An Antitubulin Agent BCFMT Inhibits Proliferation of Cancer Cells and Induces Cell Death by Inhibiting Microtubule Dynamics

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazoli din-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC50, 7.2 +/- 1.8 mu M), human breast adenocarcinoma (MCF-7) (IC50, 10.0 +/- 0.5 mu M), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC50, 6.0 +/- 1 mu M), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC50, 5.8 +/- 0.3 mu M) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC50, 6.5 +/- 1 mu M) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 mu M), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 +/- 1.8 mu M, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K-i) of 5.2 +/- 1.5 mu M suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.

Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation IMPLICATIONS FOR CANCER THERAPEUTICS

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates beta-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3'-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

Intracellular reactive oxidative stress, cell proliferation and apoptosis of Schwann cells on carbon nanofibrous substrates

Despite considerable research to develop carbon based materials for biomedical applications, the toxicity of carbon remains a major concern. In order to address this issue as well as to investigate the cell fate processes of neural cells from the perspective of neural tissue engineering applications, the in vitro cytocompatibility of polyacrylonitrile (PAN) derived continuous carbon nanofibers and PAN derived carbon thin films were investigated both quantitatively and qualitatively using in vitro biochemical assays followed by extensive flow cytometry analysis. The experimental results of Schwann cell fate, i.e. cell proliferation, cell metabolic activity and cell apoptosis on amorphous carbon substrates are discussed in reference to the time dependent evolution of intracellular oxidative stress. Apart from providing evidence that an electrospun carbon nanofibrous substrate can physically guide the cultured Schwann cells, this study suggested that continuous carbon nanofibers and amorphous carbon films are not cytotoxic in vitro and do not significantly induce apoptosis of Schwann cells, but in fact even facilitate their proliferation and growth.

Substrate conductivity dependent modulation of cell proliferation and differentiation in vitro

In designing and developing various biomaterials, the influence of substrate properties, like surface topography, stiffness and wettability on the cell functionality has been investigated widely. However, such study to probe into the influence of the substrate conductivity on cell fate processes is rather limited. In order to address this issue, spark plasma sintered HA-CaTiO3 (Hydroxyapatite-Calcium titanate) has been used as a model material system to showcase the effect of varying conductivity on cell functionality. Being electroactive in nature, mouse myoblast cells (C2C12) were selected as a model cell line in this study. It was inferred that myoblast adhesion/growth systematically increases with substrate conductivity due to CaTiO3 addition to HA. Importantly, parallel arrangement of myoblast cells on higher CaTiO3 containing substrates indicate that self-adjustable cell patterning can be achieved on conductive biomaterials. Furthermore, enhanced myoblast assembly and myotube formation were recorded after 5 days of serum starvation. Overall, the present study conclusively establishes the positive impact of the substrate conductivity towards cell proliferation and differentiation as well as confirms the efficacy of HA-CaTiO3 biocomposites as conductive platforms to facilitate the growth, orientation and fusion of myoblasts, even when cultured in the absence of external electric field.

Inhibition of cancer cell proliferation and apoptosis-inducing activity of fungal taxol and its precursor baccatin III purified from endophytic Fusarium solani

Background: Taxol (generic name paclitaxel), a plant-derived antineoplastic agent, used widely against breast, ovarian and lung cancer, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. The limited supply of the drug has prompted efforts to find alternative sources, such as chemical synthesis, tissue and cell cultures of the Taxus species both of which are expensive and yield low levels. Fermentation processes with microorganisms would be the methods of choice to lower the costs and increase yields. Previously we have reported that F. solani isolated from T. celebica produced taxol and its precursor baccatin III in liquid grown cultures J Biosci 33: 259-67, 2008. This study was performed to evaluate the inhibition of proliferation and induction of apoptosis of cancer cell lines by the fungal taxol and fungal baccatin III of F. solani isolated from T. celebica. Methods: Cell lines such as HeLa, HepG2, Jurkat, Ovcar3 and T47D were cultured individually and treated with fungal taxol, baccatin III with or without caspase inhibitors according to experimental requirements. Their efficacy on apoptotic induction was examined. Results: Both fungal taxol and baccatin III inhibited cell proliferation of a number of cancer cell lines with IC50 ranging from 0.005 to 0.2 mu M for fungal taxol and 2 to 5 mu M for fungal baccatin III. They also induced apoptosis in JR4-Jurkat cells with a possible involvement of anti-apoptotic Bcl2 and loss in mitochondrial membrane potential, and was unaffected by inhibitors of caspase-9,-2 or -3 but was prevented in presence of caspase-10 inhibitor. DNA fragmentation was also observed in cells treated with fungal taxol and baccatin III. Conclusions: The cytotoxic activity exhibited by fungal taxol and baccatin III involves the same mechanism, dependent on caspase-10 and membrane potential loss of mitochondria, with taxol having far greater cytotoxic potential.

