Nitric Oxide Prevents Glioblastoma Stem Cells' Expansion and Induces Temozolomide Sensitization.

Glioblastoma multiforme (GBM) has high mortality and recurrence rates. Malignancy resilience is ascribed to Glioblastoma Stem Cells (GSCs), which are resistant to Temozolomide (TMZ), the gold standard for GBM post-surgical treatment. However, Nitric Oxide (NO) has demonstrated anti-cancer efficacy in GBM cells, but its potential impact on GSCs remains unexplored. Accordingly, we investigated the effects of NO, both alone and in combination with TMZ, on patient-derived GSCs. Experimentally selected concentrations of diethylenetriamine/NO adduct and TMZ were used through a time course up to 21 days of treatment, to evaluate GSC proliferation and death, functional recovery, and apoptosis. Immunofluorescence and Western blot analyses revealed treatment-induced effects in cell cycle and DNA damage occurrence and repair. Our results showed that NO impairs self-renewal, disrupts cell-cycle progression, and expands the quiescent cells' population. Consistently, NO triggered a significant but tolerated level of DNA damage, but not apoptosis. Interestingly, NO/TMZ cotreatment further inhibited cell cycle progression, augmented G0 cells, induced cell death, but also enhanced DNA damage repair activity. These findings suggest that, although NO administration does not eliminate GSCs, it stunts their proliferation, and makes cells susceptible to TMZ. The resulting cytostatic effect may potentially allow long-term control over the GSCs' subpopulation.

Cripowellins Pause Plasmodium falciparum Intraerythrocytic Development at the Ring Stage.

Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B "pauses" the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.

Half-Sandwich Type Platinum-Group Metal Complexes of C-Glucosaminyl Azines: Synthesis and Antineoplastic and Antimicrobial Activities.

While platinum-based compounds such as cisplatin form the backbone of chemotherapy, the use of these compounds is limited by resistance and toxicity, driving the development of novel complexes with cytostatic properties. In this study, we synthesized a set of half-sandwich complexes of platinum-group metal ions (Ru(II), Os(II), Ir(III) and Rh(III)) with an N,N-bidentate ligand comprising a C-glucosaminyl group and a heterocycle, such as pyridine, pyridazine, pyrimidine, pyrazine or quinoline. The sugar-containing ligands themselves are unknown compounds and were obtained by nucleophilic additions of lithiated heterocycles to O-perbenzylated 2-nitro-glucal. Reduction of the adducts and, where necessary, subsequent protecting group manipulations furnished the above C-glucosaminyl heterocycles in their O-perbenzylated, O-perbenzoylated and O-unprotected forms. The derived complexes were tested on A2780 ovarian cancer cells. Pyridine, pyrazine and pyridazine-containing complexes proved to be cytostatic and cytotoxic on A2780 cells, while pyrimidine and quinoline derivatives were inactive. The best complexes contained pyridine as the heterocycle. The metal ion with polyhapto arene/arenyl moiety also impacted on the biological activity of the complexes. Ruthenium complexes with p-cymene and iridium complexes with Cp* had the best performance in ovarian cancer cells, followed by osmium complexes with p-cymene and rhodium complexes with Cp*. Finally, the chemical nature of the protective groups on the hydroxyl groups of the carbohydrate moiety were also key determinants of bioactivity; in particular, O-benzyl groups were superior to O-benzoyl groups. The IC50 values of the complexes were in the low micromolar range, and, importantly, the complexes were less active against primary, untransformed human dermal fibroblasts; however, the anticipated therapeutic window is narrow. The bioactive complexes exerted cytostasis on a set of carcinomas such as cell models of glioblastoma, as well as breast and pancreatic cancers. Furthermore, the same complexes exhibited bacteriostatic properties against multiresistant Gram-positive Staphylococcus aureus and Enterococcus clinical isolates in the low micromolar range.

ABT-751 Induces Multiple Anticancer Effects in Urinary Bladder Urothelial Carcinoma-Derived Cells: Highlighting the Induction of Cytostasis through the Inhibition of SKP2 at Both Transcriptional and Post-Translational Levels.

