Vascular endothelial growth factor antisense oligodeoxynucleotides with lipiodol in arterial embolization of liver cancer in rats

AIM:Transcatheter arterial embolization (TAE) of the hepaticartery has been accepted as an effective treatment forunresectable hepatocellular carcinoma (HCC).However,embolized vessel recanalization and collateral circulationformation are the main factors of HCC growth and recurrenceand metastasis after TAE.Vascular endothelial growth factor(VEGF) plays an important role in tumor angiogenesis.Thisstudy was to explore the inhibitory effect of VEGF antisenseoligodeoxynucleotides (ODNs) on VEGF expression incultured Walker-256 cells and to observe the anti-tumoreffect of intra-arterial infusion of antisense ODNs mixed withlipiodol on rat liver cancer.METHODS:VEGF antisense ODNs and sense ODNs wereadded to the media of non-serum cultured Walker-256 cells.Forty-eight hours later,VEGF concentrations of supernatantswere detected by ELISA.Endothelial cell line ECV-304 cellswere cultured in the supernatants.Seventy-two hours later,growth of ECV-304 cells was analyzed by MTT method.ThirtyWalker-256 cell implanted rat liver tumor models were dividedinto 3 groups.0.2 mL lipiodol (LP group,n=10),3OD antisenseODNs mixed with 0.2 mL lipiodol (LP ODNs group,n=10)and 0.2 mL normal saline (control group,n=10) were infusedinto the hepatic artery.Volumes of tumors were measuredby MRI before and 7 d after the treatment.VEGF mRNA incancerous and peri-cancerous tissues was detected by RT-PCR.Microvessel density (MVD) and VEGF expression wereobserved by immunohistochemistry.RESULTS:Antisense ODNs inhibited Walker-256 cells'VEGF expression.The tumor growth rate was significantlylower in LP ODNs group than that in LP and control groups(140.1±33.8%,177.9±64.9% and 403.9±69.4%respectively,F=60.019,P<0.01).VEGF mRNAs in cancerousand peri-cancerous tissues were expressed highest in LPgroup and lowest in LP ODNs group.The VEGF positive ratesshowed no significant difference among LP,control andLP ODNs groups (90%,70% and 50%,H=3.731,P>0.05).The MVD in LP ODNs group (53.1±18.4) was significantlyless than that in control group (73.2±20.4) and LP group(80.3±18.5) (F=5.44,P<0.05).CONCLUSION:VEGF antisense ODNs can inhibit VEGF expression of Walker-256 cells.It may be anantiangiogenesis therapy agent for malignant tumors.VEGFantisense ODNs mixed with lipiodol embolizing liver canceris better in inhibiting liver cancer growth,VEGF expressionand microvessel density than lipiodol alone.     

Hepatocyte growth factor stimulates cell growth and enhances the expression of transforming growth factor α mRNA in AsPC-1 human pancreatic cancer cells

We investigated the effects of hepatocyte growth factor (HGF) and transforming growth factor α (TGF α) on cell growth in four human pancreatic cancer cell lines. Changes in the expression of mRNAs of HGF, c-met, TGF α, and epidermal growth factor receptor (EGFR) by treatment with HGF and TGF α were observed. Cell growth with growth factors was assessed with the MTT assay and compared with basal growth without growth factors. Although HGF stimulated cell growth in AsPC-1, COLO-357, and T3M4 cells, Panc-1 cells showed no response to HGF. TGF α stimulated the growth of all the above cells. The expression of c-met mRNA under nonstimulated conditions was detected with Northern blotting in all cells. Treatment with HGF slightly enhanced the expression of c-met mRNA only in COLO-357 cells. The intensity of EGFR expression was consistent, and HGF mRNA was not detected during induction experiments in any cell type. Concomitant treatment with HGF and TGF α exerted an effect that was additive or less on the growth of all cells. Expression of TGF α was enhanced by HGF treatment only in AsPC-1 cells. These results suggested that HGF and TGF α stimulated cell growth through a final common pathway of signal transduction. Received: November 11, 1998 / Accepted: December 18, 1998     

Growth suppression of transformed cells by a human placental extract not related to transforming growth factor Β

