Mitogenic Activity Research Articles

Cancer stem cell generation by silenced MAPK enhancing PI3K/AKT signaling

Cancer stem cells, which are defined by self-renewal, differentiation potential and tumorigenicity, are proposed to be responsible for cancer initiation and maintain the tumor mass. Cancer stem cells are thought to be resistant to chemotherapy inhibiting different signaling pathways. This might sound stemness is maintained even when the growth is suppressed. Typically, growth factors are known to stimulate MEK/ERK pathways, which is responsible for mitogenic activity, while the PI3K appears more related with the maintenance of stemness. The cross-talk, even in positive or negative ways, between these two pathways could stimulate or accelerate the conversion of normal stem cells into cancer stem cells. Here, we propose a new hypothesis of a mechanism for the conversion of stem cells or progenitors including induced pluripotent stem cells (iPSCs) to tissue-specific cancer stem cells. This conversion could be prepared by inhibiting MEK/ERK pathway and enhancing PI3K/AKT pathway exploiting conditioned media derived from cancer cell lines, which are good sources of many different cytokines, chemokines, tissues-specific factors, metabolites and so on, together with some inhibitors. The feasibility of this combination will be explained in this hypothesis through the reports published somewhere. Generation of cancer stem cells using embryonic stem cells/iPSCs will bring new theory in the mechanisms of tumorigenesis and assist drug screening that applies for the precision medicines for individuals.

Antiproliferative and apoptotic effects of anti-human HB-EGF neutralizing polyclonal antibodies in vitro.

To obtain HB-EGF neutralizing polyclonal antibodies and test their anti-proliferative properties in vitro. Lab rabbits and mice were used for immunization with recombinant HB-EGF. The effect of generated polyclonal antibodies on viability and apoptosis of human epidermoid carcinoma derived A431cell line was assessed using MTT and Annexin V-propidium iodide assays. Rabbit polyclonal anti-HB-EGF serum could block binding of soluble HB-EGF to epidermal growth factor receptor/human epidermal growth factor receptor. Also, anti-HB-EGF antibodies could bind to surface of A431cells which express abnormally high levels of membrane bound proHB-EGF and its receptor. It has been shown that immune serum with polyclonal antibodies against HB-EGF was able to block the mitogenic activation of the cells with HB-EGF and cause apoptotic cell death. Inhibition of HB-EGF activity with neutralizing polyclonal antibodies can effectively inhibit mitogenic activation and cause apoptosis of cancer cells with significant epidermal growth factor receptor overexpression.

The Map3k12 (Dlk)/JNK3 signaling pathway is required for pancreatic beta-cell proliferation during postnatal development.

Unveiling the key pathways underlying postnatal beta-cell proliferation can be instrumental to decipher the mechanisms of beta-cell mass plasticity to increased physiological demand of insulin during weight gain and pregnancy. Using transcriptome and global Serine Threonine Kinase activity (STK) analyses of islets from newborn (10days old) and adult rats, we found that highly proliferative neonatal rat islet cells display a substantially elevated activity of the mitogen activated protein 3 kinase 12, also called dual leucine zipper-bearing kinase (Dlk). As a key upstream component of the c-Jun amino terminal kinase (Jnk) pathway, Dlk overexpression was associated with increased Jnk3 activity and was mainly localized in the beta-cell cytoplasm. We provide the evidence that Dlk associates with and activates Jnk3, and that this cascade stimulates the expression of Ccnd1 and Ccnd2, two essential cyclins controlling postnatal beta-cell replication. Silencing of Dlk or of Jnk3 in neonatal islet cells dramatically hampered primary beta-cell replication and the expression of the two cyclins. Moreover, the expression of Dlk, Jnk3, Ccnd1 and Ccnd2 was induced in high replicative islet beta cells from ob/ob mice during weight gain, and from pregnant female rats. In human islets from non-diabetic obese individuals, DLK expression was also cytoplasmic and the rise of the mRNA level was associated with an increase of JNK3, CCND1 and CCND2 mRNA levels, when compared to islets from lean and obese patients with diabetes. In conclusion, we find that activation of Jnk3 signalling by Dlk could be a key mechanism for adapting islet beta-cell mass during postnatal development and weight gain.

