Natural Compounds with BMI1 Promoter Inhibitory Activity from Mammea siamensis and Andrographis paniculata.

A new coumarin derivative (1) and 30 known compounds were isolated from Mammea siamensis and Andrographis paniculata, guided by B cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) promoter inhibitory activity. Among the isolated compounds, 15 compounds showed BMI1 promoter inhibitory activity, and five compounds were found to be cytotoxic. 14-Deoxy-11,12-dehydroandrographolide (18) was highly cytotoxic to DU145 cells with an IC50 value of 25.4 µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

Isolation of Various Flavonoids with TRAIL Resistance-Overcoming Activity from Blumea lacera.

Eighteen compounds, including fourteen flavonoids (1-14), one steroid (15), two fatty acids (16,17), and one nitrogen-containing compound (18), were isolated from the methanol extract of the whole Blumea lacera plant collected in Thailand. Compounds 1-11 and 15-17 exhibited tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance-overcoming activity. Among them, bonanzin (2) and cirsilineol (7) had particularly strong TRAIL resistance-overcoming activity, where the IC50 values against the human gastric adenocarcinoma cell line AGS in the presence of TRAIL (100 ng/mL) were 10.7 µM and 5.9 µM, respectively.

Evaluation of Naturally Occurring HIF-1 Inhibitors for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia-inducible factor (HIF-1). We constructed a cell-based luciferase assay system for HIF-1 inhibitors. Using this system, six HIF-1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C (1) on PASMC was investigated. Tagitinin C (1) clearly decreased the amount of HIF-1ß and the HIF-1 target PDK1. This result indicates that HIF-1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF-1 inhibitors could provide novel insights into the development of PAH medications.

Identification of BMI1 promoter inhibitors from Streptomyces sp. IFM-11958.

B-cell-specific Moloney murine leukemia virus region 1 (BMI1) is a central component of polycomb repressive complex 1 (PRC1), which maintains epigenetic repression of genes expression via chromatin condensation. BMI1 overexpression downregulates the expression of tumor suppressor genes, such as p16Ink4a and PTEN. BMI1 expression is upregulated in cancer stem cells (CSCs). Therefore, inhibitors of BMI1 expression have potential as therapeutic agents for cancer. This study aimed to identify BMI1 promoter inhibitors from actinomycetes. Using a recently constructed BMI1 promoter assay, we isolated three known compounds, elaiophylin (1), 2-methylelaiophylin (2), and nocardamin (3), from Streptomyces sp. IFM-11958 that inhibited BMI1 promoter activity with IC50 values of 30 nM, 447 nM, 22 µM, respectively. Elaiophylin (1) was the most potent. Western blot and PCR analyses revealed that elaiophylin (1) inhibited BMI1 expression at the mRNA level in human prostate cancer cells (DU145). Elaiophylin (1) also inhibited the sphere-forming activity of human hepatocellular carcinoma cells (Huh7), indicating that elaiophylin (1) suppresses the self-renewal capacity of CSCs. Elaiophylin (1) is the first BMI1 promoter inhibitor isolated from actinomycete metabolites.

Isolation of Nabscessin C from Nocardia abscessus IFM 10029T and a Study on Biosynthetic Pathway for Nabscessins.

A new aminocyclitol derivative, designated nabscessin C (1), was isolated from Nocardia abscessus IFM 10029T. Nabcessin C is an isomer of nabscessins A (2) and B (3) with different positioning of the acyl group. Absolute configuration of nabscessin A was determined by conversion into the 2-deoxy-scyllo-inosamine pentaacetyl derivative (4) by hydrolysis and acetylation of 2. The biosynthetic pathway of nabscessins is proposed based on gene expression analysis.

Sporadic PCDH18 somatic mutations in EpCAM-positive hepatocellular carcinoma.