The objective was to investigate the anti-cancer effects and underlying molecular mechanisms of cytostasis which were activated by an anti-microtubule drug, ABT-751, in two urinary bladder urothelial carcinoma (UBUC)-derived cell lines, BFTC905 and J82, with distinct genetic backgrounds. A series of in vitro assays demonstrated that ABT-751 induced G2/M cell cycle arrest, decreased cell number in the S phase of the cell cycle and suppressed colony formation/independent cell growth, accompanied with alterations of the protein levels of several cell cycle regulators. In addition, ABT-751 treatment significantly hurdled cell migration and invasion along with the regulation of epithelial-mesenchymal transition-related proteins. ABT-751 triggered autophagy and apoptosis, downregulated the mechanistic target of rapamycin kinase (MTOR) and upregulated several pro-apoptotic proteins that are involved in extrinsic and intrinsic apoptotic pathways. Inhibition of autophagosome and autolysosome enhanced apoptosis was also observed. Through the inhibition of the NFκB signaling pathway, ABT-751 suppressed S-phase kinase associated protein 2 (SKP2) transcription and subsequent translation by downregulation of active/phospho-AKT serine/threonine kinase 1 (AKT1), component of inhibitor of nuclear factor kappa B kinase complex (CHUK), NFKB inhibitor alpha (NFKBIA), nuclear RELA proto-oncogene, NFκB subunit (RELA) and maintained a strong interaction between NFKBIA and RELA to prevent RELA nuclear translocation for SKP2 transcription. ABT-751 downregulated stable/phospho-SKP2 including pSKP2(S64) and pSKP2(S72), which targeted cyclin-dependent kinase inhibitors for degradation through the inactivation of AKT. Our results suggested that ABT-751 may act as an anti-cancer drug by inhibiting cell migration, invasion yet inducing cell cycle arrest, autophagy and apoptosis in distinct UBUC-derived cells. Particularly, the upstream molecular mechanism of its anticancer effects was identified as ABT-751-induced cytostasis through the inhibition of SKP2 at both transcriptional and post-translational levels to stabilize cyclin dependent kinase inhibitor 1A (CDKN1A) and CDKN1B proteins.

Response of neuroblastoma cells to RF currents as a function of the signal frequency.

BACKGROUND: Capacitive-resistive electric transfer (CRET) is a non-invasive therapeutic strategy that applies radiofrequency electric currents within the 400-600 kHz range to tissue repair and regeneration. Previous studies by our group have shown that 48 h of intermittent exposure to a 570 kHz CRET signal at a subthermal density of 50 µA/mm2 causes significant changes in the expression and activation of cell cycle control proteins, leading to cycle arrest in human cancer cell cultures. The present study investigates the relevance of the signal frequency in the response of the human neuroblastoma cell line NB69 to subthermal electric treatment with four different signal frequency currents within the 350-650 kHz range. METHODS: Trypan blue assay, flow cytometry, immunofluorescence and immunoblot were used to study the effects of subthermal CRET currents on cell viability, cell cycle progression and the expression of several marker proteins involved in NB69 cell death and proliferation. RESULTS: The results reveal that among the frequencies tested, only a 448 kHz signal elicited both proapoptotic and antiproliferative, statistically significant responses. The apoptotic effect would be due, at least in part, to significant changes induced by the 448 kHz signal in the expression of p53, Bax and caspase-3. The cytostatic response was preceded by alterations in the kinetics of the cell cycle and in the expression of proteins p-ERK1/2, cyclin D1 and p27, which is consistent with a potential involvement of the EGF receptor in electrically induced changes in the ERK1/2 pathway. This receives additional support from results indicating that the proapototic and antiproliferative responses to CRET can be transiently blocked when the electric stimulus is applied in the presence of PD98059, a chemical inhibitor of the ERK1/2 pathway. CONCLUSION: The understanding of the mechanisms underlying the ability of slowing down cancer cell growth through electrically-induced changes in the expression of proteins involved in the control of cell proliferation and apoptosis might afford new insights in the field of oncology.

