Comparative analysis of novel autoantibody isotypes against citrullinated-inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3)(542-556) peptide in serum from Taiwanese females with rheumatoid arthritis, primary Sjögren's syndrome and secondary Sjögren's syndrome in rheumatoid arthritis.

UNLABELLED: The purpose of this study was to discover and validate inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) as novel biomarkers, and evaluate autoantibody isotypes against an unmodified and citrullinated ITIH3(542-556) peptide among Taiwanese female patients with rheumatoid arthritis (RA), primary Sjögren's syndrome (pSS), secondary Sjögren's syndrome in rheumatoid arthritis (RA-sSS), and healthy controls (HCs). We used concanavalin A (Con A) affinity chromatography, 1-D SDS-PAGE, and label-free nano-LC-MS/MS to screen biomarker candidates (serum-derived Con A-captured proteins) and then identify PTMs of validated biomarkers (serum proteins) using pooled serum from 7 RA-sSS female patients and 7 age-matched HCs (the discovery set). Furthermore, the protein level and autoantibody isotype analyses were further validated against individual serum from 18 HCs, 18 RA, 18 pSS, and 18 RA-sSS patients (the validation set). Con A-bound ITIH3 was identified and validated as the only differentially expressed protein, which was elevated. Additionally, 2 novel PTMs in ITIH3 were identified and included citrullination at arginine-(546) and arginine-(556), and hexosamine at tryptophan-(558). Further, concentrations of anti-citrullinatd-ITIH3(542-556) peptide autoantibodies significantly increased in patients with RA, pSS, and RA-sSS compared to HCs. Especially, autoantibody IgM against the citrullinated-ITIH3(542-556) peptide showed better diagnostic performance in discriminating both RA versus pSS and pSS versus RA-sSS. SIGNIFICANCE: By using comparative proteomic analysis of serum samples, the current study discovered and validates differentially expressed Con A-bound ITIH3 as a potential biomarker for secondary Sjögren's syndrome (SS) in rheumatoid arthritis (RA) patients and healthy controls (HCs). Besides, hexosamine and citrullination on ITIH3 were further identified. Through analyzing autoantibody isotypes against the citrullinated ITIH3 peptide, patients with RA, primary SS, and RA-secondary SS, and HCs can be further discriminated. The current strategy can be applied for identifying potential diagnostic and pathologic markers for autoimmune diseases.

Rana catesbeiana ribonuclease induces cell apoptosis via the caspase-9/-3 signaling pathway in human glioblastoma DBTRG, GBM8901 and GBM8401 cell lines.

Human glioblastoma multiforme is one of the most aggressive malignant brain tumor types, and the mean survival time of patients with a brain tumor is <2 years when traditional therapies are administered. Thus, numerous studies have focused on the development of novel treatments for brain tumors. Frog ribonucleases, such as Onconase and Rana catesbeiana ribonuclease (RC-RNase), exert antitumor effects on various tumor cells, including cervical cancer, breast cancer, hepatoma, leukemia, pancreatic cancer and prostate cancer cells. In addition, frog Onconase has been applied as a treatment in clinical trials. However, the antitumor effects of frog ribonucleases on brain tumors are unclear. Previous studies have indicated that RC-RNase demonstrates a decreased cytotoxic effect in normal cells compared with Onconase. Therefore, the present study investigated the ability of RC-RNase to exert antitumor activities on human glioblastoma. It was found that RC-RNase inhibits the growth of the human glioblastoma DBTRG, GBM8901 and GBM8401 cells. In addition, the present study revealed that RC-RNase induces caspase-9/-3 activity and triggers the apoptotic cell death pathway in human glioblastoma cells. Notably, it was also demonstrated that RC-RNase effectively inhibits the growth of human glioblastoma tumors in a nude mouse model. Overall, the present study indicates that RC-RNase may be a potential agent for the treatment of human glioblastoma.

Vitamin C enhances anticancer activity in methotrexate­treated Hep3B hepatocellular carcinoma cells.