BACKGROUND: The relationship between specific genome alterations and hepatocellular carcinoma (HCC) cancer stem cells (CSCs) remains unclear. In this study, we evaluated the relationship between somatic mutations and epithelial cell adhesion molecule positive (EpCAM+) CSCs. METHODS: Two patient-derived HCC samples (HCC1 and HCC2) were sorted by EpCAM expression and analyzed by whole exome sequence. We measured PCDH18 expression level in eight HCC cell lines as well as HCC1 and HCC2 by real-time quantitative RT-PCR. We validated the identified gene mutations in 57 paired of HCC and matched non-cancerous liver tissues by Sanger sequence. RESULTS: Whole exome sequencing on the sorted EpCAM+ and EpCAM- HCC1 and HCC2 cells revealed 19,263 nonsynonymous mutations in the cording region. We selected mutations that potentially impair the function of the encoded protein. Ultimately, 60 mutations including 13 novel nonsense and frameshift mutations were identified. Among them, PCDH18 mutation was more frequently detected in sorted EpCAM+ cells than in EpCAM- cells in HCC1 by whole exome sequences. However, we could not confirm the difference of PCDH18 mutation frequency between sorted EpCAM+ and EpCAM- cells by Sanger sequencing, indicating that PCDH18 mutation could not explain intracellular heterogeneity. In contrast, we found novel PCDH18 mutations, including c.2556_2557delTG, c.1474C>G, c.2337A>G, and c.2976G>T, were detected in HCC1 and 3/57 (5.3%) additional HCC surgical specimens. All four HCCs with PCDH18 mutations were EpCAM-positive, suggesting that PCDH18 somatic mutations might explain the intertumor heterogeneity of HCCs in terms of the expression status of EpCAM. Furthermore, EpCAM-positive cell lines (Huh1, Huh7, HepG2, and Hep3B) had lower PCDH18 expression than EpCAM-negative cell lines (PLC/PRL/5, HLE, HLF, and SK-Hep-1), and PCDH18 knockdown in HCC2 cells slightly enhanced cell proliferation. CONCLUSIONS: Our data suggest that PCDH18 is functionally suppressed in a subset of EpCAM-positive HCCs through somatic mutations, and may play a role in the development of EpCAM-positive HCCs.

Serum cytokine profiles predict survival benefits in patients with advanced hepatocellular carcinoma treated with sorafenib: a retrospective cohort study.

BACKGROUND: Sorafenib is a multiple receptor tyrosine kinase inhibitor known to prolong overall survival in patients with advanced hepatocellular carcinoma (HCC). Predicting this drug's survival benefits is challenging because clinical responses are rarely measurable during treatment. In this study, we hypothesized that serum cytokines levels could predict the survival of advanced HCC patients, as sorafenib targets signaling pathways activated in the tumor stromal microenvironment and potentially affects serum cytokine profiles. METHODS: Of 143 patients with advanced-stage HCC, 104 who were recruited between 2003 and 2007 received hepatic arterial infusion chemotherapy (HAIC) that mainly targets tumor epithelial cells at S-phase (cohort 1); additionally, 39 recruited between 2010 and 2012 received sorafenib, which primarily targets the stromal vascular endothelial cells. Serum samples were collected and aliquoted prior to the treatment. Serum EGF, bFGF, HGF, IFN-γ, IL-10, IL-12, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10, MIG, PDGF-BB, SCF, SDF1, TGF-ß, TGF-α, TNF-α, and VEGF-A were measured via enzyme-linked immunosorbent assays. The Modified Response Evaluation Criteria in Solid Tumors were used to assess tumor responses. RESULTS: The median survival time of HCC patients in cohorts 1 (HAIC-treated) and 2 (sorafenib-treated) were 12.0 and 12.4 months, respectively. Kaplan-Meier analysis revealed no significant survival differences between the 2 groups. Patients who survived more than 2 years after sorafenib treatment exhibited higher serum levels of IL-10, IL-12, TNF-a, IL-8, SDF-1, EGF, PDGF-BB, SCF, and TGF-α. Furthermore, cohort 2 patients with higher serum IL-5 (>12 pg/mL), IL-8 (>10 pg/mL), PDGF-BB (>300 pg/mL), and VEGF-A (>50 pg/mL) levels achieved longer survival; cohort 1 patients did not. Hierarchical cluster analysis of 6 cytokines robustly enriched for comparison analysis between cohorts 1 and 2 (IL-5, IL-8, TGF-α, PDGF-BB, CXCL9, and VEGF-A) revealed that elevation of these cytokines correlated with better survival when treated with sorafenib but not with HAIC. CONCLUSIONS: Patients who exhibited survival benefits owing to sorafenib treatment tended to present higher serum cytokines levels, potentially reflecting the activation of stromal signaling in the tumor microenvironment. Our study thus introduces novel biomarkers that may identify advanced HCC patients who may experience survival benefits with sorafenib treatment.

Nabscessins A and B, Aminocyclitol Derivatives from Nocardia abscessus IFM 10029T.

Two new aminocyclitol amide derivatives, nabscessins A (1) and B (2), were isolated from the culture broth of a pathogenic actinomycete species, Nocardia abscessus IFM 10029T. The structures of nabscessins A and B were elucidated by spectral studies, and the compounds showed antifungal activity against Cryptococcus neoformans, with IC50 values of 32 and 16 µg/mL, respectively.