Urban Occupation Increases Water Toxicity of an Important River in Central Brazil

Meia Ponte River supplies water for two million people in Goiás State, Brazil. Despite its importance, the Meia Ponte River faces serious environmental problems such as the disposal of domestic and industrial effluents, what could impact the aquatic biota and the health of people who consumes its water. In this sense, here we aimed to evaluate the environmental quality and toxicity of surface water along the course of this river. Physicochemical analyses of water at Goiânia urban perimeter were higher than the limits of Brazil environmental regulations for fresh water. In relation to the diversity of species, phytoplankton classes associated to polluted environments were detected closer to urban perimeter. Allium cepa bioassay suggested that this river may contain substances with mitogenic activity. This result is in accordance with genotoxic analysis, because it was observed a significant increase in chromosomal aberrations. This data reveal the genotoxic potential of Meia Ponte River water. This genotoxicity represents a risk for aquatic biota and humans, once the genotoxic agents in water samples might cause the loss of DNA integrity, inducing damages and DNA breaks. In this context, the water utilization from Meia Ponte River without any treatment should be avoided and public policies need to be formulated and implemented to depollute this important river for Goiás State.

Non-Mitogenic Fibroblast Growth Factor 1 Enhanced Angiogenesis Following Ischemic Stroke by Regulating the Sphingosine-1-Phosphate 1 Pathway.

Ischemic strokes account for about 80% of all strokes and are associated with a high risk of mortality. Angiogenesis of brain microvascular endothelial cells may contribute to functional restoration following ischemia. Fibroblast growth factor 1 (FGF1), a member of FGF superfamily, involved in embryonic development, angiogenesis, wound healing, and neuron survival. However, the mitogenic activity of FGF1 is known to contribute to several human pathologies, thereby questioning the safety of its clinical applications. Here, we explored the effects and mechanism of action of non-mitogenic FGF1 (nmFGF1) on angiogenesis in mice after ischemia stroke and an oxygen-glucose deprivation (OGD)-induced human brain microvascular endothelial cells (HBMECs) injury model. We found that intranasal administration nmFGF1 significantly promoted angiogenesis in mice after stroke, and significantly increased the formation of matrigel tube and promoted scratch migration in a dose-dependent manner in OGD-induced HBMECs in vitro. However, the co-administration of an FGF receptor 1 (FGFR1)-specific inhibitor PD173074 significantly reversed the effects of nmFGF1 in vitro, suggesting that nmFGF1 functions via FGFR1 activation. Moreover, nmFGF1 activated sphingosine-1-phosphate receptor 1 (S1PR1, S1P1) in mice after stroke in vivo. S1P1 protein antagonist VPC23019 and agonist FTY720 were used to confirm that nmFGF1 promotes angiogenesis in vitro partially through the S1P1 pathway. OGD induced downregulation of S1P1 expression. The S1P1 antagonist VPC23019 blocked the stimulatory effects of nmFGF1, whereas the S1P1 agonist FTY720 exerted effects comparable with those of nmFGF1. Furthermore, PD173074 reversed the effect of nmFGF1 on upregulating S1P1 signaling. In conclusion, nmFGF1 enhanced angiogenesis in mice following stroke and OGD-induced HBMECs through S1P1 pathway regulation mediated via FGFR1 activation. This new discovery suggests the potential therapeutic role of nmFGF1 for the treatment of ischemic strokes.

PEP-1-GLRX1 Protein Exhibits Anti-Inflammatory Effects by Inhibiting the Activation of MAPK and NF-κB Pathways in Raw 264.7 Cells

Glutaredoxin 1 (GLRX1) has been recognized as an important regulator of redox signaling. Although GLRX1 plays an essential role in cell survival as an antioxidant protein, the function of GLRX1 protein in inflammatory response is still under investigation. Therefore, we wanted to know whether transduced PEP-1-GLRX1 protein inhibits lipopolysaccharide (LPS)- and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced inflammation. In LPS-exposed Raw 264.7 cells, PEP-1-GLRX1 inhibited cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), activation of mitogen activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-βB) expression levels. In a TPA-induced mouse-ear edema model, topically applied PEP-1-GLRX1 transduced into ear tissues and significantly ameliorated ear edema. Our data reveal that PEP-1-GLRX1 attenuates inflammation in vitro and in vivo, suggesting that PEP-1-GLRX1 may be a potential therapeutic protein for inflammatory diseases.

