Antiproliferative activities through accelerating autophagic flux by basidalin and its analogs in human cancer cells.

Basidalin, isolated from the basidiomycete Leucoagaricus naucina, has previously demonstrated antibacterial and antitumor properties against murine cancer cells in vivo, but its effects on human cancer cells remain unknown. In this study, we found that basidalin possesses antiproliferative activity against human cancer cell lines. To elucidate the antiproliferative mechanism of basidalin, we focused on autophagy. Treatment with basidalin led to an increase in LC3-II expression level, and accelerated autophagic flux through an mTOR-independent pathway. Moreover, according to the structure-activity relationship analysis-including newly synthesized basidalin analogs-the formyl group, not the amino group, contributes to the antiproliferative activities of basidalin against human cancer cells. Additionally, the antiproliferative activity of basidalin analogs was strongly correlated with autophagy-inducing activity, indicating that basidalin exhibits antiproliferative activity through autophagy induction. These data suggest that basidalin, characterized by its ability to upregulate autophagic flux, emerges as a novel anticancer drug.

Stereodivergent synthesis of 2-oxo-oligopyrrolidines by an iterative coupling strategy.

Natural linear polyamines play diverse roles in physiological processes by interacting with receptors at the cellular level. Herein, we describe the stereodivergent synthesis of oligopyrrolidines, which are conformationally constrained polyamines. We synthesized dimeric and trimeric 2-oxo-oligopyrrolidines using an iterative coupling strategy. The key to our success is an iridium-catalyzed trans/cis-selective nucleophilic addition and subsequent threo/erythro-stereoselective reduction. The synthesized pyrrolidines show varying cytotoxicities against a human cancer cell line depending on the number of rings and their stereochemistry.

ErbB4-mediated regulation of vasculogenic mimicry capability in breast cancer cells.

ErbB4 is a member of the ErbB receptor tyrosine kinase family. It has both pro- and anti-oncogenic activities in tumors. Vasculogenic mimicry (VM), a phenomenon in which cancer cells form capillary-like structures without endothelial cells, has been recognized to be a cause of malignant phenotypes in some solid tumors. Here, we used an in vitro VM formation assay, and demonstrated that ErbB4 negatively regulated VM formation in human breast cancer cells. By using CRISPR/Cas9-mediated gene knockout, we verified that the depletion of endogenous ErbB4 improved the VM formation capability. Although treatment with neuregulin 1 (NRG1), a ligand of ErbB4, induced the phosphorylation of ErbB4 and promoted VM formation in a dose-dependent manner, it did not induce such activities in kinase-dead K751M ErbB4-overexpressing cells. Moreover, we examined the effect of the missense mutation E872K of ErbB4, which has been reported in multiple tumors, on VM formation, and found that the mutation enhanced the basal phosphorylation level and ErbB4-mediated VM formation in the absence of NRG1 stimulation. Whereas NRG1 stimulated VM formation, excessive activation of ErbB4 induced a negative effect. In E872K ErbB4-overexpressing cells, but not in wild-type ErbB4-overexpressing cells, the number of VM tubes was significantly decreased by low-dose treatment with the ErbB inhibitor afatinib. Taken together, our findings demonstrated the significance of ErbB4-mediated VM formation, and suggested the possibility of ErbB4 mutations as effective targets in breast cancer.

Elucidation of structure-activity relationship of humulanolides and identification of humulanolide analog as a novel HSP90 inhibitor.

Humulanolides are natural products isolated from Asteriscus, and the isolation and total synthesis of many types of humulanolides have been reported. In this study, we evaluated anti-proliferative activity of twelve humulanolides against various human cancer cell lines and found that humulanolide analog E, which was newly designed and synthesized, exhibited the highest anti-proliferative activity. Structure-activity relationship analysis revealed that α,ß-unsaturated carbonyl moieties in humulanolides play an important role for anti-proliferative activity. To identify molecular targets of humulanolide analog E, we investigated various cell-based and in vitro assays. Treatment with humulanolide analog E against human fibrosarcoma HT1080 cells increased the expression level of HSP70 protein and decreased the levels of AKT and CDK4, which are HSP90 client proteins. Moreover, humulanolide analog E inhibited refolding of denatured luciferase protein via suppression of HSP90 activity in vitro. These results suggest that humulanolide analog E possesses the anti-proliferative activity against human cancer cells by inhibiting HSP90 functions.