Defeating EpCAM(+) liver cancer stem cells by targeting chromatin remodeling enzyme CHD4 in human hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma is composed of a subset of cells with enhanced tumorigenicity and chemoresistance that are called cancer stem (or stem-like) cells. We explored the role of chromodomain-helicase-DNA-binding protein 4, which is encoded by the CHD4 gene and is known to epigenetically control gene regulation and DNA damage responses in EpCAM(+) liver cancer stem cells. METHODS: Gene and protein expression profiles were determined by microarray and immunohistochemistry in 245 and 144 hepatocellular carcinoma patients, respectively. The relationship between gene/protein expression and prognosis was examined. The functional role of CHD4 was evaluated in primary hepatocellular carcinoma cells and in cell lines in vitro and in vivo. RESULTS: CHD4 was abundantly expressed in EpCAM(+) hepatocellular carcinoma with expression of hepatic stem cell markers and poor prognosis in two independent cohorts. In cell lines, CHD4 knockdown increased chemosensitivity and CHD4 overexpression induced epirubicin chemoresistance. To inhibit the functions of CHD4 that are mediated through histone deacetylase and poly (ADP-ribose) polymerase, we evaluated the effect of the histone deacetylase inhibitor suberohydroxamic acid and the poly (ADP-ribose) polymerase inhibitor AG-014699. Treatment with either suberohydroxamic acid or AG-014699 reduced the number of EpCAM(+) liver cancer stem cells in vitro, and suberohydroxamic acid and AG-014699 in combination successfully inhibited tumor growth in a mouse xenograft model. CONCLUSIONS: CHD4 plays a pivotal role in chemoresistance and the maintenance of stemness in liver cancer stem cells and is therefore a good target for the eradication of hepatocellular carcinoma.

The transcription factor SALL4 regulates stemness of EpCAM-positive hepatocellular carcinoma.

BACKGROUND & AIMS: Recent evidence suggests that hepatocellular carcinoma can be classified into certain molecular subtypes with distinct prognoses based on the stem/maturational status of the tumor. We investigated the transcription program deregulated in hepatocellular carcinomas with stem cell features. METHODS: Gene and protein expression profiles were obtained from 238 (analyzed by microarray), 144 (analyzed by immunohistochemistry), and 61 (analyzed by qRT-PCR) hepatocellular carcinoma cases. Activation/suppression of an identified transcription factor was used to evaluate its role in cell lines. The relationship of the transcription factor and prognosis was statistically examined. RESULTS: The transcription factor SALL4, known to regulate stemness in embryonic and hematopoietic stem cells, was found to be activated in a hepatocellular carcinoma subtype with stem cell features. SALL4-positive hepatocellular carcinoma patients were associated with high values of serum alpha fetoprotein, high frequency of hepatitis B virus infection, and poor prognosis after surgery compared with SALL4-negative patients. Activation of SALL4 enhanced spheroid formation and invasion capacities, key characteristics of cancer stem cells, and up-regulated the hepatic stem cell markers KRT19, EPCAM, and CD44 in cell lines. Knockdown of SALL4 resulted in the down-regulation of these stem cell markers, together with attenuation of the invasion capacity. The SALL4 expression status was associated with histone deacetylase activity in cell lines, and the histone deacetylase inhibitor successfully suppressed proliferation of SALL4-positive hepatocellular carcinoma cells. CONCLUSIONS: SALL4 is a valuable biomarker and therapeutic target for the diagnosis and treatment of hepatocellular carcinoma with stem cell features.

Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma.

