Potential therapeutic strategy for non-Hodgkin lymphoma by anti-CD20scFvFc/CD28/CD3zeta gene tranfected T cells

<p>Abstract</p> <p>Background</p> <p>Anti-CD20 monoclonal antibody treatment has not only increased survival and cure rates in many non-Hodgkin lymphomas, but also has prompted an explosion in the development of novel antibodies and biologically active substances with specific cellular targets in the field of malignancies treatment. Since the robust immune responses are elicited by the gene-modified T cells, gene based T cell therapy may also provide a powerful tool for cancer immunotherapy.</p> <p>Methods</p> <p>In this study, we developed a vector construction encoding a chimeric T cell receptor that recognizes the CD20 antigen and delivers co-stimulatory signals to achieve T cell activation. One non-Hodgkin lymphoma cell line Raji cells co-cultured with peripheral blood-derived T cells were stably transfected with anti-CD20scFvFc/CD28/CD3zeta gene or anti-CD20scFvFc gene. T cells expressing anti-CD20scFvFc/CD28/CD3zeta or anti-CD20scFvFc gene co-cultured with CD20 positive Raji cells for different times. Cell lysis assay was carried by [³H]TdR release assay. The expressions of Fas, Bcl-2 and Caspase-3 of Raji cells were detected by flow cytometric. The secretion of IFN-gamma and IL-2 in co-culture medium was tested by ELISA assay. Activity of AP-1 was analyzed by EMSA.</p> <p>Results</p> <p>Following efficient transduction of peripheral blood-derived T cells with anti-CD20scFvFc/CD28/CD3zeta gene, an obvious cell lysis of Raji cells was observed in co-culture. T cells transduced anti-CD20scFvFc/CD28/CD3zeta gene had superior secretion of IFN-gamma and IL-2 compared to T cells transduced anti-CD20scFvFc gene. Also it led to a much stronger Fas-induced apoptosis signaling transduction in target cancer cells.</p> <p>Conclusion</p> <p>So adoptively T cells transduced anti-CD20scFvFc/CD28/CD3zeta gene mediates enhanced anti-tumor activities against CD20 positive tumor cells, suggesting a potential of gene-based immunotherapy for non-Hodgkin lymphoma.</p

Effects of Press Needling combined with general anesthesia on postoperative analgesia in thoracoscopic pulmonary resection for lung cancer: A randomized, single-blind, controlled trial

Objectives: To investigate the effects of press needle therapy on postoperative analgesia and other relevant complications in patients undergoing thoracoscopic pulmonary resection. Design: randomized, single-blind, controlled trial Setting: Teaching hospitals affiliated with universities. Interventions: Eighty-six patients were randomized into: the Acu group (press-needle group) and the control group Main outcome measures: Pain levels 24, 48, and three months after surgery were measured using the numeric rating scale (NRS). Perioperative hemodynamics, total and effective pressing numbers of patient-controlled intravenous analgesia (PCIA), and incidence of postoperative pulmonary complications were recorded. Peripheral blood samples were collected to measure the levels of inflammatory mediators Results: Acu group had significantly lower NRS scores at 24 and 48 h after operation (NRS scores on movement at 24 h after surgery: Acu vs. Control, 3 (2,3) vs. 3 (3,5), Z = −3.393, P < 0.01 and NRS scores on movement at 48 h after surgery: 2 (1,3) vs. 3 (2,5), Z = −3.641, P < 0.01), lower number of PCIA attempts and effective rates (mean total pressing numbers: 4(2,8) vs. 6(3,19), Z = −1.994, P = 0.046 and mean effective pressing numbers: 3(2,8) vs. 6(3,16), Z = −2.116, P = 0.034). The Acu group had significantly reduced IL-1 (14.52 ± 3.84 vs. 16.36 ± 3.30, mean difference (MD): − 1.85, 95% confidence interval (CI): − 3.46, − 0.23, P = 0.026), HIF-1α (10.15 ± 1.71 vs. 10.96 ± 1.73, MD: −0.81, 95% CI: −1.59, −0.04, P = 0.040) and the incidence of pulmonary complications after surgery. Conclusion: Press needles are a non-invasive and feasible adjunctive intervention for postoperative analgesic management in patients undergoing thoracoscopic pulmonary resection

BCAT1 decreases the sensitivity of cancer cells to cisplatin by regulating mTOR-mediated autophagy via branched-chain amino acid metabolism

