Derivation of a novel antimicrobial peptide from the Red Sea Brine Pools modified to enhance its anticancer activity against U2OS cells.

Cancer associated drug resistance is a major cause for cancer aggravation, particularly as conventional therapies have presented limited efficiency, low specificity, resulting in long term deleterious side effects. Peptide based drugs have emerged as potential alternative cancer treatment tools due to their selectivity, ease of design and synthesis, safety profile, and low cost of manufacturing. In this study, we utilized the Red Sea metagenomics database, generated during AUC/KAUST Red Sea microbiome project, to derive a viable anticancer peptide (ACP). We generated a set of peptide hits from our library that shared similar composition to ACPs. A peptide with a homeodomain was selected, modified to improve its anticancer properties, verified to maintain high anticancer properties, and processed for further in-silico prediction of structure and function. The peptide's anticancer properties were then assessed in vitro on osteosarcoma U2OS cells, through cytotoxicity assay (MTT assay), scratch-wound healing assay, apoptosis/necrosis detection assay (Annexin/PI assay), RNA expression analysis of Caspase 3, KI67 and Survivin, and protein expression of PARP1. L929 mouse fibroblasts were also assessed for cytotoxicity treatment. In addition, the antimicrobial activity of the peptide was also examined on E coli and S. aureus, as sample representative species of the human bacterial microbiome, by examining viability, disk diffusion, morphological assessment, and hemolytic analysis. We observed a dose dependent cytotoxic response from peptide treatment of U2OS, with a higher tolerance in L929s. Wound closure was debilitated in cells exposed to the peptide, while annexin fluorescent imaging suggested peptide treatment caused apoptosis as a major mode of cell death. Caspase 3 gene expression was not altered, while KI67 and Survivin were both downregulated in peptide treated cells. Additionally, PARP-1 protein analysis showed a decrease in expression with peptide exposure. The peptide exhibited minimal antimicrobial activity on critical human microbiome species E. coli and S. aureus, with a low inhibition rate, maintenance of structural morphology and minimal hemolytic impact. These findings suggest our novel peptide displayed preliminary ACP properties against U2OS cells, through limited specificity, while triggering apoptosis as a primary mode of cell death and while having minimal impact on the microbiological species E. coli and S. aureus.

Azafuramidines as potential anticancer Agents: Pro-apoptotic profile and cell cycle arrest.

The current study aimed to test the antiproliferative activity of three azafuramidines (X, Y, and Z) against three different human cell lines; liver HepG2, breast MCF-7, and bone U2OS. And to explore the molecular mechanism(s) of the antiproliferative activity of these derivatives. The three new azafuramidines demonstrated a potent cytotoxicity at < 2 µM against the three cell lines investigated. The azafuramidines were highly selective with selectivity index âˆ¼ 47 - 61 folds indicating safety to the normal cells. In the scratch assay, azafuramidines significantly reduced the percentage of wound healing indicating ability to prevent or reduce metastasis. Derivatives X and Z arrested the HepG2 cells at S and G2/M phases detected by the flow cytometry. Derivatives X, Y, and Z elevated the apoptosis of HepG2 cells by âˆ¼ 71 %, 66 %, and 59 %, respectively. Derivatives X and Z were superior to derivative Y. The potent antiproliferative, cell cycle arrest, and pro-apoptotic efficacy of these chlorophenyl derivatives could be attributed to their ability of inducing the overexpression of p53, p21, and p27. These derivatives had the potential to act as anticancer agents and merit further investigations.

Functional role and epithelial to mesenchymal transition of the miR-590-3p/MDM2 axis in hepatocellular carcinoma.

