Distinct expression of chemokine-like factor 1 in synovium of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis.

Chemokine-like factor 1 (CKLF1) is a newly cloned chemotactic cytokine with CCR4 being its functional receptor. Recent evidence demonstrates a role of CKLF1 in arthritis. The aim of this study was to quantify the expression of CKLF1 as well as assess the correlation between CKLF1 and plasma acute-phase markers. Synovium was obtained from 16 osteoarthritis (OA), 15 rheumatoid arthritis (RA) and 10 ankylosing spondylitis (AS) patients undergoing total joint arthroplasty, with other 11 patients treated for meniscal tears during sport accidents serving as normal controls. Levels of CKLF1 and CCR4 mRNA were detected by qRT-PCR, and the expression of CKLF1 was investigated by immunohistochemistry staining, subsequently analyzed with semiquantitative scores. Plasma acute-phase markers of inflammation were determined by ELISA. CKLF1 was found with a particularly up-regulated expression in synovim from AS and RA patients, and CCR4 mRNA levels increased in RA patients, not in OA or AS patients. Elevated levels of plasma markers of inflammation including CRP, ESR and D-dimer were observed in RA. Further, significantly positive correlations between relative expression levels of CKLF1 and CRP/ESR in RA patients and a positive correlation between CKLF1 and ESR in AS patients were found. There was no detectable correlation between CKLF1 and plasma D-dimer. This study confirms an increased but different level of CKLF1 in RA, OA and AS patients, all significantly higher than that in controls. Additionally, the significant positive correlations between CKLF1 levels and CRP/ESR in RA and between CKLF1 and ESR suggest that CKLF1 might contribute to the inflammation state and clinical symptoms in these rheumatic diseases. Further studies are required to investigate the utility of targeting specific CKLF1 for symptom control or disease modification in RA and AS.

Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27.

AIM: Quercetin is an effective Hsp27 inhibitor and has been reported to facilitate tumor cell apoptosis. The aim of this study was to investigate whether quercetin could sensitize human glioblastoma cells to temozolomide (TMZ) in vitro. METHODS: Both U251 and U87 human glioblastoma cells were treated with quercetin and/or TMZ for 48 h. Cell viability was detected using the MTT assay. Cell apoptosis was analyzed with caspase-3 activity kits and flow cytometry. Hsp27 expression and phosphorylation were examined using Western blot analysis. RNA interference using Hsp27 siRNA oligos was performed to knock down the gene expression of Hsp27. RESULTS: TMZ (200 or 400 µmol/L) alone effectively inhibited the viability of U251 and U87 cells. When combined with quercetin (30 µmol/L), TMZ (100 µmol/L) significantly inhibited the cell viability, and the inhibition of TMZ (200 and 400 µmol/L) was enhanced. TMZ or quercetin anole did not affect caspase-3 activity and cell apoptosis, while TMZ combined with quercetin significantly increased caspase-3 activity and induced cell apoptosis. TMZ anole significantly increased Hsp27 phosphorylation in U251 and U87 cells, while quercetin or Hsp27 siRNA oligos combined with TMZ attenuated TMZ-induced Hsp27 phosphorylation and significantly inhibited Hsp27 expression. CONCLUSION: Combined treatment with TMZ and quercetin efficiently suppressed human glioblastoma cell survival in vitro.

γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway.

AIM: Trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) is a soluble epoxide hydrolase inhibitor that suppresses glioblastoma cell growth in vitro. The aim of this study was to examine whether the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) could sensitize glioma cells to t-AUCB-induced apoptosis. METHODS: Both U251 and U87 human glioblastoma cell lines were tested. Cell growth was assessed using the cell counting kit-8. Cell apoptosis was detected with caspase-3 activity assay kits and flow cytometry. The protein levels in the p38 MAPK/MAPKAPK2/Hsp27 pathway in the cells were analyzed using Western blots. RESULTS: Pretreatment with DAPT (2 µmol/L) substantially potentiated the growth inhibition caused by t-AUCB (200 µmol/L) in U251 and U87 cells. Furthermore, pretreatment with DAPT markedly increased t-AUCB-induced apoptosis of U251 and U87 cells. T-AUCB alone did not significant affect caspase-3 activity in the cells, but t-AUCB plus DAPT pretreatment caused significant increase of caspase-3 activity. Furthermore, pretreatment with DAPT completely blocked t-AUCB-induced phosphorylation of p38 MAPK, MAPKAPK2 and Hsp27 in the cells. CONCLUSION: The γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway, suggesting that the combination of t-AUCB and DAPT may be a potentially effective strategy for the treatment of glioblastoma.

Development of clinically relevant orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma through surgical tumor tissues injection with trocar.

OBJECTIVE: Orthotopic models are important in cancer research. Here we developed orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma with a specially designed system. METHODS: Tiny fragments of surgical tumors were implanted into the mice brain with a trocar system. Immunohistochemistry was performed to detect brain tumor stem cells among glioblastoma tissues, including both the original and resulting ones with monoclonal antibody against CD133. RESULTS: Besides the constant high take rates in both models; brain transplants perfectly resembled their original tumors in biological behaviors. The brain tumor stem cells, positively stained with CD133 were found, though not frequently, in both original and resulting glioblastoma tissues. CONCLUSIONS: Orthotopic model established with a trocar system is effective and injection of tumor tissues containing stem cells promise the forming of new tumor mass when grafted.

[Transplantation and magnetic resonance imaging of transplantable human glioma tissue in the brain of nude mice].

BACKGROUND & OBJECTIVE: In previous reports, orthotopic transplantation models of glioma were produced by injecting cell suspension into the brain of mice, which is complex, time-consuming, and nearly impossible to prepare in a large scale within a short period. This study was to establish human glioma orthotopic transplantation model in nude mice by transplanting tumor tissue in the brain, and investigate magnetic resonance imaging (MRI) of the transplanted tumors. METHODS: Human glioma cells were injected subcutaneously into nude mice to form human glioma. The transplantable human glioma tissues (2 mm3) were put into trocar and directly injected into the caudate nucleus of nude mice. Thirty days later, the tumors were detected by 1.5T superconduct MR machine with micro-23 coil and measured by Dicomworks (V1.35) software. Tumor morphology was observed under light microscope with HE staining. Tumor volume was measured under stereomicroscope. The feasibility of measuring tumor volume according to MRI data was evaluated. RESULTS: MRI showed that in the 15 nude mice received orthotopic transplantation in the caudate nucleus, 14 developed glioma. Under microscope, glioma tissues were found at the same sites as where MRI indicated. Tumor volume was (23.45+/-11.64) mm3 as measured by MRI and (23.19+/-10.18) mm3 as detected under stereomicroscope (P>0.05). The successful rate of tumor model preparation was 93% (14/15). The successful rate of tumor imaging by MRI was 100% (14/14). CONCLUSIONS: Tissue quantitative transplantation via trocar is simple, time-saving, and easy to construct tumor model in a large scale with high successful rate. The 1.5T MR machine with micro-23 coil can be used to observe tumor position and size of orthotopic transplantation models of human glioma in nude mice.