P90RSK Inhibition Ameliorates TGF-β1 Signaling and Pulmonary Fibrosis by Inhibiting Smad3 Transcriptional Activity.

Idiopathic pulmonary fibrosis (IPF) is a specific form of progressive and chronic interstitial lung disease of unknown cause. IPF is characterized by excessive deposition of extracellular matrix (ECM) and destructive pathological remodeling due to epithelial-to-mesenchymal transition (EMT). Eventually, lung interstitium thickens and stiffens and breathing becomes difficult. It has been well established that the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway plays a critical role in the pathogenesis of pulmonary fibrosis. TGF-β1-mediated activation of mitogen activated protein kinase (MAPK) family affects Smad signaling. p90RSK is a serine/threonine kinase and is activated by the extracellular signal-regulated kinase (ERK) signaling pathway. However, the roles played by p90RSK in TGF-β1 signaling and the pathogenesis of pulmonary fibrosis remain unknown. We investigated whether p90RSK regulates the pathogenesis of pulmonary fibrosis using in vitro and in vivo systems and Western blotting, real-time quantitative PCR, transcriptional activity assays and immunofluorescence studies. Pharmacological inhibition of p90RSK by FMK or inhibition of p90RSK with adenoviral vector encoding a dominant negative form of p90RSK suppressed TGF-β1-induced ECM accumulation and EMT in lung epithelial cells and fibroblasts. Interestingly, FMK significantly inhibited TGF-β1-induced Smad3 nuclear translocation and smad binding element-dependent transcriptional activity, but not Smad3 phosphorylation. Furthermore, in a mouse model of bleomycin-induced lung fibrosis, FMK ameliorated pulmonary fibrosis. These findings indicate that p90RSK plays critical roles in pulmonary fibrosis, which suggests it be viewed as a novel therapeutic target for the treatment of lung fibrosis.

Antiproliferative Effect of Acridine Chalcone Is Mediated by Induction of Oxidative Stress.

Chalcones are naturally occurring phytochemicals with diverse biological activities including antioxidant, antiproliferative, and anticancer effects. Some studies indicate that the antiproliferative effect of chalcones may be associated with their pro-oxidant effect. In the present study, we evaluated contribution of oxidative stress in the antiproliferative effect of acridine chalcone 1C ((2 E)-3-(acridin-9-yl)-1-(2,6-dimethoxyphenyl)prop-2-en-1-one) in human colorectal HCT116 cells. We demonstrated that chalcone 1C induced oxidative stress via increased reactive oxygen/nitrogen species (ROS/RNS) and superoxide production with a simultaneous weak adaptive activation of the cellular antioxidant defence mechanism. Furthermore, we also showed chalcone-induced mitochondrial dysfunction, DNA damage, and apoptosis induction. Moreover, activation of mitogen activated phosphokinase (MAPK) signalling pathway in 1C-treated cancer cells was also observed. On the other hand, co-treatment of cells with strong antioxidant, N-acetyl cysteine (NAC), significantly attenuated all of the above-mentioned effects of chalcone 1C, that is, decreased oxidant production, prevent mitochondrial dysfunction, DNA damage, and induction of apoptosis, as well as partially preventing the activation of MAPK signalling. Taken together, we documented the role of ROS in the antiproliferative/pro-apoptotic effects of acridine chalcone 1C. Moreover, these data suggest that this chalcone may be useful as a promising anti-cancer agent for treating colon cancer.

