Striatal Isolated from Cyathus striatus Extracts Induces Apoptosis in Human Pancreatic Cancer Cells.

The aim of the present study was to identify the structure of active compounds in Cyathus stratus that previously demonstrated anti-pancreatic cancer activity. The active compounds were purified from a crude extract by a series of RP-18 preparative chromatography using homemade octadecyl silica gel column. HPLC injection of the crude extract revealed a chromatogram with three main peaks with retention times (RT) 15.6, 18.2, and 22.5 min. Each fraction that exhibited promising activity in vitro was further separated using various available chromatographic techniques. The purified compound with the ultimate anti-cancer activity appeared at RT of 15.8 in the HPLC chromatogram with more than 90% purity. The main peak at the mass spectra appeared at m/z = 446.2304 with the calculated molecular formula of C25H34O7. One- and two-dimensional NMR analyses indicated that the structure of the active molecule (peak 15.8 min in HPLC) was identified as striatal C. Exposure of human pancreatic cancer cells to purified striatal C resulted in induction of apoptosis. Further studies are needed in order to develop a method for the synthesis of striatal in order to use it in clinical studies for treatment of cancer.

The Effect of Natural-Based Formulation (NBF) on the Response of RAW264.7 Macrophages to LPS as an In Vitro Model of Inflammation.

Macrophages are some of the most important immune cells in the organism and are responsible for creating an inflammatory immune response in order to inhibit the passage of microscopic foreign bodies into the blood stream. Sometimes, their activation can be responsible for chronic inflammatory diseases such as asthma, tuberculosis, hepatitis, sinusitis, inflammatory bowel disease, and viral infections. Prolonged inflammation can damage the organs or may lead to death in serious conditions. In the present study, RAW264.7 macrophages were exposed to lipopolysaccharide (LPS; 20 ng/mL) and simultaneously treated with 20 µg/mL of natural-based formulation (NBF), mushroom-cannabidiol extract). Pro-inflammatory cytokines, chemokines, and other inflammatory markers were analyzed. The elevations in the presence of interleukin-6 (IL-6), cycloxygenase-2 (COX-2), C-C motif ligand-5 (CCL5), and nitrite response, following exposure to LPS, were completely inhibited by NBF administration. IL-1ß and tumor necrosis factor alpha (TNF-α) release were inhibited by 3.9-fold and 1.5-fold, respectively. No toxic effect of NBF, as assessed by lactate dehydrogenase (LDH) release, was observed. Treatment of the cells with NBF significantly increased the mRNA levels of TLR2, and TLR4, but not NF-κB. Thus, it appears that the NBF possesses anti-inflammatory and immunomodulatory effects which can attenuate the release of pro-inflammatory markers. NBF may be a candidate for the treatment of acute and chronic inflammatory diseases and deserves further investigation.

5-aza-2'-deoxycytidine induces apoptosis and inhibits tumour growth in vivo of FaDu cells, a specific HPVnegative HNSCC cell line.

Head and neck cancer squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 600,000 new diagnoses annually. Traditionally, HNCC has been related to tobacco and alcohol exposure; however, over the past decade, a growing number of head and neck cancers are attributed to human papillomavirus (HPV) infection. 5-Aza-2'-deoxycytidine (5-AzaD) was demonstrated as an effective chemotherapeutic agent for acute myelogenous leukaemia. Preclinical data revealed that 5-aza inhibits growth and increases cell death of HPV(+) cancer cells. These effects are associated with reduced expression of HPV genes, stabilization of TP53, and activation of TP53-dependent apoptosis. The aim of the present study is to test the effect of 5-AzaD on growth of human squamous cell carcinoma (FaDu), a HPV(-) and p53 mutated cells, in vitro and in vivo. The effect of 5-AzaD on cell viability, cell cycle progression and induction of apoptosis was tested in vitro. The effect of 5-AzaD on tumour growth in vivo was tested using xenograft mice inoculated with FaDu cells. The results indicated that 5-AzaD reduced cell viability and induced apoptosis in FaDu cells in vitro. In vivo studies revealed that 5-AzaD suppresses the growth of tumours in xenograft mice inoculated with FaDu cells through inhibition of proliferation and induction of apoptosis. These findings may emphasis that 5-AzaD is effective in treatment of HPV(-) HNSCC tumours through TP53 independent pathway. Future studies are needed in order to clarify the molecular mechanism of action of 5-AzaD in HPV(-) cancer cells.

Metabolomic Characterization of a cf. Neolyngbya Cyanobacterium from the South China Sea Reveals Wenchangamide A, a Lipopeptide with In Vitro Apoptotic Potential in Colon Cancer Cells.