UNLABELLED: Recent evidence suggests that hepatocellular carcinoma (HCC) is organized by a subset of cells with stem cell features (cancer stem cells; CSCs). CSCs are considered a pivotal target for the eradication of cancer, and liver CSCs have been identified by the use of various stem cell markers. However, little information is known about the expression patterns and characteristics of marker-positive CSCs, hampering the development of personalized CSC-targeted therapy. Here, we show that CSC markers EpCAM and CD90 are independently expressed in liver cancer. In primary HCC, EpCAM+ and CD90+ cells resided distinctively, and gene-expression analysis of sorted cells suggested that EpCAM+ cells had features of epithelial cells, whereas CD90+ cells had those of vascular endothelial cells. Clinicopathological analysis indicated that the presence of EpCAM+ cells was associated with poorly differentiated morphology and high serum alpha-fetoprotein (AFP), whereas the presence of CD90+ cells was associated with a high incidence of distant organ metastasis. Serial xenotransplantation of EpCAM+ /CD90+ cells from primary HCCs in immune-deficient mice revealed rapid growth of EpCAM+ cells in the subcutaneous lesion and a highly metastatic capacity of CD90+ cells in the lung. In cell lines, CD90+ cells showed abundant expression of c-Kit and in vitro chemosensitivity to imatinib mesylate. Furthermore, CD90+ cells enhanced the motility of EpCAM+ cells when cocultured in vitro through the activation of transforming growth factor beta (TGF-ß) signaling, whereas imatinib mesylate suppressed TGFB1 expression in CD90+ cells as well as CD90+ cell-induced motility of EpCAM+ cells. CONCLUSION: Our data suggest the discrete nature and potential interaction of EpCAM+ and CD90+ CSCs with specific gene-expression patterns and chemosensitivity to molecular targeted therapy. The presence of distinct CSCs may determine the clinical outcome of HCC.

New biflavonoids isolated from Xylia kerrii leaves extract with selective cytotoxicity against MH7A human rheumatoid arthritis synovial fibroblast cells.

Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9 µM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100 µM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.

Physalin H, physalin B, and isophysalin B suppress the quorum-sensing function of Staphylococcus aureus by binding to AgrA.

The virulence of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), depends on the expression of toxins and virulence factors controlled by the quorum-sensing (QS) system, encoded on the virulence accessory gene regulator (agr) locus. The aim of this study was to identify a phytochemical that inhibits Agr-QS function and to elucidate its mechanism. We screened 577 compounds and identified physalin H, physalin B, and isophysalin B--phytochemicals belonging to physalins found in plants of the Solanaceae family--as novel Agr-QS modulators. Biological analyses and in vitro protein-DNA binding assays suggested that these physalins suppress gene expression related to the Agr-QS system by inhibiting binding of the key response regulator AgrA to the agr promoters, reducing the function of hemolytic toxins downstream of these genes in MRSA. Furthermore, although physalin F suppressed gene expression in the Agr-QS system, its anti-hemolytic activity was lower than that of physalins H, B, and isophysalin B. Conversely, five physalins isolated from the same plant with the ability to suppress Agr-QS did not reduce bacterial Agr-QS activity but inhibited AgrA binding to DNA in vitro. A docking simulation revealed that physalin interacts with the DNA-binding site of AgrA in three docking states. The carbonyl oxygens at C-1 and C-18 of physalins, which can suppress Agr-QS, were directed to residues N201 and R198 of AgrA, respectively, whereas these carbonyl oxygens of physalins, without Agr-QS suppression activity, were oriented in different directions. Next, 100-ns molecular dynamics simulations revealed that the hydrogen bond formed between the carbonyl oxygen at C-15 of physalins and L186 of AgrA functions as an anchor, sustaining the interaction between the carbonyl oxygen at C-1 of physalins and N201 of AgrA. Thus, these results suggest that physalin H, physalin B, and isophysalin B inhibit the interaction of AgrA with the agr promoters by binding to the DNA-binding site of AgrA, suppressing the Agr-QS function of S. aureus. Physalins that suppress the Agr-QS function are proposed as potential lead compounds in the anti-virulence strategy for MRSA infections.

Isolation of bioactive phytochemicals from Crinum asiaticum L. along with their cytotoxic and TRAIL-resistance abrogating prospect assessment.

Crinum asiaticum L. (Amaryllidaceae) is a perennial bulbous herb, locally utilized for possessing multifaceted pharmacological properties including anticancer, immune-stimulating, analgesic, antiviral, antimalarial, antibacterial and antifungal, in addition to its popularity as an aesthetic plant. Separation of MeOH extract of C. asiaticum leaves yielded three known compounds as cycloneolitsol (1), hippeastrine (2) and ß-sitosterol (3). Among these, compounds 1 and 2 were subjected to the cytotoxic assay and found that they induced mild effect against HCT116, Huh7 and DU145 cell lines with the IC50 values from 73.76 to 132.53 µM. When tested for TRAIL-resistance abrogating activity, 1 (100 µM) along with TRAIL (100 ng/mL) showed moderate activity in AGS cells producing 25 % more inhibition than the agent alone. Whereas 2 (20 and 30 µM) in combination with TRAIL (100 ng/mL) exhibited strong activity in abrogating TRAIL-resistance and caused 34 % and 36 % more inhibition in AGS cells, respectively. The in-silico studies of compound 2 revealed high docking hits with the TRAIL-associated anti-apoptotic proteins which give a justification for the regulatory interactions to induce such abrogating activity. It is still recommended to conduct further investigations to understand their exact molecular mechanism.