BACKGROUND: There is considerable evidence that microRNAs (miRNAs) regulate several key tumor-associated genes/pathways and may themselves have a dual regulatory function either as tumor suppressors or oncogenic miRNA, depending on the tumor type. MicroRNA-590-3p (miR-590-3p) is a small non-coding RNA involved in the initiation and progression of numerous tumors. However, its expression pattern and biological role in hepatocellular carcinoma (HCC) are controversial. RESULTS: In the current work, computational and RT-qPCR analysis revealed that HCC tissues and cell lines exhibited miR-590-3p downregulation. Forced expression of miR-590-3p attenuated HepG2 cells proliferation, migration, and repressed EMT-related gene expression. Bioinformatic, RT-qPCR, and luciferase assays revealed that MDM2 is a direct functional target of miR-590-3p. Moreover, the knockdown of MDM2 mimicked the inhibitory effect of miR-590-3p in HepG2 cells. CONCLUSION: We have identified not only novel targets for miR-590-3p in HCC, but also novel target genes for miR590-3p/MDM2 pathway in HCC like SNAIL, SLUG, ZEB1, ZEB2, and N-cadherin. Furthermore, these findings demonstrate a crucial role for MDM2 in the regulatory mechanism of EMT in HCC.

Characterization of a novel peptide mined from the Red Sea brine pools and modified to enhance its anticancer activity.

Drug resistance is a major cause of the inefficacy of conventional cancer therapies, and often accompanied by severe side effects. Thus, there is an urgent need to develop novel drugs with low cytotoxicity, high selectivity and minimal acquired chemical resistance. Peptide-based drugs (less than 0.5 kDa) have emerged as a potential approach to address these issues due to their high specificity and potent anticancer activity. In this study, we developed a support vector machine model (SVM) to detect the potential anticancer properties of novel peptides by scanning the American University in Cairo (AUC) Red Sea metagenomics library. We identified a novel 37-mer antimicrobial peptide through SVM pipeline analysis and characterized its anticancer potential through in silico cross-examination. The peptide sequence was further modified to enhance its anticancer activity, analyzed for gene ontology, and subsequently synthesized. To evaluate the anticancer properties of the modified 37-mer peptide, we assessed its effect on the viability and morphology of SNU449, HepG2, SKOV3, and HeLa cells, using an MTT assay. Additionally, we evaluated the migration capabilities of SNU449 and SKOV3 cells using a scratch-wound healing assay. The targeted selectivity of the modified peptide was examined by evaluating its hemolytic activity on human erythrocytes. Treatment with the peptide significantly reduced cell viability and had a critical impact on the morphology of hepatocellular carcinoma (SNU449 and HepG2), and ovarian cancer (SKOV3) cells, with a marginal effect on cervical cancer cell lines (HeLa). The viability of a human fibroblast cell line (1Br-hTERT) was also significantly reduced by peptide treatment, as were the proliferation and migration abilities of SNU449 and SKOV3 cells. The annexin V assay revealed programmed cell death (apoptosis) as one of the potential cellular death pathways in SNU449 cells upon peptide treatment. Finally, the peptide exhibited antimicrobial effects on both gram-positive and gram-negative bacterial strains. The findings presented here suggest the potential of our novel peptide as a potent anticancer and antimicrobial agent.

Production of Bioactive Compounds from the Sulfated Polysaccharides Extracts of Ulva lactuca: Post-Extraction Enzymatic Hydrolysis Followed by Ion-Exchange Chromatographic Fractionation.

This paper describes a novel combined post-extraction process for obtaining bioactive compounds from the aqueous high molecular weight sulfated polysaccharides (SPs) extracts of the green algae, Ulva lactuca. After extracting the SPs, they were enzymatically hydrolyzed then the hydrolysate (V45) was fractionated into eight different molecular weight fractions (F1-F8) using ion exchange chromatography. Crude SPs together with V45 and (F1-F8) were examined for their carbohydrate, protein, and sulfate contents. In addition, their degree of polymerization (DP) was estimated and they were characterized by Fourier Transform Infrared Spectroscopy (FTIR). Fractions S1, F4, F5, and F8 showed promising antioxidant and antitumor activities in vitro. In particular, the remarkable antitumor activity of F5 on three types of cancer cell lines could be attributed to its comparable contents of protein, carbohydrate, and sulfate, in addition to its comparable contents of rhamnose and glucuronic acid, and the same for glucose and arabinose. F5 also possessed the highest Hill coefficient among the four promising fractions indicating a higher degree of cooperativity in ligand binding. Other influencing factors including DP, composition, and type of characteristic functional groups were also discussed. The implications of this work could potentially benefit the industries of food supplements and pharmaceuticals.

