Phytochemical Profiles and Biological Activities of Plant Extracts from Aromatic Plants Cultivated in Cyprus.

Medicinal and aromatic plants' properties, still an interesting research area, are attributed to the presence of various specialized products that possess important pharmacological activities. In the present study, six medicinal/aromatic plants (Sideritis cypria, Origanum dubium, Melissa officinalis, Mentha piperita, Thymus capitatus, and Salvia fruticosa) were evaluated for their phytochemical and nutritive composition, as well as their biological activities, including antioxidant, antimicrobial, and cytotoxic properties. The results obtained indicate that M. piperita was rich in proteins and minerals such as N and Mg, while S. cypria accumulated more K, Na, P, and Ca. The highest content of phenols and flavonoids was observed in M. piperita, followed by O. dubium and T. capitatus, which eventually influenced their high antioxidant capacity. NMR screening revealed the presence of (i) triterpenoids and hydroxycinnamic acid derivatives in M. officinalis; (ii) terpenoids, flavonoids, and phenolic acid derivatives in S. fruticosa; (iii) flavonoids and phenolic acid derivatives in M. piperita; (iv) phenolic monoterpenes in O. dubium and T. capitatus; and (v) terpenoids, flavones, and phenylethanoid glycosides in S. cypria. The results of the antimicrobial activity showed that the tested samples overall had quite good antimicrobial potential. High antibacterial activity was found in O. dubium and T. capitatus, while O. dubium and S. cypria exhibited great antifungal activities. The studied species also had an important effect on the viability of female-derived and colon cancer cells. In particular, in colon cancer cells, the extracts from T. capitatus, M. officinalis, M. piperita, and S. fruticosa exhibited a stronger effect on cell viability in the more metastatic cell line at significantly lower concentrations, indicating an important therapeutic potential in targeting highly metastatic tumors. This finding is worth further investigation. The present study unveiled interesting phytochemical profiles and biological properties of the six medicinal/aromatic plants, which should be further explored, contributing to green chemistry and the possible creation of natural health products for humans' health/nutrition and additives in cosmetics.

Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker.

Midkine (MDK) is a multifunctional secreted protein that can act as a cytokine or growth factor regulating multiple signaling pathways and being implicated in fundamental cellular processes, such as survival, proliferation, and migration. Although its expression in normal adult tissues is barely detectable, MDK serum levels are found to be elevated in several types of cancer, including hepatocellular carcinoma (HCC). In this review, we summarize the findings of recent studies on the role of MDK in HCC diagnosis and progression. Overall, studies show that MDK is a powerful biomarker for HCC early diagnosis, as it can differentiate not only between HCC patients and normal individuals but also between HCC patients and patients with other liver pathologies. It is correlated with high recurrence rates and was shown to be valuable for the diagnosis of early-stage HCC, even in patients negative for α-fetoprotein (AFP), the most commonly used biomarker for HCC diagnosis. A comparison with AFP reveals that MDK is inferior to AFP with regard to specificity but significantly superior with regard to sensitivity, which further indicates the need for using both biomarkers for more effective HCC diagnosis.

Fascin-1 in Cancer Cell Metastasis: Old Target-New Insights.

As metastasis is responsible for most cancer-related deaths, understanding the cellular and molecular events that lead to cancer cell migration and invasion will certainly provide insights into novel anti-metastatic therapeutic targets. Fascin-1 is an actin-bundling protein fundamental to all physiological or pathological processes that require cell migration. It is responsible for cross-linking actin microfilaments during the formation of actin-rich cellular structures at the leading edge of migrating cells such as filopodia, lamellipodia and invadopodia. While most epithelial tissues express low levels of Fascin-1, it is dramatically elevated in the majority of cancers and its expression has been associated with more aggressive disease and decreased overall survival. Hence, it has been proposed as a potential anti-cancer target. In the present review, we studied recent literature with regard to Fascin-1 expression in different cancers, its role in altering the mechanical properties of cancer cells, promoting cancer cell migration, invasion and metastasis and the effect of its inhibition, via various pharmacological inhibitors, in eliminating metastasis in vitro and/or in vivo. Recent studies corroborate the notion that Fascin-1 is critically involved in metastasis and prove that it is a valuable anti-metastatic target that is worth investigating further.