Biological evaluation for anti-inflammatory effect of africane-type sesquiterpenoids.

Africane-type sesquiterpenoids are a unique tricyclic carbon architecture sesquiterpenoid isolated as natural products. Δ9(15) -africanene has been reported to exhibit anti-inflammatory activity for carrageenan-induced rat foot edema. In this study, we reported structure-activity relationship study of africane-type sesquiterpenoids and found that some africane-type sesquiterpenoid analogs and their synthetic intermediate showed potent anti-inflammatory activity. To identify the mode of action of africane-type sesquiterpenoids and their synthetic intermediate, we evaluated the anti-inflammatory activity using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells. Treatment with the africane-type compounds and their synthetic intermediate suppressed LPS-induced expressions of Cox-2 protein and mRNAs of the inflammatory cytokines IL-1ß and IL-6 at the concentrations that did not affect cell viability. Interestingly, although these africane-type compounds and their synthetic intermediate suppressed the pro-inflammatory cytokines' expressions, the compounds did not modulate NF-κB activation. These results suggest that the africane-type compounds and their synthetic intermediate are anti-inflammatory compounds that suppress the expression of LPS-induced inflammatory mediators independently of NF-κB activation.

Scalable Syntheses and Biological Evaluation of Africane-Type Sesquiterpenoids.

Practical total syntheses of africane-type sesquiterpenoids were realized by reexamination of a divergent strategy employing optimized three-component coupling followed by ring-closing metathesis and substrate-controlled cyclopropanation. This sequential eight-step conversion provided Δ9(15) -africanene, a common bicyclo[5.3.0]decane intermediate for the syntheses of africane derivatives, in more than twice the yield as in the previous approach. The scalability and robustness of this improved synthetic route were confirmed by gram-scale preparation of Δ9(15) -africanene. In vitro cell-based assays of the synthesized africane-type sesquiterpenoids disclosed that ester-incorporating derivatives showed cytotoxic activity against HeLa cells. The effect of relative and absolute configuration of africane-9,15-diol monoacetates on the cytotoxicity against HeLa cells was also investigated.

Correction: Mori et al. Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts. Molecules 2021, 26, 5685.

In the original article [...].

Involvement of LH3 and GLT25D1 for glucosyl-galactosyl-hydroxylation on non-collagen-like domain of FGL1.

Glucosyl-galactosyl-hydroxylation (GGH) is one type of post-translational modification, which is mainly observed in collagen-like domain-containing proteins. Using LC-MS/MS analysis, we found a GGH-like modification at Lys65 of fibrinogen-like protein 1 (FGL1), although it does not contain a collagen-like domain. To identify the glycosyltransferases responsible for this modification, we established LH3/GLT25D1-knockout FGL1-overexpressing HT1080 cell lines. The result showed that knockout of LH3 or GLT25D1 significantly inhibited the glycosylation. Furthermore, deficiency of GGH by point mutation of the FGL1 protein or knockout of the GGH-related glycosyltransferase reduced FGL1 protein levels. Taken together, these data indicate that Lys65 of FGL1 is glucosyl-galactosyl-hydroxylated by LH3 and GLT25D1. Our results provide novel insights to regulate various FGL1 functions.

Identification of madangamine A as a novel lysosomotropic agent to inhibit autophagy.

Madangamines are marine natural products isolated from Xestospongia ingens, and madangamine A-E with a different D-ring structure have been reported. We have reported that madangamine A has strong anti-proliferative activity against various human cancer cell lines. In this study, to clarify the anti-proliferative activity of madangamine A, we searched for molecular target of the madangamine A in human cells. Treatment with madangamine A increased the levels of LC3-II and p62, autophagy-related proteins, concomitant with growth inhibition. Moreover, madangamine A resulted in lysosome enlargement and increase in lysosomal pH, which are same phenomena observed in chloroquine-treated cells. These results suggest that madangamine A is a novel lysosome inhibitor, and the anti-proliferative activity of madangamine A is due to the inhibition of lysosome function.

Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts.

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.

Seven-Step Synthesis of All-Nitrogenated Sugar Derivatives Using Sequential Overman Rearrangements.

