Protein crystals generally are stable solid protein assemblies. Certain protein crystals are suitable for use as nanovessels for immobilizing metal complexes. Here we report the preparation of ...ruthenium carbonyl-incorporated cross-linked hen egg white lysozyme crystals (Ru·CL-HEWL). Ru·CL-HEWL retains a Ru carbonyl moiety that can release CO, although a composite of Ru carbonyl-HEWL dissolved in buffer solution (Ru·HEWL) does not release CO. We found that treatment of cells with Ru·CL-HEWL significantly increased nuclear factor kappa B (NF-κB) activity as a cellular response to CO. These results demonstrate that Ru·CL-HEWL has potential for use as an artificial extracellular scaffold suitable for transport and release of a gas molecule.
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IJS, KILJ, NUK, PNG, UL, UM
Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras‐induced cancer. Therefore, the downstream effectors of Ras ...signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF‐HEV/IL‐33, a member of the interleukin‐1 family, and showed that intracellular IL‐33 was required for oncogenic Ras‐induced cellular transformation. In the present study, we demonstrated that the c‐Mer proto‐oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL‐33 signaling. The expression of MerTK was enhanced in transformed NIH‐3T3 cells by the expression of oncogenic Ras, H‐Ras (G12V), in an IL‐33‐dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL‐33 expression. The knockdown of IL‐33 or MerTK effectively attenuated the migration of NIH‐3T3 cells transformed by H‐Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K‐Ras mutation. Furthermore, the suppression of Ras‐induced cell migration by the knockdown of IL‐33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH‐3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras‐transformed cells.
Oncogenic Ras alters intracellular signals to transform cells and cause cancer. Here, we described a novel oncogenic Ras signaling where the expression of MerTK is induced by oncogenic Ras in an IL‐33‐dependent manner. MerTK is strongly phosphorylated by oncogenic Ras. Subsequently, IL‐33, MerTK, and the kinase activity of MerTK are indispensable for oncogenic Ras‐induced cell migration.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a ...proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and ...invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.
Monoclonal antibodies (mAbs) are attractive therapeutics for treating a wide range of human disorders, and bind to the antigen through their complementarity-determining regions (CDRs). Small stable ...proteins containing structurally retained CDRs are promising alternatives to mAbs. In this report, we present a method to create such proteins, named fluctuation-regulated affinity proteins (FLAPs). Thirteen graft acceptor (GA) sites that efficiently immobilise the grafted peptide structure were initially selected from six small protein scaffolds by computational identification. Five CDR peptides extracted by binding energy calculations from mAbs against breast cancer marker human epithelial growth factor receptor type 2 (HER2) were then grafted to the selected scaffolds. The combination of five CDR peptides and 13 GA sites in six scaffolds revealed that three of the 65 combinations showed specific binding to HER2 with dissociation constants (K
) of 270-350 nM in biolayer interferometry and 24-65 nM in ELISA. The FLAPs specifically detected HER2-overexpressing cancer cells. Thus, the present strategy is a promising and practical method for developing small antibody mimetics.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Lung metastasis is a major cause of mortality in patients with osteosarcoma (OS). A better understanding of the molecular mechanism of OS lung metastasis may facilitate development of new therapeutic ...strategies to prevent the metastasis. We have established high‐ and low‐metastatic sublines (LM8‐H and LM8‐L, respectively) from Dunn OS cell line LM8 by using in vivo image‐guided screening. Among the genes whose expression was significantly increased in LM8‐H compared to LM8‐L, the transcription factor lymphoid enhancer‐binding factor 1 (LEF1) was identified as a factor that promotes LM8‐H cell extravasation into the lungs. To identify downstream effectors of LEF1 that are involved in OS lung metastasis, 13 genes were selected based on LM8 microarray data and genomewide meta‐analysis of a public database for OS patients. Among them, the cytoglobin (Cygb) gene was identified as a key effector in promoting OS extravasation into the lungs. CYGB overexpression increased the extravasation ability of LM8‐L cells, whereas knocking out the Cygb gene in LM8‐H cells reduced this ability. Our results showed a novel LEF1‐CYGB axis in OS lung metastasis and may provide a new way of developing therapeutic strategies to prevent OS lung metastasis.