Ras suppressor 1 long form (RSU1L) silencing promotes apoptosis in invasive breast cancer cells.

Ras Suppressor-1 (RSU1) is a cell-extracellular matrix (ECM) adhesion protein implicated in breast cancer (BC) cell metastasis. Nevertheless, its role in apoptosis is yet unknown. In the present study, we used bioinformatics tools to evaluate the association of RSU1 expression and BC patient survival, the expression of basic pro- and anti-apoptotic genes in metastatic BC samples and their correlation with the expression of RSU1. Then, we specifically depleted RSU1 long form (RSU1L) using a short hairpin RNA (shRNA) silencing approach in two BC cell lines, the non-invasive MCF-7 and the highly invasive MDA-MB-231-LM2 cells and assessed gene expression of pro-and anti-apoptotic genes, as well as cell survival and apoptosis. Our results showed that high RSU1 expression was correlated with poor survival and significant changes were found in the expression of apoptosis-related genes (PUMA, TP53, BCL-2 and BCL-XL) in metastatic BC. Moreover, silencing of the long and most common isoform of RSU1 (RSU1L) resulted in the upregulation of PUMA and TP53 and concomitant downregulation of anti-apoptotic BCL-2 and BCL-XL, with the effect being more prominent in invasive MDA-MB-231-LM2 cells. Finally, RSU1L depletion leads to a dramatic increase in apoptosis of MDA-MB-231-LM2 cells, while no change was observed in the apoptotic rate of MCF-7 cells. This is the first study linking RSU1L with apoptosis and provides evidence for its differential role in cell lines of different invasive potential. This indicates that RSU1L represses apoptosis in aggressive BC cells helping them evade cell death and survive.

Effects of 2-methoxyestradiol on hydrogen peroxide induced neuronal cell death and tau hyperphosphorylation.

AIMS: Alzheimer's Disease (AD) is characterized by progressive cognitive impairment, and memory loss. It has been shown that depletion of estrogens renders women vulnerable to AD with menopause women presenting higher risk for AD development than men. However, women under hormone replacement therapy (HRT) with 17ß-estradiol (E2) show lower risk for AD, implying that E2 may be protective. It has been shown that E2 exerts its effects through the estrogen receptor (ER) but also via its biologically active metabolites, 2-hydroxyestradiol (2OH), and 2-methoxyestradiol (2ME). We hypothesized that the neuroprotective effects of E2 are partly attributed to its metabolites. MATERIALS AND METHODS: SH-SY5Y neuronal cells were subjected oxidative stress (OS) cell death by hydrogen peroxide (H2O2), in the presence or absence of E2, 2ME and 2OH. Viability was assessed by trypan blue and thiazolyl blue tetrazolium bromide assays, intracellular OS with the Dichlorodihydrofluorescein Diacetate (DCFDA) assay, and Bax, p53 and PUMA quantified by RT-PCR. Tau hyperphosphorylation was studied by western blot. KEY FINDINGS: E2 and its metabolites 2OH and 2ME protect from cell death as assessed by the viability assays. Their effect was partly attributed to their antioxidant properties evidenced by the reduction of intracellular OS. Treatment with 2ME resulted in a reduction of Bax, but not p53 or PUMA in cells challenged with OS. Finally, 2ME was able to inhibit tau hyperphosphorylation as well. SIGNIFICANCE: E2 protects neuron cells partly through its metabolites. Further studies are needed to fully delineate the mechanism for this protection.

LincRNAs and snoRNAs in Breast Cancer Cell Metastasis: The Unknown Players.

