Meroterpenoids from the Fungus Ganoderma sinensis and First Absolute Configuration Clarification of Zizhine H.

Five new meroterpenoids, zizhines P-S and U (1-4,7), together with two known meroterpenoids (5 and 6) were isolated from Ganoderma sinensis. Their structures including absolute configurations were assigned by using spectroscopic, computational, and chemical methods. Racemics zizhines P and Q were purified by HPLC on chiral phase. Biological evaluation found that 4, 5 and 6 are cytotoxic toward human cancer cells (A549, BGC-823, Kyse30) with IC50 values in the range of 63.43-80.83 µM towards A549, 59.2 ± 2.73 µM and 64.25 ± 0.37 µM towards BGC-823, 76.28 ± 1.93 µM and 85.42 ± 2.82 µM towards Kyse30.

RNase H meets molecular beacons: an ultrasensitive fluorometric assay for nucleic acids.

An innovative signal amplification strategy assisted by RNase H is described here for the detection of DNA targets in a universal fashion. A tailor-made RNA molecular beacon (RMB) conjugated with a fluorescence resonance energy transfer (FRET) pair (fluorophore and quencher) was designed, characterized, and combined with the employment of RNase H. Its performance is compared to that of other nucleases including Exonuclease III and T7 exonuclease. Fluorometry, performed best at excitation/emission wavelengths of 490/520 nm, gives an amazingly low detection limit of 23 fM for target DNA. The method was verified by the determination of human hemochromatosis (HFE) gene. It is perceived that the method represents a versatile tool for the detection of a wide range of targets. Graphical Abstract An RNase H-assisted signal amplification (RASA) method for the fluorometric assay of nucleic acids has been developed by using a unique RNA molecular beacon (RNA MB) conjugated with a fluorophore (F) and quencher (Q) pair for signal generation.

Novel Terpenoids with Potent Cytotoxic Activities from Resina Commiphora.

A novel sesquiterpene dimer, spirocommiphorfuran A (1); two new cadinane sesquiterpenoids, commiphorenes A (2) and B (3); along with three known terpenoids (4⁻6), were isolated from Resina Commiphora. The structures of these new compounds were characterized by NMR, HRESIMS, quantum chemical computation, and X-ray diffraction analysis. Compound 1 features a 7-oxabicyclo[2.2.1]heptane-2-ene core, representing the first example of germacrane-type sesquiterpene dimer fused via a spiro ring system. Compound 2 is a novel sesquiterpene with a completely new carbon skeleton, which is characteristic of an additional carbon attaching to the cadinane backbone via a carbon⁻carbon bond. Additionally, compounds 2 and 4 exert acceptable cytotoxicity toward normal cells and high selectivity in cancer cells, especially in HepG2 cells.

Simultaneous targeting PI3K and PERK pathways promotes cell death and improves the clinical prognosis in esophageal squamous carcinoma.

PI3K pathway is an important anti-tumor target, but its effect and mechanism is not clear in esophageal squamous cell carcinoma (ESCC). By analysis of the Cancer Genome Atlas (TCGA) datasets, we found that PI3Ks level were significantly upregulated in human esophageal cancer tissues compared with that in non-cancer tissues. The alteration of PI3K can significantly affect the overall patient survival in ESCC but not in esophageal adenocarcinoma (EAC). We found that the classic PI3K inhibitor LY294002 obviously inhibited the canonical mammalian target of rapamycin (mTOR) pathway and restrained the growth of ESCC with less toxicity to normal cells. Besides, LY294002 inhibited noncanonical PKR-like ER kinase (PERK)/elF2α/ATF4 pathway as well. Both siRNA and the small molecule inhibitor GSK2656157 against PERK/elF2α/ATF4 pathway can significantly inhibit the growth of ESCC. More importantly, GSK2656157 aggravated the inhibitory effect of LY294002 on cell growth, colony formation, and apoptosis induction of ESCC. In addition of dual high expression of PI3K and PERK pathways in the ESCC patients, the difference of overall survival (OS) was more significant than using PI3K alone. These results indicated that dual targeting of PI3K and PERK pathways might improve clinical prognosis and enhance the treatment of ESCC patients.

Sp1 and c-Myc modulate drug resistance of leukemia stem cells by regulating survivin expression through the ERK-MSK MAPK signaling pathway.

