Synthesis and in Silico Evaluation of Novel Triazolone-Derived Naphthalene-2-Sulfonates, Evaluation of Potential Antiproliferative Agents and Enzyme Inhibitory Activities.

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics.

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics.

Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells

The U251 and U87 cell lines are commonly used as experimental models of glioblastoma. However, these cells exhibit significant differences in their proliferation, invasion, and migration. The aim of the present study was to compare the protein expression profiles of the U251 and U87 cell lines in order to provide a molecular basis for the observed phenotypic differences. Isobaric tags for relative and absolute quantitation (iTRAQ) and gene ontology (GO) analyses were performed to detect differentially expressed proteins and to predict protein functions, respectively. Two hundred and forty-four proteins were highly expressed, while 263 proteins exhibited lower levels of expression, in the U251 cells compared to the U87 cells. In particular, higher expression levels of the proteins, C10orf58, FLNC, PDLIM1, TPM4, and lower expression levels of MYH10, PSIP1, SYNM, SLC9A3R2, BCAM, were verified by qPCR in the U251 cell line versus the U87 cell line. When a GO analysis was applied to the iTRAQ results, the proteins that were highly expressed in the U251 cells were found to differ in their molecular functions, biological processes, cellular distribution, and cellular pathways associated with them compared with the highly expressed proteins detected in the U87 cells. Differentially expressed proteins between the U251 and U87 cell lines are associated with regulation of nicotinamide nucleotide metabolism, RNA splicing, glycolysis, and purine metabolism pathways. Further studies on these pathways may identify whether these various pathways account for the observed phenotype differences between the U87 and U251 GBM cell lines.

Gliomas, the most common form of brain tumors, are characterized by a capacity to invade through normal brain tissue. This infiltrative character is the hallmark of poor prognosis. A greater understanding of the molecular determinants that drive cell motility may lead to improved therapy. The molecules of the extracellular matrix are involved in organization of the cytoskeleton and activation of intracellular signaling that is required for the regulation of cell adhesion and migration. Here, we show that the downregulation of matrix metalloproteinase-2 (MMP-2; gelatinase A) using an adenoviral vector expressing siRNA (Ad-MMP-2-Si) inhibited cell migration in U87 and U251 glioblastoma cell lines as determined by spheroid migration and wound-healing assays. Transcriptional inactivation of MMP-2 altered interaction of MMP-2 and the integrin-αVβ3 as determined by immunoprecipitation and co-localization studies. In addition, downregulation of MMP-2 inhibited the complex formation of FAK, integrin-αVβ3 and 14-3-3ζ, which in turn decreased the activation of GTP-bound forms of Rac1 and Cdc42. Transfection of the cells with constitutively active Rac1 and Cdc42 reversed Ad-MMP-2-Si-blocked cell migration and gelatin degrading. Further, administration of recombinant human MMP-2 reversed MMP-2 siRNA-inhibited GTP-bound forms of Rac1 and Cdc42. Additionally, functionally blocking integrin-αVβ3 antibody inhibited GTP-bound forms of Rac1 and Cdc42, thereby revealing that the interaction of MMP-2 and integrin-αVβ3 plays a critical role in cell migration. In summary, our data demonstrate that the inhibition of MMP-2 inhibits integrin-αVβ3/FAK-mediated migration by reducing the GTP-bound forms of Rac1 and Cdc42 in U87 and U251 glioblastoma cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2330.

Cholesterol oxidation products (oxysterols) are commonly found in foods of animal origin and are also produced endogenously in the body. Oxysterols are cytotoxic to certain cell lines and in some cases have been shown to induce apoptosis. The aim of this study was to investigate the effects of 7beta-hydroxy-cholesterol (7beta-OHC) and 25-hydroxycholesterol (25-OHC) on cytotoxicity and induction of apoptosis in U937 and HepG2 cells, treated in media containing either 2.5% foetal calf serum (FCS) or 10% FCS to examine the effect of increasing the cholesterol level. The cells were incubated for 24 and 48 h with 30 microM oxysterol. Viability was assessed by fluorescein diacetate/ethidium bromide staining and cell proliferation was determined by haemocytometer counting. Apoptosis was monitored by detection of DNA fragments (laddering) in 1.5% agarose gels. Cells with condensed or fragmented nuclei were identified by Hoechst 33342 staining. The percentage of cells with sub-G1 levels of DNA was measured by flow cytometry. Treatment of U937 cells with 7beta-OHC, in contrast to 25-OHC, resulted in a decrease in cell viability and proliferation at 24 and 48 h (P <.01). 25-OHC and 7beta-OHC were both equally cytotoxic to the HepG2 cell line. 7beta-OHC induced DNA laddering and an increase in the percentage of condensed or fragmented nuclei at both time points and at both serum concentrations in the U937 cell line. 25-OHC induced faint laddering in the U937 cells after 48 h in reduced serum media and resulted in a small increase in percentage condensed or fragmented nuclei which was independent of time of oxysterol exposure and serum concentration. The percentage of condensed or fragmented nuclei was low in the HepG2 cell line and no laddering was observed under any of the conditions studied. Flow cytometric analysis showed that only 7beta-OHC treated U937 cells had an increased level of hypodiploid cells. Both oxysterols appear to be equally cytotoxic to the HepG2 cell line. In U937 cells, 25-OHC is much less cytotoxic than 7beta-OHC. In addition, we have shown that 7beta-OHC induces apoptosis in U937 cells. 10% FCS displays a protective effect on cytotoxicity (as well as on 7beta-OHC induced apoptosis in U937 cells), although the data did not reach statistical significance.