Abstract OT1-01-03: Impact of RANKL inhibition on tumor microenvironment of early-stage breast cancer, a pre-surgical trial

Abstract BACKGROUND: Receptor activator of NF-kB (RANK) and its ligand (RANKL) have a well-established role in osteoclast-driven bone remodeling, however RANK/RANKL expression can also be seen on mammary epithelial cells and immune cells (IC). In breast cancer (BC) mouse models, RANKL is a paracrine effector of the mitogenic activity of progesterone and inhibition of RANKL can attenuate development of BC as well as the incidence of metastases. Pre-clinical evidence demonstrates that RANKL exerts effects on IC such as regulatory T cells (Tregs) and tumor associated macrophages (TAMs) within the tumor microenvironment (TME), thereby promoting tumor progression and metastases. Clinical studies also support a deleterious role for these IC subsets specifically in breast cancer, with high density of either Tregs or TAMs associated with increased risk for distant recurrence. Despite this preclinical support for targeting the RANK/RANKL pathway, clinical trials in early stage BC have had discordant results regarding the clinical benefit of denosumab (RANKL antagonist). As such, it is of significant clinical relevance to identify a drug response signature following treatment as this may suggest biomarkers predictive of benefit from adjuvant denosumab. This window-of-opportunity study was designed to evaluate the pharmacodynamic biomarkers of RANKL inhibition, as well as denosumab’s impact on the TME in paired tumor specimens from patients with BC following treatment with denosumab. Defining the immunomodulatory effects of denosumab may additionally provide biologic rationale for synergistic combination with specific immunotherapy agents. TRIAL DESIGN: This study is a single-arm open label Phase 0 (pre-surgical/window of opportunity) trial. Following diagnostic core needle biopsy, patients receive one dose of denosumab 120 mg given subcutaneously prior to surgical resection. ELIGIBILITY CRITERIA: ≥18 years of age with histologically-confirmed invasive BC >1 cm by radiographic or clinical criteria with available tissue from the core biopsy and planned surgery. Patients are ineligible for this study if neoadjuvant therapy is being considered. STUDY OBJECTIVES: The primary objective is to identify pharmacodynamics markers of RANKL inhibition in early BC. Planned analyses include protein and transcriptomic analyses of known targets of RANKL and compare treatment effect in both RANK/RANKL+ and RANK/RANKL- BC. As a secondary objective, the effect of RANKL inhibition on host immune response will be assessed by comparing pre-treatment biopsy specimens and surgical excision specimens from patients treated with denosumab and untreated controls matched for BC subtype and menopausal status. Planned analyses include RANK/RANKL expression on IC subsets, enumeration of tumor infiltrating lymphocytes by H&E, immunophenotype by immunohistochemistry (e.g. cytotoxic T lymphocytes, Tregs, TAMs) and expression of immune related genes (nanoString Pancancer Immune Panel). STATISTICAL METHODS: As RANK/RANKL protein expression is expected in 25-30% of BC, we plan to enroll 35 subjects to ensure at least 10 subjects with RANK/RANKL+ tumors will be included in our study cohort. With 10 subjects with tumors with high RANK/RANKL expression at baseline, a change from baseline of a single gene of >|1.7| standard deviations between subjects with and without RANK/RANKL expression is detectable based on a 2-sided two sample t-test with power of 80% and false discovery rate of 10%. Differential gene expression analysis will be performed comparing denosumab treated patients with RANK/RANKL+ vs. RANK/RANKL- BC, and between treated patients and untreated controls. PRESENT ACCRUAL: 29 of the planned 35 patients have been enrolled to date. Citation Format: Douglas Kanter Marks, Deborah Axelrod, Maryann Kwa, Nancy Tray, Karen Hiotis, Amber Guth, Yelena Novik, James Speyer, Victor Ty, Adriana Heguy, Judith Goldberg, Farbod Darvishian, Sylvia Adams. Impact of RANKL inhibition on tumor microenvironment of early-stage breast cancer, a pre-surgical trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT1-01-03.