Metabolomics can be used to study complex mixtures of natural products, or secondary metabolites, for many different purposes. One productive application of metabolomics that has emerged in recent years is the guiding direction for isolating molecules with structural novelty through analysis of untargeted LC-MS/MS data. The metabolomics-driven investigation and bioassay-guided fractionation of a biomass assemblage from the South China Sea dominated by a marine filamentous cyanobacteria, cf. Neolyngbya sp., has led to the discovery of a natural product in this study, wenchangamide A (1). Wenchangamide A was found to concentration-dependently cause fast-onset apoptosis in HCT116 human colon cancer cells in vitro (24 h IC50 = 38 µM). Untargeted metabolomics, by way of MS/MS molecular networking, was used further to generate a structural proposal for a new natural product analogue of 1, here coined wenchangamide B, which was present in the organic extract and bioactive sub-fractions of the biomass examined. The wenchangamides are of interest for anticancer drug discovery, and the characterization of these molecules will facilitate the future discovery of related natural products and development of synthetic analogues.

A Novel Follitropin Analog Inhibits Follitropin Activity In Vitro.

Follitropin (FSH) is a heterodimeric protein composed of an α subunit that is shared with the glycoprotein hormone family, including lutropin (LH), thyrotropin (TSH), human choriogonadotropin (hCG), and a unique ß specific subunit. Both α and FSHß subunits contain two sites of N-linked oligosaccharides, which are important for its function. FSH has a crucial function in the reproductive process in mammals. However, there are some clinical conditions, such as menopausal osteoporosis or adiposity, associated with increased FSH activity. Moreover, in some cases, carcinogenesis is evidently associated with activation of FSH receptor. Therefore, developing a follitropin antagonist might be beneficial in the treatment of these conditions. Here, we describe a novel, engineered, non-glycosylated single-chain FSH variant, prepared by site-directed mutagenesis and fusion of the coding genes of the α and ß subunits. The designed variant was expressed in Chinese hamster ovary (CHO) cells and successfully secreted into the culture medium. We found that the non-glycosylated single-chain FSH analog binds with high affinity to FSH receptor and efficiently inhibits FSH activity in vitro. This variant acts at the receptor level and has the potential to serve as a follitropin antagonist for clinical applications in the future.

Cyathus striatus Extract Induces Apoptosis in Human Pancreatic Cancer Cells and Inhibits Xenograft Tumor Growth In Vivo.

Pancreatic cancer is a highly lethal disease with limited options for effective therapy and the lowest survival rate of all cancer forms. Therefore, a new, effective strategy for cancer treatment is in need. Previously, we found that a culture liquid extract of Cyathus striatus (CS) has a potent antitumor activity. In the present study, we aimed to investigate the effects of Cyathus striatus extract (CSE) on the growth of pancreatic cancer cells, both in vitro and in vivo. The proliferation assay (XTT), cell cycle analysis, Annexin/PI staining and TUNEL assay confirmed the inhibition of cell growth and induction of apoptosis by CSE. A Western blot analysis demonstrated the involvement of both the extrinsic and intrinsic apoptosis pathways. In addition, a RNAseq analysis revealed the involvement of the MAPK and P53 signaling pathways and pointed toward endoplasmic reticulum stress induced apoptosis. The anticancer activity of the CSE was also demonstrated in mice harboring pancreatic cancer cell line-derived tumor xenografts when CSE was given for 5 weeks by weekly IV injections. Our findings suggest that CSE could potentially be useful as a new strategy for treating pancreatic cancer.

Author Correction: The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells.

A major challenge in chemotherapy is chemotherapy resistance in cells lacking p53. Here we demonstrate that NIP30, an inhibitor of the oncogenic REGγ-proteasome, attenuates cancer cell growth and sensitizes p53-compromised cells to chemotherapeutic agents. NIP30 acts by binding to REGγ via an evolutionarily-conserved serine-rich domain with 4-serine phosphorylation. We find the cyclin-dependent phosphatase CDC25A is a key regulator for NIP30 phosphorylation and modulation of REGγ activity during the cell cycle or after DNA damage. We validate CDC25A-NIP30-REGγ mediated regulation of the REGγ target protein p21 in vivo using p53-/- and p53/REGγ double-deficient mice. Moreover, Phosphor-NIP30 mimetics significantly increase the growth inhibitory effect of chemotherapeutic agents in vitro and in vivo. Given that NIP30 is frequently mutated in the TCGA cancer database, our results provide insight into the regulatory pathway controlling the REGγ-proteasome in carcinogenesis and offer a novel approach to drug-resistant cancer therapy.