BACKGROUND: Acute myeloid leukemia (AML) is initiated and maintained by a subset of self-renewing leukemia stem cells (LSCs), which contribute to the progression, recurrence and therapeutic resistance of leukemia. However, the mechanisms underlying the maintenance of LSCs drug resistance have not been fully defined. In this study, we attempted to elucidate the mechanisms of LSCs drug resistance. METHODS: We performed reverse phase protein arrays to analyze the expression of anti-apoptotic proteins in the LSC-enriched leukemia cell line KG-1a. Immuno-blotting, cell viability and clinical AML samples were evaluated to verify the micro-assay results. The characteristics and transcriptional regulation of survivin were analyzed with the relative luciferase reporter assay, mutant constructs, chromatin immuno-precipitation (ChIP), quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and western blotting. The levels of Sp1, c-Myc, phospho-extracellular signal-regulated kinase (p-ERK), phospho-mitogen and stress-activated protein kinase (p-MSK) were investigated in paired CD34+ and CD34- AML patient samples. RESULTS: Survivin was highly over-expressed in CD34 + CD38- KG-1a cells and paired CD34+ AML patients compared with their differentiated counterparts. Functionally, survivin contributes to the drug resistance of LSCs, and Sp1 and c-Myc concurrently regulate levels of survivin transcription. Clinically, Sp1 and c-Myc were significantly up-regulated and positively correlated with survivin in CD34+ AML patients. Moreover, Sp1 and c-Myc were further activated by the ERK/MSK mitogen-activated protein kinase (MAPK) signaling pathway, modulating survivin levels. CONCLUSION: Our findings demonstrated that ERK/MSK/Sp1/c-Myc axis functioned as a critical regulator of survivin expression in LSCs, offering a potential new therapeutic strategy for LSCs therapy.

EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of P-glycoprotein inhibition.

RATIONAL: Combination therapy to inhibit cancer stem cells may have important clinical implications. Here, we examine the molecular mechanisms by which epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, inhibits the stem cell characteristics of glioma stem-like cells (GSLCs) and synergizes with temozolomide (TMZ), a DNA-methylating agent commonly used as first-line chemotherapy in gliomas. GSLCs were enriched from the human glioblastoma cell line U87 using neurosphere culture. Cells were analyzed using flow cytometry, quantitative PCR, and western blotting. Compared to U87 cells, a higher percentage of U87 GSLCs remained in the G0/G1 phase, with downregulation of the cell-cycle protein CylinD1 and overexpression of stem cell markers CD133 and ALDH1. The drug-resistance gene ABCB1 (but not ABCG2 or MGMT) also showed high mRNA and protein expression. The resistance index of U87 GSLCs against TMZ and carmustine (BCNU) was 3.0 and 16.8, respectively. These results indicate that U87 GSLCs possess neural stem cell and drug-resistance properties. Interestingly, EGCG treatment inhibited cell viability, neurosphere formation, and migration in this cell model. EGCG also induced apoptosis, downregulation of p-Akt and Bcl-2, and cleaving PARP in a dose-dependent manner. Importantly, EGCG treatment significantly downregulated P-glycoprotein expression but not that of ABCG2 or MGMT and simultaneously enhanced sensitivity to TMZ. Our study demonstrates that the use of EGCG alone or in combination with TMZ may be an effective therapeutic strategy for glioma.

Adsorption of microcystin-LR from water with iron oxide nanoparticles.

Adsorption of microcystin-LR (MC-LR) from water using iron oxide (alpha-Fe2O3) nanoparticles was investigated in this study. Adsorption of MC-LR adsorption was well-described by a pseudo second order kinetics model and Freundlich and Langmuir isotherm equations at 15 to 35 degrees C. Thermodynamic analysis showed that the Gibbs free energy was negative, whereas standard enthalpy and entropy changes were positive at this temperature range. These findings suggest that the adsorption of MC-LR on iron oxide nanoparticles was spontaneous and endothermic. The effects of initial pH, inorganic cations, and competing compounds with carboxyl groups on absorption of MC-LR were also evaluated. Typically, adsorption efficiency decreased with increasing pH from 2 to 11. Sodium ions did not appear to significantly affect MC-LR adsorption, whereas calcium ions slightly enhanced the MC-LR adsorption capacity of the iron oxide nanoparticles. Moreover, the inhibiting effect of competing organic compounds was increased with increasing numbers of carboxyl groups, as follows: citric acid (3)>oxalic acid (2)>benzoic acid (1).

SNX-2112, a novel Hsp90 inhibitor, induces G2/M cell cycle arrest and apoptosis in MCF-7 cells.

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.