Abstract P5-01-25: Potential prognostic value of exosomal Beta Protein 1 in breast cancer

Abstract Background: Beta protein 1 (BP1), discovered in our lab, is an isoform protein of the DLX4 gene that is a member of the homeobox gene family. Previous work showed BP1 mRNA was activated in 80% of invasive ductal breast (IDC) tumors, where 100% of estrogen receptor (ER) negative tumor tissues and 73% of ER positive tumor tissues were BP1 positive. Moreover, high BP1 levels were associated with tumor cell survival, breast cancer aggressiveness and metastasis. BP1 overexpression was found to stimulate known oncogenes including BCL2 and c-Myc. Exosomes are nano-sized membrane-bound vesicles released by various cells and play important roles in intercellular communication. In addition, researchers found that exosomal proteins have unique characteristics compared to traditional biomarkers for carcinoma diagnosis and prognosis. Therefore, we hypothesized that BP1 protein may be packed in exosomes, and analysis of exosomal BP1 protein could provide a novel biomarker for diagnosis or prognosis of breast cancer. Materials and Methods: Exosomes were isolated using the commercially available Exosome Precipitation Solution. In cell line experiments, fluorescent immunohistochemistry was used to detect the location of BP1 protein. Levels of BP1 protein were determined in cell extracts (CE) and conditioned media (CM) using Western Blot analysis. The hypothesis that exosomal BP1 protein may be related to the development of breast cancer was investigated with clinical serum samples purchased from Capital Bioscience. Total exosomal protein levels in the serum were analyzed using BCA assay, and exosomal BP1 protein levels were determined using Western blot. In order to develop a method that could be easily used in the clinic, an ELISA assay was also designed to assess exosomal BP1 protein concentrations in the serum from metastatic breast cancer patients and normal controls. Results: It was observed that BP1 protein is localized to the nucleus, the cytoplasm and the conditioned media of MCF-7 cells. In addition, experiments with cell lines showed that the secreted BP1 protein could be internalized by cells and exhibited mitogenic activity, which is related to cancer metastasis. Moreover, it was observed that exosomal BP1 is included in secreted BP1 protein in the conditioned media. Experiments with serum samples demonstrated that the total exosomal protein levels have no significant differences between that in breast cancer patients and in normal controls. However, the results of Western blot and ELISA both showed exosomal BP1 protein levels were significantly higher in breast cancer samples compared to normal controls (P < 0.05). Moreover, Western blot results suggested that ER positive patients have much more (P < 0.05) exosomal BP1 protein in the serum compared to ER negative patients. Conclusion: It was shown for the first time that BP1 protein is exosomally packaged in breast cancer patients’ serum. The significant difference in the exosomal BP1 protein levels between the serum from women with metastatic breast cancer and that from normal controls indicated that exosomal BP1 protein in the serum could be a potential prognostic biomarker for breast cancer. Furthermore, the ELISA assay, designed to be easily used for exosomal BP1 protein quantification, may have many uses in the clinic through aiding in prognosis, monitoring therapy and early detection. Citation Format: Yaoxian Lou, Paul Goldsmith, Jinguen Rheey, Patricia E. Berg. Potential prognostic value of exosomal Beta Protein 1 in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-25.

Studying Properties of Prospective Biologically Active Extracts from Marine Hydrobionts

Biologically active substances (BAS) extracted from marine hydrobionts are characterized by high diversity and efficiency. They are represented by carotenoids, phospholipids, saponins and other compounds possessing high antioxidant (AOA), antitumor, immunomodulatory, radioprotective, and hypolipidemic activities. Because of extremely high marine BAS activity, the BAS dose necessary for treatment and prevention of many diseases is very small. The aim of present work was to assess biological properties of BAS complexes extracted from Black Sea marine inverterbrates for their following using in pharmacological preparations including liposomal drug delivery. BAS complexes were extracted from marine invertebrate tissues by two-phase extraction in combination with ultrasonication. Antioxidant activity of BAS extracts was determined with potassium permanganate discoloration method in an aqueous sulfuric acid medium. Antimicrobial activity was investigated by germination of BAS extracts. BAS influence on lymphocyte proliferative activity was studied with help of cytogenetic analysis. In this paper, the assessment of antioxidant, antimicrobial and mitogenic activities of biologically active complexes extracted from marine invertebrate tissues is presented. BAS properties in the oil and alcohol-water phases are compared. For the first time BAS complexes from Black Sea invertebrates are extracted by the resource-light method of two-phase extraction in combination with ultrasonication. Conditions for obtaining BAS extracts with optimal characteristics are proposed.

Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells

BackgroundThe R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic β-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation.ResultsrhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose.ConclusionTherefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

PEGylated leuprolide with improved pharmacokinetic properties

Leuprolide, a gonadotropin-releasing hormone (GnRH) agonist widely used in androgen deprivation therapy for the treatment of advanced prostate cancer, suffers from a short circulating half-life like other peptide therapeutics. As an attempt to improve its pharmacokinetic properties, two PEGylated leuprolides with different molecular weight were synthesized utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. The reaction conditions, including reaction temperature, reaction time and feed ratio of the reactants, were optimized to obtain a higher yield. Reverse-phase high performance liquid chromatography (RP-HPLC) characterization indicates a high purity of the resulting conjugates. Matrix-assisted laser desorption mass spectrometry (MALDI-MS) characterization suggests a 1:1 PEGylation. 1H NMR study reveals that the reaction occurs on the imidazolyl group on the histidine residue and the conjugates are stable in pH7.4 aqueous solutions. The in vitro bioactivity of the conjugates was evaluated using both hormone-sensitive and hormone-insensitive cell lines. It was found that the PEGylated peptides can still counteract the stimulatory action of androgens and the mitogenic action of epidermal growth factor on cell proliferation. The in vivo bioactivity of the conjugates was also tested. Like the unmodified peptide, administration of the conjugates to male rats leads to an initial testosterone surge, followed by a suppression of testosterone secretion. Pharmacokinetics of the drugs after i.v. and s.c. administrations were determined. In both cases, a prolonged circulating half-life, an increased AUC, and a decreased Cl_F were observed for the PEGylated drugs.

Antiproliferative effects of [D-Pro2, D-Trp7,9]-Substance P and aprepitant on several cancer cell lines and their selectivity in comparison to normal cells

Aneuropeptide, Substance P (SP), has mitogenic action in many types of cancer cells mediated via the neurokinin-1 receptor (NK1R). Small molecular NK1R antagonists have been frequently shown to possess anticancer activity both in vivo and in vitro, but there are only afew papers on such activity regarding peptide antagonists. In order to extend the data on this class of compounds, we have compared the effects of apeptide antagonist, [D-Pro2, D-Trp7,9]-Substance P, and asmall molecular antagonist, aprepitant on the proliferation of five cancer and three normal cell lines. The comparison was based on three assays: cell proliferation test, MTT test and assay for colony formation. Consistently with earlier reports, aprepitant potently reduced cell proliferation in cancer cell lines in all assays, but in contrast to previous works, the compound was not selective and it affected normal cell lines to asimilar degree. The studied peptide antagonist, [D-Pro2, D-Trp7,9]-Substance P, was able to decrease proliferation only in afew cell lines, and only in the highest concentration (100 µM). In alower concentration, aslight pro-proliferative effect was observed in afew cell lines. No statistically significant effects on colony formation were found for this compound.

Plasma HGF concentration in patients with brain tumors.

