphological features, histogram, and gray-level co-occurrence matrix-based texture features were obtained to produce a total of 28 features. The minimum redundancy maximum relevancy feature selection approach was implemented to rank these features and establish feature subsets, each of which was employed to build a machine learning model through the tree-based pipeline optimization tool (TPOT). Results: The feature importance ranking was obtained, and 28 feature subsets were acquired by incremental feature selection. The subset with the top 23 features was identified as the most accurate. Then stochastic gradient descent classifier optimized by the TPOT was generated with an accuracy of 96.15% in classifying normal, scar, and adjacent tissues. The area under curve of the classification results (scar versus normal and adjacent, normal versus scar and adjacent, and adjacent versus normal and scar) was 1.0, 1.0, and 0.99, respectively. Conclusions: The proposed approach has great potential for future dermatological clinical diagnosis and analysis and holds promise for the development of computer-aided systems to assist dermatologists in diagnosis and treatment.

Mapping the 3D remodeling of the extracellular matrix in human hypertrophic scar by multi-parametric multiphoton imaging using endogenous contrast.

The hypertrophic scar is an aberrant form of wound healing process, whose clinical efficacy is limited by a lack of understanding of its pathophysiology. Remodeling of collagen and elastin fibers in the extracellular matrix (ECM) is closely associated with scar progression. Herein, we perform label-free multiphoton microscopy (MPM) of both fiber components from human skin specimens and propose a multi-fiber metrics (MFM) analysis model for mapping the structural remodeling of the ECM in hypertrophic scars in a highly-sensitive, three-dimensional (3D) manner. We find that both fiber components become wavier and more disorganized in scar tissues, while content accumulation is observed from elastin fibers only. The 3D MFM analysis can effectively distinguish normal and scar tissues with better than 95% in accuracy and 0.999 in the area under the curve value of the receiver operating characteristic curve. Further, unique organizational features with orderly alignment of both fibers are observed in scar-normal adjacent regions, and an optimized combination of features from 3D MFM analysis enables successful identification of all the boundaries. This imaging and analysis system uncovers the 3D architecture of the ECM in hypertrophic scars and exhibits great translational potential for evaluating scars in vivo and identifying individualized treatment targets.

Identification of human ovarian cancer relying on collagen fiber coverage features by quantitative second harmonic generation imaging.

Ovarian cancer has the highest mortality rate among all gynecological cancers, containing complicated heterogeneous histotypes, each with different treatment plans and prognoses. The lack of screening test makes new perspectives for the biomarker of ovarian cancer of great significance. As the main component of extracellular matrix, collagen fibers undergo dynamic remodeling caused by neoplastic activity. Second harmonic generation (SHG) enables label-free, non-destructive imaging of collagen fibers with submicron resolution and deep sectioning. In this study, we developed a new metric named local coverage to quantify morphologically localized distribution of collagen fibers and combined it with overall density to characterize 3D SHG images of collagen fibers from normal, benign and malignant human ovarian biopsies. An overall diagnosis accuracy of 96.3% in distinguishing these tissue types made local and overall density signatures a sensitive biomarker of tumor progression. Quantitative, multi-parametric SHG imaging might serve as a potential screening test tool for ovarian cancer.

Highly accurate, automated quantification of 2D/3D orientation for cerebrovasculature using window optimizing method.

Significance: Deep-imaging of cerebral vessels and accurate organizational characterization are vital to understanding the relationship between tissue structure and function. Aim: We aim at large-depth imaging of the mouse brain vessels based on aggregation-induced emission luminogens (AIEgens), and we create a new algorithm to characterize the spatial orientation adaptively with superior accuracy. Approach: Assisted by AIEgens with near-infrared-II excitation, three-photon fluorescence (3PF) images of large-depth cerebral blood vessels are captured. A window optimizing (WO) method is developed for highly accurate, automated 2D/3D orientation determination. The application of this system is demonstrated by establishing the orientational architecture of mouse cerebrovasculature down to the millimeter-level depth. Results: The WO method is proved to have significantly higher accuracy in both 2D and 3D cases than the method with a fixed window size. Depth- and diameter-dependent orientation information is acquired based on in vivo 3PF imaging and the WO analysis of cerebral vessel images with a penetration depth of 800 µm in mice. Conclusions: We built an imaging and analysis system for cerebrovasculature that is conducive to applications in neuroscience and clinical fields.

The effects of a hypertension diagnosis on health behaviors: A two-dimensional regression discontinuity analysis.