Absence of herb-drug interactions of mistletoe with the tamoxifen metabolite (E/Z)-endoxifen and cytochrome P450 3A4/5 and 2D6 in vitro.

BACKGROUND: Women diagnosed with breast cancer frequently seek complementary and alternative (CAM) treatment options that can help to cope with their disease and the side effects of conventional cancer therapy. Especially in Europe, breast cancer patients use herbal products containing mistletoe (Viscum album L.). The oldest and one of the most prescribed conventional drugs for the treatment of estrogen receptor positive breast cancer is tamoxifen. Aside from positive clinical experience with the combination of tamoxifen and mistletoe, little is known about possible herb-drug interactions (HDIs) between the two products. In the present in vitro study, we investigated the effect of standardized commercial mistletoe preparations on the activity of endoxifen, the major active metabolite of tamoxifen. METHODS: The estrogen receptor positive human breast carcinoma cell line MCF-7 was treated with (E/Z)-endoxifen hydrochloride in the presence and absence of a defined estradiol concentration. Each concentration of the drug was combined with fermented Viscum album L. extracts (VAE) at clinically relevant doses, and proliferation, apoptosis and cell cycle were analyzed. In parallel, possible inhibition of CYP3A4/5 and CYP2D6 was investigated using 50-donor mixed gender pooled human liver microsomes (HLMs). RESULTS: VAE did not inhibit endoxifen induced cytostasis and cytotoxicity. At higher concentrations, VAE showed an additive inhibitory effect. VAE preparations did not cause inhibition of CYP3A4/5 and CYP2D6 catalyzed tamoxifen metabolism. CONCLUSIONS: The in vitro results suggest that mistletoe preparations can be used in combination with tamoxifen without the risk of HDIs.

Costunolide induces micronuclei formation, chromosomal aberrations, cytostasis, and mitochondrial-mediated apoptosis in Chinese hamster ovary cells.

Costunolide (CE) is a sesquiterpene lactone well-known for its antihepatotoxic, antiulcer, and anticancer activities. The present study focused on the evaluation of the cytogenetic toxicity and cellular death-inducing potential of CE in CHO cells, an epithelial cell line derived from normal ovary cells of Chinese hamster. The cytotoxic effect denoting MTT assay has shown an IC50 value of 7.56 µM CE, where 50% proliferation inhibition occurs. The oxidative stress caused by CE was confirmed based on GSH depletion induced cell death, conspicuously absent in N-acetylcysteine (GSH precursor) pretreated cells. The evaluation of genotoxic effects of CE using cytokinesis block micronucleus assay and chromosomal aberration test has shown prominent induction of binucleated micronucleated cells and aberrant metaphases bearing chromatid and chromosomal breaks, indicating CE's clastogenic and aneugenic potential. The apoptotic death in CE treated cells was confirmed by an increase in the number of cells in subG1 phase, exhibiting chromatin condensation and membranous phosphatidylserine translocation. The apoptosis induction follows mitochondrial mediation, evident from an increase in the BAX/Bcl-2 ratio, caspase-3/7 activity, and mitochondrial membrane permeability. CE also induces cytostasis in addition to apoptosis, substantiated by the reduced cytokinetic (replicative indices) and mitotic (mitotic indices and histone H3 Ser-10 phosphorylation) activities. Overall, the cellular GSH depletion and potential genotoxic effects by CE led the CHO cells to commit apoptosis and lowered cell division. The observed sensitivity of CHO cells doubts unintended adverse effects of CE on normal healthy cells, suggesting higher essentiality of further studies in order to establish its safety efficacy in therapeutic explorations.

Simultaneous Interference with HER1/EGFR and RAC1 Signaling Drives Cytostasis and Suppression of Survivin in Human Glioma Cells in Vitro.