Cisplatin is one of the most effective chemotherapy drugs and is widely used in the treatment of cancer, including hepatocellular carcinoma (HCC) and cervical cancer, but its therapeutic benefit is limited by the development of resistance. Our previous studies demonstrated that BCAT1 promoted cell proliferation and decreased cisplatin sensitivity in HCC cells. However, the exact role and mechanism of how BCAT1 is involved in cisplatin cytotoxicity remain undefined. In this study, we revealed that cisplatin triggered autophagy in cancer cells, with an increase in BCAT1 expression. The cisplatin-induced up-regulation of BCAT1 decreased the cisplatin sensitivity by regulating autophagy through the mTOR signaling pathway. In addition, branched-chain amino acids or leucine treatment inhibited cisplatin- or BCAT1-mediated autophagy and increased cisplatin sensitivity by activating mTOR signaling in cancer cells. Moreover, inhibition of autophagy by chloroquine increased cisplatin sensitivity in vivo. Also, the knockdown of BCAT1 or the administration of leucine activated mTOR signaling, inhibited autophagy, and increased cisplatin sensitivity in cancer cells in vivo. These findings demonstrate a new mechanism, revealing that BCAT1 decreases cisplatin sensitivity in cancer cells by inducing mTOR-mediated autophagy via branched-chain amino acid leucine metabolism, providing an attractive pharmacological target to improve the effectiveness of chemotherapy

LRIG2 or LRIG2ecto overexpression enhances EGFR signaling and regulates the cell cycle and apoptosis related proteins.

<p>(<b>A</b>) After synchronization for 24 h, cells were cultured in DMEM with 10% FBS for 48 h and cell lysates were subjected to western blotting for the levels of EGFR signaling related proteins. Analysis of quantification of the bands intensity was shown in the lower panel (*<i>P</i><0.05 vs con). (<b>B</b>) The indicated cells were starved for 24 h and cultured in complete medium for 48 h. The cell lysates were exacted and examined by western blotting for the cell cycle and apoptosis associated proteins. Analysis of quantification of the bands intensity was shown in the lower panel (*<i>P</i><0.05 vs con).</p

Overexpresson of LRIG2 or LRIG2ecto enhances the EGFR signaling in serum-free medium.

<p>(<b>A</b>) The stably tranduced U87 cells were synchronized in DMEM for 24 h and then stimulated with EGF (100 ng/ml) in serum-free medium for the times indicated. The expression levels of EGFR signaling proteins were analyzed by western blotting. The representative western blotting images of three independent experiments were shown and analysis of quantification of the bands intensity was shown in the lower panel (*<i>P</i><0.05 vs con). (<b>B</b>) The stably tranduced U87 cells were cultured with 10% FBS addition with gefitinib (10 µM) or DMSO for 48 h. The total lysates were extracted and subjected to western blotting. The representative images of three independent experiments were present and quantification of the bands intensity was shown in the right panel (*<i>P</i><0.05 vs con).</p

LRIG2 as well as LRIG2ecto interacts with EGFR in glioblastoma cells.

<p>(<b>A</b>) Confocal immunofluorescence laser microsopy showed colocalization of LRIG2 and EGFR in LRIG2 overexpresing U87 and U251 glioblastoma cells. Cells were stained with anti-LRIG2 and anti-EGFR antibodies, followed by using Cy3- and FITC-conjugated secondary antibodies. Yellow of merge panel indicates regions of colocalization. Scale bar, 50 µm. (<b>B</b>) Lysates from cells overexprssing LRIG2 or LRIG2ecto were immunoprecipitated with purified mouse anti-Flag monoclonal antibodies or control mouse IgG, and immunoprecipitates were blotted with antibodies to EGFR or Flag.</p

Establishment of glioblastoma cell lines stably expressing LRIG2 or LRIG2ecto.

<p>(<b>A</b>) Schematic drawing of the domain organization of the Flag tagged full-length LRIG2 and LRIG2 ectodomain. Indicated are the signal peptides (SP), Flag tag (Flag), the leucine-rich repeat domains, consisting of cysteine-rich N-flanking domain (NF), 15 leucine-rich repeats (LRR) and cysteine-rich C-flanking domain (CF), three immunoglobulin-like domains (Ig-C2), the transmembrane domain (TM) and the cytoplasmic tail (Cyto). (<b>B</b>) The total cell lysates of stably transduced cells, cultured in complete medium for 48 h, were subjected to immunoblotting using an anti-Flag antibody. β-Actin served as an internal loading control. (<b>C</b>) After maintained in complete medium for 48 h, the cells were subjected to total RNA extraction, followed by quantitative RT-PCR to measure the LRIG2 and LRIG2ecto mRNA expression levels. Expressions are shown as the fold changes of control cells (**<i>P</i><0.01, vs con). (<b>D</b>) After cultured in complete medium for 48 h, stably transduced cells were subjected to immunofluorescence analysis. Flag staining is green, and nuclear stain is blue. Representative images of three independent experiments were shown. Scale bars, 50 µm.</p