This paper explores how a diagnosis of hypertension might affect a person's health-related behaviors. The analysis uses a two-dimensional regression discontinuity design because hypertension is diagnosed when a person's systolic or diastolic blood pressure (SBP or DBP) surpasses a pre-established threshold. We find that those closely above the SBP threshold significantly adjusted their lifestyle, such as reducing daily fat intake and quitting smoking, while those just surpassing the DBP cutoff did not. Further mechanism analysis suggests that the possibility of constraints, rather than education and income gradients, does more to explain the disparate behaviors of subjects near the SBP and DBP thresholds. Those around the DBP threshold generally have tighter work schedules and undertake more competitive jobs, which hinder them from improving their lifestyle. Overall, our findings complement the existing literature by posing a new perspective for understanding people's potential reluctance to adjust their behavior.

Soil salinity regulation of soil microbial carbon metabolic function in the Yellow River Delta, China.

The ecological consequences of soil salinization, one of the major causes of soil degradation worldwide, on soil carbon (C) emissions are well known, but less is known about the related microbial C metabolic function. We conducted laboratory incubation experiments on soil samples under a salt gradient at four levels (non-saline, low, medium, and high salinity soils) from coastal saline-alkaline soil of the Yellow River Delta, China, to assess the role of soil salinity in regulating C emissions and microbial abundance. We also evaluated the associations between salt content and the read number of microbial C metabolism genes by determining the soil metagenomes. We found that soil salinity was negatively related to soil C, nitrogen (N) content, C emissions, bacterial gene copy number, and the relative abundances of Actinobacteria, Thermoleophilia, and Betaproteobacteria, but positively related to the C/N ratio and the relative abundance of Gemaproteobacteria and Halobacteria. Increases in soil salinity correlated with decreases in carbohydrate metabolism and gene abundances of glycosyl transferases and glycoside hydrolases based on the metagenomic data. In contrast, the enzyme active genes of carbohydrate esterases and auxiliary activities were positively related to soil salinity. This study provides a clear understanding of the response of soil microbial communities and their C metabolic functions to soil salinity. We offer evidence that soil salinity has significant effects on microbial communities and soil C metabolic functions, resulting in an overall negative effect on soil C emissions.

Knock-in models related to Alzheimer's disease: synaptic transmission, plaques and the role of microglia.

BACKGROUND: Microglia are active modulators of Alzheimer's disease but their role in relation to amyloid plaques and synaptic changes due to rising amyloid beta is unclear. We add novel findings concerning these relationships and investigate which of our previously reported results from transgenic mice can be validated in knock-in mice, in which overexpression and other artefacts of transgenic technology are avoided. METHODS: AppNL-F and AppNL-G-F knock-in mice expressing humanised amyloid beta with mutations in App that cause familial Alzheimer's disease were compared to wild type mice throughout life. In vitro approaches were used to understand microglial alterations at the genetic and protein levels and synaptic function and plasticity in CA1 hippocampal neurones, each in relationship to both age and stage of amyloid beta pathology. The contribution of microglia to neuronal function was further investigated by ablating microglia with CSF1R inhibitor PLX5622. RESULTS: Both App knock-in lines showed increased glutamate release probability prior to detection of plaques. Consistent with results in transgenic mice, this persisted throughout life in AppNL-F mice but was not evident in AppNL-G-F with sparse plaques. Unlike transgenic mice, loss of spontaneous excitatory activity only occurred at the latest stages, while no change could be detected in spontaneous inhibitory synaptic transmission or magnitude of long-term potentiation. Also, in contrast to transgenic mice, the microglial response in both App knock-in lines was delayed until a moderate plaque load developed. Surviving PLX5266-depleted microglia tended to be CD68-positive. Partial microglial ablation led to aged but not young wild type animals mimicking the increased glutamate release probability in App knock-ins and exacerbated the App knock-in phenotype. Complete ablation was less effective in altering synaptic function, while neither treatment altered plaque load. CONCLUSIONS: Increased glutamate release probability is similar across knock-in and transgenic mouse models of Alzheimer's disease, likely reflecting acute physiological effects of soluble amyloid beta. Microglia respond later to increased amyloid beta levels by proliferating and upregulating Cd68 and Trem2. Partial depletion of microglia suggests that, in wild type mice, alteration of surviving phagocytic microglia, rather than microglial loss, drives age-dependent effects on glutamate release that become exacerbated in Alzheimer's disease.