The Hepatocyte Growth Factor is a strong mitogenic factor and seems to play important role in tumor angiogenesis. The purpose of this study was to analyse the plasma concentration of this factor in patients treated surgically because of intracranial tumors. The study included 47 patients, both sexes treated surgically for intracranial tumors and 30 adult volunteers of both sexes, without cancer diagnosis. In study group 4 measurements of plasma HGF were taken: measurement 1: within 24 hours to 1 hour before the operation (preoperative), measurement 2: on the first day after the operation, i.e. after 24 hours, measurement 3: between the third and fifth day following the treatment, i.e. within 72–120 hours, and measurement 4: on the seventh day after the operation, i.e. after 840 hours. In control group only one measurement was taken. The distribution of the analyzed parameters was different from the normal distribution, therefore nonparametric statistics were used. The result values are presented in the form of a median (Me). The analysis revealed that HGR plasma levels in the patients with intracranial tumors in all 4 measurements (Me1 = 543.16 pg/ml, Me2 = 762.59 pg/ml, Me3 = 819.82 pg/ml, Me4 = 804.82 pg/ml) in the perioperative period were elevated in comparison to healthy subjects (Me = 361.04 pg/ml). The association has been shown to exist between postoperative HGF plasma levels and the clinical condition of patients with intracranial tumors (p = 0.0342). Postoperative HGF levels correlated negatively with the patients' postoperative condition. It was also found that in patients with supratentorial tumors HGF plasma levels were higher (Me = 557.74 pg/ml) in comparison to patients with posterior fossa tumors (Me = 325.00 pg/ml). These results suggest increased angiogenic and mitogenic activity in patients with intracranial tumors and its even greater intensity in the postoperative period. Greater angiogenic activity appears to occur in patients with supratentorial tumors.

CHANGES OF CYCLIN D1-DEPENDENT REGULATION OF CELL CYCLE IN PERIPHERAL BLOOD LYMPHOCYTES OF CHORNOBYL CLEAN-UP WORKERS AT A REMOTE PERIOD AFTER RADIATION EXPOSURE.

To study proliferative potential of peripheral blood lymphocytes of Chornobyl clean-up workers by levelof expression of cyclin D1 and quantitative parameters of cell cycle at a remote period after radiation exposure. The research subject was the peripheral blood lymphocytes (PB) of Chornobyl clean-upworkers 30-33 years after radiation exposure. A total of 207 men were surveyed, 164 of them were clean-up workers exposed in the dose range 10.43-3623.31 mSv and 43 persons of the control group. Analysis of proliferationpotential (cell cycle initiation) and cyclin D1 expression in PB lymphocytes were performed in vitro by a micro methodof whole blood leukocytes culture with phytohemagglutinine-P (PHA). Sample preparation was performed by a standard immunofluorescent assay for intracellular proteins using the FITC labelled Mouse Anti-Human Cyclin D1Antibody Set. Cell distribution by cell cycle phases studied by propidium iodide DNA staining and analysis onFACSCalibur laser flow cytometer in histogram mode with separation of G0/G1-, S- and G2/M-regions and Sub-G0/G1-region (apoptotic cells). An increase in the level of spontaneous сyclin D1 expression and disturbance of сyclinD1-dependent regulation of cell cycle of PB lymphocytes after mitogen activation were determined in a remote period after radiation exposure. An increase in the level of cyclin D1 expression was accompanied by increase in pool ofcells in the S- and G2/M-phases of cell cycle which characterizes the high proliferative potential of PB lymphocytes.Mitogen-induced delay of cell cycle of lymphocytes in G1/S check point and reduction of S-phase was revealed.These changes are a manifestation of genomic instability caused by the effect of radiation and depend on the radiation dose. The results confirm the hypothesis about the significance of levels of cyclin D1 expression, as a criterion for manifestations of genome instability and risks of oncogenesis in a remote period after irradiation.

The mechanism of dysbiosis in alcoholic liver disease leading to liver cancer

Currently, alcoholic liver disease (ALD) is one of the most prevalent chronic liver diseases worldwide, representing one of the main etiologies of cirrhosis and hepatocellular carcinoma (HCC). Although we do not know the exact mechanisms by which only a selected group of patients with ALD progress to the final stage of HCC, the role of the gut microbiota within the progression to HCC has been intensively studied in recent years. To date, we know that alcohol-induced gut dysbiosis is an important feature of ALD with important repercussions on the severity of this disease. In essence, an increased metabolism of ethanol in the gut induced by an excessive alcohol consumption promotes gut dysfunction and bacterial overgrowth, setting a leaky gut. This causes the translocation of bacteria, endotoxins, and ethanol metabolites across the enterohepatic circulation reaching the liver, where the recognition of the pathogen-associated molecular patterns via specific Toll-like receptors of liver cells will induce the activation of the nuclear factor kappa-B pathway, which releases pro-inflammatory cytokines and chemokines. In addition, the mitogenic activity of hepatocytes will be promoted and cellular apoptosis will be inhibited, resulting in the development of HCC. In this context, it is not surprising that microbiota-regulating drugs have proven effectiveness in prolonging the overall survival of patients with HCC, making attractive the implementation of these drugs as co-adjuvant for HCC treatment.