Recent advances in research have led to earlier diagnosis and targeted therapies against breast cancer, which has resulted in reduced breast cancer-related mortality. However, the majority of breast cancer-related deaths are due to metastasis of cancer cells to other organs, a process that has not been fully elucidated. Among the factors and genes implicated in the metastatic process regulation, non-coding RNAs have emerged as crucial players. This review focuses on the role of long intergenic noncoding RNAs (lincRNAs) and small nucleolar RNAs (snoRNAs) in breast cancer cell metastasis. LincRNAs are transcribed between two protein-coding genes and are longer than 200 nucleotides, they do not code for a specific protein but function as regulatory molecules in processes such as cell proliferation, apoptosis, epithelial-to-mesenchymal transition, migration, and invasion while most of them are highly elevated in breast cancer tissues and seem to function as competing endogenous RNAs (ceRNAs) inhibiting relevant miRNAs that specifically target vital metastasis-related genes. Similarly, snoRNAs are 60-300 nucleotides long and are found in the nucleolus being responsible for the post-transcriptional modification of ribosomal and spliceosomal RNAs. Most snoRNAs are hosted inside intron sequences of protein-coding and non-protein-coding genes, and they also regulate metastasis-related genes affecting related cellular properties.

The focal adhesion protein Integrin-Linked Kinase (ILK) as an important player in breast cancer pathogenesis.

Cell-extracellular matrix interactions, or focal adhesions (FA), are crucial for tissue homeostasis but are also implicated in cancer. Integrin-Linked Kinase (ILK) is an abundantly expressed FA protein involved in multiple signaling pathways. Here, we reviewed the current literature on the role of ILK in breast cancer (BC). Articles included in vitro and in vivo experiments as well as studies in human BC samples. ILK attenuation via silencing or pharmaceutical inhibition, leads to apoptosis or inhibition of epithelial-to-mesenchymal transition, and cell invasion whereas ILK overexpression suppresses anoikis and promotes tumor growth and metastasis. Finally, ILK is upregulated in BC tumors and its expression is associated with grade, and metastasis. Therefore, ILK should be evaluated as a potential anti-cancer pharmaceutical target.

Ras Suppressor-1 (RSU1) in Cancer Cell Metastasis: A Tale of a Tumor Suppressor.

Cancer is a multifactorial disease responsible for millions of deaths worldwide. It has a strong genetic background, as mutations in oncogenes or tumor suppressor genes contribute to the initiation of cancer development. Integrin signaling as well as the signaling pathway of Ras oncogene, have been long implicated both in carcinogenesis and disease progression. Moreover, they have been involved in the promotion of metastasis, which accounts for the majority of cancer-related deaths. Ras Suppressor-1 (RSU1) was identified as a suppressor of Ras-induced transformation and was shown to localize to cell-extracellular matrix adhesions. Recent findings indicate that its expression is elevated in various cancer types, while its role in regulating metastasis-related cellular processes remains largely unknown. Interestingly, there is no in vivo work in the field to date, and thus, all relevant knowledge stems from in vitro studies. In this review, we summarize recent studies using breast, liver and brain cancer cell lines and highlight the role of RSU1 in regulating cancer cell invasion.

ILK silencing inhibits migration and invasion of more invasive glioblastoma cells by downregulating ROCK1 and Fascin-1.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor and it is associated with poor survival. Integrin-linked kinase (ILK) is a serine/threonine protein pseudo-kinase that binds to the cytoplasmic domains of ß1 and ß3 integrins and has been previously shown to promote invasion and metastasis in many cancer types, including GBM. However, little is known regarding the exact molecular mechanism implicating ILK in GBM aggressiveness. In this study, we used two brain cell lines, the non-invasive neuroglioma H4 cells, and the highly invasive glioblastoma A172 cells, which express ILK in much higher levels than H4. We studied the effect of ILK silencing on the metastatic behavior of glioblastoma cells in vitro and elucidate the underlying molecular mechanism. We showed that siRNA-mediated silencing of ILK inhibits cell migration and invasion of the highly invasive A172 cells while it does not affect the migratory and invasive capacity of H4 cells. These data were also supported by respective changes in the expression of Rho-associated kinase 1 (ROCK1), fascin actin-bundling protein 1 (FSCN1), and matrix metalloproteinase 13 (MMP13), which are known to regulate cell migration and invasion. Our findings were further corroborated by analyzing the Cancer Genome Atlas Glioblastoma Multiforme (TCGA-GBM) dataset. We conclude that ILK promotes glioblastoma cell invasion through activation of ROCK1 and FSCN1 in vitro, providing a more exact molecular mechanism for its action.

