Reduction of HIF-1α/PD-L1 by Catalytic Topoisomerase Inhibitor Induces Cell Death Through Caspase Activation in Cancer Cells Under Hypoxia.

BACKGROUND/AIM: Under severe hypoxia, cellular apoptosis is induced through hypoxia-inducible factor 1, alpha subunit (HIF-1α)-dependent P53 accumulation and P53 phosphorylation via ataxia telangiectasia mutated and ataxia telangiectasia and RAD3-related (ATR) activation via replication stress-induced DNA damage response (DDR) activation. We previously demonstrated that the topoisomerase I catalytic inhibitor, 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (3EZ,20Ac-ingenol), induced apoptosis in Jeko-1 and Panc-1 cells, both of which show cyclin D1 overexpression. After progression to the S phase facilitated by nuclear cyclin D1, an intra S phase checkpoint was induced in the presence of 3EZ,20Ac-ingenol, by ATR activation in response to replication stress-induced DDR. MATERIALS AND METHODS: In this study, we examined whether 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (3EE,20Ac-ingenol) might induce a higher degree of P53 phosphorylation and additional HIF-1α and P53 accumulation in response to replication stress-induced DDR activation under hypoxic conditions than under normoxic conditions, by controlling ATR activation. RESULTS: In the Panc-1 cells, 3EE,20Ac-ingenol induced P53 activation and HIF-1α-dependent P53 accumulation through cooperative ATR activation via hypoxia-induced DDR activation. Jeko-1 cells showed slight HIF-1α accumulation under hypoxia, but HIF-1α-dependent 53 accumulation was not observed in the presence of 3EE,20Ac-ingenol, so that the cells remained resistant to hypoxia. CONCLUSION: 3EE,20Ac-ingenol induces an intricate interplay between P53 and HIF-1α accumulation via ATR activation that results in a high P53 accumulation, which promoted transient expression and early disappearance of HIF-1α, accelerating cell death. Strong P53 accumulation and consequent phosphatase and tensin homolog deleted on chromosome 10 activation in Panc-1 cells also reduced HIF-1α accumulation and programmed death-ligand 1 expression, which resulted in intense apoptosis.

Ingenol mebutate inhibits the growth of pancreatic cancer cells in vitro via STING with an efficacy comparable to that of clinically used anticancer agents.

Pancreatic cancer is associated with a poor prognosis; thus, there is an urgent need to develop new and effective treatments. Ingenol mebutate (IM), which is isolated from the latex of Euphorbia peplus, was recently shown to be effective against pancreatic cancer cell lines; however, its mechanism of action has not been fully elucidated. In this study, we focused on the less drug-sensitive pancreatic cancer cell line Panc-1 and compared IM to commercially available anticancer drugs using cell survival assays. In addition, we aimed to identify novel biomolecules that may be involved in the mechanism of action of IM using RNA sequencing, western blotting, and inhibition assays. The IC50 values after 72 h of exposure to IM and SN-38, drugs to which the Panc-1 cells are most sensitive among the tested anticancer agents, were 43.1 ± 16.8 nM and 165 ± 37 nM, respectively. IM showed a cytostatic effect equal to or greater than that of the clinically used pancreatic cancer therapeutic drugs. RNA sequencing and protein expression analysis revealed that expression of stimulator of interferon genes (STING) increased at low IM concentration, whereas cell viability decreased. Co-exposure of IM and STING inhibitor, H-151, to Panc-1 or MIA PaCa-2 cell lines canceled the growth-inhibitory effects of IM alone. In conclusion, IM may have an efficacy comparable to that of existing pancreatic cancer therapeutic agents on the less drug-sensitive Panc-1 cell line and the immune-related molecule STING plays a role in the mechanism of action of IM.

3EZ, 20Ac-ingenol-induced Apoptosis in Chemoresistant Cancers With Cyclin D1 Accumulation.

BACKGROUND/AIM: The topoisomerase 1 catalytic inhibitor 3EZ, 20Ac-ingenol specifically induces apoptosis through the activation of ATR and the up-regulation of PTEN by enhancing the DNA damage response (DDR) in human B lymphoma (BALL-1) cells. The accumulation of cyclin D1 in cancer is known to be related to chemoresistance to DNA damage agents and nuclei of BALL-1 cells exhibit high levels of cyclin D1. However, 3EZ, 20Ac-ingenol effectively induced apoptosis of BALL-1 cells. MATERIALS AND METHODS: Cell growth, protein levels, and apoptosis were determined by an MTT assay, immunoblotting and DNA fragmentation assay, respectively. RESULTS: 3EZ, 20Ac-ingenol strongly induced inhibition of cell proliferation and apoptosis in Jeko-1 and Panc-1 cell lines through the activation of tumor suppressor proteins and caspase 3. CONCLUSION: 3EZ, 20Ac-ingenol-induced apoptosis might occur in cells with cyclin D1 accumulation through enhancing DDR, regardless of the cancer cell type.