Inconsistent response of bacterial phyla diversity and abundance to soil salinity in a Chinese delta.

Soil salinization is an increasingly serious problem and decreases crop yields in the Yellow River Delta (YRD), but its effects on bacterial community and diversity at the phylum level are not well known. We used high-throughput sequencing of soil bacterial 16S rRNA to identify soil bacterial communities and diversity across a gradient of soil salinity (electrical conductivity), namely, S1: low salinity level (1.78 ds/m), S2: medium salinity level (3.16 ds/m), S3: high salinity level (17.26 ds/m), S4: extreme salinity level (34.41 ds/m), and a non-salted site as the control (CK, 0.92 ds/m). Our results indicated the significantly higher values of soil C/N ratio in S2, S3, and S4 compared with that in CK. Significantly lower values of the Shannon and Chao 1 indexes were observed in S4 compared with the CK (p < 0.05). High salinity decreased the relative abundance of Actinobacteria and Acidobacteria, but increased that of Gemmatimonadetes and Bacteroidetes. Additionally, the Shannon diversity of Bacteroidetes increased by 15.5% in S4 compared with that in the CK. Our results indicate that soil salt is a main factor regulating bacterial phyla diversity and community in the extremely saline-alkaline soils of YRD. The high abundance and diversity of Bacteroidetes can be used for saline-alkali land restoration.

GPC3 affects the prognosis of lung adenocarcinoma and lung squamous cell carcinoma.

BACKGROUND: Glypican 3 (GPC3) is a heparin sulphate proteoglycan whose expression is associated with several malignancies. However, its expression in non-small-cell lung carcinoma (NSCLC) is limited and ambiguous. This study aimed to comprehensively evaluate the expression of GPC3 in NSCLC and develop a risk-score model for predicting the prognosis of NSCLC. METHODS: The gene expression profiles of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were downloaded from the UCSC Xena database. Using the limma package, the differentially expressed genes (DEGs) between different comparison groups were analysed and the differential expression of GPC3 was calculated. A functional enrichment analysis was conducted for GPC3-associated genes using the DAVID tool. For the GPC3-associated genes shared by the four comparison groups, a protein-protein interaction network was built using the Cytoscape software. After conducting a survival analysis and a Cox regression analysis, the genes found to be significantly correlated with prognosis were selected to construct a risk-score model. Besides, the gene and protein levels of GPC3 were examined by quantitative reverse transcriptase-PCR (qRT-PCR) and immunohistochemistry (IHC) in LUSC tissues and paracancer tissues. RESULTS: The differential expression of GPC3 was significant (adjusted P < 0.05) in the NSCLC vs. normal, LUAD vs. normal, LUSC versus normal, and LUAD versus. LUSC comparison groups. GPC3 directly interacted with SERPINA1, MFI2, and FOXM1. Moreover, GPC3 expression was significantly correlated with pathologic N, pathologic T, gender, and tumour stage in LUAD samples. Finally, the risk-score model (involving MFI2, FOXM1, and GPC3) for LUAD and that (involving SERPINA1 and FOXM1) for LUSC were established separately. The qRT-PCR result showed that GPC3 expression was much higher in the LUSC tissues than that in the normal group. The IHC results further showed that GPC3 is highly expressed in LUSC tissues, but low in paracancer tissues. CONCLUSION: The three-gene risk-score model for LUAD and the two-gene risk-score model for LUSC might be valuable in improving the prognosis of these carcinomas.

Interleukin-35 stimulates tumor necrosis factor-α activated osteoblasts differentiation through Wnt/ß-catenin signaling pathway in rheumatoid arthritis.

Interleukin (IL)-35 plays an important role in the pathogenesis of rheumatoid arthritis (RA), which is characterized by tumor necrosis factor (TNF)-α activated bone loss beginning early and persisting over time. The aim of this study was to explore the effects and signaling pathway of IL-35 on osteoblasts differentiation in MC3T3E1 cells and TNF-α activated MC3T3E1 cells. A microenvironment was established with low concentration and short-term treatment of TNF-α to mimic inflammatory activated osteoblasts of RA in vitro. The role of IL-35 on osteoblasts proliferation and apoptosis were assessed using cell counting kit (CCK)-8 assay and flow cytometry, respectively. Alkaline phosphatase (ALP) activity was measured by p-nitrophenyl phosphate assay. Extracellular matrix mineralization was measured by Alizarin red S staining. Osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in response to IL-35 were investigated using real-time polymerase chain reaction and western blot analysis. Wnt/ß-catenin signaling pathway in osteoblasts was investigated. In basal and TNF-α activated osteoblasts, IL-35 promoted proliferation and inhibited apoptosis. Basal and TNF-α activated ALP activity and mineralization in vitro was increased stimulated by IL-35. Furthermore, IL-35 increased the basal and TNF-α activated OPG expression and decreased basal and TNF-α activated RANKL expression. Blocking Wnt/ß-catenin signaling pathway with Dickkopf (Dkk)-1 inhibited the osteogenic effects of IL-35. IL-35 stimulates basal and TNF-α activated osteoblasts differentiation through the Wnt/ß-catenin signaling pathway, thus highlighting the IL-35 for pharmaceutical and medicinal applications for treating RA bone loss.

