Engineering of an EPHA2-Targeted Monobody for the Detection of Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third most common cancer worldwide, and is second only to lung cancer with respect to cancer-related deaths. Noninvasive molecular imaging using established markers is a new emerging method to diagnose CRC. The human ephrin receptor family type-A 2 (hEPHA2) oncoprotein is overexpressed at the early, but not late, stages of CRC. Previously, we reported development of an E1 monobody that is specific for hEPHA2-expressing cancer cells both in vitro and in vivo. Herein, we investigated the ability of the E1 monobody to detect hEPHA2 expressing colorectal tumors in a mouse model, as well as in CRC tissue. MATERIALS AND METHODS: The expression of hEPHA2 on the surface of CRC cells was analyzed by western blotting and flow cytometry. The targeting efficacy of the E1 monobody for CRC cells was examined by flow cytometry, and immunofluorescence staining. E1 conjugated to the Renilla luciferase variant 8 (Rluc8) reporter protein was used for in vivo imaging in mice. Additionally, an enhanced green fluorescence protein (EGFP) conjugated E1 monobody was used to check the ability of the E1 monobody to target CRC tissue. RESULTS: The E1 monobody bound efficiently to hEPHA2-expressing CRC cell lines, and E1 conjugated to the Rluc8 reporter protein targeted tumor tissues in mice transplanted with HCT116 CRC tumor cells. Finally, E1-EGFP stained tumor tissues from human CRC patients, showing a pattern similar to that of an anti-hEPHA2 antibody. CONCLUSION: The E1 monobody has utility as an EPHA2 targeting agent for the detection of CRC.

The anticancer effect of PASylated calreticulin-targeting L-ASNase in solid tumor bearing mice with immunogenic cell death-inducing chemotherapy.

L-Asparaginase (L-ASNase), a bacterial enzyme that degrades asparagine, has been commonly used in combination with several chemical drugs to treat malignant hematopoietic cancers such as acute lymphoblastic leukemia (ALL). In contrast, the enzyme was known to inhibit the growth of solid tumor cells in vitro, but not to be effective in vivo. We previously reported that two novel monobodies (CRT3 and CRT4) bound specifically with calreticulin (CRT) exposed on tumor cells and tissues during immunogenic cell death (ICD). Here, we engineered L-ASNases conjugated with monobodies at the N-termini and PAS200 tags at the C-termini (CRT3LP and CRT4LP). These proteins were expected to possess four monobody and PAS200 tag moieties, which did not disrupt the L-ASNase conformation. These proteins were expressed 3.8-fold more highly in E. coli than those without PASylation. The purified proteins were highly soluble, with much greater apparent molecular weights than expected ones. Their affinity (Kd) against CRT was about 2 nM, 4-fold higher than that of monobodies. Their enzyme activity (∼6.5 IU/nmol) was similar to that of L-ASNase (∼7.2 IU/nmol), and their thermal stability was significantly increased at 55 °C. Their half-life times were > 9 h in mouse sera, about 5-fold longer than that of L-ASNase (∼1.8 h). Moreover, CRT3LP and CRT4LP bound specifically with CRT exposed on tumor cells in vitro, and additively suppressed the tumor growth in CT-26 and MC-38 tumor-bearing mice treated with ICD-inducing drugs (doxorubicin and mitoxantrone) but not with a non-ICD-inducing drug (gemcitabine). All data indicated that PASylated CRT-targeted L-ASNases enhanced the anticancer efficacy of ICD-inducing chemotherapy. Taken together, L-ASNase would be a potential anticancer drug for treating solid tumors.

N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide is a novel inhibitor of resorptive bone loss in mice.

The dynamic balance between bone formation and bone resorption is vital for the retention of bone mass. The abnormal activation of osteoclasts, unique cells that degrade the bone matrix, may result in many bone diseases such as osteoporosis. Osteoporosis, a bone metabolism disease, occurs when extreme osteoclast-mediated bone resorption outstrips osteoblast-related bone synthesis. Therefore, it is of great interest to identify agents that can regulate the activity of osteoclasts and prevent bone loss-induced bone diseases. In this study, we found that N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide (PPOAC-Bz) exerted a strong inhibitory effect on osteoclastogenesis. PPOAC-Bz altered the mRNA expressions of several osteoclast-specific marker genes and blocked the formation of mature osteoclasts, suppressing F-actin belt formation and bone resorption activity in vitro. In addition, PPOAC-Bz prevented OVX-induced bone loss in vivo. These findings highlighted the potential of PPOAC-Bz as a prospective drug for the treatment of osteolytic disorders.

PMSA prevents osteoclastogenesis and estrogen-dependent bone loss in mice.

Excessive bone resorption mediated by mature osteoclasts can cause osteoporosis, leading to fragility fractures. Therefore, an effective therapeutic strategy for anti-osteoporosis drugs is the reduction of osteoclast activity. In this study, the osteoclast inhibitory activity of a novel compound, N-phenyl-methylsulfonamido-acetamide (PMSA), was examined. PMSA treatment inhibited receptor activator of nuclear factor kappa B ligand (RNAKL)-induced osteoclast differentiation in bone marrow-derived macrophage cells (BMMs). We investigated two PMSAs, N-2-(3-acetylphenyl)-N-2-(methylsulfonyl)-N-1-[2-(phenylthio)phenyl] glycinamide (PMSA-3-Ac), and N-2-(5-chloro-2-methoxyphenyl)-N-2-(methylsulfonyl)-N-1-[2-(phenylthio)phenyl]glycinamide (PMSA-5-Cl), to determine their effects on osteoclast differentiation. PMSAs inhibited the signaling pathways at the early stage. PMSA-3-Ac inhibited tumor necrosis factor receptor-associated factor 6 (TRAF6) expression, whereas PMSA-5-Cl suppressed the mitogen-activated protein kinase (MAPK) signaling pathways. However, both PMSAs inhibited the master transcription factor, nuclear factor of activated T cell cytoplasmic-1 (NFATc1), by blocking nuclear localization. An in vivo study of PMSAs was performed in an ovariectomized (OVX) mouse model, and PMSA-5-Cl prevented bone loss in OVX mice. Therefore, our results suggested that PMSAs, specifically PMSA-5-Cl, may serve as a potential therapeutic agent for postmenopausal osteoporosis.

Expression of c-Myc is related to host cell death following Salmonella typhimurium infection in macrophage.

It has been known that ornithine decarboxylase (ODC) induced by the binding of c-Myc to odc gene is closely linked to cell death. Here, we investigated the relationship between their expressions and cell death in macrophage cells following treatment with Salmonella typhimurium or lipopolysaccharide (LPS). ODC expression was increased by bacteria or LPS and repressed by inhibitors against mitogen-activated protein kinases (MAPKs) in Toll-like receptor 4 (TLR4) signaling pathway. In contrast, c-Myc protein level was increased after treatment with bacteria, but not by treatment with LPS or heat-killed bacteria although both bacteria and LPS increased the levels of c-myc mRNA to a similar extent. c-Myc protein level is dependent upon bacterial invasion because treatment with cytochalasin D (CCD), inhibitors of endocytosis, decreased c-Myc protein level. The cell death induced by bacteria was significantly decreased after treatment of CCD or c-Myc inhibitor, indicating that cell death by S. typhimurium infection is related to c-Myc, but not ODC. Consistent with this conclusion, treatment with bacteria mutated to host invasion did not increase c-Myc protein level and cell death rate. Taken together, it is suggested that induction of c-Myc by live bacterial infection is directly related to host cell death.