Inhibition of migration and invasion of colorectal cancer cells via deletion of Rac1 with RNA interference.

Cell migration and invasion are triggered by a number of chemoattractants that stimulate intracellular signaling pathways through regulating reorganization of the actin cytoskeleton. Rac1, an intracellular signal transducer, regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. However, currently, very little is known about the roles of Rac1 in the cytoskeleton formation and invasion of human colorectal cancer cells. In our study, Rac1-shRNA was used to silence the Rac1 to reduce its expression specifically in Lovo cells. Our studies showed that RNA interference-mediated deletion of Rac1 strongly inhibited lamellipodia formation, cell migration, and invasion of Lovo cells in vitro. The deletion of Rac1 can serve as an alterative therapy to inhibit the invasion and metastasis of colorectal cancer cells.

[Spheres isolated from Colo205 cell line possess cancer stem-like cells under serum-free culture condition].

OBJECTIVE: Isolation and expansion tumor spheres from colorectal cancer cell line Colo205 cultured in serum-free medium(SFM) supplemented with human recombinant EGF and bFGF. METHODS: Colo205 cells were cultivated in SFM,while cells cultivated in serum-supplemented medium(SSM) served as the control. Cells morphology were observed by optical microscope, and expression of intestinal stem cells marker Musashi-1 was detected by immunocytochemical. To induce cell differentiation, tumour spheres were cultivated without EGF and bFGF in the presence of 10% serum. Then we analysed expressions of stem cell surface markers CD133 and CD44 among undifferentiated cell, post-differentiated cells and routine Colo205 cells under serum-supplemented culture condition by flow cytometry. At last we compared cell cycle and spectral karyotype between two groups. RESULTS: In SFM consisting of EGF and bFGF, a minority of Colo205 cells could survive, proliferate and form the suspended tumor spheres. We detected high Musashi-1 expression in these cells. Compared with the SSM group and the post-differentiation SFM group, the expressions of CD133 and CD44 were significantly increased in the undifferentiated SFM group (P<0.05). There was no statistical difference in the expression of CD133 and CD44 between the post-differentiation SFM group and the SSM group (P>0.05). Cell cycle analysis indicated that tumor spheres were of a high proliferation state.We could not find any noticeable difference in the number of chromatosomes between the SFM group and the SSM group. CONCLUSION: Tumor spheres in which enriched cancer stem cells can be generated under serum-free culture condition with EGF and bFGF.

[Peripheral blood p53 single nucleotide polymorphism analysis for early diagnosis of colorectal carcinoma].

OBJECTIVE: To evaluate the role of p53 gene mutation in colorectal carcinoma and assess the value of peripheral blood p53 single nucleotide polymorphism (SNP) in early diagnosis of colorectal carcinoma. METHODS: NSP in axons 5-8 of p53 gene was detected using ligase detection reaction-polymerase chain reaction (LDR-PCR) in the peripheral blood of 100 patients with colorectal cancer and 100 healthy subjects. RESULTS: The mutation rate of p53 gene was 24% (24/120) in colorectal carcinoma patients and 0% (0/120) in the healthy subjects (P<0.05). CONCLUSION: p53 plays an important role in the carcinogenesis of colorectal carcinoma, and SNP analysis for p53 gene can be helpful in early diagnosis of colorectal carcinoma.

[Expression and significance of ADP-ribosylation factor 1 (ARF1) in colorectal laterally spreading tumor].

BACKGROUND & OBJECTIVE: Laterally spreading tumor (LST) behaves close to malignant tumor. This study was to search for the genes and functional proteins correlated to growth and development of colorectal LST (CLST), and to reveal the molecular mechanisms of CLST growth pattern and malignant transformation. METHODS: cDNA microarray was used to screen differentially expressed genes among CLST cell line and other 2 colorectal cell lines LoVo and SW480. Different mRNA and protein expressions of the genes were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Compared with LoVo and SW480 cells, 58 genes were up-regulated and 39 genes were down-regulated in CLST cells. mRNA level of ADP-ribosylation factor 1 (ARF1) was significantly higher in CLST cells than in SW480 and LoVo cells (0.93 vs. 0.74 and 0.47, P = 0.04); protein level of ARF1 was significantly higher in CLST cells than in LoVo cells. CONCLUSIONS: CLST has specific gene expression profile. ARF1 is overexpressed in CLST, and may mediate specific biological function of CLST.