We have previously reported that combined inhibition of the epidermal growth factor receptor by erlotinib and of RAC1 by NSC23766 yielded a synergistic antiproliferative effect on established and primary cultured glioblastoma cells. The current study aimed at identifying the molecular mechanism. Staining for annexin V/PI or carboxyfluorescein succinimidyl ester was performed in order to determine the induction of apoptosis, necrosis or cytostasis in established and primary cultured glioblastoma cells. Moreover, expression of Ki-67 was determined by immunofluorescence, and the expression of cell cycle proteins was analysed by Western blot. Our data show that combined treatment with erlotinib and NSC23766 resulted in a reduced number of cell divisions, a significantly decreased Ki-67 expression, increased apoptosis and autophagy when compared to single agent treatments. On the molecular level, concomitant treatment with both agents resulted in a pronounced downregulation of cyclin D1, cyclin-dependent kinases 2, 4 and 6, as well as of survivin when compared to treatments with either agent alone. In conclusion, we demonstrate that combined treatment of human glioma cell lines in vitro with erlotinib and NSC23766 markedly inhibits cell division, induces apoptosis independent of caspase-3 activation and induces autophagy concomitant with suppression of survivin.

Synthesis and biological evolution of hydrazones derived from 4-(trifluoromethyl)benzohydrazide.

Reflecting the known biological activity of isoniazid-based hydrazones, seventeen hydrazones of 4-(trifluoromethyl)benzohydrazide as their bioisosters were synthesized from various benzaldehydes and aliphatic ketones. The compounds were screened for their in vitro activity against Mycobacterium tuberculosis, nontuberculous mycobacteria (M. avium, M. kansasii), bacterial and fungal strains. The most antimicrobial potent derivatives were also investigated for their cytostatic and cytotoxic properties against three cell lines. Camphor-based molecule, 4-(trifluoromethyl)-N'-(1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide, exhibited the highest and selective inhibition of M. tuberculosis with the minimum inhibitory concentration (MIC) of 4 µM, while N'-(4-chlorobenzylidene)-4-(trifluoromethyl)benzohydrazide was found to be superior against M. kansasii (MIC = 16 µM). N'-(5-Chloro-2-hydroxybenzylidene)-4-(trifluoromethyl)benzohydrazide showed the lowest MIC values for gram-positive bacteria including methicillin-resistant Staphylococcus aureus as well as against two fungal strains of Candida glabrata and Trichophyton mentagrophytes within the range of ≤0.49-3.9 µM. The convenient substitution of benzylidene moiety at the position 4 or the presence of 5-chloro-2-hydroxybenzylidene scaffold concomitantly with a sufficient lipophilicity are essential for the noticeable antimicrobial activity. This 5-chlorosalicylidene derivative avoided any cytotoxicity on two mammalian cell cultures (HepG2, BMMΦ) up to the concentration of 100 µM, but it affected the growth of MonoMac6 cells.

Comparative study on the induction of complex genomic alterations after exposure of mammalian cells to carboplatin and oxaliplatin.

Metal complexes are still broadly used as the first line of the treatment for different types of tumors nowadays. Carboplatin and oxaliplatin were authorized for clinical use, even though there is little information on the mutagenic profile associated to their usage. This study evaluated the cytostatic effects and the induction of complex genomic alterations after 24-h treatment of CHO-K1 cells to concentrations of 12.5-800 µM of carboplatin and oxaliplatin in the cytokinesis-block micronucleus assay (CBMN-Cyt). The results demonstrated that carboplatin and oxaliplatin significantly increased the frequency of micronuclei (MN), nucleoplasmatic bridges (NPBs), and nuclear buds (NBUDs). On one hand, oxaliplatin induces significantly more chromosomal abnormalities than carboplatin at concentrations of 12.5 and 25 µM. On the other hand, carboplatin, in cells exposed to concentrations of 50 and 100 µM, is more efficient than oxaliplatin in the induction of chromosomal instability events. Both drugs cause significant reduction in the cytokinesis-block proliferation index, demonstrating their cytostatic effects at concentrations 50-800 µM. The results of this study shed more light on the characterization of biological effects associated with the exposure to carboplatin and oxaliplatin.