Correction to: Ectopic delivery of miR-200c diminishes hepatitis C virus infectivity through transcriptional and translational repression of Occludin.

The author would like to correct the errors in the online published article.

Ectopic delivery of miR-200c diminishes hepatitis C virus infectivity through transcriptional and translational repression of Occludin.

Occludin (OCLN) is an essential factor for HCV entry through interacting with other surface receptors. The aim of this study was to investigate the epigenetic regulation of Occludin expression and to study its impact on viral infectivity. microRNAs expression was assessed using qRT-PCR, while OCLN protein expression was investigated by indirect immunofluorescence and Western blotting. Viral infectivity was assessed by measuring viral-load using qRT-PCR. In silico analysis predicted that miR-200c targeted the OCLN 3'UTR, which was further experimentally confirmed. miR-122 was previously validated to target the 3'UTR of OCLN and was used as a control. We report a significant down-regulation of miR-200c in liver tissues of HCV-infected patients. Ectopic expression of both miR-122 and miR-200c in Huh7 cells reduced OCLN mRNA and protein levels. Viral infectivity was significantly reduced by miR-200c but enhanced by miR-122. This work sheds light on miR-200c as a novel regulator of HCV infectivity through the regulation of OCLN.

Computational comparative analysis identifies potential stemness-related markers for mesenchymal stromal/stem cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that reside in multiple tissues are capable of self-renewal and differentiation into various cell types. These properties make them promising candidates for regenerative therapies. MSC identification is critical in yielding pure populations for successful therapeutic applications; however, the criteria for MSC identification proposed by the International Society for Cellular Therapy (ISCT) are inconsistent across different tissue sources. This study aimed to identify potential markers to be used together with the ISCT criteria to provide a more accurate means of MSC identification. Thus, we carried out a computational comparative analysis of the gene expression in human and mouse MSCs derived from multiple tissues to identify the differentially expressed genes that are shared between the two species. We show that six members of the proteasome degradation system are similarly expressed across MSCs derived from bone marrow, adipose tissue, amnion, and umbilical cord. Additionally, with the help of predictive models, we found that the expression profile of these genes correctly validated the identity of the MSCs across all the tissue sources tested. Moreover, using genetic interaction networks, we showed a possible link between these genes and antioxidant enzymes in the MSC antioxidant defense system, thereby pointing to their potential role in prolonging the life span of MSCs. According to our findings, members of the proteasome degradation system may serve as stemness-related markers.

Role of NELF-B in supporting epithelial-mesenchymal transition and cell proliferation during hepatocellular carcinoma progression.

Negative elongation factor-B (NELF-B), also known as cofactor of BRCA1 (COBRA1), is one of the four subunits of the NELF complex. It interacts with BRCA1, in addition to other transcription complexes in various tissues. The NELF complex represses the transcription of several genes by stalling RNA polymerase II during the early phase of transcription elongation. The role of NELF-B in liver cancer and hepatocellular carcinoma (HCC), the most prevalent type of liver cancer, remains to be elucidated. It has been previously demonstrated that silencing of NELF-B inhibits the proliferation and migration of HepG2 cells. The present study aimed to investigate the consequences of ectopic expression and silencing of NELF-B in liver cancer HepG2 and SNU449 cell lines. Functional assays were performed to examine the effects on gene and protein expression, viability, migration and invasion of cells. Overexpression of NELF-B did not alter the proliferation and migration of HepG2 cells, or the expression of tested genes, indicating that overexpression alone may not be sufficient for altering these features in HepG2 cells. By contrast, knockdown of NELF-B in SNU449 cells resulted in decreased cell proliferation, together with induction of apoptosis and decreased expression levels of Ki-67 and survivin, which are markers of proliferation and inhibition of apoptosis, respectively. Additionally, silencing of NELF-B resulted in a significant decrease in the hallmarks of epithelial-mesenchymal transition (EMT), including cell migration and invasion, and decreased the expression levels of EMT markers, such as N-cadherin, vimentin and ß-catenin. Decreased expression levels of forkhead box F2 transcription factor and increased mRNA levels of trefoil factor 1, a putative tumor suppressor, were also detected following the silencing of NELF-B. The current results demonstrated that NELF-B enhanced the manifestation of most hallmarks of cancer, including cell proliferation, migration, invasion and inhibition of apoptosis, indicating its critical role in the progression of HCC.