Inula Viscosa Extract Inhibits Growth of Colorectal Cancer Cells in vitro and in vivo Through Induction of Apoptosis.

Colorectal cancer (CRC) is the second most common cancer in females and the third in males worldwide. Conventional therapy of CRC is limited by severe side effects and by the development of resistance. Therefore, additional therapies are needed in order to combat the problem of selectivity and drug resistance in CRC patients. Inula viscosa (IV) is a well-known medicinal perennial herb in traditional medicine. It is used for different therapeutic purposes, such as; topical anti-inflammatic, diuretic, hemostatic, antiseptic, antiphlogistic, and in the treatment of diabetes. Several studies attempted to reveal the anti-cancer activity of different extracts prepared by different organic solvents from different parts of the IV plant. The aim of the present study is to examine the potential beneficial effects of IV leaf aqueous extract on the growth of colon cancer cells in vitro and in vivo. The results indicated that exposure of colorectal cancer cells to IV extract, significantly reduced cell viability in a dose and time dependent manner. Moreover, treatment of cells with 300 µg/ml of IV extract induced apoptosis, as it was detected by Annexin V/FITC/PI, TUNEL assay, and the activation of caspases. In vivo studies revealed that treatment with 150 or 300 mg/kg IV extract inhibited tumor growth in mice transplanted with MC38 cells. Tumors' weight and volume were significantly (P < 0.001) reduced when compared to untreated-control group. Staining of the paraffin section of tumors revealed that IV treatment inhibited cell proliferation and induced apoptosis. Additionally, no side effects such as; weight loss, behavior changes, ruffled fur or changes in kidney, and liver functions were observed. These results may indicate that active doses of IV extract are not toxic. Further studies are needed in order to identify the structure of the active compounds. Results from this study may contribute to the development of new and efficient strategies for treatment of human colon cancer.

Epigenetic modification in 4T1 mouse breast cancer model by artificial light at night and melatonin - the role of DNA-methyltransferase.

Currently, one of the most disputed hypotheses regarding breast cancer (BC) development is exposure to short wavelength artificial light at night (ALAN) as multiple studies suggest a possible link between them. This link is suggested to be mediated by nocturnal melatonin suppression that plays an integral role in circadian regulations including cell division. The objective of the research was to evaluate effects of 1 × 30 min/midnight ALAN (134 µ Wcm-2, 460 nm) with or without nocturnal melatonin supplement on tumor development and epigenetic responses in 4T1 tumor-bearing BALB/c mice. Mice were monitored for body mass (Wb) and tumor volume for 3 weeks and thereafter urine samples were collected at regular intervals for determining daily rhythms of 6-sulfatoxymelatonin (6-SMT). Finally, mice were sacrificed and the tumor, lungs, liver, and spleen were excised for analyzing the total activity of DNA methyltransferases (DNMT) and global DNA methylation (GDM) levels. Mice exposed to ALAN significantly reduced 6-SMT levels and increased Wb, tumor volume, and lung metastasis compared with controls. These effects were diminished by melatonin. The DNMT activity and GDM levels showed tissue-specific response. The enzymatic activity and GDM levels were lower in tumor and liver and higher in spleen and lungs under ALAN compared with controls. Our results suggest that ALAN disrupts the melatonin rhythm and potentially leading to increased BC burden by affecting DNMT activity and GDM levels. These data may also be applicable to early detection and management of BC by monitoring melatonin and GDM levels as early biomarker of ALAN circadian disruption.

Development of long-acting recombinant glycoprotein hormones by increasing the carbohydrate content.

Therapeutic recombinant glycoproteins are important for both the biotechnological industry and clinical purposes. Given the rapid clearance of these proteins from the circulation, they have to be injected frequently to obtain optimal therapy. Several strategies have been developed to overcome this limitation, aiming to increase the half-life of such proteins in the circulation. These strategies included chemical attachment of polyethylene glycol, nanocapsulation, fusion to immunoglobulins or to albumin as protein carriers, or enrichment of the carbohydrate content. Here, we describe a strategy for increasing the half-life of recombinant proteins using gene fusion to increase the carbohydrate content of the protein backbone.

Pathogenic variants in glutamyl-tRNAGln amidotransferase subunits cause a lethal mitochondrial cardiomyopathy disorder.

Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.

Whole-Exome Sequencing Identifies a Novel Homozygous Frameshift Mutation in the MTMR2 Gene as a Causative Mutation in a Patient with Charcot-Marie-Tooth Disease Type 4B1.