Interaction of a standardized mistletoe (Viscum album) preparation with antitumor effects of Trastuzumab in vitro.

BACKGROUND: Besides conventional anticancer therapy many breast cancer patients use complementary and alternative medicine (CAM) like the medicinal herb mistletoe (Viscum album L.). To gain more knowledge about possible herb-drug interactions between CAM and conventional anticancer medications, in the present in vitro study we investigated the effect of a standardized mistletoe preparation on the action of Trastuzumab, a drug used for the treatment of Her-2 positive breast cancer. METHODS: The Her-2 positive human breast carcinoma cell line SK-BR-3 was treated with Trastuzumab. Different doses of the drug were combined with Viscum album extract (VAE) in clinically relevant doses. Proliferation, apoptosis, cell cycle and the secretion of vascular endothelial growth factor (VEGF) were analyzed. RESULTS: No inhibition of antitumor efficacy of Trastuzumab by VAE was detected. VAE and Trastuzumab, either alone or in combination, inhibited proliferation of SK-BR-3 cells in vitro. At higher concentrations VAE induced apoptosis, which was not observed for Trastuzumab. Cells treated with Trastuzumab underwent a G0/G1 cell cycle arrest and cells treated with VAE a G2/M arrest. After application of the two drugs in combination both G0/G1 and G2/M arrest was observed. VEGF secretion of SK-BR-3 cells was significantly inhibited by sole treatment with Trastuzumab or VAE. Combined treatment of Trastuzumab and VAE at clinically relevant doses showed additive inhibitory effects on VEGF secretion. CONCLUSIONS: VAE did not interfere with cytostatic effects of Trastuzumab on SK-BR-3 cells in vitro. Our in vitro results suggest that no risk of safety by herb drug interactions has to be expected from the exposition of cancer cells to Trastuzumab and VAE simultaneously. In contrast, VAE and Trastuzumab seem to exhibit complementary anti-cancer effects in vitro.

Synthesis and in vitro biological evaluation of 2-(phenylcarbamoyl)phenyl 4-substituted benzoates.

Based on the previously described antimicrobial activity of salicylanilide derivatives, we designed and synthesized novel 2-(phenylcarbamoyl)phenyl 4-substituted benzoates. The most active salicylanilides were selected for esterification by various 4-substituted benzoic acids. These compounds were evaluated in vitro against Mycobacterium tuberculosis, including multidrug-resistant strains, nontuberculous mycobacteria (Mycobacterium avium and Mycobacterium kansasii), and eight bacterial and fungal strains. We also investigated the cytostatic and cytotoxic actions of the esters. The minimum inhibitory concentrations (MICs) against mycobacteria ranged from 0.125 to 8µM. Interestingly, the drug-resistant strains exhibited the highest susceptibility without any cross-resistance with established drugs. 4-Bromo-2-[4-(trifluoromethyl)phenylcarbamoyl]phenyl 4-nitrobenzoate showed the most potent inhibition with MIC values ranging from 0.25 to 2µM. Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, were inhibited by two derivatives with MIC values of at least 0.49µM, whereas Gram-negative bacteria and most of the tested fungi did not display any marked susceptibility. Benzoates exhibited no cytotoxicity at concentrations up to 50µM but most caused significant cytostasis with IC50 values lower than 10µM. Some cytotoxicity-based selectivity indexes for drug-susceptible and drug-resistant M. tuberculosis as well as Staphylococci were higher than 100. These values indicate that some of these derivatives are promising candidates for future research.

Vitality, viability, long-term clonogenic survival, cytotoxicity, cytostasis and lethality: what do they mean when testing new investigational oncology drugs?