Propionylation has been identified recently as a new type of protein post-translational modification. Bacterial propionyl-CoA synthetase and human histone H4 are propionylated at specific lysine residues that have been known previously to be acetylated. However, other proteins subject to this modification remain to be identified, and the modifying enzymes involved need to be characterized. In this work, we report the discovery of histone H3 propionylation in mammalian cells. Propionylation at H3 lysine Lys(23) was detected in the leukemia cell line U937 by mass spectrometry and Western analysis using a specific antibody. In this cell line, the propionylated form of Lys(23) accounted for 7%, a level at least 6-fold higher than in other leukemia cell lines (HL-60 and THP-1) or non-leukemia cell lines (HeLa and IMR-90). The propionylation level in U937 cells decreased remarkably during monocytic differentiation, indicating that this modification is dynamically regulated. Moreover, in vitro assays demonstrated that histone acetyltransferase p300 can catalyze H3 Lys(23) propionylation, whereas histone deacetylase Sir2 can remove this modification in the presence of NAD(+). These results suggest that histone propionylation might be generated by the same set of enzymes as for histone acetylation and that selection of donor molecules (propionyl-CoA versus acetyl-CoA) may determine the difference of modifications. Because like acetyl-CoA, propionyl-CoA is an important intermediate in biosynthesis and energy production, histone H3 Lys(23) propionylation may provide a novel epigenetic regulatory mark for cell metabolism.

IgA nephropathy (IgAN) is characterized by raised serum IgA and predominant mesangial IgA deposits of polymeric nature. The expression of IgA receptor molecules in white blood cells and glomerular mesangial cells has recently attracted much attention in relation to the uptake of IgA by these cells. This study investigates the expression of IgA Fc receptor (Fc alphaR1 or CD89), asialoglycoprotein receptor (ASGPR), and polymeric Ig receptor (pIgR) in cultured glomerular mesangial cells. Using a sensitive nested reverse transcription-PCR, mRNA encoding for Fc alphaR1, pIgR, or the H2 chain of ASGPR was not demonstrated on human mesangial cells. U937, HepG2, and HT29 cell lines, used as positive controls, strongly expressed the Fc alphaR1, ASGPR, and pIgR mRNA, respectively, under similar experimental conditions. Flow cytometry also demonstrated the presence of surface proteins for Fc alphaR1, ASGPR, and pIgR on the respective control cell lines but not on human mesangial cells. Expression of Fc alphaR1 mRNA on cultured U937 cells was upregulated by tumor necrosis factor-alpha. However, tumor necrosis factor-alpha, interleukin-1beta, or transforming growth factor-beta failed to induce the expression of Fc alphaR1 on human mesangial cells. Human serum IgA or secretory IgA bound to human mesangial cells, HepG2, or the U937 cell line in a dose-dependent manner. The binding of purified IgA to human mesangial cells was not blocked by preincubation with human IgG, IgM, orosomucoid, asialo-orosomucoid, anti-CD89 antibody (My43), or anti-secretory component antibody. The present study concluded that there was an absence of Fc alphaR1, ASGPR, or pIgR on human mesangial cells. These findings suggest that the predominant binding of human IgA to human mesangial cells is mediated by other mechanisms.