Growth differentiation factor 15 (GDF15) in cancer cell metastasis: from the cells to the patients.

Growth differentiation factor 15 (GDF15), a member of the transforming growth factor ß superfamily, has been postulated to be implicated in cancer cell metastasis although its role has not been fully elucidated yet. The purpose of this review is to clarify the role of GDF-15 in cancer cell metastasis based on current advances in the field. The studies were divided into those involving evaluation of GDF15 expression in the serum or tissue of cancer patients, and those involving in vitro experiments in cancer cell lines or in vivo experiments in animal models. GDF15 was shown to be elevated in the serum or tissues of cancer patients with its expression being correlated with decreased survival. Moreover, most in vitro and in vivo studies also corroborated a metastasis-promoting role for GDF15. However, there were a few studies, where GDF15 was shown to suppress the metastatic properties of cells. As, GDF15 has been known for its pleiotropic effects, it is not surprising to behave differently in different types of cancer. Thus, GDF15 has the potential of not only being a useful metastasis biomarker, but also a promising therapeutic target against cancer cell metastasis in many cancer types.

Silencing of Growth Differentiation Factor-15 Promotes Breast Cancer Cell Invasion by Down-regulating Focal Adhesion Genes.

BACKGROUND/AIM: As metastasis accounts for most breast cancer (BC)-related deaths, identifying key players becomes research priority. Growth differentiation factor-15 (GDF15), a member of the transforming growth factor-ß superfamily, is affected by the actin cytoskeleton and has been associated with cancer. However, its exact role in BC cell invasiveness is vague. MATERIALS AND METHODS: GDF15 short-hairpin (shRNA)-mediated silencing was used to inhibit GDF15 expression in MCF-7 and MDA-MB-231 BC cells and gene expression of relevant focal adhesion (FA) genes, cell migration, invasion and tumor spheroid invasion were subsequently analyzed. RESULTS: GDF15 silencing promoted cell migration, cell invasion as well as tumor spheroid invasion and up-regulated urokinase plasminogen activator (uPA) and FA genes, integrin-linked kinase (ILK), LIM zinc finger domain containing 1 (LIMS1), α-parvin (PARVA), and RAS suppressor-1 (RSU1). Computational analysis of Cancer Genome Atlas BC dataset however, revealed no significant correlation between GDF15 expression and metastasis pointing towards a more complex molecular interplay between GDF15, actin cytoskeleton and FA-related genes which ultimately affects their expression pattern, in vivo. CONCLUSION: GDF15 suppresses BC cell invasion in vitro through down-regulation of FA genes but its role in BC is more complicated in vivo and warrants further investigation.

Coordinated Expression of Ras Suppressor 1 (RSU-1) and Growth Differentiation Factor 15 (GDF15) Affects Glioma Cell Invasion.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor due to its invasive phenotype. Ras suppressor 1 (RSU-1) is a cell-extracellular matrix adhesion protein and we recently found that it promotes cell invasion in aggressive cells and inhibits it in non-invasive. Growth differentiation factor-15 (GDF15) is known to be involved in actin cytoskeleton reorganization and metastasis. In this study, we used three brain cell lines (H4, SW1088 and A172) with increasing RSU-1 expression levels and invasive capacity and decreasing GDF15 levels to investigate the interplay between RSU-1 and GDF15 with regard to cell invasion. Four experimental approaches were used: (a) GDF15 treatment, (b) Rsu-1 silencing, (c) GDF15 silencing, and (d) combined GDF15 treatment and RSU-1 silencing. We found that the differential expression of RSU-1 and GDF15 in H4 and A172 cells leading to inhibition of cell invasion in H4 cells and promotion in A172 through respective changes in PINCH1, RhoA and MMP-13 expression. Interestingly SW1088, with intermediate RSU-1 and GDF15 expression, were not affected by any treatment. We conclude that there is a strong connection between RSU-1 and GDF15 in H4, SW1088 and A172 cells and the relative expression of these two proteins is fundamental in affecting their invasive fate.