We found that the cytoglobin (Cygb) gene is an important effector that promotes extravasation of osteosarcoma (OS) to the lungs. Our data showed a novel function of the LEF1‐CYGB axis in OS lung metastasis and may provide novel targets for drugs that prevent OS lung metastasis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Hypoxic stress is a risk factor of ocular neovascularization. Hypoxia visualization may provide clues regarding the underlying cause of angiogenesis. Recently, we developed a hypoxia-specific probe, ...protein transduction domain-oxygen-dependent degradation domain-HaloTag-Rhodamine (POH-Rhodamine). In this study, we observed the localization of HIF-1α proteins by immunohistochemistry and the fluorescence of POH-Rhodamine on RPE-choroid flat mounts. Moreover, we compared the localization of POH-Rhodamine with pimonidazole which is a standard reagent for detecting hypoxia. Next, we investigated the effects of triamcinolone acetonide (TAAC) against visual function that was evaluated by recording electroretinogram (ERG) and choroidal neovascularization (CNV) development. Mice were given laser-induced CNV using a diode laser and treated with intravitreal injection of TAAC. Finally, we investigated POH-Rhodamine on CNV treated with TAAC. In this study, the fluorescence of POH-Rhodamine and HIF-1α were co-localized in laser-irradiated sites, and both the POH-Rhodamine and pimonidazole fluorescent areas were almost the same. Intravitreal injection of TAAC restored the reduced ERG b-wave but not the a-wave and decreased the mean CNV area. Furthermore, the area of the POH-Rhodamine-positive cells decreased. These findings indicate that POH-Rhodamine is useful for evaluating tissue hypoxia in a laser-induced CNV model, suggesting that TAAC suppressed CNV through tissue hypoxia improvement.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Pancreatic cancer is one of the most lethal digestive system cancers with a 5‐year survival rate of 4–7%. Despite extensive efforts, recent chemotherapeutic regimens have provided only limited ...benefits to pancreatic cancer patients. Gemcitabine and TS‐1, the current standard‐of‐care chemotherapeutic drugs for treatment of this severe cancer, have a low response rate. Hypoxia is one of the factors contributing to treatment resistance. Specifically, overexpression of hypoxia‐inducible factor, a master transcriptional regulator of cell adaption to hypoxia, is strongly correlated with poor prognosis in many human cancers. TAT‐ODD‐procaspase‐3 (TOP3) is a protein prodrug that is specifically processed and activated in hypoxia‐inducible factor‐active cells in cancers, leading to cell death. Here, we report combination therapies in which TOP3 was combined with gemcitabine or TS‐1. As monotherapy, gemcitabine and TS‐1 showed a limited effect on hypoxic and starved pancreatic cancer cells, whereas co‐treatment with TOP3 successfully overcame this limitation in vitro. Furthermore, combination therapies of TOP3 with these drugs resulted in a significant improvement in survival of orthotopic pancreatic cancer models involving the human pancreatic cancer cell line SUIT‐2. Overall, our study indicates that the combination of TOP3 with current chemotherapeutic drugs can significantly improve treatment outcome, offering a promising new therapeutic option for patients with pancreatic cancer.
We recently developed a fusion protein drug TOP3, which is designed to be specifically stabilized and activated in hypoxia‐inducible factor (HIF)‐active cancers. HIF is strongly correlated with poor prognosis of pancreatic cancer patients. In this study, we reported that the combination of TOP3 with current chemotherapeutic drugs can significantly improve treatment outcome, offering a promising new therapeutic option for patients with pancreatic cancer.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The ubiquitin-proteasome system (UPS) is a selective protein degradation system that plays a critical role in many essential biological processes by regulating the existence of various cellular ...proteins. The target proteins of UPS are recognized and tagged with polyubiquitin chains by E3 ubiquitin ligases, which have high substrate-specific activities. Here we present a novel injectable imaging probe POL-N that can detect the UPS-regulated hypoxia-inducible factor (HIF) activity in vivo. Because the luciferase is fused to the E3 ligase-recognition domain of the HIF-1α, POL-N is intact only in the HIFα-overexpressing cells, that is, HIF-active cells, generating signals via an intramolecular bioluminescence resonance energy transfer (BRET) between luciferase and a near-infrared (NIR) fluorescent dye at the C-terminal end of the probe. Off-target signals of the NIR-BRET were so low that we could achieve highly sensitive and fast detection of intratumoral HIF-activity. Notably, we successfully detected hypoxic liver metastasis, which is extremely difficult to detect by injectable imaging probes due to strong off-target signals, as early as 1 h after systemic injection of POL-N. Our probe design can be widely adapted to UPS-target proteins and may contribute to the exploration of their roles in animal disease models.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Bone metastasis is a multistep process that includes cancer cell dissemination, colonization, and metastatic growth. Furthermore, this process involves complex, reciprocal interactions between cancer ...cells and the bone microenvironment. Bone resorption is known to be involved in both osteolytic and osteoblastic bone metastasis. However, the precise roles of the bone resorption in the multistep process of osteoblastic bone metastasis remain unidentified. In this study, we show that bone resorption plays important roles in cancer cell colonization during the initial stage of osteoblastic bone metastasis. We applied bioluminescence/X‐ray computed tomography multimodal imaging that allows us to spatiotemporally analyze metastasized cancer cells and bone status in osteoblastic bone metastasis models. We found that treatment with receptor activator of factor‐κB ligand (RANKL) increased osteoblastic bone metastasis when given at the same time as intracardiac injection of cancer cells, but failed to increase metastasis when given 4 days after cancer cell injection, suggesting that RANKL‐induced bone resorption facilitates growth of cancer cells colonized in the bone. We show that insulin‐like growth factor‐1 released from the bone during bone resorption and hypoxia‐inducible factor activity in cancer cells cooperatively promoted survival and proliferation of cancer cells in bone marrow. These results suggest a mechanism that bone resorption and hypoxic stress in the bone microenvironment cooperatively play an important role in establishing osteoblastic metastasis.
Bone resorption facilitates cancer cell colonization of osteoblastic bone metastasis. We revealed that insulin‐like growth factor‐1 released from the bone during bone resorption and hypoxia‐inducible factor activity cooperatively promote cancer cell colonization in hypoxic bone marrow.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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