A new 1,2-diketone physalin isolated from Physalis minima and TRAIL-resistance overcoming activity of physalins.

A new 1,2-diketone physalin, physalin XII (1), and 13 known compounds were isolated from the methanol extract of Physalis minima whole plant collected in Thailand. Among them, five physalins (2-6) had tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance overcoming activity, and physalin F (3) was the most active with an IC50 value of 0.39 µM against human gastric adenocarcinoma cell line AGS in the presence of TRAIL (100 ng/mL). An investigation of the TRAIL-resistance overcoming activity of physalins using western blot analysis showed that 3 promoted TRAIL-induced apoptosis by suppressing anti-apoptotic proteins c-FLIP and Bcl-2.

Thannilignan glucoside and 2-(ß-glucopyranosyl)-3-isoxazolin-5-one derivative, two new compounds isolated from Terminalia bellirica.

Two new compounds, thannilignan 9-O-ß-glucoside (1) and 2-(ß-glucopyranosyl)-3-isoxazolin-5-one derivative (2), and seven known compounds were isolated from the methanol extract of Terminalia bellirica leaves, collected in Bangladesh. The structures of the compounds were elucidated using spectroscopic analysis. Among these isolated compounds, corilagin (3) was cytotoxic against human gastric adenocarcinoma cell line AGS at an IC50 of 20.8 µM, and ß-D-glucopyranose 1,3,6-trigallate (4) exhibited the ability to overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance.

Two Bioactive Compounds, Uniformides A and B, Isolated from a Culture of Nocardia uniformis IFM0856T in the Presence of Animal Cells.

Two new peptides named uniformides A and B (1 and 2, respectively) were isolated from the cultured extracts of Nocardia uniformis IFM0856T in the presence of mouse macrophage-like cell line J774.1, in modified Czapek-Dox medium. These compounds were not produced in a culture containing only N. uniformis but in one that also included J774.1. Compounds 1 and 2 showed high cytotoxicity against J774.1 and suppressed the production of nitric oxide, IL-6, and IL-1ß by inhibiting the NF-κB pathway.

Isolation of nocobactin NAs as Notch signal inhibitors from Nocardia farcinica, a possibility of invasive evolution.

Notch signaling inhibitors with the potential of immune suppressor production by pathogenic bacteria for easy host infection were searched from extracts of Nocardia sp. Nocobactin NA-a (compound 1) and nocobactin NA-b (compound 2), which have been suggested as pathogenesis factors, were isolated from N. farcinica IFM 11523 isolated from the sputum of a Japanese patient with chronic bronchitis. Compounds 1 and 2 showed Notch inhibitory activities with IC50 values of 12.4 and 17.6 µM, respectively. Compound 1 and 2 decreased of Notch1 protein, Notch intracellular domain, and hairy and enhancer of split 1, which is a Notch signaling target protein. In addition, compounds 1 and 2 showed cytotoxicity against mouse macrophage-like cell line RAW264.7 with IC50 values of 18.9 and 21.1 µM, respectively. These results suggested that the Notch inhibitors production by pathogenic bacteria may increase pathogen infectivity.

Acacienone, a terpenoid-like natural product having an unprecedented C20 framework isolated from Acacia mangium leaves.

A novel C20 natural product, acacienone (1), was isolated from the leaves of Acacia mangium collected in Bangladesh. The structure of compound 1 was elucidated by spectral studies and X-ray crystallographic analysis. Acacienone (1) possesses a terpenoid-related tetracyclic framework containing 20 carbons with biogenetically unusual structural features: (i) vicinal C1-branches at the C-3 and C-4 positions in the A ring, and (ii) a cyclopentenone D ring in an androsterone-like assembly, lacking a methyl group at the C-13 position.

Cadinane sesquiterpenoids isolated from Santalum album using a screening program for Wnt signal inhibitory activity.

Upon screening compounds having Wnt signal inhibitory activity through evaluating TCF/ß-catenin transcriptional (TOP) activity, eight cadinane sesquiterpenoids, including three new compounds (1-3), were isolated from wood extracts of Santalum album (Santalaceae). Structures of compounds 1-3 were elucidated by spectral data to have a cadinane skeleton with an aromatic ring. Of the eight compounds isolated, compound 4, identified as mansonone I, was found to be active against TOP, having an IC50 of 1.2 µM.