Depletion of Ras Suppressor-1 (RSU-1) promotes cell invasion of breast cancer cells through a compensatory upregulation of a truncated isoform.

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.

Ras suppressor-1 (RSU-1) promotes cell invasion in aggressive glioma cells and inhibits it in non-aggressive cells through STAT6 phospho-regulation.

Most gliomas are invasive tumors formed from glial cells and associated with high mortality rates. In this study, we characterized four glioma cell lines of varying degree of aggressiveness (H4, SW1088, A172 and U87-MG) in terms of morphology, cytoskeleton organization and stiffness, and evaluated their invasive potential by performing invasion, colony forming and spheroid invasion assays. Cells were divided into two distinct groups: aggressive cell lines (A172 and U87-MG) with more elongated, softer and highly invasive cells and less aggressive cells (H4 and SW088). Interestingly, we found that Ras Suppressor-1 (RSU-1), a cell-matrix adhesion protein involved in cancer cell invasion, was significantly upregulated in more aggressive glioma cells compared to less aggressive. Importantly, RSU-1 silencing had opposing effects on glioma cell invasion depending on their aggressiveness, inhibiting migration and invasion of aggressive cells and promoting those of less aggressive cells. Finally, we found that RSU-1 silencing in aggressive cells led to decreased Signal Transducer and Activator of Transcription6 (STAT6) phosphorylation and Matrix Metalloproteinase13 (MMP13) expression in contrast to less invasive cells. Our study demonstrates that RSU-1 promotes invasion of aggressive glioma cells and inhibits it in the non-aggressive cells, indicating that it could serve as a predictor of gliomas progression.

Collagen content and extracellular matrix cause cytoskeletal remodelling in pancreatic fibroblasts.

In many solid tumours a desmoplastic reaction takes place, which results in tumour tissue stiffening due to the extensive production of extracellular matrix (ECM) proteins, such as collagen, by stromal cells, mainly fibroblasts (FBs) and cancer-associated fibroblasts (CAFs). In this study, we investigated the effect of collagen stiffness on pancreatic FBs and CAFs, particularly on specific cytoskeleton properties and gene expression involved in tumour invasion. We found that cells become stiffer when they are cultured on stiff substrates and express higher levels of alpha-smooth muscle actin (α-SMA). Also, it was confirmed that on stiff substrates, CAFs are softer than FBs, while on soft substrates they have comparable Young's moduli. Furthermore, the number of spread FBs and CAFs was higher in stiffer substrates, which was also confirmed by Ras-related C3 botulinum toxin substrate 1 ( RAC1) mRNA expression, which mediates cell spreading. Although stress fibres in FBs become more oriented on stiff substrates, CAFs have oriented stress fibres regardless of substrate stiffness. Subsequently, we demonstrated that cells' invasion has a differential response to stiffness, which was associated with regulation of Ras homologue family member ( RhoA) and Rho-associated, coiled-coil containing protein kinase 1 ( ROCK-1) mRNA expression. Overall, our results demonstrate that collagen stiffness modulates FBs and CAFs cytoskeleton remodelling and alters their invasion properties.

Inhibition of Breast Cancer Cell Invasion by Ras Suppressor-1 (RSU-1) Silencing Is Reversed by Growth Differentiation Factor-15 (GDF-15).