Three new sesquiterpenoids with cytotoxic activity from Artemisia argyi.

A new eudesmane sesquiterpenoid, artemisargin A (1), two new guaianolide sesquiterpenoids, artemisargins B (2) and C (3), along with three known sesquiterpenoids (4-6), were isolated from the leaves of Artemisia argyi. Their structures were determined by extensive spectroscopic methods and electronic circular dichroism calculations. Biological evaluation showed that 1 could inhibit the growth of cancer cells, especially in BGC-823 cells with an IC50 value of 49.87 µM.

Populeuphrines A and B, two new cembrane diterpenoids from the resins of Populus euphratica.

Two new cembrane diterpenoids, named populeuphrines A and B (1 and 2), together with three known analogues (3-5) were isolated from the resins of Populus euphratica. The planar structures and relative configurations of 1 and 2 were elucidated by detailed 1 D and 2 D NMR spectroscopic analyses. The absolute configurations of 1 and 2 were determined by X-ray diffraction analysis and quantum chemical computation. Biological activities of all the isolates against proliferation of human cancer cells and umbilical cord mesenchymal stem cells were evaluated.

Commipholactam A, a cytotoxic sesquiterpenoidal lactam from Resina Commiphora.

Diverse terpenoids including a novel sesquiterpenoidal lactam, commipholactam A (1), and a structurally related new cadinane sesquiterpenoid, commiphorane H (2), a new eudesmane sesquiterpenoid, commiphorane I (4), a new guaiane sesquiterpenoid, commiphorane J (5), and two new nor-abietane diterpenoids, commiphoranes K1 and K2 (6 and 7) along with two known terpenoids (3 and 8), were isolated from Resina Commiphora. Their structures with absolute configurations were characterized by spectroscopic methods and calculated electronic circular dichroism (ECD). Notably, commipholactam A represents the first example of cadinane sesquiterpene alkaloids isolated from Resina Commiphora. Biological assessment toward human cancer cells showed that the IC50 values of 1 against HepG2 and A549 cells were 21.73 µM and 128.50 µM, respectively.

[Detection of alpha-fetoprotein-L3 using agglutinin-coupled spin column to be used in diagnosis of hepatocellular carcinoma].

OBJECTIVE: To explore the effect of use of lens culinaris agglutinin (LCA)-coupled spin column (ACSC) in detection of alpha-fetoprotein (AFP) isoform AFP-L3 and to evaluate the value of AFP-L3 as a biomarker in diagnosis of hepatocellular-carcinoma (HCC). METHODS: The serum samples of 132 patients with elevated AFP level (20-1000 microg/L), 79 diagnosed as with HCC and 53 with benign liver diseases (35 with liver cirrhosis and 18 with chronic hepatitis) underwent ACSC to isolate the fraction of AFP-L3. The contents of AFP and AFP-L3 were detected by micro-particle immunoassay. The ratio of AFP-L3 to total AFP, AFP-L3%, was calculated. Correlation between the abnormally elevated AFP-L3% and HCC was analyzed. RESULTS: Detection of AFP-L3% using ACSC method was operating friendly. The average value of AFP-L3% in the patients with HCC was 36.4%, significantly higher than those of the patients with benign liver diseases (5.3% respectively, P < 0.01). The area under the receiver operating characteristic (ROC) curve of AFP-L3% was 0.807. Taking AFP-L3% > or = 10% as diagnostic criteria, the sensitivity of AFP-L3% in HCC diagnosis was 84.8% (67/79) and the specificity was 92.5% (49/53), with a total conformity rate of 87.9% compared to the confirmed clinical diagnosis. Conclusion ACSC is of clinical value in detecting AFP-L3. AFP-L3% is a valuable biomarker in diagnosis and prediction of prognosis of HCC.

Tetrandrine inhibits glioma stem-like cells by repressing ß-catenin expression.

