Proliferation Of NIH3T3 Cells Research Articles

FGF-2-Zinc-Apatite Composite Layers on External Fixation Rod for Promoting Cell Activity

FGF-2-apatite and FGF-2-zinc-apatite composite layers were formed on commercially available anodically oxidized Ti external fixation rods using FGF-2-and ZnCl2-containing supersaturated calcium phosphate solutions. The FGF-2-zinc-apatite composite layers precipitated on the Ti external fixation rods significantly enhanced proliferation of fibroblastic NIH3T3 and osteoblastic MC3T3-E1 cells in vitro.

Superoxide dismutase induces G1‐phase cell cycle arrest by down‐regulated expression of Cdk‐2 and cyclin‐E in murine sarcoma S180 tumor cells

As an efficient reactive oxygen species-scavenging enzyme, superoxide dismutase (SOD) has been shown to inhibit tumor growth and interfere with motility and invasiveness of cancer cells. In this study, the molecular mechanisms of cell cycle arrest when S180 tumor cells were exposed to high levels of SOD were investigated. Here, both murine sarcoma S180 tumor cells and NIH-3T3 mouse fibroblasts were respectively treated with varying concentrations of Cu/Zn-SOD for 24, 48 and 72 h to determine optimal dose of SOD, which was a concentration of 800 U/ml SOD for 48 h. It is found that SOD induced S180 cell cycle arrest at G1-phase with decreasing level of superoxide production, whereas SOD had less effect on proliferation of NIH-3T3 cells. Moreover, the expression rate of Proliferating Cell Nuclear Antigen (PCNA) in S180 tumor cells was suppressed after SOD treatment, which indicated the inhibition of DNA synthesis in S180 cells. Besides, there were significant down-regulations of cyclin-E and Cdk-2 in S180 cells after SOD treatment, which contributed to the blockage of G1/S transition in S180 cell cycle. Together, our data confirmed that SOD could notably inhibit proliferation of S180 tumor cell and induce cell cycle arrest at G1-phase by down-regulating expressions of cyclin-E and Cdk-2.

Film-form dye adsorbents of NiO: Synthesis, biological activity and application on dye sorption

Nowadays dye and agrichemical contaminations are becoming a severe environmental problem and subsequently a low-cost and effective method to eliminate the contaminations is becoming impending. In this study, a facile chemical precipitation method was developed to fabricate series of NiO coatings on porous alumina matrix. Scanning electron microscope, energy dispersive X-ray and powder X-ray diffraction confirmed the formation of meshy and crystalline NiO coatings. Moreover, the pore size, specific surface area and hydrophilic/hydrophobic properties could be adjusted by changing the matrix pore sizes. The biological assays revealed that NiO coatings could restrain the attachment of Aspergillus niger and exhibit a toxicity to the metabolism, proliferation and spread of NIH 3T3 cells. Additionally, the dye sorption capacity of coatings was investigated by employing Congo red as the model compound. The results indicated that the dye sorption capacity of coatings was affected by its surface features, including specific surface area and hydrophilic/hydrophobic properties. It could be concluded that film-form NiO products with special surface features had the potential as dye adsorbents for wastewater treatment.

Effects of Panax notoginseng saponins on proliferation and differentiation in NIH3T3 cells

To investigate the effects of Panax notoginseng saponins (PNS) on the proliferation and differentiation in NIH3T3 cells. NIH3T3 cells were treated by various concentrations of PNS 0, 0.05, 0.10, 0.20, and 0.40 g/L. The vitality and proliferation potential of cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the alkaline phosphatase (ALP) activity was measured by p-nitrophenyl phosphate (pNPP) assay, and the mineralization formation ability was tested for the cellular differentiation toward osteoblast, as well as the expression level of phosphorylated extracellular signal-regulated kinase1/2(P-ERK1/2), extracellular signal-regulated kinase1/2 (ERK1/2) protein kinase was analyzed by Western blot with total cell lysate of NIH3T3 cells treated by PNS. Both MTT and pNPP assay showed that optical density (OD) values were increased in response to PNS treatment at a dose-dependent pattern. The mineralization formation ability was enhanced in PNS-treated NIH3T3 cells compared with untreated cells. Meanwhile, the expression level of P-ERK1/2 protein kinase was up-regulated in PNS-treated NIH3T3 cells, while, the expression level of ERK1/2 protein kinase revealed no obvious difference with or without PNS treated cells. PNS could pay a role to promote the proliferation and differentiation in NIH3T3 cells by means of up-regulation of P-ERK1/2 protein kinase.

An integrin from shrimp Litopenaeus vannamei mediated microbial agglutination and cell proliferation.

BackgroundIntegrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps.MethodologyA shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner.ConclusionsLvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp.

Vaccination with Platelet-Derived Growth Factor B Kinoids Inhibits CCl4-Induced Hepatic Fibrosis in Mice

Platelet-derived growth factor B (PDGF-B) plays an essential role in hepatic fibrosis. Inhibition of the PDGF-B signaling in chronically injured livers might represent a potential therapeutic measure for hepatic fibrosis. In this study, we assessed the effects of vaccination against PDGF-B on CCl₄-induced liver fibrosis in BALB/c mice. The PDGF-B kinoid immunogens were prepared by cross-linking two PDGF-B-derived B-cell epitope peptides [PDGF-B¹⁶-(23-38) and PDGF-B¹⁶-(72-83)] to ovalbumin and keyhole limpet hemocyanin, respectively. Enzyme-linked immunosorbent assay, Western blotting, and NIH3T3 cell proliferation assay verified that immunization with the PDGF-B kinoids elicited the production of high levels of neutralizing anti-PDGF-B autoantibodies. The vaccination markedly alleviated CCl₄-induced hepatic fibrosis, as indicated by the lessened morphological alternations and reduced hydroxyproline contents in the mouse livers. Moreover, immunohistochemical staining for proliferating cell nuclear antigen, α-smooth muscle actin, and desmin demonstrated that neutralization of PDGF-B inhibited both the proliferation and the activation of hepatic stellate cells in the fibrotic mouse livers. Taken together, this study demonstrated that vaccination with PDGF-B kinoids significantly suppressed CCl₄-induced hepatic fibrosis in mice. Our results suggest that vaccination against PDGF-B might be developed into an effective, convenient, and safe therapeutic measure for the treatment of hepatic fibrosis.

A Novel Solid-Phase Site-Specific PEGylation Enhances the In Vitro and In Vivo Biostabilty of Recombinant Human Keratinocyte Growth Factor 1

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1.

Functional expression of mouse insulin-like growth factor-I with food-grade vector in Lactococcus lactis NZ9000

To functionally express the recombinant mouse insulin-like growth factor-I (rtmIGF-I) in Lactococcus lactis NZ9000 with a food-grade vector. The rtmIGF-I encoding sequence was inserted into secreted food-grade vector pLEB688 and transformed into L. lactis NZ9000. The expression of the recombinant protein rtmIGF-I was confirmed by tricine-SDS-PAGE analysis and Western blot. The concentration of this recombinant protein was 3 mg l(-1) in the medium fraction. Further experiment demonstrated that the recombinant protein was biologically active and promoted NIH3T3 cell proliferation in a concentration-dependent manner. The rtmIGF-I was expressed in L. lactis and located into the medium fraction. The optimal final concentration which could promote NIH3T3 cell proliferation after incubation was 100 ng ml(-1) . The rtmIGF-I was functionally expressed in L. lactis NZ9000 with a food-grade vector. Thus, the recombinant L. lactis NZ9000 could act as a host for the production of rtmIGF-I for further study. The recombinant strain could serve as an IGF-I delivery system.

Suppressed proliferation of NIH3T3 cells by stable expression of perlecan shRNA lentiviral particles

Objective To investigate the impact of stable expression of perlecan shRNA lentiviral particles on proliferation of NIH3T3 cells. Methods Mouse fibroblasts were cultured.Lentiviral particles-green fluorescent protein (LV-GFP) was used to transfect the cultured NIH3T3 cells with multiplicity of infection (MOI) of 10,30 and 50.The GFP expression was observed with fluorescence microscopy after transfection for one week to estimate the proper MOI and the time of GFP expression needed.The transfection efficiency of LV-GFP with the proper MOI by fluorescence-activated cell sorting was detected.The stably transfected cell lines were developed by puromycin screening for more than 2 weeks.The third generation HFF in good condition was randomly divided into 3 groups:GFP group,shRNA group and control group.RT-PCR,Western blot and MTT assays were used to detect the expressions of perlecan mRNA and protein and cell proliferation in the 3 groups. Results Perlecan mRNA and protein showed high expressions in the control and GFP groups but low expressions in the shRNA group,with significant differences respectively between the shRNA group and the other 2 groups ( P ＜ 0.05).There was no significant difference between the 3 groups in the optical density at the first 2 days ( P ＞ 0.05).On 3 to 6 days the cells in the control and GFP groups grew normally while the cells in the shRNA group proliferated in a weak manner.the transfected cells in the shRNA group showed a significantly reduced proliferation rate compared with the other 2 groups ( P ＜ 0.05 ). Conclusion The growth of NIH3T3 cells can be inhibited significantly by transfection with perlecan shRNA lentiviral particles. Key words: RNA; Transfection; Fibroblast; Cicatrix; Lentivirus

Stress-Induced Cell-Cycle Activation in Tip60 Haploinsufficient Adult Cardiomyocytes

BackgroundTat-interactive protein 60 (Tip60) is a member of the MYST family of histone acetyltransferases. Studies using cultured cells have shown that Tip60 has various functions including DNA repair, apoptosis and cell-cycle regulation. We globally ablated the Tip60 gene (Htatip), observing that Tip60-null embryos die at the blastocyst stage (Hu et al. Dev.Dyn.238:2912;2009). Although adult heterozygous (Tip60+/−) mice reproduce normally without a haploinsufficient phenotype, stress caused by Myc over-expression induced B-cell lymphoma in Tip60+/− adults, suggesting that Tip60 is a tumor suppressor (Gorrini et al. Nature 448:1063;2007). These findings prompted assessment of whether Tip60, alternative splicing of which generates two predominant isoforms termed Tip60α and Tip60β, functions to suppress the cell-cycle in adult cardiomyocytes.Methodology/Principal FindingsWestern blotting revealed that Tip60α is the predominant Tip60 isoprotein in the embryonic heart, transitioning at neonatal stages to Tip60β, which is the only isoprotein in the adult heart wherein it is highly enriched. Over-expression of Tip60β, but not Tip60α, inhibited cell proliferation in NIH3T3 cells; and, Tip60-haploinsufficient cultured neonatal cardiomyocytes exhibited increased cell-cycle activity. To address whether Tip60β suppresses the cardiomyocyte cell-cycle in the adult heart, hypertrophic stress was induced in Tip60+/+ and Tip+/− littermates via two methods, Myc over-expression and aortic banding. Based on immunostaining cell-cycle markers and western blotting cyclin D, stress increased cardiomyocyte cell-cycle mobilization in Tip60+/− hearts, in comparison with Tip60+/+ littermates. Aortic-banded Tip60+/− hearts also exhibited significantly decreased apoptosis.Conclusions/SignificanceThese findings provide evidence that Tip60 may function in a tumor suppressor pathway(s) to maintain adult cardiomyocytes in replicative senescence.

Design, Synthesis and Anti-fibrosis Activity Study of N1-Substituted Phenylhydroquinolinone Derivatives

Pirfenidone (5-methyl-1-phenyl-2(1H)-pyridone, PFD) is a small-molecule compound acting on multiple targets involved in pathological fibrogenesis and is effective to increase the survival of patients with fibrosis, such as idiopathic pulmonary fibrosis. However, PFD is not active enough, requiring a high daily dose. In this study, to keep the multiple target profiles, N1-substituted phenylhydroquinolinone derivatives, which retain the 1-phenyl-2(1H)-pyridone scaffold were designed and synthesized. The preliminary anti-fibrosis activities for all target compounds were evaluated on a NIH3T3 fibroblast cell line using MTT assay methods. Most compounds showed significant inhibition on NIH3T3 cell proliferation with a IC50 range of 0.09–26 mM, among which 5-hydroxy-1-(4'-bromophenyl)-5,6,7,8-tetrahydroquinolin-2(1H)-one (6j) displayed 13 times higher potency (IC50 = 0.3 mM) than that of AKF-PD (IC50 = 4.2 mM). These results suggest that N1-substituted phenylhydroquinolinone is a promising scaffold which can be applied for further investigation and for developing novel anti-fibrosis agents.

In vitro 및 in vivo에서 검은콩 추출물의 육모 효과

본 연구에서는 검은콩의 육모 촉진 효과를 평가하기 위하여 검은콩 추출물을 이용하여 모유두 세포의 성장 촉진과 모세혈관 확장에 미치는 효과를 평가하였으며, C57BL/6 마우스 모델을 이용하여 검은콩 추출물의 경구 섭취가 모발 성장에 미치는 효과를 2% minoxidil과 비교하여 평가하였다. 그 결과 검은콩 추출물이 모유두 세포의 증식을 촉진하고 모세혈관을 확장 시키는 효과가 있으며, 우수한 성장기 모낭 유도 촉진 효과를 통해 모발의 성장을 촉진하는 것을 확인하였다. 또한, 혈액의 항산화능을 증가시키고 혈중 중성 지질을 감소하는 효과를 가져 혈액을 맑게 하고 혈액 순환에 도움을 주어 육모 및 모발 건강 증진에 기여할 수 있음을 확인하였다. 검은콩 추출물의 우수한 육모 효과를 확신하기 위해서는 사람을 대상으로 6개월 이상의 인체 효능 평가와 검은콩 추출물 내의 유효 성분을 규명하는 연구가 후속으로 진행되어야 할 것이다. 그러나 본 연구는 민간에서 널리 알려진 검은콩의 육모 촉진 효과를 체계적으로 평가하였다는 것에 큰 의의가 있으며, 이를 통하여 검은콩 추출물이 혈액 순환 개선 및 모발 성장 주기 조절을 통해 모발 건강 개선 및 육모 촉진제로써 이용될 수 있다고 기대할 수 있다. It is commonly believed that black soybean (Glycine max: BB) prevents and alleviates hair loss. However, few studies have directly assessed the effect of BB on hair growth. We presently evaluated the mitosis induction on dermal papillae cell and mitogenic effect on NIH3T3 cells in vitro. To elucidate the hair growth promoting effect in vivo, anagen induction and hair restoration were examined in a shaving model of C57BL/6 mice. We also conducted biochemical analysis of blood plasma. Significant stimulation of dermal papillae and NIH3T3 cell proliferation were observed by treatment of BB in a dose-dependent manner. BB markedly promoted hair growth and significantly improved blood total antioxidant capacity and reduced lipid peroxidation and triglyceride level. These results suggest that BB has hair growth promoting effect and it is a potent candidate for the prevention of hair loss.

Cooperative Role of Nectin-Nectin and Nectin-Afadin Interactions in Formation of Nectin-based Cell-Cell Adhesion

The nectin cell adhesion molecules interact in trans with each other through their extracellular regions and with afadin through their cytoplasmic tails, forming adherens junctions in cooperation with cadherins. In a single cell, Necl-5 (nectin-like molecule-5) localizes at the leading edge and regulates directional cell movement in response to a chemoattractant. In such a single cell, afadin also localizes at the leading edge without interacting with nectins or Necl-5. It remains unknown how the nectin-nectin and nectin-afadin interactions are initiated when moving cells contact each other to initiate the formation of adherens junctions. We show here that the Necl-5-nectin interaction induced by cell-cell contact enhances the nectin-afadin interaction. This interaction then enhances the nectin-nectin interaction, which further enhances the nectin-afadin interaction in a positive feedback manner. Thus, the Necl-5-nectin, nectin-nectin, and nectin-afadin interactions cooperatively increase the clustering of the nectin-afadin complex at the cell-cell contact sites, promoting the formation of the nectin-based cell-cell adhesion.

Noninvasive Transdermal Iontophoretic Delivery of Biologically Active Human Basic Fibroblast Growth Factor

Human basic fibroblast growth factor (hbFGF; 17.4 kDa) has shown promise in the treatment of several dermatological conditions; symptomatic improvement was also observed in patients with peripheral arterial disease after arterial infusion. The objective of this study was to demonstrate the feasibility of using transdermal iontophoresis to deliver biologically active hbFGF noninvasively into and across the skin. The protein was cloned, expressed and purified in-house. Porcine skin was used to investigate transdermal iontophoretic transport of hbFGF as a function of current density (0.15, 0.3, and 0.5 mA/cm(2)); results were subsequently confirmed using human skin. Cumulative hbFGF permeation and skin deposition were quantified by ELISA. The absence of proteolytic degradation during skin transit was confirmed by SDS-PAGE. Biological activity postdelivery was determined using cell proliferation assays in human foreskin fibroblast (HFF) and NIH 3T3 cell lines. Confocal laser scanning microscopy (CLSM) was used to visualize the distribution of rhodamine-tagged hbFGF in the skin. Cumulative iontophoretic permeation at 0.3 mA/cm(2) was statistically superior to that at 0.15 mA/cm(2); however, there was no further improvement at 0.5 mA/cm(2). Significant skin deposition of hbFGF was observed, and this dominated transport; for example, after iontophoresis for 8 h at 0.5 mA/cm(2), skin deposition (77.74 ± 37.36 μg/cm(2)) was 4.4-fold higher than cumulative permeation (17.64 ± 5.18 μg/cm(2)). The superior skin deposition may be advantageous for dermatological applications. The HFF and NIH 3T3 cell proliferation assays confirmed that biological activity of hbFGF was retained postdelivery. Coiontophoresis of acetaminophen showed that the dominant transport mechanism switched from electroosmosis to electromigration upon increasing current density from 0.15 to 0.3 mA/cm(2). Experiments using human skin confirmed that iontophoretic permeation of hbFGF across porcine and human membranes was statistically equivalent. CLSM images of rhodamine-tagged hbFGF postiontophoresis indicated that the protein was evenly distributed throughout the epidermis and dermis. In conclusion, the results confirmed that transdermal iontophoresis was indeed able to deliver structurally intact, functional hbFGF noninvasively into and across the skin. The amounts of protein delivered were similar to those in reports from preclinical and clinical studies.

Expression and Production of Therapeutic Recombinant Human Platelet-Derived Growth Factor-BB in Pleurotus eryngii

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is widely used in many therapeutic applications. Until now, there has been no report on rhPDGF-BB expressed in fungi. In this study, we tested whether Pleurotus eryngii could support the expression of human therapeutic rhPDGF-BB protein. A binary vector pCAMBIA1304 containing the hPDGF-BB gene was constructed and introduced into P. eryngii via Agrobacterium tumefaciens-mediated transformation. The transformation of hPDGF-BB gene was confirmed by Southern blot and PCR, whereas the expression was confirmed by Western blot analysis. The recombinant hPDGF-BB reached a maximum expression level of 1.98% of total soluble protein in transgenic mycelia and was in dimeric form. A bioassay revealed that hPDGF-BB expressed in P. eryngii increased proliferation of NIH-3T3 cells similarly to standard material. These results suggest that P. eryngii can be a robust system for the production of human therapeutic proteins including the hPDGF-BB.

Computational modeling of in vitro biological responses on polymethacrylate surfaces

The objective of this research was to examine the capabilities of QSPR (Quantitative Structure Property Relationship) modeling to predict specific biological responses (fibrinogen adsorption, cell attachment and cell proliferation index) on thin films of different polymethacrylates. Using 33 commercially available monomers it is theoretically possible to construct a library of over 40,000 distinct polymer compositions. A subset of these polymers were synthesized and solvent cast surfaces were prepared in 96 well plates for the measurement of fibrinogen adsorption. NIH 3T3 cell attachment and proliferation indices were measured on spin coated thin films of these polymers. Based on the experimental results of these polymers, separate models were built for homo-, co-, and terpolymers in the library with good correlation between experiment and predicted values. The ability to predict biological responses by simple QSPR models for large numbers of polymers has important implications in designing biomaterials for specific biological or medical applications.

Abstract 1352: Mechanism of action of AL622, a molecule rationally designed to release an EGFR and a Src kinase inhibitor

Abstract In many solid tumors, including prostate and breast carcinomas, it has now been shown that Src synergizes with EGFR to promote cell proliferation and invasion. While molecules with EGFR and Src targeting functions have been designed, prodrugs capable of releasing optimized inhibitors of the two targets are scant. We designed AL622, which contains a quinazoline head targeted to EGFR and a hydrolysable linker that connects it to the PP2-like structure. HPLC analysis showed that this molecule is capable of releasing an EGFR inhibitor and intact PP2 inside the cells. The growth inhibition of AL622 in a panel of isogenic NIH3T3 cell lines (wt, Neu/ErbB2 and Her14/EGFR transfected) in comparison with Gefitinib (a clinically approved strong EGFR inhibitor) and PP2 (a Src-inhibitor) was assessed using the SRB assay. Like Gefitinib, AL622 blocked the proliferation of NIH3T3 cells transfected with the EGFR or Her2 gene. Although Gefitinib was twice as potent, AL622 showed a higher selectivity (fold selectivity = 1.77 for Neu-transfected and 1.63 for HER14-transfected cells). On the other hand, AL622 showed a 5-fold greater potency than PP2 in the EGFR and Her2 transfected cells. It was also much more selective than PP2 (fold selectivity = 35.9 for Neu-transfected and 37.8 for HER14-transfected cells). Western blot analysis in MDA-MB231 cells known to be characterized by Src-EGFR interaction showed that AL622 was capable of inducing a dose-dependent inhibition of EGFR and Src. In contrast, PP2 at an equidose was incapable of blocking EGFR but induced potent Src-inhibition. While Gefitinib induced some Src inhibition, it was less potent than AL622. Results from the wound healing assay showed that AL622 was capable of blocking the motility of 4T1 mouse breast cancer cells at doses comparable with those of PP2. However, a Boyden Chamber invasion assay showed that AL622 was capable of inducing more significant inhibition of invasion when compared with PP2 or the combination of PP2 and Gefitinib. The results in toto suggest that designing molecules to inhibit Src and EGFR can be an effective strategy to block aggressive proliferation and invasion in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1352. doi:10.1158/1538-7445.AM2011-1352

Abstract 500: Activation of 5’-flanking sequences of the NANOG pseudogene (NANOGP8) in prostate cancer cell lines increases NANOG RNA expression, sphere formation, and clonogenicity

Abstract In cancer cells, in contrast to normal embryonic stem cells, NANOG is transcribed from the NANOG pseudogene, NANOGP8. The over-expression of NANOGP8 has been documented to promote the proliferation of NIH3T3 cells, the invasion of prostate PC3 cells, and the development of prostate tumors. In this study, we hypothesized that NANOGP8 transcription could be controlled by a promoter-like region in its 5’-flanking sequences and cells with activation of these sequences should exhibit NANOGP8 function, namely sphere formation and increased cell growth. To test this hypothesis, we first verified if the expressed NANOG in prostate cancer comes from NANOGP8. RT-PCR was conducted from RNA collected from the prostate cell lines PC3 and DU145 and primary culture of normal and cancerous prostate cells with primers spanning across both the whole coding region and the 3’UTR. The 1.6kb PCR products were sequenced verifying that the NANOG RNA was transcribed from NANOGP8 and not from NANOG in cancer cells and normal prostate cells. To examine if the 5’-flanking sequences of NANOGP8 have promoter-like function, we cloned a 5kb fragment of the 5’-flanking region of NANOGP8 from a PAC clone containing the whole NANOGP8 gene and inserted the fragment into the pEGFP vector to replace the CMV promoter. The EGFP expression plasmid driven by the 5’-flanking sequence was then transfected into PC3 and DU145 cells. After selection with G418, the EGFP positive cells were separated from EGFP negative cells by flow cytometry and expression of NANOG was analyzed with real-time RT-PCR in the two populations. The RNA expression of NANOG in EGFP positive DU145 and PC3 cells was 3.5 fold and 1.5 fold higher than in EGFP negative cells, respectively. In a DU145 stably transfected cell line the colony formation in the EGFP positive subset was 24% compared to 13.4% in the EGFP negative subset. Similarly, sphere formation in the EGFP positive DU145 cell subset was 40% more than that in the EGFP negative subset. Interestingly, in the spheres formed from the EGFP negative subset about 50% became EGFP positive and of the total spheres formed from both subsets, 82% were EGFP positive and only 18% of spheres were EGFP negative, suggesting that culture in serum-free medium without cell attachment promoted activation of the 5’-flanking region of NANOGP8. In conclusion, NANOG P8 is transcribed in both normal and cancerous prostate cells. The 5’-flanking sequence of NANOGP8 could play a role in expression of the NANOGP8 gene in prostate cancer cells and in regulation of stem-like properties of cancer stem cells. The mechanisms of the activation of the promoter-like 5’-flanking sequence of NANOGP8 is under investigation but could provide potential therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2011-500

Caffeic acid and quercitrin purified from Houttuynia cordata inhibit DNA topoisomerase I activity

A methanol extract of Houttuynia cordata showed an inhibitory effect on mammalian DNA topoisomerase I. Two topoisomerase I inhibitory compounds were purified and identified as caffeic acid and quercitrin. Caffeic acid and quercitrin inhibited the activity of topoisomerase I with IC50 values of about 0.15 and 0.05 mM, respectively. A concentration of 45 µM caffeic acid caused 50% growth inhibition in human leukaemia U937 cells, but not on those of normal fibroblast NIH3T3 cells. However, quercitrin mysteriously stimulated proliferation of U937 and NIH3T3 cells. Caffeic acid-induced cell death was characterised with the cleavage of poly (ADP-ribose) polymerase and procaspase-3, indicating that this inhibitor triggered apoptosis. The apoptotic induction by caffeic acid was also confirmed using flow cytometry analysis. Because DNA topoisomerase I is an important target for tumour chemotherapy, the present study suggests that caffeic acid, but not quercitrin, may function by suppressing oncogenic disease through the inhibition of cellular topoisomerase I activity.

Control of Surface Chemistry, Substrate Stiffness, and Cell Function in a Novel Terpolymer Methacrylate Library

A focused library of methacrylate terpolymers was synthesized to explore the effects of varying surface chemistry and adhesive peptide ligands on cell function. The chemical diversity of methacrylate monomers enabled construction of a library of polymers in which one can systematically vary the chemical composition to achieve a wide range of contact angle, Young's modulus, and T(g) values. Furthermore, the materials were designed to allow surface immobilization of bioactive peptides. We then examined the effects of these material compositions on protein adsorption and cell attachment, proliferation, and differentiation. We observed that chemical composition of the polymers was an important determinant for NIH 3T3 cell attachment and proliferation, as well as human mesenchymal stem cell differentiation, and correlated directly with the ability of the polymers to adsorb proteins that mediate cell adhesion. Importantly, functionalization of the methacrylate terpolymer library with an adhesive GRGDS peptide normalized cellular responses. RGD-functionalized polymers uniformly exhibited robust attachment, proliferation, and differentiation irrespective of the underlying substrate chemistry. These studies provide a library-based approach to rapidly explore the biological functionality of biomaterials with a wide range of compositions and highlight the importance of cell and protein cell adhesion in predicting their performance.