[Effects of angiotensin II and aldosterone on NF-kappaB binding activity in hepatic stellate cells].

OBJECTIVE: To investigate the signal transduction mechanism underlying the effects of angiotensin II (AngII) and aldosterone (Aldo) on nuclear factor-kappaB (NF-kappaB) DNA binding activity. METHODS: Sixty male Wistar rats were randomly divided into 4 groups: model group (Mo group), injected with CCl(4) subcutaneously twice a week to establish a model of hepatic fibrosis; perindopril group (Pe group), injected with CCl(4) subcutaneously twice a week and perfused with perindopril once a day; losartan group (Lo group), injected with CCl(4) subcutaneously twice a week and perfused with losartan once a day; and control group (Nc group), injected with olive oil subcutaneously. The rats were killed in batches respectively 4 and 6 weeks after and their livers were collected to undergo Masson staining and be observed by light microscope. Electrophoretic gel mobility shift assay (EMSA) was used to detect the NF-kappaB DNA binding activity in the liver tissues. Western blotting was used to detect the expression of IkappaBalpha in the plasma protein. Hepatic stellate cells (HSCs)-T6 were cultured and preincubated for 1 h or not with U0126 (an inhibitor of MAPK/ERK kinase MEK), irbesartan (an AT-1 receptor blocker), and N-acetylcysteine (NAC, an antioxidant), angiotensin-converting enzyme inhibitor (ACEI), or tumor necrosis factor alpha (TNFalpha) prior to exposure to AngII or Aldo for 0.5 h, 1 h, 2 h, and 4 h respectively. The binding activities of NF-kappaB DNA were observed by EMSA. The expression of IkappaBalpha protein was detected with Western blotting. Histochemistry was used to detect the expression of NF-kappaB p65. RT-PCR was used to detect the expression of TNFalpha mRNA in HSC-T6 cells. RESULTS: The binding activity to NF-kappaB of the liver tissues was the strongest in the Mo group, followed by the Pe and Lo groups and Nc group. The IkappaBalpha expressions in liver tissues 4 and 6 weeks after the beginning of experiment in the Pe and Lo groups were significantly stronger than that in the Mo group (both P < 0.05). 0.5 hour after the intervention of AngII the DNA binding activity of the HSCs began to increase and peaked 1 hour later and then gradually decreased. The increase of NF-kappaB activity induced by AngII could be inhibited by irbesartan, ACEI and NAC pretreatment and could not be inhibited by U0126 pretreatment. Combined action of AngII and TNFalpha significantly increased the NF-kappaB DNA binding activity. The IkappaBalpha expression began to decrease 0.5 hour after the intervention of AngII and reached the lowest value 2 hours after. The expression of IkappaBalpha protein was increased by ACEI (P < 0.05), irbesartan and NAC (both P < 0.01). EMSA showed that 0.5 hour after the intervention of Aldo the DNA binding activity began to be increased and peaked by 1 hour and then began to be decreased. NAC, but not U0126 partly inhibited the increased of NF-kappaB activity induced by Aldo. Combined action of Aldo and TNFalpha significantly increased the NF-kappaB activity. Aldo increased the expression of IkappaBalpha protein in the HSCs at different time points (all P < 0.05). 0.5 hour after the AngII intervention the IkappaBalpha protein expression began to decrease and reach the lowest value 1 hour later and then began to increase 2 hours later. the IkappaBalpha protein expression was significantly decreased in the NAC and NAC+ Aldo intervention groups (both P < 0.05). There was no significant difference in IkappaBalpha protein expression between the Aldo intervention group and U0126 + Aldo, TNFalpha, and Aldo + TNFalpha treatment groups (all P > 0.05). Before stimulation, NF-kappaB was expressed in the plasma of HSCs, however, after the stimulation of AngII or Aldo for 1 hour it was expressed in the nuclei, and then transferred from the nuclei to the plasma 4 hours after the stimulation. However, little nuclear transfer was observed after pretreatment of NAC followed by AngII or Aldo intervention. The TNFalpha mRNA expression was significantly increased in the AngII and Aldo treatment groups in comparison with the control group (both P < 0.05). The TNFalpha mRNA expression was significantly weaker in the irbesartan + AngII, NAC + AngII, and ACEI groups in comparison with the AngII group (all P < 0.05). CONCLUSION: Stimulation of NF-kappaB activity mediates hepatic fibrosis induced by intrahepatic renin-angiotensin-aldosterone system (RAAS).