Elevation of Serum IL-35 in Patients with Primary Sjögren's Syndrome.

Interleukin-35 (IL-35) is a critical anti-inflammatory cytokine in autoimmune disease. In the current study, we aimed to investigate the serum IL-35 levels and its clinical association in patients with primary Sjögren's syndrome (pSS) and to investigate whether or not IL-35 participates in the pathogenesis of pSS. One hundred seventy-six pSS patients and 60 healthy controls (HCs) were recruited. Disease activity was assessed according to EULAR SS disease activity index. Serum IL-35 levels were quantified by enzyme-linked immunosorbent assay. The correlations between the serum IL-35 levels with the clinical parameters were analyzed by a Spearman's correlation test. The serum IL-35 levels in the patients with pSS were significantly increased compared with the HCs. The serum IL-35 levels were elevated in the patients with pSS. Strikingly, the serum IL-35 levels in pSS patients with short disease duration (<1 year) were significantly lower compared with HCs. However, the serum IL-35 levels in pSS patients with medium (1-2 year) and long disease duration (>2 years) were higher compared with HCs. We also found a positive correlation between expression of IL-35 and erythrocyte sedimentation rate, disease activity, and immunoglobulin G. Furthermore, the pSS patients with RF-positive showed high serum IL-35 levels. These findings suggest that IL-35 could play a key role in pSS pathogenesis. In addition, our results highlight the potential exploitation of IL-35 as a biomarker of disease activity and may represent a novel therapeutic agent for pSS.

Interleukin-35 inhibits angiogenesis through STAT1 signalling in rheumatoid synoviocytes.

OBJECTIVES: We studied the anti-angiogenic effect of interleukin-35 (IL-35) by investigating its effects on signal transmission through the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in fibroblast-like synoviocytes (FLS). METHODS: Using the collagen-induced arthritis (CIA) model of rheumatoid arthritis (RA), we derived and cultured FLS, stimulated FLS with IL-35 at different concentrations and examined the expression levels of mRNA and protein of both vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), endostatin, TNF-α, and IL-6 using reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. We used Western blotting to study the effects of IL-35 on the function of the JAK-STAT pathway in FLS. RESULTS: IL-35 treatment inhibited the expression of VEGF, FGF-2, TNF-α and IL-6, and increased the expression of endostatin in FLS. Western blotting showed that IL-35 treatment of CIA-derived FLS resulted in signalling through STAT1, but not through STAT3 or STAT5. CONCLUSIONS: IL-35 signalling through STAT1 and inhibition of the expression of mediators of angiogenesis and inflammation in FLS provide a likely mechanism for anti-angiogenic effects seen in experimental models of RA. Our data suggest that IL-35 and its signalling pathway represent a therapeutic target for the treatment of RA and other angiogenesis-related diseases.

Expression of cleaved caspase-3 predicts good chemotherapy response but poor survival for patients with advanced primary triple-negative breast cancer.