Summary We have examined whether human placental extracts contain tumour-growth-inhibitory factors. One fraction (EAP) from such extracts inhibited growth, in soft agar, ofHa-ras-transformed BALB/c 3T3 cells and human squamous lung carcinoma A-2182 cells. However, this fraction had no effect on the anchorage-dependent growth of these cells, although there was a slight mitogenic activity on nontransformed cells. These data together with those on plating efficiency indicated no significant cytotoxicity of EAP on transformed cell lines. Although this fraction contained transforming growth factor (TGF), this cannot account for its inhibitory activity, since (a) pure TGF does not inhibit anchoragedependent growth of Ha-ras-transformed BALB/c 3T3 cells, (b) EAP retains its inhibitory activity in the presence of antibodies against TGF and (c) the inhibitory activity did not copurify with TGF. Partial characterization of our inhibitory factor suggests that the inhibitory factor is a new tumour-growth-inhibitory factor.Abbreviations SDS sodium dodecyl sulphate - TGF or transforming growth factor or - TIF1 or 2 transformed inhibitory factor 1 or 2 - A-2182 human squamous lung carcinoma cell line - BALB/c 3T3 1-1 mouse fibroblast cell line, clone A31 1-1 - BALB/c 3T3 Ha-ras, BALB/c 3T3 1-1 cells transformed by Ha-ras oncogene - CHO Chinese hamster ovary; MEM, minimum essential medium This research was supported by a training grant from Groupement d'Etude et de Recherche sur le Placenta.     

Scleroderma dermal fibroblasts overexpress vascular endothelial growth factor due to autocrine transforming growth factor β signaling

Abstract

Objectives Overexpression of vascular endothelial growth factor (VEGF) in scleroderma (SSc) skin may play a role in the pathogenesis of the disease. Our study was undertaken to evaluate whether dermal fibroblasts function as one of the sources of the increased VEGF in SSc, and to clarify its mechanism.

Methods Protein and mRNA levels of VEGF were analyzed using immunoblotting, enzyme-linked immunosorbent assay, and real-time PCR. The DNA-binding ability of Smad3 was evaluated by DNA affinity precipitation.

Results VEGF mRNA expression in vivo was increased in SSc skin compared to skin with other collagen diseases. Expression of VEGF protein and mRNA in cultured SSc dermal fibroblasts was constitutively and significantly upregulated. Ectopic TGF-β stimulation induced VEGF synthesis in normal fibroblasts, and TGF-β knockdown normalized the upregulated VEGF levels in SSc fibroblasts. Furthermore, Smad3 overexpression induced VEGF levels. We found that bp ?532 to ?521 on the VEGF promoter is a putative binding site for Smads, and that the binding activity of Smad3 to VEGF promoter was constitutively increased in SSc fibroblasts as well as in normal fibroblasts treated with exogenous TGF-β1.

Conclusions We demonstrated that VEGF were overexpressed due to autocrine TGF-β/Smad signaling in SSc. TGF-β signaling may contribute to the pathogenesis of angiopathy as well as tissue fibrosis.     

Hepatic stem cells and transforming growth factor β in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. It arises from modulation of multiple genes by mutations, epigenetic regulation, noncoding RNAs and translational modifications of encoded proteins. Although >40% of HCCs are clonal and thought to arise from cancer stem cells (CSCs), the precise identification and mechanisms of CSC formation remain poorly understood. A functional role of transforming growth factor (TGF)-β signalling in liver and intestinal stem cell niches has been demonstrated through mouse genetics. These studies demonstrate that loss of TGF-β signalling yields a phenotype similar to a human CSC disorder, Beckwith-Wiedemann syndrome. Insights into this powerful pathway will be vital for developing new therapeutics in cancer. Current clinical approaches are aimed at establishing novel cancer drugs that target activated pathways when the TGF-β tumour suppressor pathway is lost, and TGF-β itself could potentially be targeted in metastases. Studies delineating key functional pathways in HCC and CSC formation could be important in preventing this disease and could lead to simple treatment strategies; for example, use of vitamin D might be effective when the TGF-β pathway is lost or when wnt signalling is activated.     