[Effects of combined use of curcumin and catechin on cyclooxygenase-2 mRNA expression in dimethylhydrazine-induced rat colon carcinogenesis].

OBJECTIVE: To examine the effect of combined use of curcumin and catechin on the number of aberrant crypt foci (ACF) and expression levels of cyclooxygenase-2 (COX-2) mRNA in rat colon carcinogenesis. Methods Dimethylhydrazine (DMH)-induced rats colon carcinogenesis model was used for evaluation of the synergistic inhibitory effect between curcumin and catechin in light of ACF formation and tumor incidence. COX-2 mRNA expression was also detected in rat colon carcinogenesis. RESULTS: Curcumin, catechin and their co-treatment caused significant inhibition of DMH-induced ACF and colon carcinogenesis as compared with untreated DMH-induced rat models (P<0.01). Co-treatment with curcumin and catechins caused greater inhibition of DMH-induced ACF and colon carcinogenesis than the single use of curcumin or catechin (P<0.05). A synergistic inhibitory effect between curcumin and catechin on the expression of COX-2 mRNA was observed in the early stage of rat colon carcinogenesis but not in colon tumor tissues. CONCLUSION: Curcumin and catechin have synergistic effect on ACF and COX-2 mRNA expression in rat colon carcinogenesis, suggesting their potential value in the prevention of human colon cancers.

[Modulation of intestinal mucosal inflammatory factors by curcumin in rats with colitis].

OBJECTIVE: To explore the mechanism of modulation of intestinal mucosal inflammatory factors by curcumin, the inhibitor of the transcriptional factor nuclear factor -kappaB (NF-kappaB), in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis, and screen for a targeted therapeutic agent for treatment of inflammatory bowel disease (IBD). METHODS: Rats with TNBS-induced colitis were fed with diet containing 2.0% curcumin (treatment group), 0.5% sulfasalazine (SASP, positive control group), and normal diet (model group and negative control group). Changes in colonic mucosal histological scores were evaluated and the cytokine mRNA expressions in the colonic tissue assessed by semiquantitative reverse transcriptional PCR (RT-PCR). RESULTS: Treatment with curcumin ameliorated the histopathologic signs in rats with TNBS-induced intestinal inflammation. Curcumin and sulfasalazine obviously suppressed the high expression of proinflammatory cytokine interleukin (IL)-1beta mRNA and increased the low expression of IL-10 mRNA in the colonic mucosa. Expression of the anti-inflammatory cytokine IL-4 mRNA was detected in none of the groups. CONCLUSIONS: Curcumin could modulate the expressions of IL-1beta and IL-10 mRNA in murine model of IBD, which suggests the potential of curcumin as a targeted therapeutic agent for IBD.

[Screening of differentially expressed genes related to laterally spreading tumor by cDNA microarray].

OBJECTIVE: To screen differentially expressed genes between laterally spreading tumor (LST) cell line and common colon carcinoma cell lines, and identify new targets and strategies for exploring the pathogenesis of colorectal tumor. METHODS: The total RNA was extracted from the LST, SW480 and LoVo cells, from which purified mRNAs were obtained. The PCR products of 18 816 genes were blotted onto a fibrous membrane to generate the microarray. The mRNAs from the 3 cell lines were reversely transcribed into cDNA probes and labeled with (33)P before hybridization with the cDNA microarray. After thorough washing, the cDNA microarray was scanned and the 3 samples compared. RESULTS AND CONCLUSIONS: A series of differentially expressed genes were found between the 3 samples, and 58 up-regulated and 39 down-regulated genes were identified among the 97 differentially expressed genes, which suggest different pathogeneses of the laterally spreading tumor. Further analysis of the obtained genes can be helpful in understanding the molecular mechanism of colorectal tumors.

[Magnifying endoscope and stereomicroscope in diagnosing colon tumor: experience with 139 cases].