OBJECTIVE: To assess cleaved caspase-3 (CC3), Ki-67, and E-cadherin (E-cad) expression in relation to chemotherapy response and prognosis of patients with advanced primary triple-negative breast cancer (TNBC). METHODS: CC3 expression was detected immunohistochemically in 67 pre-chemotherapy biopsy samples. Ki67 and E-cad levels were obtained from patients' medical records. RESULTS: CC3-positivity (N = 32; 47.8%) was associated with a higher first-line chemotherapy overall response rate (ORR; P = 0.028) and second-line chemotherapy clinical benefit rate (CBR; P = 0.033). The Ki-67 high-risk group (N = 51; 76.1%) exhibited a reduced second-line chemotherapy CBR (P = 0.024). The E-cad negative group (N = 25; 37.3%) exhibited a lower first-line chemotherapy ORR (P = 0.044) and CBR (P<0.001), and a lower second-line chemotherapy CBR (P = 0.020). CC3, Ki-67, and E-cad were significant predictors of third-line chemotherapy ORR or CBR. Similar numbers of chemotherapy cycles were completed by the CC3-positive and -negative groups. The Ki-67 high-risk and E-cad negative groups completed fewer second-line chemotherapy cycles (P = 0.038) and fewer first-line chemotherapy cycles, respectively (P = 0.001). Kaplan-Meier analyses identified worse outcomes for the CC3-positive, Ki-67 high-risk, and E-cad negative groups than for their corresponding comparison groups (P<0.05). Multivariate Cox regression analysis identified CC3 expression and an absence of E-cad expression as independent survival factors (P<0.05). CONCLUSIONS: Our CC3-positive group exhibited a better chemotherapy response, but a worse prognosis. The Ki-67 high-risk and E-cad negative groups exhibited both a worse chemotherapy response and worse prognosis.

The association between the TP53 Arg72Pro polymorphism and colorectal cancer: An updated meta-analysis based on 32 studies.

Several previous studies evaluated the association between the Arg72Pro (rs1042522) polymorphism in the TP53 tumor suppressor gene and colorectal cancer (CRC). However, the results are conflicting. This meta-analysis aimed to shed new light on the precise association between TP53 variants and CRC. We analyzed 32 published case-control studies involving 8,586 cases and 10,275 controls using crude odd ratios (ORs) with 95% confidence intervals (CIs). The meta-analysis was performed using a fixed-effect or random-effects model, as appropriate. We found that the TP53 Arg72Pro polymorphism was not significantly associated with CRC risk in the overall population. However, subgroup analysis based on ethnicity revealed an increased risk of CRC among Asians (CC vs. GC+GG: OR=1.22, 95% CI: 1.02-1.45), and similar results were found for rectal cancer (CC vs. GC+GG: OR=1.34, 95% CI: 1.120-1.62). These results suggest that the TP53 Arg72Pro polymorphism CC genotype may contribute to an increased risk of CRC, especially for rectal cancer and among Asians.

IL-35 Inhibits Angiogenesis through VEGF/Ang2/Tie2 Pathway in Rheumatoid Arthritis.

BACKGROUND/AIMS: The pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins (Angs) play a prominent role in synovial angiogenesis, an early and critical event in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-35 is an anti-inflammatory cytokine that attenuates collagen-induced arthritis, however, the mechanisms involved are not fully understood. METHODS: The effects of IL-35 on endothelial cell migration, adhesion, and tube formation were examined using human umbilical vein endothelial cells (HUVEC) in vitro. The effects of IL-35 on vessel formation in vivo were examined using a murine Matrigel plugs model. MMP2/MMP9 and IL-6/IL-8 secretion were assessed by zymography and ELISA, respectively. The crosstalk between IL-35, VEGF, and Ang2 in HUVECs and RA synovial tissue explants was investigated. RESULTS: IL-35 inhibited basal and VEGF-induced HUVEC migration and adhesion in vitro as well as tube formation in vitro and in vivo. VEGF increased Ang2 secretion by HUVECs and RA synovial tissue explants, and exogenous Ang2 promoted HUVEC migration, adhesion, and tube formation with similar potency to VEGF. Blocking the Ang/Tie2 pathway with a Tie2 kinase antibody inhibited the proangiogenic effects of exogenous Ang2 and VEGF in HUVECs. IL-35 inhibited basal and VEGF-induced Ang2 secretion by HUVECs and RA synovial tissue explants; it also antagonized the proangiogenic effects of exogenous Ang2 in HUVECs. Moreover, IL-35 reduced basal and VEGF/Ang2-induced MMP2/MMP9 and IL-6/IL-8 secretion. CONCLUSION: These results suggested that IL-35 restrains RA angiogenesis and inflammation by downregulating basal and VEGF-induced Ang2 secretion as well as disrupting Ang2/Tie2 signal transduction. Our findings extend current understanding of mechanisms regulating RA angiogenesis and may support development of novel angiogenesis-targeting therapeutics for RA treatment.

Inhibition of MDM2 Re-Sensitizes Rapamycin Resistant Renal Cancer Cells via the Activation of p53.