Cancer stem cells (CSCs) in glioma are often responsible for relapse and resistance to therapy. The purpose of the present study was to confirm the self-renewal and migration inhibitory effects of tetrandrine (Tet), which is a compound extracted from the dried root of Stephania tetrandra S. Moore, toward glioma stem-like cells (GSLCs) and to examine the associated molecular mechanisms. Using a neurosphere culture technique, we enriched the GSLC population from the human glioblastoma cell lines U87 and U251. Cells were analyzed using cell counting kit-8 (CCK-8), western blotting, flow cytometry, transwell assay and immunofluorescence staining. GSLCs displayed properties of neural stem cells, including elevated expression of the cancer stem cell marker ALDH1 and ß-catenin. We found that Tet treatment decreased sphere formation in GSLCs in a dose-dependent manner using tumor spheroid formation assay. The GSK3ß inhibitor BIO maintained sphere formation and migration capacity in GSLCs, whereas the ß-catenin/TCF transcription inhibitor ICG-001 decreased sphere formation and the migration capacity of GSLCs. The proportion of apoptotic GSLCs also increased in response to ICG-001 treatment. These results indicate that ß-catenin activity is vital in maintaining neural stem cell traits of GSLCs. Tet inhibits cell viability, neurosphere formation and migration of GSLCs in vitro. Importantly, Tet treatment significantly repressed the nuclear translocation and expression of ß-catenin and induced apoptosis in GSLCs, as indicated in part by the upregulation of Bax, the cleavage of PARP and the downregulation of Bcl-2. The present study demonstrates that the inhibition of ß-catenin in CSCs by Tet could be an effective strategy for the treatment of glioma.

The heat shock protein 90 inhibitor SNX-2112 inhibits B16 melanoma cell growth in vitro and in vivo.

SNX-2112 is a selective heat shock protein 90 (Hsp90) inhibitor which can exert a potent anticancer activity. In this study, we investigated the effects of SNX-2112 on B16 melanoma cells in vitro and in vivo. The 3-(4,5-dimetrylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis demonstrated that SNX-2112 dose-dependently inhibited the growth of B16 cells, and induced G0/G1 cell cycle arrest and apoptosis. Western blotting revealed that SNX-2112 lead to the degradation of Hsp90 client proteins including Akt, IKKα, NF-κB, B-Raf and GSK3ß. Furthermore, we assessed the antitumor effect of SNX-2112 in vivo, using a xenograft model in C57BL/6 mice. Oral administration of SNX-2112 significantly inhibited the growth of B16 tumors in mice, with a 47% inhibition observed at dose of 80 mg/kg/day for 15 days, compared to control tumors. Hematoxylin-eosin (H&E) staining of xenograft tissues showed that SNX-2112 also inhibited angiogenesis and lead to a lower blood vessel density in the tumors, compared to the control group. These findings demonstrate that SNX-2112 can exhibit a potent anticancer activity against B16 melanoma cells both in vitro and in vivo, by inhibiting cell proliferation and inducing cell cycle arrest and apoptosis in a mechanism dependent on the degradation of Hsp90 client proteins.

SNX-2112, an Hsp90 inhibitor, induces apoptosis and autophagy via degradation of Hsp90 client proteins in human melanoma A-375 cells.

SNX-2112 is an Hsp90 inhibitor which is currently undergoing multiple phase 1 clinical trials; however, its mechanism of action needs to be further elaborated. Here we investigated the effects of SNX-2112 in A-375 cells. SNX-2112 induced the degradation of multiple Hsp90 client proteins, activated both the mitochondrial-mediated and death receptor-mediated apoptotic pathways, downregulated Bcl-2 and Bcl-xL, upregulated Bid, cleaved caspase-9, caspase-7, caspase-3 and PARP, and activated caspase-8. The general caspase inhibitor, z-VAD-fmk, did not completely abolish SNX-2112-induced cell death. SNX-2112 induced autophagy in a time- and dose-dependent manner via Akt/mTOR/p70S6K inhibition. SNX-2112 induces significant apoptosis and autophagy in human melanoma A-375 cells, and may be an effective targeted therapy agent.

The selective Hsp90 inhibitor BJ-B11 exhibits potent antitumor activity via induction of cell cycle arrest, apoptosis and autophagy in Eca-109 human esophageal squamous carcinoma cells.

