Ibuprofen­derived nitric oxide donors with a high affinity to human serum albumin induce cell death in pancreatic cancer cells through a non­caspase 3/7­mediated pathway.

Nitric oxide (NO) has been reported to have a cytotoxic effect on various types of cancer. However, the efficient delivery of NO donors to tumors remains challenging. The present study used ibuprofen, which has a high binding affinity to human serum albumin (HSA). A total of two types of nitrated forms of ibuprofen, 4-[(nitrooxy)methyl]benzyl 2-(4-isobutylphenyl)propanoate [nitrated ibuprofen benzyl linker (NIB)] and 2-(nitrooxy)ethyl 2-(4-isobutylphenyl) propanoate [nitrated ibuprofen ethyl linker (NIE)], were synthesized. It was demonstrated that both NIB and NIE bound to the ibuprofen-binding site of HSA. Although NOx release was observed from NIB, but not NIE, intracellular NO release was detected from both NIB and NIE, which indicated that the mechanisms of NO release may be different for NIB and NIE. Both NIB and NIE induced concentration- and time-dependent cell death in human pancreatic cancer cells, whereas this cell death was not observed with ibuprofen, which could suggest that these cell death-inducing effects may be mediated by NO. The non-specific caspase inhibitor, z-VAD-FMK, inhibited cell death induced by NIB and NIE, but activation of caspase 3/7 was not observed. These results suggested that both NIB and NIE induced cell death through a non-caspase 3/7 pathway. The findings of the present study demonstrated that both NIB and NIE, as NO donors that could be retained in blood, may potentially be useful anti-cancer agent candidates in the future.

Novel Diclofenac-NO Donor With High Affinity for Human Serum Albumin Induces Endoplasmic Reticulum Stress-mediated Cell Death in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: Nitric oxide (NO) has various physiological activities. In this study, diclofenac (DF) which has a high affinity for human serum albumin (HSA) was nitrosylated to a novel NO donor (NDF). The cytotoxic effects and the mechanism of NDF were investigated. MATERIALS AND METHODS: Binding experiments of NDF to HSA were performed by the ultrafiltration method. NO was measured by the Griess method. The number of dead cells were measured using annexin V. Apoptosis and endoplasmic reticulum stress were evaluated by western blotting. RESULTS: NDF competitively inhibits the binding of DF to HSA, suggesting that NDF and DF have equivalent binding characteristics. NDF rapidly released NOx after being dissolved. At 200 µM, NDF induced cell death in human pancreatic cancer cells. Western blotting showed that NDF promoted the cleavage of PARP, caspase-3, and caspase-7. Inhibitors of caspase-1 and caspase-9 significantly suppressed NDF-induced cell death, as did a non-specific caspase inhibitor (Z-VAD). In addition, NDF significantly increased the expression of the endoplasmic reticulum stress marker, CHOP. CONCLUSION: NDF induces apoptotic cell death by causing endoplasmic reticulum stress. The findings of this study suggest that NDF may become a promising compound for the treatment of pancreatic cancer.

Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the ß2-microglobulin (ß2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized ß2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with ß2m, thus accounting for ß2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.

Crystal structure of the lamprey variable lymphocyte receptor C reveals an unusual feature in its N-terminal capping module.

Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to γδ T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.

Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL.

Mincle [macrophage inducible Ca(2+)-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6'-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca(2+)-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.

Distinct HIV-1 escape patterns selected by cytotoxic T cells with identical epitope specificity.

Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.

Applicability of press needles to a double-blind trial: a randomized, double-blind, placebo-controlled trial.

OBJECTIVES: Owing to a lack of a suitable needle procedure, it has been impossible to evaluate the efficacy of acupuncture in clinical studies using double-blind testing. We evaluated the applicability of a new kind of press needle (Pyonex) to a double-blind trial by comparing the press needle with a placebo (lacking the needle element). METHODS: The purpose of the study consisted of 2 phases. In the phase 1, to evaluate the applicability and efficacy of the press needles, 90 participants who had never been treated using acupuncture were randomly assigned to receive either the press needle (n=45) or a placebo (n=45). The applicability was measured using a questionnaire regarding the perception of penetration, and efficacy was measured using a visual analog scale of low back pain (LBP). When the applicability and efficacy of the press needles were confirmed in phase 1, the mechanism of LBP relief by the press needles was examined in phase 2. RESULTS: In phase 1, intergroup comparisons showed no significant differences concerning the perception of penetration. In addition, for patients with LBP, the press needles reduced the subjective evaluation of LBP compared with the placebo (P<0.05). In phase 2, visual analog scale results indicated that LBP was reduced significantly more in the press needle group than in the local anesthesia group (P<0.05). DISCUSSION: The participants could not distinguish between the press needle and a placebo, and the data from the press needle group suggested a specific influence on patients with LBP. These findings imply that the press needle and a placebo provide an effective means of realizing a double-blind setting for clinical studies of acupuncture.