BACKGROUND/AIMS: Rapamycin is a potential anti-cancer agent, which modulates the activity of mTOR, a key regulator of cell growth and proliferation. However, several types of cancer cells are resistant to the anti-proliferative effects of rapamycin. In this study, we report a MDM2/p53-mediated rapamycin resistance in human renal cancer cells. METHODS: Trypan blue exclusion tests were used to determine the cell viability. Changes in mRNA and protein expression were measured using real-time PCR and western blot, respectively. Xenograft models were established to evaluate the in vivo effects of rapamycin combined with a MDM2 inhibitor. RESULTS: Rapamycin treatment suppresses the expression of MDM2 and exogenous overexpression of MDM2 in A498 cells contributes to rapamycin resistance. By establishing a rapamycin resistant cell line, we observed that MDM2 was significantly upregulated in rapamycin resistant cells than that in rapamycin sensitive cells. Importantly, the rapamycin resistant cells demonstrated attenuated accumulation of p53 in the nucleus in response to rapamycin treatment. Moreover, the inhibition of MDM2 by siMDM2 sensitizes A498 cells to rapamycin through the activation of p53. In both in vitro and in vivo models, the combination of rapamycin with the MDM2 inhibitor, MI-319, demonstrated a synergistic inhibitory effect on rapamycin resistant cells. CONCLUSION: Our study reports a novel mechanism for rapamycin resistance in human renal cancer and provides a new perspective for the development of anti-cancer drugs.

Lentivirus-mediated knockdown of NLK inhibits small-cell lung cancer growth and metastasis.

Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, has been recognized as a critical regulator of various cancers. In this study, we investigated the role of NLK in human small-cell lung cancer (SCLC), which is the most aggressive form of lung cancer. NLK expression was evaluated by quantitative real-time polymerase chain reaction in 20 paired fresh SCLC tissue samples and found to be noticeably elevated in tumor tissues. Lentivirus-mediated RNAi efficiently suppressed NLK expression in NCI-H446 cells, resulting in a significant reduction in cell viability and proliferation in vitro. Moreover, knockdown of NLK led to cell cycle arrest at the S-phase via suppression of Cyclin A, CDK2, and CDC25A, which could contribute to cell growth inhibition. Furthermore, knockdown of NLK decreased the migration of NCI-H446 cells and downregulated matrix metalloproteinase 9. Treatment with NLK short hairpin RNA significantly reduced SCLC tumor growth in vivo. In conclusion, this study suggests that NLK plays an important role in the growth and metastasis of SCLC and may serve as a potential therapeutic target for the treatment of SCLC.

Interleukin-35 (IL-35) inhibits proliferation and promotes apoptosis of fibroblast-like synoviocytes isolated from mice with collagen-induced arthritis.

Rheumatoid arthritis (RA) is an inflammatory disorder of the joints that affects 0.5-1 % of adults. Excessive growth of the fibroblast-like synoviocytes (FLS) promotes hyperplasia of synovial tissues and causes its invasion into the bone and cartilage, which eventually causes deformity and dysfunction of affected joints. Interleukin 35 (IL-35) was shown to suppress the inflammatory responses to collagen-induced arthritis (CIA) via upregulation of T regulatory cells and suppression of T helper type 17 cells in a mouse model. To study the effects of IL-35 on the proliferation and apoptosis frequency of cultured FLS isolated from mice with CIA as well as to examine the effects of IL-35 on CIA in vivo. Thirty DBA/1 J mice, which are used as an animal model for RA, were divided randomly (ten mice per group) to a CIA group (collagen treatment), a CIA + IL-35 group (collagen and IL-35 treatments), and a control group (no treatment). Starting on the 24th day after collagen administration, IL-35 was injected intraperitoneally into mice of the CIA + IL-35 group once per day for 10 days. An arthritis index was calculated, and pathological analysis of synovial tissue was performed. FLS isolated from CIA mice were treated with various concentrations of IL-35 (12.5-100 ng/ml). The MTT assay was used to examine FLS proliferation, and apoptosis frequency of FLS was detected by flow cytometry. On day 24, the CIA mice began to exhibit arthritis symptoms, and the symptoms rapidly progressed with time. Treatment with IL-35 significantly alleviated arthritis symptoms and reduced the synovial tissue inflammation. In addition, IL-35 treatment inhibited proliferation and promoted apoptosis in cultured FLS from CIA mice in a dose-dependent manner. IL-35 could ameliorate the symptoms of arthritis in the CIA mouse model in vivo and inhibited FLS proliferation while promoting FLS apoptosis in vitro, thereby exhibited the potential in inhibiting the progression of RA.