In- vitro evaluation of the bioactivity and the biocompatibility of a novel coated UHMWPE biomaterial for biomedical applications.

Ultra-High Molecular Weight Polyethylene (UHMWPE) is the gold standard biomaterial used as a bearing surface in total joint replacement surgeries. However, osteolysis and subsequent implant failure as a result of the production of wear debris may occur at the contacting surfaces. One potential solution to overcome this problem is to strengthen the surface of UHMWPE which can be achieved by adding a thin coating layer made of Polyamide. In this article, a combination of biological and biochemical tests, including cell viability, antibacterial activity (using Escherichia coli and Staphylococcus aureus), and wound healing assays were performed to assess the bioactivity and the biocompatibility of the coated specimens. Additional tests, such as simulated body fluid absorption, Alizarin Red Staining, Scanning Electron Microscopy, Energy-dispersive X-ray spectroscopy, and Fourier-transform infrared spectroscopy techniques were conducted to evaluate the moisture uptake, osteogenic activity, and the morphology of the coated samples. The antibacterial activity test results after 24 h incubation showed that the nylon-coated UHMWPE has significantly higher antibacterial activity compared to the uncoated UHMWPE. The results of wound closure showed that nylon-coated UHMWPE promotes more wound healing compared to the uncoated material that exhibits a similar percentage of wound closure as the control. This is the first study to demonstrate the superiority of the proposed coated biomaterial for wound healing applications with improved antibacterial capabilities.

Potential Anticancer Activity of Crude Ethanol, Ethyl Acetate, and Water Extracts of Ephedra foeminea on Human Osteosarcoma U2OS Cell Viability and Migration.

Medicinal plants are potential sources for a wide range of complex compounds with probable anticancer activity. Ephedra foeminea Forssk. (E. foeminea), a medicinal plant found in the Eastern Mediterranean, has recently been gaining popularity as a cancer remedy; there is, however, a paucity of empirical evidence supporting this claim. In this study, the effect of E. foeminea ethyl acetate, ethanol, and water crude extracts on viability, migratory ability, and the steady-state mRNA levels of genes involved in these processes was, respectively, examined using MTT assay, wound healing assay, and reverse transcriptase PCR (RT-PCR). The study concludes that all extracts significantly reduce human osteosarcoma U2OS percentage viability in a dose- and time-dependent manner, with varying potencies. The least half-maximal inhibitory concentration (IC50) was observed in the water extract after 48 h incubation (30.761 ± 1.4 µg/mL) followed by the ethyl acetate extract after 72 h incubation (80.35 ± 1.233 µg/mL) and finally the ethanol extract after 48 h incubation (97.499 ± 1.188 µg/mL). Ethanol extract significantly reduced U2OS percentage wound closure. On the other hand, both ethanol and water extracts considerably reduced the steady-state mRNA expression of beta-catenin, promoting both cell proliferation and migration in osteosarcoma by regulating target genes. Additionally, E. foeminea showed no hemolytic activity. These effects suggest that E. foeminea decreases U2OS cell viability and migratory ability by modulating the expression of critical genes involved in regulating these processes and is likely cytocompatible with human erythrocytes.