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Transcriptional Repression of Tumor Suppressor CDC73, Encoding an RNA Polymerase II Interactor, by Wilms Tumor 1 Protein (WT1) Promotes Cell Proliferation IMPLICATION FOR CANCER THERAPEUTICS

The Wilms tumor 1 gene (WT1) can either repress or induce the expression of genes. Inconsistent with its tumor suppressor role, elevated WT1 levels have been observed in leukemia and solid tumors. WT1 has also been suggested to act as an oncogene by inducing the expression of MYC and BCL-2. However, these are only the correlational studies, and no functional study has been performed to date. Consistent with its tumor suppressor role, CDC73 binds to RNA polymerase II as part of a PAF1 transcriptional regulatory complex and causes transcriptional repression of oncogenes MYC and CCND1. It also represses beta-catenin-mediated transcription. Based on the reduced level of CDC73 in oral squamous cell carcinoma (OSCC) samples in the absence of loss-of-heterozygosity, promoter methylation, and mutations, we speculated that an inhibitory transcription factor is regulating its expression. The bioinformatics analysis predicted WT1 as an inhibitory transcription factor to regulate the CDC73 level. Our results showed that overexpression of WT1 decreased CDC73 levels and promoted proliferation of OSCC cells. ChIP and EMSA results demonstrated binding of WT1 to the CDC73 promoter. The 5-azacytidine treatment of OSCC cells led to an up-regulation of WT1 with a concomitant down-regulation of CDC73, further suggesting regulation of CDC73 by WT1. Exogenous CDC73 attenuated the protumorigenic activity of WT1 by apoptosis induction. An inverse correlation between expression levels of CDC73 and WT1 was observed in OSCC samples. These observations indicated that WT1 functions as an oncogene by repressing the expression of CDC73 in OSCC. We suggest that targeting WT1 could be a therapeutic strategy for cancer, including OSCC.

In vitro osteogenic cell proliferation, mineralization, and in vivo osseointegration of injection molded high-density polyethylene-based hybrid composites in rabbit animal model

The present work reports the biocompatibility property of injection molded HDPE-HA-Al2O3 hybrid composites. In vitro cytocompatibility results reveal that osteogenic cell viability and bone mineralization are favorably supported in a statistically significant manner on HDPE-20% HA-20% Al2O3 composite, in comparison to HDPE-40 wt.% HA or HDPE-40 wt.% Al2O3. The difference in cytocompatibility property is explained in terms of difference in substrate wettability/surface energy and importantly, both the cell proliferation at 7 days or bone mineralization at 21 days on HDPE-20% HA-20% Al2O3 composite are either comparable or better than sintered HA. The progressive healing of cylindrical femoral bone defects in rabbit animal model was assessed by implantation experiments over 1, 4 and 12 weeks. Based on the histological analysis as well as histomorphometrical evaluation, a better efficacy of HDPE-20% HA-20% Al2O3 over high-density polyethylene (HDPE) for bone regeneration and neobone formation at host bone-implant interface was established. Taken together, the present study unequivocally establishes that despite the presence of 20% Al2O3, HDPE-based hybrid composites are as biocompatible as HA in vitro or better than HDPE in vivo.

MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor alpha IMPLICATIONS FOR CANCER THERAPEUTICS

Estrogen-related receptor (ESRRA) functions as a transcription factor and regulates the expression of several genes, such as WNT11 and OPN. Up-regulation of ESRRA has been reported in several cancers. However, the mechanism underlying its up-regulation is unclear. Furthermore, the reports regarding the role and regulation of ESRRA in oral squamous cell carcinoma (OSCC) are completely lacking. Here, we show that tumor suppressor miR-125a directly binds to the 3UTR of ESRRA and represses its expression. Overexpression of miR-125a in OSCC cells drastically reduced the level of ESRRA, decreased cell proliferation, and increased apoptosis. Conversely, the delivery of an miR-125a inhibitor to these cells drastically increased the level of ESRRA, increased cell proliferation, and decreased apoptosis. miR-125a-mediated down-regulation of ESRRA impaired anchorage-independent colony formation and invasion of OSCC cells. Reduced cell proliferation and increased apoptosis of OSCC cells were dependent on the presence of the 3UTR in ESRRA. The delivery of an miR-125a mimic to OSCC cells resulted in marked regression of xenografts in nude mice, whereas the delivery of an miR-125a inhibitor to OSCC cells resulted in a significant increase of xenografts and abrogated the tumor suppressor function of miR-125a. We observed an inverse correlation between the expression levels of miR-125a and ESRRA in OSCC samples. In summary, up-regulation of ESRRA due to down-regulation of miR-125a is not only a novel mechanism for its up-regulation in OSCC, but decreasing the level of ESRRA by using a synthetic miR-125a mimic may have an important role in therapeutic intervention of OSCC and other cancers.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-gamma treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced H-3]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses. (C) 2014 Elsevier B.V. All rights reserved.

Enhanced Efficacy of Pluronic Copolymer Micelle Encapsulated SCR7 against Cancer Cell Proliferation(a)

5,6-Bis(benzylideneamino)-2-mercaptopyrimidin-4-ol (SCR7) is a new anti cancer molecule having capability to selectively inhibit non-homologous end joining (NHEJ), one of the DNA double strand break (DSB) repair pathways inside the cells. In spite of the promising potential as an anticancer agent, hydrophobicity of SCR7 decreases its bioavailability. Herein the entrapment of SCR7 in Pluronic copolymer is reported. The size of the aggregates was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS) which yields an average diameter of 23 nm. SCR7 encapsulated micelles (ES) were also characterized by small-angle neutron scattering (SANS). Evaluation of its biological properties by using a variety of techniques, including Trypan blue, MTT and Live-dead cell assays, reveal that encapsulated SCR7 can induce cytotoxicity in cancer cell lines, being more effective in breast cancer cell line. Encapsulated SCR7 treatment resulted in accumulation of DNA breaks within the cells, resulting in cell cycle arrest at G1 phase and activation of apoptosis. More importantly, we found approximate to 5 fold increase in cell death, when encapsulated SCR7 was used in comparison with SCR7 alone.

BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly

We have identified a potent antibacterial agent N-(4-sec-butylphenyl)-2-(thiophen-2-yl)-1H-benzod]imidazole-4-carboxa mide (BT-benzo-29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT-benzo-29 disassembled the cytokinetic Z-ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT-benzo-29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid-cell. Further, BT-benzo-29 bound to FtsZ with a dissociation constant of 24 +/- 3 m and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT-benzo-29 may bind to FtsZ at the C-terminal domain near the T7 loop. BT-benzo-29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT-benzo-29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT-benzo-29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT-benzo-29 did not affect the membrane integrity of mammalian red blood cells. BT-benzo-29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.

RPRDC: Reliable Proliferation Routing with low Duty-Cycle in Wireless Sensor Networks

Ensuring reliable energy efficient data communication in resource constrained Wireless Sensor Networks (WSNs) is of primary concern. Traditionally, two types of re-transmission have been proposed for the data-loss, namely, End-to-End loss recovery (E2E) and per hop. In these mechanisms, lost packets are re-transmitted from a source node or an intermediate node with a low success rate. The proliferation routing(1) for QoS provisioning in WSNs low End-to-End reliability, not energy efficient and works only for transmissions from sensors to sink. This paper proposes a Reliable Proliferation Routing with low Duty Cycle RPRDC] in WSNs that integrates three core concepts namely, (i) reliable path finder, (ii) a randomized dispersity, and (iii) forwarding. Simulation results demonstrates that packet successful delivery rate can be maintained upto 93% in RPRDC and outperform Proliferation Routing(1). (C) 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models

Background: Statins may have therapeutic effects on hepatocarcinoma (HCC). This type of disorder is the most common malignant primary tumour in the liver. Our objective was to determine whether pravastatin had a therapeutic effect in vitro and in vivo models. Method: We design in vitro and in vivo model. In vitro we used PLC and determine cell proliferation. In vivo, we used and animal model to determined, PCNA and MAT1A expression and transaminases levels. Results: We found that pravastatin decreases cell proliferation in vitro (cell proliferation in pravastatin group was 82%, in sorafenib group 51% and in combined group 40%) and in vivo (in pravastatin group 80%, in sorafenib group 76.4% and in combined group 72.72%). The MAT1A levels, was significantly higher in Pravastatin group (D 62%, P 94%, S 71%, P + S 91%). The transaminases levels, decreased significantly in Pravastatin group (GOT and GPT levels D 619.5 U/L; 271 U/L) (P 117.5 U/L; 43.5 U/L) (S 147 U/L; 59 U/L) (P + S 142 U/L; 59 U/L). Conclusion: The combination of pravastatin + sorafenib were more effective than Sorafenib alone.

Substrate Strain and Proliferation & Differentiation of Bone Marrow Mesenchymal Stem Cells

New methods of surface modification of transparent silicone substrate were developed, and a new set of cell culture devices that provide homogeneous substrate strain was designed. Using the new device, effects of cyclic substrate strain on bone marrow mesenchymal stem cells(MSCs) were studied. It was found that cyclic strain influenced proliferation and differentiation of bone marrow MSCs in different ways.