Rapid mitogen-activated protein kinase activation by transforming growth factor α in wounded rat intestinal epithelial cells

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Fibroblast growth factor 2 and transforming growth factor β1 interactions in human liver myofibroblasts

During liver fibrogenesis, myofibroblastic liver cells proliferate and synthesize components of fibrosis. Fibroblast growth factor 2 (FGF-2) is expressed in vivo in myofibroblastic liver cells (MFLCs) during fibrogenesis, and exogenous FGF-2 is mitogenic for MFLCs. The aim of this study was to study the expression and role of endogenous FGF-2 in cultured human MFLCs. FGF-2 and FGF-2 receptors were studied using immunoblotting. All RNA studies used ribonuclease protection. Growth of MFLCs was studied using [³H]thymidine incorporation and direct cell counting. MFLCs expressed FGF-2 and its receptors FGF receptor 1 and FGF receptor 2. An antibody to FGF-2 blocked the mitogenic effect of transforming growth factor β1 (TGF-β1) for MFLCs but not TGF-β1-induced increase in cellular fibronectin messenger RNA (mRNA). TGF-β1 increased levels of FGF-2 and FGF receptor mRNAs in MFLCs. We have previously shown that TGF-β1 also increased platelet-derived growth factor (PDGF) A chain mRNA in these cells and that anti-PDGF antibody blunted the mitogenic effect of TGF-β1. The present results show that anti-FGF-2 and anti-PDGF-AA are not additive and that FGF-2 and PDGF-AA are not sequentially induced by TGF-β1. FGF-2 mediates the mitogenic but not the profibrogenic effect of TGF-β1 for human MFLCs, and autocrine FGF-2 and PDGF-A interact in the mediation of the mitogenic effect of TGF-β1.     

Expression of growth factors and their receptors in human esophageal carcinomas: regulation of expression by epidermal growth factor and transforming growth factor α

The expression of mRNAs for epidermal growth factor (EGF), transforming growth factor (TGF), EGFR, platet-derived growth factor (PDGF) A and B chain, PDGF receptor (PDGFR), transforming growth factor (TGF),erbB-2 and estrogen receptor (ER) genes was first examined in 6 human esophageal carcinoma cell lines, 6 xenoplanted and 15 surgically resected esophageal carcinomas. Secondly, the effect of EGF and TGF on the expression of these genes by the TE-1 esophageal carcinoma cell line was investigated. The expression of EGF mRNA was detected in 8 (29.6%) of 27 tumors including the cell lines, whereas the TGF and EGFR genes were expressed in 21 (77.8%) and 24 (88.9%) tumors respectively. PDGF B chain and PDGFR were detected in 18 (66.7%) and 20 (74.1%), respectively, and ER mRNA was observed in 16 (59.3%) tumors. Genes for PDGF A chain and TGF and theerbB-2 gene were commonly expressed. On the other hand, exogenous EGF and TGF stimulated the expressions offos andmyc genes by TE-1 cells. The expression of mRNAs for TGF, PDGF A and B chain and theerbB-2 genes was also increased after treatment with EGF. TGF increased the accumulation of mRNAs for EGF, Moreover, the expression of mRNAs for interstitial collagenase, stromelysin and type IV collagenase was increased after EGF or TGF treatment. These results indicate that EGF and TGF may regulate the multi-growth-factor receptor expression and may play a central role for tumor invasion and metastasis as autocrine modulators for human esophageal carcinoma.Abbreviations EGF epidermal growth factor - TGF transforming growth factor - PDGF platelet-derived growth factor - ER estrogen receptor - R receptor     

Survivin antisense oligodeoxynucleotide inhibits growth of gastric cancer cells

AIM:To investigate the effect of transfected survivin antisenseoligonucleotide (ASODN) on proliferation and apoptosis ofgastric cancer ceils.METHODS:The authors designed ASODNs targetingdifferent regions of survMn mRNA,including survivingASODN1,ASODN2 and ASODN3.ASODNs were transfectedinto gastric cancer cell line SGC 7901,cell growth was detectedby MTT assay.Cells exposed to the potent oligonucleotidewere also examined for apoptosis induction by FCM andfluorescence microscopy.Semiquantitive RT-PCR andWestern blot examinations were carried for expression ofsurvivin mRNA and protein.RESULTS:ASODN3 caused a statistically significantreduction of cell viability to 60.6% (±2.9%) (P<0.01),whileASODN1 and ASODN2 had no such changes (P>0.05).Thecell growth was also significantly inhibited by ASODN3,compared with reversal and scrambled sequence.A significantloss of survivin mRNA was presented in ASODN3 treatedceils and this was not seen in treatment with sense ODNor scramble ODN.Protein level was significantly decreased48 h after survivin ASODN trasfected by approximately 2-folddecrease comparedwith untreated controls.However.ASODN3 did not induce significant apoptosis response until48 h after transfection (P>0.05).CONCLUSION:ASODN3,which targets translation initiationpart,can be identified as a most potent antisense compound.Srvivin ASODN3 may provide a novel approach to therapyof gastric cancer.     