Identification of Two Phenanthrene Derivatives from Australasian Allied Species in Genus Dendrobium.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.

Identification of Lycopodium Alkaloids Produced by an Ultraviolet-Irradiated Strain of Paraboeremia, an Endophytic Fungus from Lycopodium serratum var. longipetiolatum.

12- epi-Lycopodine (1), a Lycopodium alkaloid, along with lycopodine (2) and huperzine A (3), were discovered in the mycelium of Paraboeremia sp. Lsl3KI076, a UV-irradiated strain of Paraboeremia sp. Lsl3, an endophytic fungus from Lycopodium serratum Thunb. var. longipetiolatum Spring. Additionally, a trace of 1 was isolated from Phlegmariurus nummulariifolius (Blume) Ching, and the structure was elucidated on the basis of spectroscopic data. This is the first report proving that a new naturally occurring Lycopodium alkaloid can be obtained from an endophytic fungus.

3EZ, 20Ac-ingenol induces cell-specific apoptosis in cyclin D1 over-expression through the activation of ATR and downregulation of p-Akt.

Acute lymphoblastic leukemia (ALL) samples exhibit an activated PI3K/Akt pathway, which suggests a general role of Akt in the development of leukemia. We have previously used western blot analysis to show that the catalytic topoisomerase (topo) inhibitor, 3EZ, 20Ac-ingenol, induced DNA damage response (DDR), which activated ATR, downregulated p-Akt through upregulation of PTEN level, and led to cell cycle arrest or apoptosis. In this study, we used ATR or PTEN siRNA and observed that the specific cell arrest and apoptosis of BALL-1 cells in DDR caused by 3EZ, 20Ac-ingenol was dependant on activation of ATR and downregulation of nuclear p-Akt through upregulation of PTEN. Moreover, some B cell lymphomas among ALLs overexpress cyclin D1. The DDR induced during the S-phase with 3EZ, 20Ac-ingenol treatment was increased by the intra S-phase checkpoint response that was triggered by the loss of nuclear cyclin D1 regulation in BALL-1 cells overexpressing cyclin D1. Although topo 1 catalytic inhibitors induce a decatenation checkpoint and subsequent G2/M phase arrest, the decatenation checkpoint caused by 3EZ, 20Ac-ingenol induced apoptosis only in the BALL-1 cells that accumulated cyclin D1.

Stereospecific inhibition of nitric oxide production in macrophage cells by flavanonols: Synthesis and the structure-activity relationship. Part 2.

To explore the structure-activity relationships of flavanonols on the inhibition of nitric oxide (NO) production in RAW 264.7 cells, we have prepared a series of synthetic flavanonols. In our previous study, the 2',3'-dihydroxyphenyl substructure was found to be the most potent B ring substructure among the flavanonols having 3,5,7-trihydroxychroman-4-one as the A/C ring. In this study, we examined the effect of diverse substitutions on the A ring of the 2-(2,3-dihydroxyphenyl)-3-hydroxychroman-4-one scaffold, i.e., by fixing the B ring to the 2',3'-dihydroxyphenyl substructure. Eighteen stereoisomers and 4 racemic mixtures were prepared, and their inhibitory potency on NO production in RAW 264.7 cells was tested. We observed higher inhibitory activity in the (2R,3R) stereoisomers than in the (2S,3S) stereoisomers. The presence of a hydroxy or a methoxy group at the 7-postiion enhanced the inhibitory potency, and the additional substitutions at the 6- or 8-position in the A ring increased potency and stereospecificity. A representative compound, (2R,3R)-2',3',7,8-tetrahydroxyflavanonol 5e, had an IC50 value of 17µM, whereas its (2S,3S) stereoisomer did not inhibit NO production at all at a concentration of 100µM. In this study, it was necessary to determine the absolute configuration of the stereoisomers of the synthesized flavanonols that carry methoxy substitutions in the A ring. The procedure to determine their absolute configuration by the CD excitation chirality method is also discussed.

Structure-activity relationship of the inhibitory effects of flavonoids on nitric oxide production in RAW264.7 cells.