OBJECTIVE: To explore the practical means for identifying early large bowel carcinoma and precancerous lesions with magnifying endoscope. METHODS: We examined 139 patients with polyp using colonoscopy and mucosal staining, and observed the pit patterns (proposed by Kudo) with magnifying endoscope and stereomicroscope to identify the relations between the pit patterns and the pathologic diagnosis. RESULTS: Polyps were identified in 124 patients and advanced cancers in 9. Five laterally spreading tumors (LST) ranging from 10 to 50 mm in diameter, including 1 of pit IIIL, and 4 of pit IV. were found. The findings of the pit patterns by magnifying endoscope were highly consistent with those by stereomicroscope in these patients. CONCLUSIONS: Pit patterns are crucial to distinguish the cancerous lesions form non-cancerous one and helpful for early detection of colorectal cancers. Pit V may serve as warning sign of early cancerous lesions.

[Preparation and characterization of monoclonal antibody against Clostridium difficile toxin A].

AIM: To prepare monoclonal antibodies (mAbs) against Clostridium difficile toxin A and identify their properties. METHODS: BALB/c mice were immunized with C.difficile toxin A. The splenocytes from immunized mice were fused with myeloma cells Sp2/0. The hybridoma cells were screened by indirect ELISA and limiting dilution method. The titer and relative affinity of ascitic mAbs were determined by ELISA. Specificity of mAbs was analyzed by Western blot. RESULTS: Six hybridoma cell lines (2H7, 3E9, 4B5, 5C10, 6G8 and 8A1) secreting mAbs against C.difficile toxin A were obtained. The Ig classes and subclasses of mAbs 2H7, 3E9 and 6G8 were IgM, mAbs 4B5 and 8A1 were IgG1, and mAb 5C10 was IgG2a. All 6 mAbs had no neutralization activity. Epitope recognized by 5 mAbs(2H7, 4B5, 5C10, 6G8 and 8A1) differed from that by mAb 3E9. Relative affinities of mAbs 8A1 and 4B5 were all above 10(5), and those of other 4 mAbs were 10(4). Western blot analysis no-denatured PAGE showed that all 6 mAbs reacted to C.difficile toxin A with M(r) being 55 x 10(4), and under the condition of denatured SDS-PAGE, Western blot analysis showed that all 6 mAbs reacted to subunits of C.difficile toxin A with M(r) being 5 x 10(4)-24 x 10(4). CONCLUSION: Six mAbs against C.difficile toxin A with high titers were obtained successfully with satisfactory specificity and relative affinity, which will be useful for detection of C.difficile toxin A.

Competitive inhibition of adherence of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027.

AIM: To observe competitive inhibition of adherence of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC) and Clostridium difficile (C. difficile) to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027 (B. ado 1027). METHODS: The binding of bacteria to intestinal epithelial cell line Lovo was counted by adhesion assay. The inhibition of adherence of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo by purified adhesin of B. ado 1027 was evaluated quantitatively by flow cytometry. RESULTS: The purified adhesin at the concentration of 10 microg/mL, 20 microg/mL and 30 microg/mL except at 1 microg/mL and 5 microg/mL could inhibit significantly the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo. Moreover, we observed that a reduction in bacterial adhesion was occurred with increase in the concentration of adhesin, and MFI (Mean fluorescent intensity) was decreased with increase in the concentration of adhesin. CONCLUSION: The purified adhesin of B. ado 1027 can inhibit the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo in a dose-dependent manner.

[Effect of Bifidobacterial adhesin on lipopolysaccharide- and H2O2-induced proliferation and apoptosis of intestinal epithelial cells in vitro].

OBJECTIVE: To study the effect of Bifidobacterial adhesin on proliferation and apoptosis of intestinal epithelial cell induced by lypopolysaccharide (LPS) and H(2)O(2) in vitro. METHODS: With (3)H-TdR incorporation method, flow cytometry and fluorochrome staining, the proliferation and apoptosis of intestinal epithelial cells induced by LPS and H(2)O(2) in vitro were studied. RESULTS: LPS at the dose of 100 microg/L effectively stimulated the proliferation and apoptosis of cells, whereas H(2)O(2) at the dose of 200 micromol/L obviously restrained the proliferative ability while enhanced the apoptosis of the cells. Fluorochrome staining showed cell shrinkage, nuclear condensation and fragmentation under microscope. After treatment with Bifidobacterial adhesin, the cell proliferation and apoptosis decreased significantly in LPS group, and in H(2)O(2) group, cell apoptosis was significantly decreased. CONCLUSIONS: Bifidobacterial adhesin can protect intestinal epithelial cells from the damage by LPS and H(2)O(2), and maintain the balance between the proliferation and apoptosis of the cells.