Lipopolysaccharide enhances transforming growth factor β1-induced platelet-derived growth factor-B expression in bile duct epithelial cells

Background and Aim: Platelet‐derived growth factor (PDGF)‐B is a potent profibrogenic mediator expressed by bile duct epithelial cells (BDECs) that contributes to liver fibrosis after bile duct ligation. However, the mechanism of PDGF‐B induction in BDECs during cholestasis is not known. Transforming growth factor β (TGFβ) and lipopolysaccharide (LPS) also contribute to the profibrogenic response after bile duct ligation. We tested the hypothesis that LPS and TGFβ1 synergistically induce PDGF‐B expression in BDECs. Methods: Transformed human BDECs (MMNK‐1 cells) and primary rat BDECs were stimulated with LPS and/or TGFβ1, and signaling pathways through which LPS potentiates TGFβ1‐induced PDGF‐B mRNA expression were investigated. Results: Stimulation of MMNK‐1 cells with LPS alone did not significantly induce PDGF‐B mRNA expression. However, LPS co‐treatment enhanced TGFβ1 induction of PDGF‐B mRNA in MMNK‐1 cells and also in primary rat BDECs. Importantly, co‐treatment of MMNK‐1 cells with LPS and TGFβ1 also significantly increased PDGF‐BB protein expression. Interestingly, LPS did not affect TGFβ1 activation of a SMAD‐dependent reporter construct. Rather, stimulation of MMNK‐1 cells with LPS, but not TGFβ1, increased JNK1/2 phosphorylation. Expression of dominant negative JNK2, but not dominant negative JNK1, inhibited the LPS potentiation of TGFβ1‐induced PDGF‐B mRNA expression in MMNK‐1 cells. In addition, LPS treatment caused IκBα degradation and activation of a nuclear factor κB (NFκB)‐dependent reporter construct. Expression of an IκBα super repressor inhibited activation of NFκB and attenuated LPS potentiation of TGFβ1‐induced PDGF‐B mRNA. Conclusions: The results indicate that LPS activation of NFκB and JNK2 enhances TGFβ1‐induced PDGF‐B expression in BDECs.     

Effect of matrine on transforming growth factor β1 and hepatocyte growth factor in rat liver fibrosis model

Objective:To observe the preventive and control effect of matrine on transforming growth factor(TCF- β1) and hepatocyte.growth factor(HCF) of liver fibrosis tissue in rals.Methods:A total of48 SD rats were randomly divided into A,B,C,D groups with 12 in each,group A as the normal control group and groups B.C,D as liver fibrosis models using composite modulus method with carbon tetrachloride(CCL_4).Group B was the model group,group C adopted γ— interferon lavage therapy in the second day of modeling,and group D adopted matrine lavage treatment,at 4 and8 weeks after treatment.Six rats were executed for detection of TGF- β1 and HGF,liver tissue histology and comparison fibrosis degree changes of rat liver tissue between groups.Results:Croups B,C,D showed a more significantly increased TCF- β1 at each time point compared with group A(P0.05);Group B showed a more significantly increased TGF- β1 than groups C and D at weeks 4 and 8(P0.05);group D showed a lowest level of TGF-β1,followed by groups C and B.HGF of group B decreased more significantly than A group at weeks 4 and 8(P0.05);HGF of groups C and D was significantly elevated at 4 and 8 weeks than groups A and B(P0.05),in which the group D showed the highest level of HGF.According to tissue histologic observation,rat liver tissue structure of group A was clear and normal,tissue structure of group B was destroyed with obvious fibrous tissue hyperplasia and fatty change of hepatic cells;groups C and D showed a slighter liver tissue damage,cell necrosis and connective tissue hyperplasia in collect abbacy than group B with a trend of obvious improvement.Conclusions:Matrine can reduce TGF- β1expression and enhance the activity of HGF,so as to realize the inhibition effect on liver fibrosis in rats.     

E-cadherin antagonizes transforming growth factor β1 gene induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation

Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fibrosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECAD's potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1-inducible or Smad3-inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Conclusion: Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFβ1 and its target genes, and facilitates liver fibrosis.