Abstract Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor. Immunotherapy research has led to advances in its treatment, but further research is necessary to identify its effective biomarkers. This study investigated the expression, pathological and prognostic significance, protein interactions, pathway enrichment, immune microenvironment, correlations between immune regulators and infiltration of immune cells, associations with drug resistance genes, and chemosensitivity of the immune-related biomarker leupaxin (LPXN) in ESCC using bioinformatics. The relative expression levels of LPXN mRNA and protein were evaluated and verified in both healthy and ESCC tissues using quantitative polymerase chain reaction and immunohistochemistry. The potential role of LPXN in ESCC was investigated using cell proliferation, apoptosis, clonogenic, and migration assays. The co-expression of LPXN and programmed cell death-ligand 1 (PD-L1) at the protein level in ESCC lines was determined by western blotting. We validated the expression of the LPXN gene in ESCC using clinical samples and investigated the correlation between LPXN gene expression and the efficacy of immune therapy for ESCC. Functional experiments demonstrated that inhibiting LPXN led to decreased cell proliferation, increased apoptosis, and impaired cell migration and invasion in ESCC cells. Our results indicate the involvement of the immune-related biomarker LPXN in the proliferation and migration processes of ESCC, establishing a novel framework for treatment.
Esophageal cancer is of high prevalence and poor prognosis. Hesperetin has been reported to exert antitumor ability by inducing apoptosis in many cancers in vitro and in vivo without obvious toxicity. However, there is no study concerning about the effect of hesperetin on esophageal cancer. In this study, we aimed to investigate whether hesperetin could induce apoptosis in esophageal cancer cells and explore its potential mechanism. We found that hesperetin induced esophageal cancer cells apoptosis in a concentration-dependent and time-dependent manner compared with the untreated cells. Hoechst 33258 staining and flow cytometry analysis showed more apoptotic cells in the hesperetin-treated group (p < 0.05, respectively). The intracellular reactive oxygen species (ROS) increased significantly, and glutathione (GSH) was depleted. The loss of △Ψ m was more tremendous in the hesperetin-treated cells. N-acetylcysteine (NAC) reduced the proapoptotic ability of hesperetin, while DL-buthionine-S, R-sulfoximine (BSO) enhanced the anticancer effect. Western blotting showed that the expression levels of cytochrome C (Cyt C) and apoptosis-inducing factor (AIF) decreased in mitochondria and increased in cytoplasm (p < 0.05). The levels of intracellular cleaved caspase-9, cleaved caspase-3, Apaf-1, Bcl-2-associated X protein (Bax), and suppressor of fused (SuFu) increased, while B cell lymphoma 2 (Bcl-2) and Survivin decreased. What is more, in xenograft tumor model, hesperetin inhibited the tumor growth significantly via induction of cell apoptosis which was detected by TUNEL assay (p < 0.05). Taken together, our study demonstrated that hesperetin could induce cell apoptosis in esophageal cancer cells via mitochondrial-mediated intrinsic pathway by accumulation of ROS.
Acute lymphoblastic leukemia (ALL) is one of the most common pediatric cancers in the world. Several single-nucleotide polymorphisms (SNPs) locating at PIP4K2A locus were identified to be associated with ALL susceptibility through genome-wide association studies, however, followed by inconsistent reports in replication studies. In this study, we conducted a meta-analysis to investigate the association status of the top independent SNPs (rs7088318 and rs4748793) with ALL susceptibility by combining the data from 6 independent studies, totally including 3508 cases and 12,446 controls with multiethnic populations. Consistent association with ALL risk of both SNPs were observed (odds ratio [OR] 1.28 and 1.29, 95% confidence interval [CI] 1.20–1.36 and 1.19–1.40, respectively). Considering clinic characteristics, rs7088318 is more related to patients with African ancestry (OR 1.48, 95% CI 1.21–1.80) and hyperdiploid subtype (OR 1.42, 95% CI 1.25–1.61). Moreover, several SNPs (eg, rs45469096) were identified to be in high linage disequilibrium with rs7088318, and affected PIP4K2A expression in lymphocytes probably by altering the binding affinity of some transcriptional factors. In conclusion, we systematically investigated the relationship between SNPs at PIP4K2A locus and ALL susceptibility, and further found potential causal variant candidates, thus better elucidating the role of PIP4K2A gene in leukemogenesis.