In the field of experimental therapeutics for oncology purposes researchers are continuously evaluating the toxicity of novel treatment approaches against cancer cells. Within this topic of research, it is highly critical to define parameters of toxicity that denote when cancer cells are perturbed in their functionality by a new investigational drug. As the goal for these approaches is to achieve cellular demise, then what approaches to use and what do they mean in terms of assessing such cell death is of critical importance. In this comment article we highlight the definition of vitality and differentiate it from viability, and further define clonogenic survival in a chronic fashion. Additionally, we highly recommend the use of the term cytotoxicity as a general descriptor indicating toxicity towards a cell, but within that we encourage to sub-classify it as either cytostasis (i.e., when a treatment does not allow a cell to grow but it does not kill it either), or lethality (when a cell dies in response to the treatment). A more precise use of these terms should help advance the field of experimental therapeutics in oncology towards better defining the mechanisms of action of novel investigational drugs.

Biological potential, chemical profiling, and molecular docking study of Morus alba L. extracts.

Morus alba L. is a plant with a long history of dietary and medicinal uses. We hypothesized that M. alba possesses a significant biological potential. In that sense, we aimed to generate the chemical, antimicrobial, toxicological, and molecular profile of M. alba leaf and fruit extracts. Our results showed that extracts were rich in vitamin C, phenols, and flavonoids, with quercetin and pterostilbene concentrated in the leaf, while fisetin, hesperidin, resveratrol, and luteolin were detected in fruit. Extracts exhibited antimicrobial activity against all tested bacteria, including multidrug-resistant strains. The widest inhibition zones were in Staphylococcus aureus ATCC 33591. The values of the minimum inhibitory concentration ranged from 15.62 µg/ml in Enterococcus faecalis to 500 µg/ml in several bacteria. Minimum bactericidal concentration ranged from 31.25 µg/ml to 1000 µg/ml. Extracts impacted the biofilm formation in a concentration-dependent and species-specific manner. A significant difference in the frequency of nucleoplasmic bridges between the methanolic extract of fruit (0.5 µg/ml, 1 µg/ml, 2 µg/ml), as well as for the frequency of micronuclei between ethanolic extract of leaf (2 µg/ml) and the control group was observed. Molecular docking suggested that hesperidin possesses the highest binding affinity for multidrug efflux transporter AcrB and acyl-PBP2a from MRSA, as well as for the SARS-CoV-2 Mpro. This study, by complementing previous research in this field, gives new insights that could be of great value in obtaining a more comprehensive picture of the Morus alba L. bioactive potential, chemical composition, antimicrobial and toxicological features, as well as molecular profile.

Inhibition of thioredoxin reductase 1 by vulpinic acid suppresses the proliferation and migration of human breast carcinoma.

AIMS: It was aimed to investigate the thioredoxin reductase 1 (TrxR1)-targeted anticancer effect of vulpinic (VA) and lecanoric (LA) acids, which are lichen secondary metabolites, on breast cancer MCF-7 and MDA-MB-453 cell lines, and to compare the effectiveness of this potential effect against commercial chemotherapeutic drugs carboplatin and docetaxel. MAIN METHODS: The anticancer effects of both lichen metabolites were evaluated by XTT, flow cytometry analysis, cell scratch, and transwell migration assays. Apoptotic results were also confirmed by qPCR and western blot. Changes in TrxR1 were investigated in gene and protein expressions and enzyme activity levels. KEY FINDINGS: VA suppressed the proliferation of MCF-7 and MDA-MB-453 cells in a dose- and time-dependent manner, and the IC50 values were calculated as 22.92 µg/ml and 95.65 µg/ml, respectively. As for LA, it did not have a considerable antiproliferative effect on both cell lines. VA had stronger cytotoxicity than both chemotherapeutic drug in MCF-7 cells and showed antiproliferative activity closer to carboplatin in MDA-MB-453 cells. qPCR, western blot, and flow cytometry analysis results revealed that VA did not induce apoptosis in both cell lines. In contrast, VA caused cell cycle arrest, significantly. Migration assay results showed that VA suppressed migration in both cells. VA induced the gene expression of TrxR1 while inhibiting its protein expression and enzymatic activity in both cell lines. SIGNIFICANCE: The findings reveal that vulpinic acid may be a novel inhibitor candidate on TrxR1 and could be considered a potential chemotherapeutic agent for breast cancer treatment, especially in MCF-7 cells.