miR-590-3p and Its Downstream Target Genes in HCC Cell Lines.

miRNAs are small non-coding RNA sequences of 18-25 nucleotides. They can regulate different cellular pathways by acting on tumor suppressors, oncogenes, or both. miRNAs are mostly tissue-specific, and their expression varies depending on the cancer or the tissue in which they are found. hsa-miR-590-3p was found to be involved in several types of cancers. In this study, we identified potential downstream target genes of hsa-miR-590-3p computationally. Several bioinformatics tools and more than one approach were used to identify potential downstream target genes of hsa-miR-590-3p. CX3CL1, SOX2, N-cadherin, E-cadherin, and FOXA2 were utilized as potential downstream target genes of hsa-miR-590-3p. SNU449 and HepG2, hepatocellular carcinoma cell lines, were used to carry out various molecular techniques to further validate our in silico results. mRNA and protein expression levels of these genes were detected using RT-PCR and western blotting, respectively. Co-localization of hsa-miR-590-3p and its candidate downstream target gene, SOX2, was carried out using a miRNA in situ hybridization combined with immunohistochemistry staining through anti-SOX2. The results show that there is an inverse correlation between hsa-miR-590-3p expression and SOX2 protein expression in SNU449. Subsequently, we suggest that SOX2 can be a direct downstream target of has-miR-590-3p indicating that it may have a role in the self-renewal and self-maintenance of cancer cells. We also suggest that CX3CL1, E-cadherin, N-cadherin, and FOXA2 show a lot of potential as downstream target genes of hsa-miR-590-3p signifying its role in epithelial-mesenchymal transition. Studying the expression of hsa-miR-590-3p downstream targets can enrich our understanding of the cancer pathogenesis and how it can be used as a therapeutic tool.

Ubiquitination and proteasome-mediated degradation of BRCA1 and BARD1 during steroidogenesis in human ovarian granulosa cells.

Germ-line mutations in BRCA1 predispose women to early-onset, familial breast and ovarian cancers. However, BRCA1 expression is not restricted to breast and ovarian epithelial cells. For example, ovarian BRCA1 expression is enriched in ovarian granulosa cells, which are responsible for ovarian estrogen production in premenopausal women. Furthermore, recent tissue culture and animal studies suggest a functional role of BRCA1 in ovarian granulosa cells. Although levels of BRCA1 are known to fluctuate significantly during folliculogenesis and steroidogenesis, the mechanism by which BRCA1 expression is regulated in granulosa cells remains to be elucidated. Here we show that the ubiquitin-proteasome degradation pathway plays a significant role in the coordinated protein stability of BRCA1 and its partner BARD1 in ovarian granulosa cells. Our work identifies the amino-terminal RING domain-containing region of BRCA1 as the degron sequence that is both necessary and sufficient for polyubiquitination and proteasome-mediated protein degradation. Interestingly, mutations in the RING domain that abolish the ubiquitin E3 ligase activity of BRCA1 do not affect its own ubiquitination or degradation in ovarian granulosa cells. The proteasome-mediated degradation of BRCA1 and BARD1 also occurs during the cAMP-dependent steroidogenic process. Thus, the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells provide an excellent paradigm for investigating the regulation of this protein complex under physiological conditions.

Disruption of Claudin-1 Expression by miRNA-182 Alters the Susceptibility to Viral Infectivity in HCV Cell Models.

HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3'UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection.

miR-590-3p Promotes Ovarian Cancer Growth and Metastasis via a Novel FOXA2-Versican Pathway.