Arresten as a endogenous inhibitor of angiogenesis originated from the carboxyl-terminal 223 amino acids fragment of the non-collagen domain in alpha1 chain of human collagen IV. In order to get the soluble arresten with biological activity, the cDNA of arresten was cloned and expressed in Pichia pastoris. The produced arresten cDNA was amplified by PCR using primer P1:5'-AGGCCCCGATGGGTTGC-3', primer P2:5'-CTATAAG GCACTTTACGGTTTC-3'. The PCR products was cloned into pGEM-T vector, and sequenced. The arresten cDNA from pGEM-T vector was recombined with vector pPIC9 as pPIC9-arresten, used to transform E. coli DH5alpha, and the inserted arresten cDNA confirmed by agarose electrophoresis and sequencing. pPIC9-arresten was linearized by Sac I. Pichia pastoris GS115 was treated with PEG1000 (followed Invitrogen' s specification); transformed with linear recombined pPIC9-arresten. Pichia pastoris GS115 was culured on MD mediun, single clone was selected and the DNA from the single clone was extracted, used as template, characterized by PCR using the second pair primers P3:5'-CGCTCGAGAAAAGATCTGTTGATC-3', P4:5'-GCCCCGG ATCCTTATGTTCTFCTCATACAG-3'. The polynucleotides CTCGAGAAAAGA used as marker sequence was inserted into primer P3 for signal peptidase to cleave off the signal sequence correctively. The recombined Pichia pastoris GS115 was selected according to the results of PCR, cultured on MM and MD media and then in the BMGY media using methanol as inducer. Expressed arresten was analysed by SDS-PAGE. The soluble arresten expressed by Pichia pastoris gave apparent molecular weight in SDS-PAGE consistent with that calculated, and in matrigel gel it showed inhibitary activity on the tubulation of endothelial cell ECV-304 induced by tumor cell MDA-MB-435S. These results showesd arresten with biological activity is expressed successfully in Pichia pastoris GS115.
A non-profit organization (NPO) is the third type of department, which exists simultaneously with the government one and the profit one (enterprise). The discipline of Management of still remains unformed in the foreign theories of public management. Being a late comer in this field, China has introduced many foreign theories in relevant documents though they are not always applicable owing to the non-profit organizations' characteristics in China. The above-mentioned factors have caused a great theoretical blank in the study of NPO in China. This article mainly aims to analyze the similarities and differences between the strategic management of a profit organization (enterprise) and that of a non-profit organization. The author makes the conclusion that some management theories of a profit organization can be directly applied to the management of a non-profit organization; some must be transformed before its application and some can not be applied at all.
To investigate the effect of ascorbic acid (VC) on relaxation of ex vivo Bufo gastrocnemius during sustained isometric contraction.Dynamic tension of the muscle was recorded under constant voltage stimulation within 7.0 min at 2 s intervals. The rest tension and relaxation rate of the muscle was obtained by weighted fitting to the relaxation process of tension <90% of its peak with a mono-exponential model to characterize the muscular relaxation.VC at 2.0 mmol/L alone or in combination with the inhibitors of the antixoidation enzymes (surperoxide dismutase, glutathione peroxidase and catalase) resulted in negligible alterations in the muscular relaxation kinetics. VC combined with the inhibitor of surperoxide dismutase resulted in significantly lowered relaxation rate while increased rest tension, but VC with the inhibitor of either catalase or glutathione peroxidase showed negligible action. VC combined with the inhibitors of all the 3 enzymes also caused significant effect on the muscular relaxation kinetics, which was similar the effect of VC with superoxide dismutase inhibitor.VC at high concentration may result in oxidative toxicity to the biological system rich in transitional metal ion complexes but with low antioxidation capacity by causing superoxide-mediated oxidative damages.