Cytostatic Effects of Polyethyleneimine Surfaces on the Mesenchymal Stromal Cell Cycle.

Polyelectrolytes assembled layer-by-layer (PEMs) are commonly used as functional coatings to build-up biological interfaces, particularly suitable as compatible layers for the interaction with a biological medium, providing suitable conditions to promote or prevent cell seeding while maintaining the phenotype. The proper assessment of the biocompatibility of PEMs and the elucidation of the related mechanisms are therefore of paramount importance. In this study, we report in detail the effect of two different PEM endings, polystyrene sulfonate (PSS) and polyethylenimine (PEI), respectively, on the cell adhesion, growth, and viability of human bone mesenchymal stromal cells (MSCs). The results have shown that PSS-ended substrates appear to be the most suitable to drive the cell adhesion and phenotype maintenance of MSCs, showing good biocompatibility. On the contrary, while the cells seem to adhere more quickly and strongly on the PEI-ended surfaces, the interaction with PEI significantly affects the growth and viability, reducing the cell spreading capability, by sequestering the adhesion molecules already in the very early steps of cell-substrate contact. These results point to the promotion of a cytostatic effect of PEI, rather than the often-claimed cytotoxicity.

Lycorine inhibits cell proliferation, migration and invasion, and primarily exerts in vitro cytostatic effects in human colorectal cancer via activating the ROS/p38 and AKT signaling pathways.

Colorectal cancer (CRC) is a life­threatening malignant tumor of the digestive tract. Diverse gene mutations and complicated alterations to the signaling pathways in CRC lead to heterogeneity in response to chemotherapy. Moreover, anticancer drugs for CRC chemotherapy are limited due to adverse events. Therefore, developing more effective, tolerable and safe drugs for the treatment of CRC is important. The present study aimed to investigate the effect of lycorine on human CRC cell proliferation, migration, invasion, apoptosis, cell cycle distribution, as well as the underlying molecular mechanism. The crystal violet staining and MTT assay results demonstrated that lycorine suppressed cell proliferation in a dose­ and time­dependent manner in the three CRC cell lines, HCT116, LoVo and SW480. Similarly, verified by performing wound healing and Transwell assays, lycorine significantly inhibited HCT116 and LoVo cell migration and invasion in vitro compared with the control group. In LoVo cells, the protein expression levels of matrix metallopeptidases, snail family transcriptional repressor 1, Vimentin and N­cadherin were significantly downregulated, whereas the protein expression levels of E­cadherin were significantly upregulated by lycorine treatment compared with the control group. The Hoechst 33258 staining and flow cytometry assay results indicated that lycorine mediated its cytostatic effect on CRC cells potentially via inducing cell cycle arrest, but not apoptosis. Compared with the control group, lycorine significantly induced HCT116 cell cycle arrest at the G2/M phase, but significantly induced LoVo cell cycle arrest at the S and G2/M phases. Furthermore, lycorine significantly downregulated the protein expression levels of cyclin D1 and cyclin E1, but significantly increased p21 and Smad4 protein expression levels in HCT116 and LoVo cells compared with the control group. The intracellular reactive oxygen species (ROS) measurement results also indicated that compared with the control group, lycorine significantly induced ROS accumulation, and increased phosphorylated­p38 expression levels and AKT phosphorylation. Collectively, the present study suggested that lycorine might induce cell cycle arrest and exert cytostatic effects potentially via activating ROS/p38 and AKT signaling pathways in CRC cells.

Mutagenic and cytostatic activities of the Xylopia laevigata essential oil in human lymphocytes.

Hydro-distilled essential oil from leaves of Xylopia laevigata was characterized by GC-MS. Twenty-seven components were identified and the oil's major constituents comprised germacrene D, bicyclogermacrene, (E)-caryophyllene and germacrene B. The cytotoxicity of the essential oil of X. laevigata (EOXL), determined by MTT and mitotic index methods in cultured human lymphocytes was observed in all tested concentrations. Cultures treated with EOXL demonstrated significant increase in the frequencies of micronuclei in the cytokinesis-block micronucleus assay (CBMN) and reduction of the cytokinesis-block proliferation index (CBPI) rates. Results demonstrated the cytostatic and mutagenic effects of EOXL, the latter for the first time.