miRNAs play important roles in gene regulation, and their dysregulation is associated with many diseases, including epithelial ovarian cancer (EOC). In this study, we determined the expression and function of miR-590-3p in EOC. miR-590-3p levels were higher in high-grade carcinoma when compared with low-grade or tumors with low malignant potential. Interestingly, plasma levels of miR-590-3p were significantly higher in patients with EOC than in subjects with benign gynecologic disorders. Transient transfection of miR-590-3p mimics or stable transfection of mir-590 increased cell proliferation, migration, and invasion. In vivo studies revealed that mir-590 accelerated tumor growth and metastasis. Using a cDNA microarray, we identified forkhead box A2 (FOXA2) and versican (VCAN) as top downregulated and upregulated genes by mir-590, respectively. miR-590-3p targeted FOXA2 3' UTR to suppress its expression. In addition, knockdown or knockout of FOXA2 enhanced cell proliferation, migration, and invasion. Overexpression of FOXA2 decreased, whereas knockout of FOXA2 increased VCAN mRNA and protein levels, which was due to direct binding and regulation of the VCAN gene by FOXA2. Interrogation of the TCGA ovarian cancer database revealed a negative relationship between FOXA2 and VCAN mRNA levels in EOC tumors, and high FOXA2/low VCAN mRNA levels in tumors positively correlated with patient survival. Finally, overexpression of FOXA2 or silencing of VCAN reversed the effects of mir-590. These findings demonstrate that miR-590-3p promotes EOC development via a novel FOXA2-VCAN pathway.Significance: Low FOXA2/high VCAN levels mediate the tumor-promoting effects of miR-590-3p and negatively correlate with ovarian cancer survival. Cancer Res; 78(15); 4175-90. ©2018 AACR.

Enrichment, Propagation, and Characterization of Mouse Testis-Derived Mesenchymal Stromal Cells.

The therapeutic potential of multipotent stromal cells (MSCs) largely depends on the isolation and expansion methods used. In this study, we propose a laminin-based technique to select and enrich for MSCs isolated from the mouse testis. Primary cell cultures were prepared from juvenile mouse testes and the capacity to generate colony forming units together with population doubling time (PDT) during expansion were determined. The identity of MSCs was assayed using reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry for the active expression of cell surface markers, such as CD44, CD73, and CD29; absence of the CD45 hematopoietic cell marker; and in vitro differentiation of the cells into osteoblasts and adipocytes. Testis-derived MSCs (tMSCs) displayed self-renewal properties and in the early passages, exhibited high proliferation patterns with an average PDT of 44.1 hours. The lack of Vasa expression implied that the tMSCs were not of germ cell origin. The RT-PCR data, which were confirmed by immunophenotyping, revealed high expression of CD44 and the absence of CD45 expression in tMSCs. The strong Alizarin Red stain in tMSCs that were stimulated into making bone cells was indicative of the presence of calcium-producing cells (osteoblasts). Likewise, the adipogenic potential of tMSCs was demonstrated based on Oil Red O staining of lipid vacuoles in differentiated cells. Loss of fibroblast-like morphology in late passage cells along with the increase in PDT and the decrease in the mRNA levels of CD73 and CD29 suggested that the tMSCs developmental program is reformed at this stage.

Knockdown of COBRA1 decreases the proliferation and migration of hepatocellular carcinoma cells.

Cofactor of BRCA1 (COBRA1) is one of the four subunits that make up the negative elongation factor (NELF) complex that is involved in the stalling of RNA polymerase II early during transcription elongation. As such, it regulates the expression of a substantial number of genes involved in cell cycle control, cellular metabolism and DNA repair. With no DNA binding domain, its capacity to modulate gene expression occurs via its ability to interact with different transcription factors. In the field of cancer, its role is not yet fully understood. In this study, we demonstrate the frequent overexpression of COBRA1 along with the remaining NELF subunits in hepatocellular carcinoma (HCC) tissues relative to non-cancerous liver tissues. To elucidate its biological significance in HCC, RNA interference was utilized to silence COBRA1 expression in the HCC cell line, HepG2. Interestingly, COBRA1 knockdown resulted in a significant decrease in cell proliferation and migration, accompanied by a concomitant reduction in the expression of the proliferation marker, Ki-67. Survivin, a proto-oncogene that is commonly upregulated in almost all human malignancies including HCC, was also significantly downregulated following COBRA1 silencing. This suggests that it might be one of the mechanisms by which COBRA1 mediates its role in HCC. Taken together, our data findings collectively highlight an important role for COBRA1 in supporting HCC proliferation and migration.