Methotrexate (MTX) has been widely used for rheumatoid arthritis therapy for a long time. MTX is also used as an anticancer drug for various tumors. However, many studies have shown that high-dose MTX treatment for cancer therapy may cause liver and renal damage. Alhough the mechanisms involved in MTX-induced liver and renal damage require further research, many studies have indicated that MTX-induced cytotoxicity is associated with increases in oxidative stress and caspase activation. In order to reduce MTX-induced side-effects and increase anticancer efficiency, currently, combination treatments of low-dose MTX and other anticancer drugs are considered and applied for various tumor treatments. The present study showed that MTX induces increases in H2O2 levels and caspase-9/-3 activation leading to cell death in hepatocellular carcinoma Hep3B cells. Importantly, this study is the first to demonstrate that vitamin C can efficiently aid low-dose MTX in inducing cell death in Hep3B cells. Therefore, the present study provides a possible powerful therapeutic method for tumors using a combined treatment of vitamin C and low-dose MTX.

Dual role of acetaminophen in promoting hepatoma cell apoptosis and kidney fibroblast proliferation.

Acetaminophen (APAP), is a safe analgesic and antipyretic drug at therapeutic dose, and is widely used in the clinic. However, high doses of APAP can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high­dose APAP­induced acute liver and kidney injury. So far, few studies have investigated the effects of the therapeutic dose (1/10 of the high dose) or of the low dose (1/100 of the high dose) of APAP on the cells. The aim of this study was to investigate the cellular effects of therapeutic- or low­dose APAP treatment on hepatoma cells and kidney fibroblasts. As expected, high­dose APAP treatment inhibited while therapeutic and low­dose treatment did not inhibit cell survival of kidney tubular epithelial cells. In addition, therapeutic-dose treatment induced an increase in the H2O2 level, activated the caspase­9/­3 cascade, and induced cell apoptosis of hepatoma cells. Notably, APAP promoted fibroblast proliferation, even at low doses. This study demonstrates that different cellular effects are exerted upon treatment with different APAP concentrations. Our results indicate that treatment with the therapeutic dose of APAP may exert an antitumor activity on hepatoma, while low­dose treatment may be harmful for patients with fibrosis, since it may cause proliferation of fibroblasts.

RC-6 ribonuclease induces caspase activation, cellular senescence and neuron-like morphology in NT2 embryonal carcinoma cells.

Frog ribonucleases have been demonstrated to have anticancer activities. However, whether RC-6 ribonuclease exerts anticancer activity on human embryonal carcinoma cells remains unclear. In the present study, RC-6 induced cytotoxicity in NT2 cells (a human embryonal carcinoma cell line) and our studies showed that RC-6 can exert anticancer effects and induce caspase-9 and -3 activities. Moreover, to date, there is no evidence that frog ribonuclease-induced cytotoxicity effects are related to cellular senescence. Therefore, our studies showed that RC-6 can increase p16 and p21 protein levels and induce cellular senescence in NT2 cells. Notably, similar to retinoic acid-differentiated NT2 cells, neuron-like morphology was found on some remaining live cells after RC-6 treatment. In conclusion, our study is the first to demonstrate that RC-6 can induce cytotoxic effects, caspase-9/-3 activities, cellular senescence and neuron-like morphology in NT2 cells.

The NS3 protease and helicase domains of Japanese encephalitis virus trigger cell death via caspase­dependent and ­independent pathways.

Japanese encephalitis virus (JEV), a mosquito­borne flavivirus, causes acute encephalitis and nervous damage. Previous studies have demonstrated that JEV induces apoptosis in infected cells. However, to date the mechanisms of JEV­induced apoptosis are unclear. In order to identify the viral proteins associated with JEV­induced apoptosis, pEGFP­non­structural protein 3 (NS3) 1­619 (expressing the JEV NS3 intact protein, including the protease and helicase domains), pEGFP­NS3 1­180 (expressing the protease domain) and pEGFP­NS3 163­619 (expressing the helicase domain) were transfected into target cells to study cell death. Results demonstrate that the JEV NS3 intact protein and protease and helicase domains induce cell death. In addition, cell death was identified to be significantly higher in cells transfected with the NS3 protease domain compared with the intact protein and helicase domain. Caspase activation was also analyzed in the current study. NS3 intact protein and NS3 protease and helicase domains activated caspase­9/­3­dependent and ­independent pathways. However, caspase­8 activity was not found to be significantly different in NS3­transfected cells compared with control. In summary, the present study demonstrates that the NS3 helicase and protease domains of JEV activate caspase­9/­3­dependent and ­independent cascades and trigger cell death.