Charcot-Marie-Tooth (CMT) disease refers to a heterogeneous group of axonal and demyelinating polyneuropathies, characterized by chronic motor and sensory dysfunction. CMT is the most common genetic cause of neuropathy. The present study aimed to identify the gene mutation responsible for CMT in Ashkenazi Jew (AJ) patient. Genomic DNA was extracted from whole blood leukocytes of affected family and normal subject. Whole-exome sequencing was performed using the Illumina HiSeq2500. The DNA region containing the identified mutation was amplified by PCR and sequenced using dye-terminator chemistry and the forward primer. Physical examination of the patient revealed weakness and atrophy of the lower extremity muscles and Pes cavus foot deformity. Whole-exome sequencing indicated that the patient is homozygous for a novel frameshift mutation (c.1877_1878insAGAG, p.Arg630fs) in the myotubularin-related protein-2 gene (MTMR2), which resulted in an erroneous C-terminal sequence and extension by 15 amino acids. Patients' parents are healthy, and DNA sequencing analysis indicated that both are heterozygotes to the described mutation. The clinical feature of the patient may indicate a complete co-segregation of the p.Arg630fs mutation in MTMR2 gene with the CMT type 4B1 phenotype. Further studies are needed in order to estimate the prevalence of this mutation among AJ.

Estrogen receptor and laminin genetic polymorphism among women with pelvic organ prolapse.

OBJECTIVE: Laminin is a connective tissue component. The LAMC1 gene encodes for gamma-1 chain of laminin, which is associated with familial clustering of POP. The ERα gene which encodes for cellular estrogen receptor has also been associated with POP. The aim of this study was to evaluate a possible correlation between polymorphism in these genes and the risk for developing POP. MATERIALS AND METHODS: Blood samples were drawn from 33 women with advanced POP (study group) and 33 women without POP (control group). DNA was extracted, and the presence of the rs10911193 C/T mutation in LAMC1 and of the rs2228480 G/A mutation in ERα was detected using the PCR technique. RESULTS: 26 samples were available for each group regarding ERα. 33 samples were available for each group, regarding LAMC1. The prevalence of homozygotes for the ERα rs2228480 G/A mutation was 19.2% and 0% among women with and without POP, respectively (OR 39.77, 95% CI 1.93-817.0, P = 0.00046). The prevalence of heterozygotes for this mutation was 83.3% and 11.5%, respectively (OR 19.2, 95% CI 4.15-88.6, P < 0.0001). The prevalence of homozygotes for the LAMC1 gene rs10911193 C/T mutation was 3.6% and 6.1% among women with and without POP (NS), while the respective for heterozygotes for this mutation was 21.4% and 33.3% (NS). CONCLUSIONS: Polymorphism in the ERα gene is associated with an increased risk for advanced POP. However, polymorphism in the LAMC1 gene does not seem to be associated with such risk.

Endothelial nitric oxide synthase polymorphism and prognosis in systolic heart failure patients.

BACKGROUND: The endothelial nitric oxide synthase (eNOS) gene single nucleotide polymorphism G894T is associated with thrombotic vascular diseases. However, its functional significance is controversial and data are scarce concerning its influence in heart failure (HF). METHODS: We studied 215 patients with chronic systolic HF. DNA was analyzed for eNOS gene G894T polymorphism using PCR and DNA sequencing. Evaluation of clinical characteristics and analysis of factors associated with 2-year mortality were performed for the homozygous G-allele G894T variant (GG), relative to the TT and GT variants. RESULTS: The genotype distributions of eNOS G894T alleles were: GG 135 patients (63%) and TT/GT 80 (37%). Two-year mortality was significantly higher in the GG variant (48%) than the combined TT/GT group (32%). The usage of nitrates was associated with increased 2-year mortality (HR 2.0, 95% CI 1.28-3.17; p = 0.003), which was most significant in the GG group treated with nitrates (73.5%) in comparison to the TT/GT group not treated with nitrates (34%); HR 2.75, 95% CI 1.57-4.79, P < 0.001. CONCLUSIONS: Homozygosity for the G allele of the eNOS G894T polymorphism was associated with worse survival in systolic HF patients, especially in those treated with nitrates. ENOS polymorphism may result in different mechanistic interactions in HF than in thrombotic vascular diseases, suggesting that overexpression of NO may be associated with deleterious effects in systolic HF.

The Shaggy Ink Cap Medicinal Mushroom, Coprinus comatus (Higher Basidiomycetes) Extract Induces Apoptosis in Ovarian Cancer Cells via Extrinsic and Intrinsic Apoptotic Pathways.