In this study, we synthesized and evaluated novel histone deacetylase (HDAC) inhibitors derived from the clinical candidate quisinostat. A library of 16 compounds categorized in three novel chemotypes was rapidly generated using multicomponent reactions (MCRs), enabling efficient structure-activity relationship studies. First, the compounds were evaluated for their activity against the Plasmodium falciparum strains 3D7 and Dd2, the main malaria-causing parasite, identifying compound 18b of the type C series as the most potent. It demonstrated low nanomolar IC50 values (IC50 (3D7) = 0.023 μM; IC50 (Dd2) = 0.047 μM) and high parasite selectivity (SIMRC−5/Pf3D7 > 2174). HDAC inhibition assays confirmed substantial inhibition of the P. falciparum enzyme PfHDAC1 (IC50 = 0.037 μM) as well as of human HDAC1 (IC50 = 0.021 μM) and HDAC6 (IC50 = 0.25 μM). Docking studies suggested distinct binding modes of 18b in P. falciparum and human HDAC1. Additionally, the in vitro anticancer activity was evaluated in Cal27 (head-neck carcinoma), HepG2 (hepatocellular carcinoma), A2780 (ovarian carcinoma), and U87 (glioblastoma) cell lines. Compounds 9b, 9d, and 13f showed potent antiproliferative activity and caspase 3/7 activation, in contrast to 18b. Furthermore, these compounds caused hyperacetylation of histone H3 and α-tubulin, indicating robust cellular target engagement. Overall, in this work we have identified the HDAC inhibitor 18b with selective antiplasmodial and 9b, 9d, and 13f with selective anticancer activities, providing valuable hits for further drug development efforts aimed at creating derivatives with reduced cytotoxicity against non-cancer cells compared to quisinostat.

Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide.

Development and In vitro and In vivo efficacy investigation of multifunctional, targeted, theranostic liposomes for imaging and photodynamic therapy of glioblastoma

Cancer is a debilitating disease that is on the increase in both developed and developing countries. Anticancer drugs are often expensive, have narrow spectrum of activities, and are associated with toxicities and side effects such as myelosuppression, immunosuppression, gastrointestinal disturbance, alopecia, skin toxicity, and hepatotoxicity. Plants have been the major source of anticancer drugs both in orthodox and traditional medicine. Many of the plants claimed by the traditional medicine practitioners (TMPs) to be effective in the treatment of cancer are yet to be evaluated scientifically. In this work, five medicinal plants used by TMPs in Borno State, Nigeria, were tested against two brain tumor cell lines. Ethanol extracts of Securidaca longepedunculata, Andira inermis subsp. rooseveltii, Annona senegalensis, Carissa edulis, and Parinari polyandra were used. U87 and U231 brain tumor cell lines were used for proliferation assay, U251 cell line was used for the invasion assay in collagen V coated inserts, and U87 cell line was used for the western blot detection of cleaved Poly-ADP-Ribose-Polymerase (PARP). The result revealed that all tested extracts significantly (p < 0.05) inhibited the proliferation of U87 and U231 cell lines with the respective IC50 values ranging between 8 and 20 μg/ml for S. longepedunculata and 100 and 90 μg/ml for P. polyandra. The five extracts significantly (p < 0.05) inhibited the invasion of U251 cell line at the concentration of 10 μg/ml (S. longepedunculata), 20 μg/ml (A. inermis), 50 μg/ml (A. senegalensis), 50 μg/ml (C. edulis), and 50 μg/ml (P. polyandra). Securidaca longepedunculata extract induced the cleavage of PARP. It was concluded that these medicinal plants have antiproliferative and anti-invasive activities and possess good prospects as source of anticancer agents especially S. longepedunculata which induced apoptosis in U87 cell line.

BACKGROUND Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) pathogenesis by affecting stemness, angiogenesis and immune pathways. Clinical trials targeting CMV in GBM patients have shown some early promise, and CMV seropositivity has been associated with poorer outcomes. However, the underlying mechanisms remain unclear. Here we investigated the effects of CMV infection on critical signaling pathways in GBM (IL6/STAT3, and Akt signaling) based on the hypothesis that CMV may contribute to poorer outcomes in GBM by increasing oncogenic signaling. MATERIALS AND METHODS Human U251 and LN229 GBM cell lines were infected with mCherry expressing human CMV TB40 strain at a multiplicity of infection (MOI) of 0.5. We performed basic phenotypic studies in vitro and investigated the status of IL6/STAT3, and Akt signaling by Western blotting. RESULTS Both LN229 and U251 cell lines were readily infectable by CMV. Infection of U251 cells was over 90% and in LN229 cells was approximately 60%. Interestingly expression of CMV was sustained over many passages in these cell lines. Infection was also verified by the presence of the CMV pp65 protein assessed by Western blotting. Measurement of cell proliferation indicated faster growth after CMV infection. Interrogation of key pro-oncogenic signaling pathways in GBM revealed a robust upregulation of IL6, as well as phospho(Tyr705)-STAT3 and phospho(Thr308)-Akt. This elevation of pro-oncogenic signaling pathways was observed in both cell lines compared with controls. CONCLUSIONS Here we show for the first time that infection of human GBM cell lines with CMV results in a more aggressive phenotype associated with upregulation of IL6/STAT3 signaling as well as a significant increase in phospho-Akt levels. We are currently studying sensitivity to standard therapies and additional molecular changes in these cells. These data support the hypothesis that CMV causes more aggressive disease and that CMV is a relevant therapeutic target in GBM.