We isolated flavonoids from herbal specimens from the Tibetan region (Sophora yunnanensis and Rhodiola sacra) that suppress nitric oxide (NO) production in macrophages stimulated by lipopolysaccharide and interferon-γ. The isolated flavonoids carry symmetric substitutions in the B ring (R3'=R5'). We analyzed the quantitative structure-activity relationship of the inhibitory activity by comparative molecular field analysis (CoMFA) using this series of flavonoids. Use of flavonoids with symmetrical substitutions in the B ring made it simpler to align molecules because it was not necessary to consider a huge number of combinations due to the B-ring conformation. The CoMFA model, whose cross-validated q2 value was 0.705, suggested the existence of a hydroxy group at the 5-position, the choice of the A/C-ring scaffold (chromane or chromene) and electrostatic field around the B ring are important for NO inhibitory activity. Flavonoids synthesized based on the CoMFA model exhibited significant inhibitory potential against NO production, validating the predictive capability of the CoMFA model.

Chemical Compounds in Natural Medicines That Affect Macropharges and Adipocyte Cells.

Macrophages play major roles in inflammation, immunity and host defense mechanisms. Once activated they produce and release cytokines, oxygen and nitrogen species, and eicosanoids. The best characterized stimuli to induce the transcription of genes encoding pro-inflammatory proteins in macrophages in vitro is bacterial lipopolysaccharide (LPS). LPS could be used alone or in combination with recombinant mouse interferon-γ (IFN-γ). Such stimulation results in cytokine release and the synthesis of enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The nitric oxide (NO) radical is known to play a central role in inflammatory and immune reactions for self-protection. However, the excessive production of NO may lead to tissue damage. In inflammatory diseases such as rheumatoid arthritis, excessive NO production by activated macrophages has been observed. Adipose tissue is composed of various cell types such as mature adipocytes, preadipocytes, fibroblasts, endothelial cells, vascular cells, and macrophages. Recent studies indicate that obesity is associated with low-grade chronic inflammation of adipose tissues, and that such inflammation is one of the potential mechanisms leading to the insulin resistance. It has been demonstrated that obese adipose tissue is characterized by the increased infiltration of macrophages. Therefore, we attempted to identify natural anti-inflammatory compounds that not only inhibit the secretion of NO from RAW 264.7 cells, but also inhibit triglyceride accumulation in 3T3-L1 adipocytes. This review describes the NO prpduction inhibitory activity or the TG accumulation inhibitory activity of the compounds obtained from 18 plants and a fungi that have been used as traditional medicines.

Metabolome Analysis of Oryza sativa (Rice) Using Liquid Chromatography-Mass Spectrometry for Characterizing Organ Specificity of Flavonoids with Anti-inflammatory and Anti-oxidant Activity.

Oryza sativa L. (rice) is an important staple crop across the world. In the previous study, we identified 36 specialized (secondary) metabolites including 28 flavonoids. In the present study, a metabolome analysis using liquid chromatography-mass spectrometry was conducted on the leaf, bran, and brown and polished rice grains to better understand the distribution of these metabolites. Principal component analysis using the metabolome data clearly characterized the accumulation patterns of the metabolites. Flavonoids, e.g., tricin, tricin 7-O-rutinoside, and tricin 7-O-ß-D-glucopyranoside, were mainly present in the leaf and bran but not in the polished grain. In addition, anti-inflammatory and anti-oxidant activity of the metabolites were assayed in vitro. Tricin 4'-O-(erythro-ß-guaiacylglyceryl)ether and isoscoparin 2″-O-(6‴-(E)-feruloyl)-glucopyranoside showed the strongest activity for inhibiting nitric oxide (NO) production and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging, respectively.

Serralongamines B-D, three new Lycopodium alkaloids from Lycopodium serratum var. longipetiolatum, and their inhibitory effects on foam cell formation in macrophages.

Three new Lycopodium alkaloids, serralongamines B-D (1-3), have been isolated from the club moss Lycopodium serratum var. longipetiolatum, and the structures were elucidated on the basis of spectroscopic data and chemical transformation. 1 and 3 significantly exhibited the inhibitory activity against foam cell formation in human macrophages, one of characteristic features of early atherosclerotic lesions.

Stereospecific inhibition of nitric oxide production in macrophage cells by flavanonols: Synthesis and the structure-activity relationship.