Extracellular matrix (ECM)-related adhesion proteins are important in metastasis. Ras suppressor-1 (RSU-1), a suppressor of Ras-transformation, is localized to cell⁻ECM adhesions where it interacts with the Particularly Interesting New Cysteine-Histidine rich protein (PINCH-1), being connected to Integrin Linked Kinase (ILK) and alpha-parvin (PARVA), a direct actin-binding protein. RSU-1 was also found upregulated in metastatic breast cancer (BC) samples and was recently demonstrated to have metastasis-promoting properties. In the present study, we transiently silenced RSU-1 in BC cells, MCF-7 and MDA-MB-231. We found that RSU-1 silencing leads to downregulation of Growth Differentiation Factor-15 (GDF-15), which has been associated with both actin cytoskeleton reorganization and metastasis. RSU-1 silencing also reduced the mRNA expression of PINCH-1 and cell division control protein-42 (Cdc42), while increasing that of ILK and Rac regardless of the presence of GDF-15. However, the downregulation of actin-modulating genes PARVA, RhoA, Rho associated kinase-1 (ROCK-1), and Fascin-1 following RSU-1 depletion was completely reversed by GDF-15 treatment in both cell lines. Moreover, complete rescue of the inhibitory effect of RSU-1 silencing on cell invasion was achieved by GDF-15 treatment, which also correlated with matrix metalloproteinase-2 expression. Finally, using a graph clustering approach, we corroborated our findings. This is the first study providing evidence of a functional association between RSU-1 and GDF-15 with regard to cancer cell invasion.

The role of fibroblast growth factors and their receptors in gliomas: the mutations involved.

The central nervous system (CNS) comprises of neurons, which are responsible for impulse transmission, and glial cells, which surround neurons providing protection and nutrition. Glial cells are categorized into astrocytes, oligodendrocytes, microglial cells, and ependymal cells. Tumors forming from glial cells are called gliomas, and they are classified accordingly into astrocytomas, oligodendrogliomas, and ependymomas. Gliomas are characterized by high mortality rates and degree of malignancy, heterogeneity, and resistance to treatment. Among the molecular players implicated in glioma pathogenesis are members of the fibroblast growth factor (FGF) superfamily as well as their receptors (FGFRs). In the present study, we provide a review of the literature on the role of FGFs and FGFRs in glioma pathogenesis. We also demonstrate that FGFs, and particularly FGF1 and FGF2, bear a variety of mutations in gliomas, while FGFRs are also crucially involved. In fact, several studies show that in gliomas, FGFRs bear mutations, mainly in the tyrosine kinase domains. Specifically, it appears that FGFR1-TACC1 and FGFR3-TACC3 fusions are common in these receptors. A better understanding of the mutations and the molecular players involved in glioma formation will benefit the scientific community, leading to the development of more effective and innovative therapeutic approaches.

Atomic force microscopy nano-characterization of 3D collagen gels with tunable stiffness.

As extracellular matrix (ECM) nano-characteristics play a crucial role in cell behavior, including cancer development and metastasis, several ECM in vitro models have been used in order to study cells behavior under different biochemical and mechanical conditions. Among the ECM constituents, collagen (especially collagen type I) has been extensively used as an essential component of ECM models, since it is one of the most abundant ECM protein. Use of three-dimensional (3D) collagen gels provides the advantage of allowing the cells to grow in a 3D environment that bears strong similarities to their natural, in vivo setting. Thus, the ability to form collagen gels with tunable stiffness and well defined naturally occurring nano-characteristics is crucial for these studies. Atomic Force Microscopy (AFM) is a unique tool that is ideal for the complete characterization of such models, in terms of morphology and mechanical properties without destroying the collagen fiber structure. In this protocol, the development and the AFM nano-scale characterization of 3D collagen type I gels is presented. The protocol includes: •The formation of 3D collagen type I gels with tunable stiffness•The preparation of histological sections from collagen gels•The AFM-based morphological and mechanical nano-characterization of the gels.