1,2,4-Triazines exhibit various pharmacological properties due to their strong biological activities, such as anticancer, anti-inflammatory, antimicrobial, antiviral, and antioxidant, and also occupy an important place in the pharmaceutical field. The tested compounds were synthesized and characterized. 1,2,4-Triazines were evaluated for their inhibitory and cytotoxicity effects on acetylcholinesterase (AChE), glutathione S-transferase (GST), human liver cancer cell line (HepG2), and human colorectal adenocarcinoma cell line (HT-29). 1,2,4-Triazines (3a-c, 5, 6, 10) were found to inhibit these enzymes with IC50 values ranging from 1.41 to 4.28 μM for AChE and from 1.58 to 4.13 μM for GST. 1,2,4-Triazine compounds were tested in range of 12.5-100 µM concentrations against HepG2 and HT-29 cell lines. Among the 1,2,4-triazines tested, 10 was the most effective substance on HepG2 cells, especially at high doses, while 3b was effective on HT-29. Furthermore, molecular docking results indicated that the 1,2,4-triazines showed strong binding and stability at the active site of enzymes and selected proteins. 1,2,4-Triazines may serve as promising precursors for the design of potent enzyme inhibitors and anticancer agents.

Background: Activation of telomerase is essential for the indefinite replication potential of most cancer cells. Inhibition of telomerase is expected to lead to loss of telomere maintenance resulting in cell cycle arrest and/or cancer cell death, making telomerase inhibition an attractive anti-cancer approach. Cancer stem cells (CSCs) are rare cells in tumors implicated in cancer initiation and potentiation, as well as persistence or recurrence after standard treatment. Unlike normal tissue counterparts, all CSCs tested to date have upregulated telomerase activity. Recent identification of several non-canonical roles of telomerase components may expand the functionality of telomerase-inhibiting drugs. Imetelstat, a potent telomerase inhibitor currently in Phase II clinical trials, has been shown to reduce proliferative potential in multiple cancer models. Several recent studies have demonstrated that imetelstat depletes CSCs in various tumor types. The relatively rapid onset of CSC depletion could probably indicate a mechanism independent of telomere shortening. To understand the contributions of non-canonical pathways in the response of cancer cells to imetelstat treatment, we analyzed modulations of key signaling pathways in imetelstat-treated glioblastoma multiforme cancer cell lines (U87-MG and U118) and their CSC and bulk subsets. Results: Imetelstat inhibited telomerase activity, proliferative capacity and colony-forming potential in the U87 and U118 cell lines. Imetelstat treatment (2 weeks at 3uM) resulted in decreased expression of casein kinase 2 (CK2) subunits alpha and beta, and a reduction in phosphorylation of downstream CK2 substrates such as the DNA repair protein XRCC1. In addition, imetelstat reduced the transcriptional activity of beta-catenin, which is regulated by members of the CK2 family, in U87-MG cells. Cyclin D1, which is regulated by beta-catenin activity, was found to be down regulated by imetelstat treatment. These pathway modulations were not observed in T98G, a cell line resistant to imetelstat-mediated proliferation effects. Reducing CK2-alpha expression had an additive effect on cell growth inhibition in combination with imetelstat in U87-MG cells. Knock-down of the CK2-alpha, but not CK2-beta subunits, with siRNAs rapidly reduced the numbers of CSCs in U87-MG and U118 cell lines. Concomitant with the down-regulation of CK2-alpha signaling, imetelstat treatment depleted the number of CSCs in U78-MG and U118 cell lines. Preliminary data indicate a more marked reduction of CK2-alpha levels in the CSC subset of U87-MG cells compared to the bulk cells upon short term imetelstat treatment (1 week at 3uM). These in-vitro exposure concentrations of imetelstat are comparable to those attainable intratumorally in a xenograft mouse model. Conclusions: Our results suggest that CK2-alpha signaling and dysregulation of its downstream targets may play a key role in survival of CSCs as well as bulk tumor cells in U87 and U118 glioblastoma cell lines. These novel insights may help identify drugs that can act synergistically with imetelstat in treating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A56.