In a previous study, ethyl acetate extract of Coprinus comatus was found to reduce viability of human ovarian cancer cells. The objective of the current research was to clarify the mechanism of action of this extract. Ovarian cancer cells (ES-2) were subjected to ethyl acetate extract of C. comatus for different concentrations or exposure times. Cell cycle analysis and annexin V staining were performed using an automated flow cytometer. DNA fragmentation was detected using the TUNEL assay. Western blot analysis was performed for the assessment of activation of caspases -3, -8, and -9. Results revealed that treatment of ES-2 cells with ethyl acetate extract of C. comatus (100 µg/ml medium), for 48 h or for 72 h resulted in an increased number of cells at the sub-G1 phase of the cell cycle. These treatments also resulted in an increased number of apoptotic cells (positively stained by annexin and positively labeled by TUNEL), in comparison to the control. Reduced levels of procaspases -3, -8, and-9 were also detected in treated cells. In conclusion, ethyl acetate extract of C. comatus induces apoptosis in ovarian cancer cells (ES-2), via both extrinsic and intrinsic pathways. Meanwhile, more investigations are needed to demonstrate weather the apoptotic effect on ovarian cancer cells is accomplished by one active compound, or combined activities of different compounds that exist in the extract.

Zebularine induces prolonged apoptosis effects via the caspase-3/PARP pathway in head and neck cancer cells.

Zebularine, a potent DNA methyltransferase inhibitor, is potentially able to influence gene regulation and thereby alters cell behavior. This study illustrates the effect of zebularine on human squamous cell carcinoma (SCC-9 and SCC-25) in vitro. The results indicated that zebularine significantly (P<0.05) reduced viability and DNA synthesis of treated cancer cells, by induction of cell cycle arrest at G2/M phase and apoptosis in both tested cell lines. This effect was confirmed to be mediated through p21/CHK1- and caspase 3/PARP­dependent pathways, respectively. However, no methylation was observed in the promoter region of the upregulated p21 and CHK1 genes. This may indicate that the alteration of p21 and CHK1 following zebularine administration was not due to inhibition of methylation of their promoter. Interestingly, it was observed that zebularine continued to influence cell viability for a week following its withdrawal. This may indicate feasibility of novel drug administration strategies, in which, daily administration of the drug replaced by weekly use, leading to improved therapeutic process and cost-effectiveness of the treatment in head and neck cancer.

Benzene-poly-carboxylic acid complex, a novel anti-cancer agent induces apoptosis in human breast cancer cells.

Some cases of breast cancer are composed of clones of hormonal-independent growing cells, which do not respond to therapy. In the present study, the effect of Benzene-Poly-Carboxylic Acid Complex (BP-C1) on growth of human breast-cancer cells was tested. BP-C1 is a novel anti-cancer complex of benzene-poly-carboxylic acids with a very low concentration of cis-diammineplatinum (II) dichloride. Human breast cancer cells, MCF-7 and T47D, were used. Cell viability was detected by XTT assay and apoptosis was detected by Flow Cytometry and by annexin V/FITC/PI assay. Caspases were detected by western blot analysis and gene expression was measured by using the Applied Biosystems® TaqMan® Array Plates. The results showed that exposure of the cells to BP-C1 for 48 h, significantly (P<0.001) reduced cell viability, induced apoptosis and activated caspase 8 and caspace 9. Moreover, gene expression experiments indicated that BP-C1 increased the expression of pro-apoptotic genes (CASP8AP1, TNFRSF21, NFkB2, FADD, BCL10 and CASP8) and lowered the level of mRNA transcripts of inhibitory apoptotic genes (BCL2L11, BCL2L2 and XIAP. These findings may lead to the development of new therapeutic strategies for treatment of human cancer using BP-C1 analog.

Light at night and melatonin have opposite effects on breast cancer tumors in mice assessed by growth rates and global DNA methylation.

Light-at-night (LAN) is a worldwide problem co-distributed with breast cancer prevalence. We hypothesized that exposure to LAN is coincided with a decreased melatonin (MLT) secretion level, followed by epigenetic modifications and resulted in higher breast cancer tumors growth-rate. Accordingly, we studied the effect of LAN exposure and exogenous MLT on breast cancer tumors growth-rate. 4T1 cells were inoculated into BALB/c short day-acclimated mice, resulting in tumors growth. Growth rates were followed under various light exposures and global DNA methylations were measured. Results demonstrated the positive effect of LAN on tumors growth-rate, reversed by MLT through global DNA methylation.