Modulation of aromatase expression by BRCA1: a possible link to tissue-specific tumor suppression.

Mutations in BRCA1 increase risks of familial breast and ovarian cancers, particularly among premenopausal women. While BRCA1 plays an active role in DNA repair, this function alone may not be sufficient to explain why BRCA1-associated tumors predominantly occur in estrogen-responsive tissues. Aromatase is the rate-limiting enzyme in estrogen biosynthesis and a key target in breast cancer treatment. Aromatase expression in ovarian granulosa cells dictates levels of circulating estrogen in premenopausal women, and its aberrant overexpression in breast adipose tissues promotes breast cancer growth. Here, we show that BRCA1 modulates aromatase expression in ovarian granulosa cells and primary preadipocytes. The cyclic AMP-dependent expression of aromatase in ovarian granulosa cells is inversely correlated with the protein level of BRCA1. Importantly, transient knockdown of BRCA1 enhances aromatase expression in both ovarian granulosa cells and primary preadipocytes. We propose that BRCA1 deficiency in epithelial and certain nonepithelial cells may result in combined effects of aberrant estrogen biosynthesis and compromised DNA repair capability, which in turn may lead to specific cancers in the breast and ovary.

Mechanical stress promotes cisplatin-induced hepatocellular carcinoma cell death.

Cisplatin (CisPt) is a commonly used platinum-based chemotherapeutic agent. Its efficacy is limited due to drug resistance and multiple side effects, thereby warranting a new approach to improving the pharmacological effect of CisPt. A newly developed mathematical hypothesis suggested that mechanical loading, when coupled with a chemotherapeutic drug such as CisPt and immune cells, would boost tumor cell death. The current study investigated the aforementioned mathematical hypothesis by exposing human hepatocellular liver carcinoma (HepG2) cells to CisPt, peripheral blood mononuclear cells, and mechanical stress individually and in combination. HepG2 cells were also treated with a mixture of CisPt and carnosine with and without mechanical stress to examine one possible mechanism employed by mechanical stress to enhance CisPt effects. Carnosine is a dipeptide that reportedly sequesters platinum-based drugs away from their pharmacological target-site. Mechanical stress was achieved using an orbital shaker that produced 300 rpm with a horizontal circular motion. Our results demonstrated that mechanical stress promoted CisPt-induced death of HepG2 cells (~35% more cell death). Moreover, results showed that CisPt-induced death was compromised when CisPt was left to mix with carnosine 24 hours preceding treatment. Mechanical stress, however, ameliorated cell death (20% more cell death).

Translational Initiation at a Non-AUG Start Codon for Human and Mouse Negative Elongation Factor-B.

Negative elongation factor (NELF), a four-subunit protein complex in metazoan, plays an important role in regulating promoter-proximal pausing of RNA polymerase II (RNAPII). Genetic studies demonstrate that the B subunit of mouse NELF (NELF-B) is critical for embryonic development and homeostasis in adult tissue. We report here that both human and mouse NELF-B proteins are translated from a non-AUG codon upstream of the annotated AUG. This non-AUG codon sequence is conserved in mammalian NELF-B but not NELF-B orthologs of lower metazoan. The full-length and a truncated NELF-B that starts at the first AUG codon both interact with the other three NELF subunits. Furthermore, these two forms of NELF-B have a similar impact on the transcriptomics and proliferation of mouse embryonic fibroblasts. These results strongly suggest that additional amino acid sequence upstream of the annotated AUG is dispensable for the essential NELF function in supporting cell growth in vitro. The majority of mouse adult tissues surveyed express the full-length NELF-B protein, and some contain a truncated NELF-B protein with the same apparent size as the AUG-initiated version. This result raises the distinct possibility that translational initiation of mouse NELF-B is regulated in a tissue-dependent manner.