BJ-B11 is a selective heat shock protein 90 (Hsp90) inhibitor that has been reported to possess significant antitumor activity in multiple types of cancer cells; however, the mechanism of action needs to be further clarified. We investigated, for the first time, the antitumor activity and the molecular mechanism underlying growth inhibition in Eca-109 cells. The results revealed that BJ-B11 inhibited the proliferation of Eca-109 cells in a time- and concentration-dependent manner, with 50% inhibitory concentration (IC(50)) values of 0.31±0.01 µM after 48-h incubation. BJ-B11 induced concentration-dependent G2/M cell cycle arrest and apoptosis. The cleavage of caspase 3 and PARP signals detected might originate from mitochondrial dysfunction, which was supported by the results of reactive oxygen species (ROS) production, cytochrome c release and the mitochondrial membrane potential (MMP) reduction. The general caspase inhibitor Z-VAD-fmk did not completely abolish BJ-B11-induced cell death. Furthermore, inhibition of the Akt/mTOR/p70S6K signaling pathway might be involved in the process of BJ-B11-induced autophagy, which was characterized by the production of autophagic vacuoles and upregulation of LC3-II protein in a time- and concentration-dependent manner. Meanwhile, the general autophagy inhibitor 3-MA decreased the apoptotic ratio. Furthermore, BJ-B11 induced the polymerization of cytoskeleton ß-tubulin and F-actin. Taken together, our results suggest that the growth inhibition of Eca-109 cells induced by BJ-B11 may result from the induction of G2/M cell cycle arrest, apoptosis and autophagy.

Induction of apoptosis in K562 cells by dicyclohexylammonium salt of hyperforin through a mitochondrial-related pathway.

Hyperforin is an abundant phloroglucinol-type constituent isolated from the extract of the flowering upper portion of the plant Hypericum perforatum L. The dicyclohexylammonium salt of hyperforin (DCHA-HF) has exhibited antitumor and antiangiogenic activities in various cancer cells. Here, the antitumor effects of DCHA-HF on the chronic myeloid leukemia K562 cell line were investigated for the first time. DCHA-HF exhibited dose- and time-dependent inhibitory activities against K562 cells, with IC(50) values of 8.6 and 3.2 µM for 48 h and 72 h of treatment, respectively, which was more effective than that of the hyperforin. In contrast, little cytotoxic activity was observed with DCHA-HF on HUVECs. DCHA-HF treatment resulted in induction of apoptosis as evidenced from DNA fragmentation, nuclear condensation and increase of early apoptotic cells by DAPI staining analysis, TUNEL assay and Annexin V-FITC/PI double-labeled staining analysis, respectively. Moreover, DCHA-HF elicited dissipation of mitochondrial transmembrane potential that commenced with the release of cytochrome c through down-regulation of expression of anti-apoptotic proteins and up-regulation of expression of pro-apoptotic proteins. DCHA-HF treatment induced activation of the caspase 3, 8, and 9 cascade and subsequent PARP cleavage, and DCHA-HF-induced apoptosis was significantly inhibited by caspase inhibitors. Treated cells were arrested at the G1 phase of the cell cycle and the expression of p53 and p27(Kip1), two key regulators related to cell cycle and apoptosis, was up-regulated. These results suggest that DCHA-HF inhibits K562 cell growth by inducing caspase-dependent apoptosis mediated by a mitochondrial pathway and arresting the cell cycle at the G1 phase. Therefore, DCHA-HF is a potential chemotherapeutic antitumor drug for chronic myeloid leukemia therapy.

BJ-B11, a novel Hsp90 inhibitor, induces apoptosis in human chronic myeloid leukemia K562 cells through the mitochondria-dependent pathway.

In the past few years heat shock protein 90 (Hsp90) inhibitors have been reported to possess significant antitumor activity. We investigated, for the first time, the antitumor activity of a novel Hsp90 inhibitor 2-(4-acetyloxycyclohexylamino)-4-(3, 6, 6-trimethyl-4-oxo-4, 5, 6, 7-tetrahydro-1H-indazol-1-yl)-benzamide (BJ-B11) and the molecular mechanism underlying the apoptosis it induces in human chronic myeloid leukemia K562 cells. The results revealed that BJ-B11 triggered growth inhibition in K562 cells and other malignant cell lines in vitro with only minor toxicity in a normal human cell line. BJ-B11 inhibited the proliferation of K562 cells in a concentration- and time-dependent manner, with IC(50) values of 1.1 ± 0.2 µM and 0.4 ± 0.1 µM after 48 and 72 h incubations respectively. This most likely results from cell cycle arrest at the G(0)/G(1) phase and the induction of apoptosis. In addition, BJ-B11 degraded the Hsp90 client proteins Bcr-Abl and Akt, induced activation of caspase-9 and caspase-3, and subsequent cleavage of PARP. The caspase signals may originate from mitochondrial dysfunction, which is supported by the finding of cytochrome c release. In addition, inactivation of the Akt signaling pathway may be involved in the process of BJ-B11-induced apoptosis. Taken together, our data provide a putative molecular mechanism for the anticancer effect of BJ-B11 on K562 cells, and suggest a potential application for BJ-B11 in chronic myeloid leukemia therapy.