Paracrine Fibroblast Growth Factor 1 Functions as Potent Therapeutic Agent for Intrahepatic Cholestasis by Downregulating Synthesis of Bile Acid.

Endocrine fibroblast growth factor (FGF) 19 has been shown to be capable of maintaining bile acid (BA) homeostasis and thus hold promise to be a potential therapeutic agent for cholestasis liver disease. However, whether paracrine FGFs possess this BA regulatory activity remains to be determined. In our study, we identified that paracrine fibroblast growth factor 1 (FGF1) was selectively downregulated in the liver of alpha naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis mice, suggesting a pathological relevance of this paracrine FGF with abnormal BA metabolism. Therefore, we evaluated the effects of engineered FGF1 mutant - FGF1ΔHBS on the metabolism of hepatic BA and found that this protein showed a more potent inhibitory effect of BA biosynthesis than FGF19 without any hepatic mitogenic activity. Moreover, the chronic administration of FGF1ΔHBS protected liver against ANIT-induced injury by reducing hepatic BA accumulation. Taken together, these data suggest that FGF1ΔHBS may function as a potent therapeutic agent for intrahepatic cholestasis liver disease.

Isosteroid alkaloids with different chemical structures from Fritillariae cirrhosae bulbus alleviate LPS-induced inflammatory response in RAW 264.7 cells by MAPK signaling pathway

Isosteroid alkaloids, natural products from Fritillariae Cirrhosae Bulbus, are well known for its antitussive, expectorant, anti-asthmatic and anti-inflammatory properties. However, the anti-inflammatory effect and its mechanism have not been fully explored. In this study, the anti-inflammatory activitives and the potential mechanisms of five isosteroid alkaloids from F. Cirrhosae Bulbus were investigated in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. The pro-inflammatory mediators and cytokines were measured by Griess reagent, ELISA and qRT-PCR. The expression of MAPKs was investigated by western blotting. Treatment with the five isosteroid alkaloids in appropriate concentrations could reduce the production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in supernatant, and suppressed the mRNA expressions of TNF-α and IL-6. Meanwhile, the five isosteroid alkaloids significantly inhibited the phosphorylated activation of mitogen activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). These results demonstrated that isosteroid alkaloids from F. Cirrhosae Bulbus exert anti-inflammatory effects by down-regulating the level of inflammatory mediators via mediation of MAPK phosphorylation in LPS-induced RAW264.7 macrophages, thus could be candidates for the prevention and treatment of inflammatory diseases.

Modulation of the leptin receptors expression in breast cancer cell lines exposed to leptin and tamoxifen

One of the factors that has increased the incidence and worse prognosis of breast cancer is obesity. In this condition, high amounts of leptin are secreted, which have proliferative, mitogenic, antiapoptotic, and proinflammatory activity that may be antagonistic to treatment with tamoxifen, considered the first choice. The modulation evaluation of leptin receptor expression in the presence of leptin and tamoxifen stimuli was performed in breast cancer cell lines MCF 7, MDA MB 231 and HCC 1937 as a model of initial approach for the study of breast cancer subtypes and their behavior to the action response of adipokines and their possible relationship with the mechanism of resistance to chemotherapeutics such as tamoxifen in ER positive cell lines and triple negative marker. It was determined that leptin increases the proliferation of the three breast cancer cell lines and tamoxifen is able to exert an antiproliferative effect on them, however, it was identified that the ability of tamoxifen to decrease the proliferation of cancer cells is diminished in the presence of leptin, in addition to changes in the modulation of the expression of its receptor. It was determined that tamoxifen induces a greater modulation of the expression of ObRb in cell lines, which may be related to the decrease of its antiproliferative activity, while leptin generates a proliferative effect in the three cell lines and could participate in the tamoxifen treatment resistance mechanism.