Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals.

CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3ß1/α6ß1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

Leustroducsin B activates nuclear factor-kappaB via the acidic sphingomyelinase pathway in human bone marrow-derived stromal cell line KM-102.

The novel colony-stimulating factor (CSF) inducer leustroducsin B (LSN-B), which was isolated from Streptomyces platensis, has been shown to have potent cytokine-inducing activities in clonal human bone marrow-derived stromal cell line KM-102 and in primary human bone marrow-derived stromal cells. In this study, we investigated the signal transduction pathway of LSN-B using luciferase expression plasmids linked to the 5'-flanking region of interleukin-8 (IL-8) and that of the IL-11 gene. In KM-102 cells, LSN-B induced luciferase activity both in the wild-type and in the activated protein 1 (AP-1) site point-mutated IL-8 promoter. The mutation in the nuclear factor-kappaB (NF-kappaB) site abrogated LSN-B-stimulated induction of the reporter gene. LSN-B-inducing activity was inhibited by (1) N-acetyl-L-cysteine, a well-characterized antioxidant, (2) cationic amphiphilic drugs, inhibitors of acidic sphingomyelinase (A-SMase), and (3) D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). These observations suggest that LSN-B potentiates the A-SMase-mediated signaling pathway to stimulate NF-kappaB. In contrast, LSN-B did not induce IL-11 promoter-driven luciferase activity. The observed increase in IL-11 mRNA stability by LSN-B indicates that the inducible production of IL-11 by LSN-B is regulated at the posttranscriptional level. In addition, inhibition of LSN-B-mediated induction of IL-11 production by cationic amphiphilic drugs and D609 in KM-102 cells demonstrates that increased IL-11 mRNA stability by LSN-B might be mediated via NF-kappaB activation. From these results, we suggest that LSN-B induces cytokine production through at least two separate mechanisms, at the transcriptional level and at the posttranscriptional level via NF-kappaB activation.

Identification of 2'-phosphodiesterase, which plays a role in the 2-5A system regulated by interferon.

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2',5'-oligoadenylate synthetases (2',5'-OASs), inactivated by 5'-phosphatase and completely degraded by 2'-phosphodiesterase (2'-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2',5'-OASs and RNase L have been molecularly cloned and studied well, the identification of 2'-PDE has remained elusive. Here, we describe the first identification of 2'-PDE, the third key enzyme of the 2-5A system. We found a putative 2'-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2'-PDE by mass spectrometry. Based on the full-length sequence of the human 2'-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2'-PDE expressed in mammalian cells certainly cleaved the 2',5'-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2'-PDE were found, the human 2'-PDE was considered to be a unique enzyme without isoform. Suppression of 2'-PDE by a small interfering RNA and a 2'-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2'-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2'-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.