EMP2 induces cytostasis and apoptosis via the TGFß/SMAD/SP1 axis and recruitment of P2RX7 in urinary bladder urothelial carcinoma.

PURPOSE: Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease, and its high recurrence rates impose a heavy clinical burden. The objective of this study was to identify signaling pathways downstream of epithelial membrane protein 2 (EMP2), which induces cytostasis and apoptosis in UBUC. METHODS: A series of in vitro and in vivo assays using different UBUC-derived cell lines and mouse xenograft models were performed, respectively. In addition, primary UBUC specimens were evaluated by immunohistochemistry. RESULTS: Exogenous expression of EMP2 in J82 UBUC cells significantly decreased DNA replication and altered the expression levels of several TGFß signaling-related proteins. EMP2 knockdown in BFTC905 UBUC cells resulted in opposite effects. EMP2-dysregulated cell cycle progression was found to be mediated by the TGFß/TGFBR1/SP1 family member SMAD. EMP2 or purinergic receptor P2X7 (P2RX7) gene expression upregulation induced apoptosis via both intrinsic and extrinsic pathways. In 242 UBUC patient samples, P2RX7 protein levels were found to be significantly and positively correlated with EMP2 protein levels. Low P2RX7 levels conferred poor disease-specific and metastasis-free survival rates, and significantly decreased apoptotic cell rates. EMP2 was found to physically interact with P2RX7. In the presence of a P2RX7 agonist, BzATP, overexpression of both EMP2 and P2RX7 significantly increased apoptotic cell rates compared to overexpression of EMP2 or P2RX7 alone. CONCLUSIONS: EMP2 induces cytostasis via the TGFß/SMAD/SP1 axis and recruits P2RX7 to enhance apoptosis in UBUC. Our data provide new insights that may be employed for the design of UBUC targeting therapies.

Amplification-driven BCL6-suppressed cytostasis is mediated by transrepression of FOXO3 and post-translational modifications of FOXO3 in urinary bladder urothelial carcinoma.

Muscle-invasive urinary bladder urothelial carcinoma (UBUC) is a lethal disease for which effective prognostic markers and potential therapy targets are still lacking. Previous array comparative genomic hybridization identified that 3q27 is frequently amplified in muscle-invasive UBUCs, one candidate proto-oncogene, B-cell CLL/lymphoma 6 (BCL6), mapped to this region. We therefore aimed to explore its downstream targets and physiological roles in UBUC progression. Methods: Specimens from UBUC patients, NOD/SCID mice and several UBUC-derived cell lines were used to perform quantitative RT-PCR, fluorescence in situ hybridization immunohistochemistry, xenograft, gene stable overexpression/knockdown and a series of in vitro experiments. Results: Amplification of the BCL6 gene lead to upregulation of BCL6 mRNA and protein levels in a substantial set of advanced UBUCs. High BCL6 protein level significantly predicted poor disease-specific and metastasis-free survivals. Knockdown of the BCL6 gene in J82 cells inhibited tumor growth and enhanced apoptosis in the NOD/SCID xenograft model. In vitro experiments demonstrated that BCL6 inhibited cytostasis, induced cell migration, invasion along with alteration of the expression levels of several related regulators. At molecular level, BCL6 inhibited forkhead box O3 (FOXO3) transcription, subsequent translation and upregulation of phosphorylated/inactive FOXO3 through phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) and/or epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase 1/2 (MAP2K1/2) signaling pathway(s). Two BCL6 binding sites on the proximal promoter region of the FOXO3 gene were confirmed. Conclusion: Overexpression of BCL6 served a poor prognostic factor in UBUC patients. In vivo and in vitro studies suggested that BCL6 functions as an oncogene through direct transrepression of the FOXO3 gene, downregulation and phosphorylation of the FOXO3 protein.