The cytotoxic protein can induce autophagocytosis in addition to apoptosis in MCF-7 human breast cancer cells.

Phagocytic clearance of dying cells is found in many phagocytes. It has been shown that dying cells can be phagocytosed by other phagocytic cells through autophagic or apoptotic cellular death. To date, whether cancer cells have such phagocytic activity has not been studied. In this study, our data shows that RC-RNase can trigger cell death in human breast cancer MCF-7 cells through the apoptotic pathway. Interestingly, when treated with cytotoxic protein, the remaining MCF-7 cells can phagocytose the dying MCF-7 cells via autophagocytic activity, demonstrated directly by real-time image observation and electron microscopy analysis. To sum up, this study demonstrates for the first time that RC-RNase can trigger apoptosis and autophagocytosis in MCF-7 cancer cells.

Microrheology of human synovial fluid of arthritis patients studied by diffusing wave spectroscopy.

The viscoelastic properties of synovial fluid (SF) are critical to its functions of lubrication and shock-absorption of joints in human body; a change in the viscoelastic properties, even of only a few percents, is often concomitant with arthritis. In this work, the elastic modulus G '(f) and the viscous modulus G ''(f) of SF from patients suffering from three kinds of joint diseases, namely, osteoarthritis (OA), rheumatoid arthritis (RA), and gouty arthritis (GA), were determined as a function of frequency "f " (in the low frequency range from f ∼ 0.1 to 10 Hz) by Diffusing Wave Spectroscopy (DWS) and correlated with the white blood cell (WBC) count. A strong correlation was observed, showing a higher WBC count corresponding to lower elastic and viscous moduli, G ' and G ''; further details depend on inflammatory vs. non-inflammatory, and on the severity of inflammation. Different types of arthritis lead to different degrees of decreasing viscoelasticity. Identical measurements were carried out with a commercial visco-supplementation (or artificial SF) to serve as reference. In general, the reduction in both G ' and G '' was most severe in the case of GA and least severe in the case of OA. Besides, in all cases, the reduction in G ' was more prominent than the reduction in G '', indicating that in general, the deterioration in the elasticity of SF by inflammation is more severe than that in the viscosity. This simple method for quantitative physical characterization of synovial fluid may serve as a useful complementary metric to the conventional biochemical analysis in clinical diagnosis of arthritis.

Immunotherapy for SV40 T/t antigen-induced breast cancer by recombinant adeno-associated virus serotype 2 carrying interleukin-15 in mice.

Human interleukin-15 (hIL15) exerts anticancer effects through the activities of lymphokine-activated killer (LAK) cells. However, its short half-life hinders its clinical application. Recombinant adeno-associated virus serotype 2 (rAAV2) is used for hIL15 gene transfer vectors, because of its low immunogenicity and long-term gene expression in human clinical trials. SV40 T/t antigens are related with many human epithelial cancers and are generally found in human breast cancer. In order to demonstrate the anticancer effects of hIL15 on SV40 T/t antigen-induced breast cancer, rAAV2-hIL15 was constructed and an SV40 T/t antigen-induced transgenic mouse breast cancer model was established. Our study showed that rAAV2-hIL15 could express the hIL15 protein with anticancer bioactivity. In addition, rAAV2-hIL15 could activate the cytotoxic activity of LAK cells in vivo. Furthermore, the rAAV2-hIL15 successfully delayed cancer growth and eventually led to cancer cell death in SV40 T/t antigen-induced breast cancer transgenic mice. In summary, our study indicates that rAAV2-hIL15 may be applied for cancer immunotherapy of SV40 T/t antigen-induced breast cancer.