To explore the structure-activity relationships on the inhibitory activity of flavanonols against nitric oxide (NO) production in inflammatory cells, we synthesized 19 flavanonols which shared a common 3,5,7-trihydroxychroman scaffold. A range of substitutions was included in the B ring in order to investigate the structure-activity relationship. We also succeeded in isolating stereoisomers from 16 of the flavanonols using chiral column chromatography. The inhibitory effects of these compounds on NO production were examined in RAW 264.7 cells (a murine macrophage-like cell line), which were activated by lipopolysaccharide (LPS). We only observed inhibitory activity against NO production in (2R,3R) stereoisomers, while the inhibitory activities of (2S,3S) stereoisomers were significantly weaker. We also evaluated the free radical scavenging potential of the flavanonols using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Each stereoisomer indicated the equivalent DPPH scavenging potential as expected. The radical scavenging activity was not correlated with the inhibitory activity against NO. The inhibition of NO production by flavanonols is stereospecific and cannot simply be explained by their radical scavenging activity. We propose the possible existence of a 'target' molecule for flavanonols which is involved in the production and/or regulation of NO in RAW 264.7 cells.

Mechanism of the inhibition of leukemia cell growth and induction of apoptosis through the activation of ATR and PTEN by the topoisomerase inhibitor 3EZ, 20Ac-ingenol.

The PI3K/Akt signaling pathway is constitutively activated in various leukemias. In the present study, the topoisomerase inhibitor, 3EZ, 20Ac-ingenol, was more effective in inhibiting the growth of BALL-1 cells than that of normal lymphocyte cells. ATM/ATR protein levels were increased, PTEN protein was upregulated, and p-Akt protein was downregulated at early time points after treatment with 3EZ, 20Ac-ingenol. In further experiments, p53 protein expression was increased, and H2AX phosphorylation and p21 protein expression were induced after treatment with 3EZ, 20Ac-ingenol. Moreover, the activation of caspase 3 followed decrease in the Bcl-2/Bax ratio after treatment with 3EZ, 20Ac-ingenol, and accumulation of sub-G1 phase cells was observed in flow cytometry analyses. These data suggest that 3EZ, 20Ac-ingenol-induced DNA damage downregulates p-Akt and upregulates ATR leading to cell cycle arrest and increased apoptosis in BALL-1 cells.

Chemical constituents isolated from Juncus effusus induce cytotoxicity in HT22 cells.

Effususol A (1), a new 9,10-dihydrophenanthrene, has been isolated from the medullae of Juncus effusus along with ten known compounds, effusol (2), dehydroeffusol (3), juncusol (4), dehydrojuncusol (5), juncuenin B (6), dehydrojuncuenin B (7), juncuenin D (8), luteolin (9), luteolin 5-methyl ether (10), and 4-hydroxy-2,3-dimethyl-2-nonen-4-olide (11). The structure of 1 was elucidated on the basis of spectroscopic data. 2, 4, 6, 7, and 8 have induced caspase-3-mediated cytotoxicity in HT22 cells.

[Isoflavonoids from Caragana changduensis and their nitric oxideinhibitory activities].

Ten isoflavonoids were isolated from the heartwoods of Caragana changduensis Lion f. by means of various column chromatographic techniques. Based on the detailed spectral data analysis (MS and NMR), as well as comparison with the literatures, their chemical structures were determined as 7,2'-dihydroxy-8,4'-dimethoxyisoflavone (1), 4'-hydroxy-7,3'-dimethoxyisoflavone (2), 5, 7, 4'-trihydroxy-2',5'-dimethoxyisoflavone (3), prunetin (4), afrormosin (5), odoratin (6), genistein (7), texasin (8), pratensein (9), and 6,7,3'-trihydroxy-4'-methoxyisoflavone (10). Among them, compounds 1-3 and 9-10 were isolated from the Caragana genus for the first time. All the compounds were obtained from this species for the first time. In the preliminary assays, compounds 1, 2, 6, and 7 possessed significant inhibitory effects on NO production, with IC50 values of 48.12, 25.32, 62.71, 43.59 µmol x L(-1), respectively.

Molecular phylogenetics and character evolution of morphologically diverse groups, Dendrobium section Dendrobium and allies.

It is always difficult to construct coherent classification systems for plant lineages having diverse morphological characters. The genus Dendrobium, one of the largest genera in the Orchidaceae, includes ∼1100 species, and enormous morphological diversification has hindered the establishment of consistent classification systems covering all major groups of this genus. Given the particular importance of species in Dendrobium section Dendrobium and allied groups as floriculture and crude drug genetic resources, there is an urgent need to establish a stable classification system. To clarify phylogenetic relationships in Dendrobium section Dendrobium and allied groups, we analysed the macromolecular characters of the group. Phylogenetic analyses of 210 taxa of Dendrobium were conducted on DNA sequences of internal transcribed spacer (ITS) regions of 18S-26S nuclear ribosomal DNA and the maturase-coding gene (matK) located in an intron of the plastid gene trnK using maximum parsimony and Bayesian methods. The parsimony and Bayesian analyses revealed 13 distinct clades in the group comprising section Dendrobium and its allied groups. Results also showed paraphyly or polyphyly of sections Amblyanthus, Aporum, Breviflores, Calcarifera, Crumenata, Dendrobium, Densiflora, Distichophyllae, Dolichocentrum, Holochrysa, Oxyglossum and Pedilonum. On the other hand, the monophyly of section Stachyobium was well supported. It was found that many of the morphological characters that have been believed to reflect phylogenetic relationships are, in fact, the result of convergence. As such, many of the sections that have been recognized up to this point were found to not be monophyletic, so recircumscription of sections is required.