All-nitrogenated sugars (ANSs), in which all hydroxy groups in a carbohydrate are replaced with amino groups, are anticipated to be privileged structures with useful biological activities. However, ANS synthesis has been challenging due to the difficulty in the installation of multi-amino groups. We report herein the development of a concise synthetic route to peracetylated ANSs in seven steps from commercially available monosaccharides. The key to success is the use of the sequential Overman rearrangement, which enables formal simultaneous substitution of four or five hydroxy groups in monosaccharides with amino groups. A variety of ANSs are available through the same reaction sequence starting from different initial monosaccharides by chirality transfer of secondary alcohols. Transformations of the resulting peracetylated ANSs such as glycosylation and deacetylation are also demonstrated. Biological studies reveal that ANS-modified cholesterol show cytotoxicity against human cancer cell lines, whereas each ANS and cholesterol have no cytotoxicity.

Identification of vibsanin A analog as a novel HSP90 inhibitor.

Vibsanin A is the first natural product isolated from Viburnum awabuki and has several biological activities. We have reported that a vibsanin A analog, obtained from process of total synthesis of vibsanin A, has anti-proliferative activity against human cancer cell lines. In this study, we evaluated anti-proliferative effect of the vibsanin A analogs against various human cancer cell lines and examined molecular target of the analog in human cells. Among the vibsanin A analogs, vibsanin A analog C (VAC) showed anti-proliferative effect against various cancer cell lines, and the anti-proliferative activity was strongest among the vibsanin A analogs. Additionally, VAC fluctuated amounts of HSP90-related proteins in cells and inhibited HSP90-mediated protein refolding of luciferase in vitro. These results suggest that the anti-proliferative activity of VAC is due to HSP90 inhibition, and VAC has a potential as novel anti-cancer drug as HSP90 inhibitor.

Identification and characterization of collagen-like glycosylation and hydroxylation of CCN1.

CCN1 is a secreted protein and belongs to the CCN family of matricellular proteins. CCN1 binds to various cell surface receptors; thus, CCN1 has important functions in cell proliferation, migration and angiogenesis through a variety of signaling pathways. We have reported that CCN1 is O-fucosylated and that this O-fucosylation regulates the secretion of CCN1 into the extracellular region. In this study, we detected collagen-like glycosylation and hydroxylation at Lys203 of recombinant CCN1 by mass spectrometry. We then examined the role of collagen-like glycosylation in the functions of CCN1. As a result, we found that a deficiency in collagen-like glycosylation decreased the secretion of CCN1 using wild-type CCN1- and collagen-like glycosylation-defective mutant CCN1-overexpressing cell lines. Further, knockout of lysyl hydroxylase3, a multifunctional protein with hydroxylase and glucosyltransferase activities, impaired the secretion and glycosylation level of recombinant CCN1. Previous studies reported that collagen glycosylation of Lys residues mediated by lysyl hydroxylase3 is glucosyl-galactosyl-hydroxylation, presuming that this collagen-like glycosylation detected at Lys203 of recombinant CCN1 in this study might be glucosyl-galactosyl-hydroxylation. Taken together, our results demonstrate the novel function of the collagen-like glycosylation of CCN1 and suggest that lysyl hydroxylase3-mediated glycosylation is important for CCN1 secretion.

Identification of topoisomerases as molecular targets of cytosporolide C and its analog.

Cytosporolide (Cytos) A-C, isolated from the fungus Cytospora sp., have anti-microbial activity, but their molecular targets in mammalian cells are unknown. We have previously reported the total synthesis of Cytos A by biomimetic hetero-Diels-Alder reaction. In this study, to examine the novel bioactivity of Cytos, we synthesized Cytos C and measured cell growth-inhibiting activities of 7 compounds, including Cytos A and C, in several human cancer cell lines. Among these compounds, Cytos C and tetradeoxycytosporolide A (TD-Cytos A), a model compound for the synthesis of Cytos A, had anti-proliferative effects on cancer cells, and TD-Cytos A exhibited stronger activity than Cytos C. In vitro topoisomerase-mediated DNA relaxing experiments showed that TD-Cytos A inhibited the activities of topoisomerase I and II, whereas Cytos C targeted only topoisomerase I. These data suggest that the anti-proliferative activities of Cytos correlate with the inhibition of topoisomerases and implicated TD-Cytos A as a novel anti-cancer drug that suppresses the activities of topoisomerase I and II.