Abstract We reported that adoptive transfer of tumor reactive B cells could mediate tumor regression, and that anti-PD-L1 administration significantly augments the therapeutic efficacy of cancer stem cell (CSC) vaccine. However, whether B cells are involved in the anti-tumor immunity mediated by anti-PD-L1 has not been identified. In the present study, we used two murine models: breast tumor 4T1 and colon cancer CT26. Tumor-bearing mice were administered with anti-CD20 mAb (MB20.11) to deplete B cells prior to anti-PD-L1 mAb administration. Depletion of B cells as compared to the control resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced (p=0.041 and p=0.0011 for 4T1 and CT26 respectively), indicating the involvement of host B cells in the function of anti-PD-L1. Mechanistically, the administration of anti-PD-L1 partially recovered the humoral immune response, suggesting that the blockage of PD-L1/PD1 pathway could contribute to the rescue of B cell immune function. We detected higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs or on unsorted tumor cells. On the other hand, we detected elevated expression of PD-1 on activated B cells. While the suppressive effect of CSCs on T cells has well been recognized, the interactions between CSCs and B cells is unknown. We hypothesize that CSCs may suppress B cells either directly via the PD-L1/PD1 immune check point interaction or indirectly via CSC suppression on Th cells. To test this hypothesis, we co-cultured purified B cells with ALDHhigh CSCs with or without the addition of anti-PD-L1 mAb. We found that the reduction of IgG secretion of B cells suppressed by CSCs could be rescued by anti-PD-L1 in a dose-dependent manner. These experiments indicate that CSCs can directly suppress the IgG production by B cells via PD-L1/PD-1 interaction. Moreover, when we co-cultured purified B cells plus purified CD4+ Th cells with ALDHhigh CSCs with or without the addition of anti-PD-L1 mAb, we found that the IgG production was further and significantly (p=0.0155) reduced as compared to the co-culture of CSCs and B cells alone. The addition of anti-PD-L1 to the cultures rescued IgG production by T/B/CSC co-cultures to a greater (p=0.0429) extent than in just B/CSC co-culture. These results indicate that CSCs can also indirectly suppress IgG production by B cells via the PD-L1/PD-1 interaction with Th cells. Together, this study demonstrates that the anti-tumor function of anti-PD-L1 mAb involves host B cells, and that CSCs can both directly and indirectly inhibit B cell function through the PD-L1/PD-1 axis on both B cells and Th cells, respectively. Citation Format: Leiming Xia, FEI LIAO, Jing Zhang, Ming Lin, Ronald Herbst, Elaine Hurt, Alfred Chang, Max Wicha, Qiao Li. Involvement of B cells in the efficacy of anti-PD-L1 antibodies: Suppression of B cells by cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1676.
Radiotherapy resistance is one of the main causes for treatment failure in colorectal cancer (CRC), and it is associated with the deregulation of certain microRNAs. In this study, we constructed the microRNA-mRNA network consisting of 2275 microRNAs and 7045 target genes, collected the known microRNAs related to CRC-radiosensitivity (CRCR) (n=18) as the seed nodes, and applied the algorithm of random walk with restart (RWR) to the network to identify novel CRCR-related microRNAs (n=263). In functional analysis, 263 novel microRNAs shared a high proportion of the same biological processes and pathways with the known microRNAs. In topological analysis of the sub-network of the 263 microRNAs and their targets, hsa-mir-506-3p and hsa-mir-140-5p were identified as network hub nodes. In plasma, radiosensitive patients had a higher expression level of hsa-mir-506-3p and hsa-mir-140-5p than radioresistant patients. In experimental validation, both hsa-mir-506-3p and hsa-mir-140-5p over-expression could obviously decrease the cell proliferation, survival rate and colonality in CRC cells after radiation. In conclusion, this study combined the novel network-based method with experimental validation, and identified two novel radiosensitive biomarkers of hsa-mir-506-3p and hsa-mir-140-5p in CRC.
Maximum activities of 6His-tagged enzyme/mutants from lysates adsorbed on immobilized anti-tag antibody were predicted as specific activities for comparison.
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