Toward better annotation in plant metabolomics: isolation and structure elucidation of 36 specialized metabolites from Oryza sativa (rice) by using MS/MS and NMR analyses.

Metabolomics plays an important role in phytochemical genomics and crop breeding; however, metabolite annotation is a significant bottleneck in metabolomic studies. In particular, in liquid chromatography-mass spectrometry (MS)-based metabolomics, which has become a routine technology for the profiling of plant-specialized metabolites, a substantial number of metabolites detected as MS peaks are still not assigned properly to a single metabolite. Oryza sativa (rice) is one of the most important staple crops in the world. In the present study, we isolated and elucidated the structures of specialized metabolites from rice by using MS/MS and NMR. Thirty-six compounds, including five new flavonoids and eight rare flavonolignan isomers, were isolated from the rice leaves. The MS/MS spectral data of the isolated compounds, with a detailed interpretation of MS fragmentation data, will facilitate metabolite annotation of the related phytochemicals by enriching the public mass spectral data depositories, including the plant-specific MS/MS-based database, ReSpect.

Inhibitory effect of chemical constituents from Artemisia scoparia Waldst. et Kit. on triglyceride accumulation in 3T3-L1 cells and nitric oxide production in RAW 264.7 cells.

We investigated the anti-obesity effect of the aerial part of Artemisia scoparia Waldst. et Kit. (Compositae). An 80 % aqueous EtOH extract of the aerial part inhibited triglyceride (TG) accumulation and the nitric oxide (NO) production activity. A new chromane derivative was isolated from the aerial part of A. scoparia Waldst. et Kit. along with 18 known compounds. The structure of the new chromane, scopariachromane (1), was elucidated by spectroscopic analyses. The inhibitory effects of the compounds on TG accumulation activity were examined. Among these, cirsiliol (11) inhibited TG accumulation in 3T3-L1 preadipocytes. Jaceosidin (12) inhibited NO production in a murine macrophage-like cell line (RAW 264.7). These results indicate that the 80 % aqueous EtOH extract and compounds isolated from the aerial part of A. scoparia Waldst. et Kit. may improve obesity-related insulin resistance.

Nortriterpene Saponins from Akebia trifoliate.

Three new nortriterpene saponins, 2α,3ß,20α-trihydroxy-30-norolean-12-en-28-oic acid O-ß-D-xylopyranosyl-(1-->3)-α-L-rhamnopyranosyl-(1-->4)-ß-D- glucopyranosyl-(1-->6)-ß-D-glucopyranosyl ester (3), 2α,3ß,20α-trihydroxy-30-norolean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1-->4)-ß-D- glucopyranosyl-(1-->6)-ß-D-glucopyranosyl ester (4), and 2α,3ß,23-trihydroxy-30-noroleana-12,19-dien-28-oic acid O-ß-D-xylopyranosyl-(1-->3)-α-L- rhamnopyranosyl-(1-->4)-ß-D-glucopyranosyl-(1-->6)-ß-D-glucopyranosyl ester (6), were isolated from the methanol extract of the pericarps of Akebia trifoliata Koidzumi (Lardizabalaceae), together with five known nortriterpene saponins and three phenolic glycosides. Their structures were elucidated by spectroscopic analyses and acid hydrolysis.

Sesquiterpene coumarins from Ferula fukanensis and their pro-inflammatory cytokine gene expression inhibitory effects.

Six new sesquiterpene coumarin derivatives, fukanefuromarin H-M (1-6), were isolated from an 80% aqueous methanol extract of the roots of Ferula fukanensis. The structures were elucidated on the basis of spectroscopic evidence, particularly heteronuclear multiple-bond connectivity (HMBC) and high-resolution MS. The sesquiterpene coumarin derivatives inhibited nitric oxide (NO) and inducible NO synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) gene expression in a murine macrophage-like cell line (RAW264.7), which was activated by lipopolysaccharide (LPS) and recombinant mouse interferon-γ (IFN-γ).