Integrin ß1 is an essential factor in vasculogenic mimicry of human cancer cells.

Vasculogenic mimicry (VM) formation by cancer cells is known to play a crucial role in tumor progression, but its detailed mechanism is unclear. In the present study, we focused on integrin ß1 (ITGB1) and assessed the role of ITGB1 in VM formation. We used in vitro methods to seed cancer cells on Matrigel to evaluate the capability of VM formation. We carried out ITGB1 gene deletion using the CRISPR/Cas9 system, and these ITGB1-knockout cells did not show a VM-like network formation. Further, reintroduction of ITGB1 rescued VM-like network formation in ITGB1-knockout cells. In conclusion, ITGB1 is a critical factor in VM of human cancer cells, and inhibition of ITGB1 may be a novel therapeutic approach for malignant cancer.

Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling.

Regulation of secretion and enzymatic activity of lipoprotein lipase by C-mannosylation.

Lipoprotein lipase (LPL) is a crucial enzyme in lipid metabolism and transport, and its enzymatic deficiency causes metabolic disorders, such as hypertriglyceridemia. LPL has one predicted C-mannosylation site at Trp417. In this study, we demonstrated that LPL is C-mannosylated at Trp417 by mass spectrometry. Furthermore, by using wild-type and a C-mannosylation-defective mutant of LPL-overexpressing cell lines, we revealed that both secretion efficiency and enzymatic activity of C-mannosylation-defective mutant LPL were lower than those of wild-type. These data suggest the importance of C-mannosylation for LPL functions.

Synthesis and evaluation of biological activities of vibsanin A analogs.

Vibsanin A is an 11-membered vibsane diterpenoid and is reported to induce myeloid cell differentiation via activation of protein kinase C (PKC) without tumor-promoting activity. Therefore, vibsanin A is thought to be an attractive compound for acute myeloid leukemia (AML) therapy. In this study, we synthesized vibsanin A analogs and compared the activity of these compounds for PKC activation and myeloid cell differentiation. We found that the hydroxymethyl group in vibsanin A is an important substituent to induce differentiation of AML cells. Collectively, our results showed the biochemical features of vibsanin A and provided new insights into the development of new antileukemic drugs.

The Mammalian Sterile 20-like 1 Kinase Controls Selective CCR7-Dependent Functions in Human Dendritic Cells.

The chemokine receptor CCR7 directs mature dendritic cells (mDCs) to the lymph nodes where these cells control the initiation of the immune response. CCR7 regulates chemotaxis, endocytosis, survival, migratory speed, and cytoarchitecture in mDCs. The molecular mechanisms used by CCR7 to regulate these functions in mDCs are not completely understood. The mammalian sterile 20-like 1 kinase (Mst1) plays a proapoptotic role under stress conditions; however, recently, it has been shown that Mst1 can also control homeostatic cell functions under normal conditions. In this study, we show that stimulation of CCR7 in mDCs induces Gαi-dependent activation of Mst1, suggesting the involvement of this kinase in the control of CCR7-dependent functions. Analysis of the mDCs in which Mst1 expression levels were reduced with small interfering RNA shows that this kinase mediates CCR7-dependent effects on cytoarchitecture, endocytosis and migratory speed but not on chemotaxis or survival. In line with these results, biochemical analysis indicates that Mst1 does not control key signaling regulators of CCR7-dependent chemotaxis or survival. In contrast, Mst1 regulates downstream of CCR7 and, of note, independently of Gα13, the RhoA pathway. Reduction of Mst1 inhibits CCR7-dependent phosphorylation of downstream targets of RhoA, including cofilin, myosin L chain, and myosin L chain phosphatase. Consistent with the role of the latter molecules as modulators of the actin cytoskeleton, mDCs with reduced Mst1 also displayed a dramatic reduction in actin barbed-end formation that could not be recovered by stimulating CCR7. The results indicate that the kinase Mst1 controls selective CCR7-dependent functions in human mDCs.

Conophylline protects cells in cellular models of neurodegenerative diseases by inducing mammalian target of rapamycin (mTOR)-independent autophagy.

Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD.