;~lasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs （shRNA） can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 （GRIM-19）. Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium （S. typhimurium） to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitroand in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.

Effects of Vascular Endothelial Cell Growth Factor on Fibrovascular Ingrowth into Rabbit's Hydroxyapatite Orbital Implant

Summary: The effects of different concentrations of vascular endothelial cell growth factor (VEGF) on the fibrovascular ingrowth into rabbits hydroxyapatite orbital implant were investigated. Twelve New Zealand white rabbits were divided into 3 groups and received hydroxyapatite orbital implant surgery in their right eyes. Before and after the operation, the implants were treated with 10 ng/ml VEGF, 100 ng/ml VEGF, or normal saline as control group. The animals received technetium bones scan at 2, 4, and 6 weeks postoperatively. The mean radioactivity counts within region of interest (ROI) of the surgery eye (R) and the non-surgery eye (L) in the same animal were tested, and the R/L ratios were calculated. The implants were harvested at 6th weeks and examined histopathologically. The results showed that at second week, there was no significant difference in mean R/L ratios between VEGF group and control group (F=2.83, P=0.111); At 4th week (F=7.728, P=0.011) and 6th week (F=7.831, P=0.011) postoperatively, the mean ratios in VEGF groups were significantly higher than that in control group. At 6th week postoperatively, the fibrovascularization rates in VEGF groups were higher than in control group significantly (F=8.711, P=0.008). It was suggested that VEGF could promote the fibrovascular ingrowth into hydroxyapatite orbital implant, thus might shorten the time required for complete vascularization of the HA orbital implant.

IPN's PVA AND ITS CELL GROWTH PROPERTY

6 kinds of IPN's PVA were synthesized by using different monomers of HEMA, MAA and MMA. The effect of adding polysaccharide was also studied. The cell growth on them were determined and compared. The results showed that the cell growth property of the IPN's PVA with hydrophobic monomer of MMA is better, and it can be improved further by adding hyaluronic acid.

Suppression of cell growth and invasion by miR-205 in breast cancer

MicroRNAs (miRNAs ) 内长的、小、非编码的 RNA，它能够在 post-transcriptional 的 silencing 基因表示铺平。在这研究，我们报导 miR-205 是显著地在与匹配的正常的胸织物相比的胸肿瘤的 underexpressed。同样，包括 MCF-7 和 MDA-MB-231，乳癌房间行比非恶意的 MCF-10A 房间表示低级 miR-205。兴趣， miR-205 的宫外的表示显著地禁止房间增长和抛锚独立人士生长，以及房间侵略。而且， miR-205 被显示在一个动物模型压制肺转移。最后，西方的污点与试金表明的酶记者结合了那 ErbB3 和脉管的 endothelial 生长因素 A ( VEGF --一)是为 miR-205 的直接目标，并且这 miR-205-mediated 抑制通过和在 ErbB3 和 VEGF 的 3 鈥? untranslated 区域( 3 鈥? UTR )的通常认为的 miR-205 绑定地点的直接相互作用是可能的--一。一起，这些结果建议 miR-205 是在乳癌的肿瘤 suppressor。

Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro

AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction(realtime RTPCR),Western blotting and immunofluorescence.Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction(MSP)and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2'deoxycytidine(5azadC)treatment.The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96?AQueous One Solution Cell Proliferation Assay(MTS)after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells.The expression ofβcatenin,phosphorylated extracellular signalregulated protein kinases(pERK)and extracellular signalregulated protein kinases(ERK)was also examined by realtime RTPCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.RESULTS:The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested.The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines,which showed reduced Dkk3 expression.These two cell lines,which initially had a methylated Dkk3 promoter,showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent,5azadC,treatment(P<0.05 or P<0.01).When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells,the ability of cells to proliferate decreased(P<0.01),and the expression ofβcatenin and pERK was downregulated(P<0.01).Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmidtransfected cells.CONCLUSION:Our findings,for the first time,implicate Dkk3 as a tumor suppressor in human pancreatic cancer,through the downregulation ofβcatenin expression via the ERKmediated pathway.

Onco-microRNA miR-130b promoting cell growth in children APL by targeting PTEN

Objective:To study the expression of microRNA-130b(miR-l 30b) in children acute promyelocytic leukemia(APL) and its role for regulating PTEN expression.Methods:A total of SO children APL marrow tissues and IS normal marrow tissues between January and December in 2012 were collected into our study.The expression of miR-l30 b in APL and normal marrow tissues were detected by quantitative real-time polymerase chain reaction.MiR-l30 b inhibitor was transfected into HL-60 cells.Cell Counting Kit-8 assay and flow cytometry were used to measure cell proliferation and apoptosis.respectively.The expression of PTEN,a potential target of miR-130 b,and its downstream genes,Bcl-2 and Box,in transformed cells were detected by quantitative real-time polymerase chain reaction and western-blot Results:The expression of miR-l30 b was significantly higher in children APL marrow tissues than in normal marrow tissues(P<0.05).Down-regulation of miR-1 30 b could significantly suppress cell proliferation and induce apoptosis in HL-60 cells(P<0.05).PTEN expression was upregulated when miR-130 b was knocking-down(P<0.05).As downstream genes of PTEN,the expression of Bcl-2 and Box were regulated as well.Conclusions:MiR-130 b is overexpressed in children APL marrow tissues and associated with cell growth.MiR-130 b may promote children APL progression by inducing cell proliferation and inhibiting apoptosis.

Evidence for the involvement of NOD2 in regulating colonic epithelial cell growth and survival

AIM: To investigate the function of NOD2 in colonic epithelial cells (CEC). METHODS: A combination of in vivo and in vitro analyses of epithelial cell turnover in the presence and absence of a functional NOD2 protein and, in response to enteric Salmonella typhimurium infection, were used. shRNA interference was also used to investigate the consequences of knocking down NOD2 gene expression on the growth and survival of colorectal carcinoma cell lines. RESULTS:In the colonic mucosa the highest levels of NOD2 expression were in proliferating crypt epithelial cells. Muramyl dipeptide (MDP), that is recognized by NOD2, promoted CEC growth in vitro . By contrast,the growth of NOD2-deficient CECs was impaired. In vivo CEC proliferation was also reduced and apoptosis increased in Nod2-/- mice, which were also evident following enteric Salmonella infection. Furthermore, neutralization of NOD2 mRNA expression in human colonic carcinoma cells by shRNA interference resulted in decreased survival due to increased levels of apoptosis. CONCLUSION: These findings are consistent with the involvement of NOD2 protein in promoting CEC growth and survival. Defects in proliferation by CECs in cases of CD may contribute to the underlying pathology of disrupted intestinal homeostasis and excessive inflammation.

Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth

AIM: To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth. METHODS: The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry, and the results were correlated with clinicopathological param- eters, including the mitotic count, proliferative index (Ki-67 immunohistochemical staining), mitotic index (phospho-histone H3 immunohistochemical staining) and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling). Using primary cultured GIST cells, the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting, methyl thiazolyl tetrazolium (MTT), and apoptosis assays. RESULTS: We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples, respectively, and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis, including larger tumor size (P = 0.0118), higher mitotic count (P = 0.0058), higher proliferative index (P = 0.0012), higher mitotic index (P = 0.0282), lower apoptosis index (P = 0.0484), and increased National Institutes of Health risk level (P = 0.0012). We also found that the introduction of exogenous SCF potently increased KIT kinase activity, stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation, while a KIT immunoblocking antibody suppressed proliferation (P = 0.01) and promoted apoptosis (P < 0.01) in cultured GIST cells. CONCLUSION: SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth. The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.

The Effects of Liquor Spirits on RNA Pol III Genes and Cell Growth of Human Cancer Lines

Alcohol consumption is a major health issue and associated with human cancers, such as liver and breast cancers. Alcohol was classed as carcinogen to human by IARC. We have performed in vivo and in vitro studies which demonstrate that diluted ethanol promotes cell proliferation and transformation and tumor formation. Consumption of liquor spirits (white wines) is a popular behavior. However, it is unclear whether liquor spirits affect cellular phenotypes of human cancers. At present study, we used diluted ethanol and liquor spirits (Sample #1 and Sample #2) to determine the changes in RNA polymerase III-dependent gene (Pol III gene) transcription, cell growth and colony formation in the different human cancer lines. The results indicate that low concentration of ethanol increases RNA Pol III gene transcription and rate of cell growth. However, both liquor spirits (Sample #1 and Sample #2) inhibit the activity of RNA Pol III genes and repress cell proliferation of the cancer lines, compared to diluted ethanol. The liquor spirits reduce the rate of colony formation of human breast cancer cells and esophageal carcinoma cells. The inhibitions of the liquor spirits to RNA Pol III genes, cell growth and colony formation are in a dose-dependent manner. These new findings suggest that the liquor spirits contain some active components to repress Pol III gene transcription and cell growth caused by ethanol in different human cancer cells.

Inhibition of human gastric carcinoma cell growth by atofluding derivative N_3-o-toluyl-fluorouracil

AIM: To evaluate the growth inhibition efficacy of atofluding derivative N3-o -toluyl-fluorouracil (TFU) on human gastric carcinoma cell lines SGC-7901 and MKN-45. METHODS: Cell growth inhibition by TFU was measured by MTT and clonogenic assays without or with liver microsomal enzymes. Xenografts of cancer cells in nude mice were employed to study the anti-proliferative effects of TFU in vivo. RESULTS: TFU inhibited the growth of SGC-7901 and MKN-45 cells. However, the inhibitory effects of TFU on cell growth were not significant. The inhibition rates were enhanced in the presence of liver microsomal enzymes, ranging 4.73%-48.57% in SGC-7901 cells and 9.0%-62.02% in MKN-45 cells. In vivo, TFU delayed the growth of SGC-7901 and MKN-45 cells in nude mice. The inhibition rates were 40.49%, 63.24%, and 75.98% in SGC-7901 cells and 40.76%, 61.41%, and 82.07% in MKN-45 cells when the oral doses were 25, 50, and 100 mg/kg, respectively. TFU treatment was generally welltolerated by mice with less than 20% reduction in body weight. CONCLUSION: TFU inhibits the growth of human gastric carcinoma cells. The inhibition rates are increased in the presence of liver microsomal enzymes. The efficacy of TFU may be associated with the sustaining release of 5-fluorouracil (5-FU) mediated by the enzymes.

Inhibition of Obtusifolin on retinal pigment epithelial cell growth under hypoxia

AIM: To explore the effect of Obtusifolin on retinal pigment epithelial cell growth under hypoxia.METHODS: In vitro chemical hypoxia model of ARPE-19 cells was established using cobalt chloride(CoCl2). Cell viability was tested by cell counting kit-8(CCK-8) assay. Western blot and real-time quantitative polymerase chain reaction were applied to detect proteins and mRNAs respectively. Flow cytometry was used to examine the cell cycle. Secretion of vascular endothelial growth factor(VEGF) was tested by using enzyme linked immunosorbent assay(ELISA).RESULTS: Under the chemical hypoxia model established by CoCl2, hypoxia inducible factor-1α(HIF-1α) mRNA and protein levels was up-regulated. Cell viability was increased and the proportion of S phase was higher. Obtusifolin could reduce cell viability under hypoxic conditions and arrest cells in G1 phase. Obtusifolin reduced the expression of Cyclin D1 and proliferating cell nuclear antigen(PCNA) in the hypoxic environment and increased the expression of p53 and p21. The levels of VEGF, VEGFR2 and eNOS proteins and mRNA were significantly increased under hypoxia while Obtusifolin inhibited the increasing.CONCLUSION: Obtusifolin can inhibit cell growth under hypoxic conditions and down-regulate HIF-1/VEGF/eNOS secretions in ARPE-19 cells.

ESTABLISHMENT OF A HUMAN B CELL LINE THAT RESPONDS SPECIFICALLY TO B CELL GROWTH FACTOR

A human B cell line （3D5） that responds specifically to B cell growth factor （BCGF） hasbeen developed by a sequence of Staphylococcus aureus Cowen I activation,EB virus im-mortalization,and cloning.Proliferative response to PHA-stimulated T cell supernatant（PHA-T-Sup） and nonresponsiveness to rIL-2 stimulation were factors used to screen positivecells.Phenotype analysis with a flow cytometer indicated that:1) 3D5 is a B cell line:100% of the cells were positive for B1 marker and 59% were positive for sIg,while T3and Mo 1 were negative:2) 3D5 is an activated B cell line:both Tac and 4F2 markersof activated （but not of resting） B cells were 100% positive:3) 3D5 expresses high molecularweight BCGF （HMW-BCGF） receptor-associated epitope BA5.3D5 cells proliferated inresponse to cpBCGF stimulation in a dose-dependent manner.HMW-BCGF also induced3D5 cells to proliferate.Interestingly.no proliferation could be detected in the presenceof rIL-2,rIL-4,or rIFN-r.The data show that 3D5 cells are specifically BCGF-responsiveB cells.Using 3D5 cells as target,BCGF activity was detected in crude BCGF preparationsedimented by 85% （NH4）2SO4 and chromatographed in a DEAE-Sephadex A-25 column fromPHA-T-Sup.T24 cell supernatant with B cell differentiation factor （BCDF） activity couldnot induce 3D5 cells to differentiate into immunoglobulin-secreting cells.

Inhibitory Effects of AURKB Gene on Apoptosis and Cancer Cell Growth in HCT 116 Cells

[Objectives]To explore the inhibitory effect of AURKB gene in apoptosis and cancer cell growth in HCT 116 cells.[Methods]The in vitro cytology studies were carried out to confirm the expression of the AURKB gene in HCT 116 cells and make clear its role in cell activity,cell cycle control and apoptosis,and investigate the effect of AURKB gene in colorectal cancer(CRC).Quantitative reverse transcription/polymerase chain reaction(PCR)analysis and immunofluorescence(IF)staining for markers of AURKB gene were used to examine the effect of AURKB on HCT 116.The AURKB gene target was examined using western blot analysis.In addition,inhibition of AURKB expression was examined using RNA interference(RNAi)on HCT 116 cells in vitro.[Results]HCT 116 cells infected with AURKB shRNA virus suppressed expression of AURKB in vitro.AURKB gene knockdown HCT 116 cells showed reducing cell apoptosis in vitro.Finally,it demonstrated that AURKB function can induce apoptosis of HCT cells.[Conclusions]AURKB is a key regulator of colorectal cancer.AURKs are potential novel molecular targets for the prevention of cancer cell proliferation.

Sodium Nitroprusside inhibits HEK293 Cell Growth by cGMP-Dependent and Independent Mechanisms

The acute and chronic effects of sodium nitroprusside (SNP) are well characterized for vascular smooth muscle cells (VSMC). Stimulation of soluble guanylyl cyclase (sGC) gives a rapid elevation of intracellular cGMP levels and relaxation of VSMC. The antiproliferative effect of SNP needs days to develop. In the present study human embryonic kidney (HEK 293) cells were used to study the growth after repeated exposure to SNP. A dose-dependent antiproliferative effect was evident and after 5 days with an IC50 value of 108 μM. Cyclic GMP was able to mimic the antiproliferative effect of SNP on HEK293 cells. When cGMP (1000 μM) was added to the cell culture medium for 5 days the cell densities were reduced with 37% below baseline and cGMPin increased from 5.3 to 195 pmol/107cells. The interaction with the non-selective PDE (cyclic nucleotide phosphodiesterase) inhibitor 3-isobutyl-1-methylxanthine (IBMX) was tested after three days. IBMX alone (1000 μM) reduced cell densities with 48% and elevated cGMPin (from 5.2 to 9.3 pmol/107cells). The effect of 10 μM SNP was reinforced on proliferation (from 13% to 90%) and elevation of cGMP levels (from 7.6 to 13.5 pmol/107cells). A corresponding effect was observed after addition of 1000 μM cGMP and 1000 μM IBMX for 3 days. The antiproliferative effect of cGMP increased from 30% to 89% and the cGMPin increased from 240 to 480 pmol/107cells. However, additional mechanisms exist for the antiproliferative effect of SNP. One of these is the intracellular oxidative effect which includes production of S-nitrosoglutathione. The fall in ratios between GSH and GSSG from 260 to 85 after 100 μM SNP exposure is compatible with such a mechanism since cGMP (1000 μM) added to the culture medium did not change the ratio. This study shows that the antiproliferative effects of SNP on HEK293 cells are mediated through cGMP-dependent and cGMP-independent mechanisms. The concentration-dependent effects develop over time. HEK293 cells had an efficient efflux system for cGMP and the use of inside-out vesicles (IOVs) showed high affinity ATP-dependent cGMP transport with a Km value of 2.3 μM. The antiproliferative effect of SNP was correlated to cGMPex/in.

Effect of Vitamin K1 on Cell Growth Inhibition and Apoptosis on the U937 Cell Line

This experiment was conducted in order to verify the role of Vitamin K1 as a cell growth inhibitor on the U937 cell line. This experiment was performed in two parts—one with a lesser concentration of Vitamin K1, and the other with a range of concentrations from low-to-high. Through the remaining number of U937 cells, as well as cell areas, it was concluded that the presence of Vitamin K1 reduces the number of cancer cells. It was also concluded that as Vitamin K1 concentration increases, so does the frequency and effects of apoptosis.

<i>In-Vitro</i>Effect of Steroids on Melanoma Cell Growth—A Prelude to Melanoma Treatment?

Skin is not only a target organ for various sex steroids and hormones, but also an endocrine organ, which produces sex steroids. It has been suggested by Nikolakis et al. that impairment in skin steroidogenesis may result in inflammatory or autoimmune or other skin disorders. Melanoma is one such skin disease or disorder, which is believed to be caused by UV rays. But, epidemiological, clinical, in-vivo and in-vitro studies suggested the involvement of steroids in the regulation of melanoma growth. However, these studies either did not identify the steroid involved or did not relate to the protective function of the steroid in menstruating females in melanoma, as reported by the clinical studies. In this context, our studies with mouse and human melanoma cell lines showed that female sex steroid progesterone not only inhibited melanoma cell growth, but also affected adhesion and migration functions. In addition, our studies also showed that the effect of progesterone was not a toxic or spurious, but a specific effect on melanoma cells. Hence, our in-vitro studies along with previous other studies subscribed to the idea proposed earlier by Slominski et al. that modulation of local steroids could be a new therapeutic approach for treatment of skin disease or disorder, melanoma.

Cloning and characterization of proliferating cell nuclear antigen gene of Alexandrium catenella (Dinoflagellate) with respect to cell growth

Harmful algal blooms （HABs） have been affecting negatively the shellfish and aquaculture industries around the world. Though a lot of efforts have been made to disclose the changes of environmental factors involved and their effects on the HABs events, the molecular mechanism of this process remains unclear. To address this problem, proliferating cell nuclear antigen gene （pcna） was isolated and characterized from Alexandrium catenella. It showed high homology to those of other dinoflagellates （89% and 91% homology to Pfiesteria piscicid and Pyrocystis lunula, respectively）, and also 42%–43% homology to those of plant and animals. The expression level of pcna revealed by quantitative real time PCR was the lowest at the late lagging cell growth phase, increased to the highest at the late exponential phase, and then decreased at the stationary phase. Though the cell growth rate was also changing, no positive correlation between pcna expression level and cell growth rate was displayed throughout the whole cell growth stages （r 2 =0.024 6）. However, the pcna expression level had the similar trend with the change of cell growth rate throughout the whole growing process, e.g., from increasing at the earlier cell growth stage to decreasing at the following stages, though slightly lagging to the latter.

Vaccinia-related kinase 2 variants differentially affect breast cancer growth by regulating kinase activity

Genetic information is transcribed from genomic DNA to mRNA,which is then translated into threedimensional proteins.mRNAs can undergo various post-transcriptional modifications,including RNA editing that alters mRNA sequences,ultimately affecting protein function.In this study,RNA editing was identified at the 499th base(c.499)of human vaccinia-related kinase 2(VRK2).This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine(with adenine base)to valine(with guanine base).Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2,which leads to an increase in tumor cell proliferation.Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein(dysbindin)and results in reducing its stability.Herein,we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valinecontaining VRK2.Dysbindin interacts with cyclin D and thereby regulates its expression and function.The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression,resulting in increased tumor growth and reduction in survival rates.It has also been observed that in patient samples,VRK2 level was elevated in breast cancer tissue compared to normal breast tissue.Additionally,the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue.Therefore,it is concluded that VRK2,especially dependent on the 167th variant amino acid,can be one of the indexes of tumor progression and proliferation.