[Electron microscopic observation of primary cultured laterally spreading tumor cell line].

OBJECTIVE: To observe the microscopic characteristics of laterally spreading tumor (LST) cell line in primary culture. METHODS: The cells isolated from a rectum LST specimen obtained by endoscopic mucosal resection was primary cultured, followed by observation with scanning and transmission electron microscope in comparison with the cells of adenocarcinoma and normal mucosa of the rectum. RESULTS: Scanning and transmission electron microscopes both revealed numerous microvilli covering the surface of the LST cells, and the cytoplasm contained large quantity of lysosomes, mitochondria and phagosomes. Obviously heterogeneous cell nuclei were present with abnormal nuclear fossa and huge nucleoli. CONCLUSION: The cultured LST cells are highly malignant.

[Contribution of eukaryotic initiation factor-4E inhibition to heparanase expression and activity in human colon adenocarcinoma cell line LS-174T].

OBJECTIVE: To determine whether the eukaryotic initiation factor-4E (eIF-4E) is involved in the cap-dependent translational regulation of heparanase and study the correlation between heparanase expression and metastatic potential of LS-174T cells. METHODS: The protein and mRNA levels of inhibited eIF-4E were tested by Western blot and RT-PCR. Heparanase activity was defined as the ability to degrade high molecular weight (40-100 000) radiolabeled ((35)S) heparan sulfate (HS) substrate into low molecular weight (5-15 000) HS fragments. The invasive potential of tumor cells in vitro was observed by Matrigel invasion assay system. RESULTS: The 20-mer antisense oligonucleotide (asODN) against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels. The expression and the activity of heparanase were effectively lowered, which further decreased the invasive potential of LS-174T. CONCLUSION: eIF-4E, probably being involved in translational regulation of heparanase in colon adenocarcinoma cell line LS-174T, can be a particularly interesting target for heparanase regulation, based on of its critical function.

[Expression of pro-inflammation cytokines and activation of nuclear factor kappab in the intestinal mucosa of mice with ulcerative colitis].

OBJECTIVE: To investigate the expression of pro-inflammation cytokines and activation of nuclear factor kappaB (NF-kappaB) in mouse models of ulcerative colitis. METHODS: Mouse models of ulcerative colitis were established by oral administration of 5% dextran sulfate sodium for 7 d, and the expression of tumor necrosis factor (TNF)- alpha and interleukin (IL)-1beta in the intestinal mucosa were detected by semi-quantitative reverse transcriptional (RT) PCR. The activation of NF-kappaB in the intestinal mucosa was evaluated by electrophoretic mobility shift assay (EMSA). RESULTS: The expressions of TNF-alpha and IL-1beta were increased in the intestinal mucosa (P=0.009), and the nuclear binding activity of NF-kappaB was also up-regulated after the onset of colitis. CONCLUSION: Pro-inflammatory cytokines play important roles in the pathogenesis of UC, and may exacerbate the inflammation of the intestinal mocosa and cause apoptosis of the epithelial cells, possibly under the regulation of NF-kappaB activation.

[Role of eukaryotic initiation factor-4E (eIF-4E) in regulation of expression of NF-kappaB and its subsequent influence on transcription and activity of heparanase in human colon adenocarcinoma cell line].

BACKGROUND & OBJECTIVE: Recent studies have revealed that some transcription factors may be translationally regulated by eukaryotic initiation factor-4E (eIF-4E) in human cancer. These modifications in the expression levels of the key transcription factors will alter the expression of some malignancy-related gene products at the transcriptional levels. The current study was designed to investigate the effect of eIF-4E on the expression and activity of NF-kappaB and observe how the activity level of NF-kappaB contributes, as a secondary effect, to the transcription of heparanase in human colon adenocarcinoma LS-174T cells. METHODS: In order to repress the expression of eIF-4E, a 20-mer antisense oligodeoxynucleotide (ASODN) targeted against the translation start site of eIF-4E mRNA was transfected into human colorectal cancer cell line LS-174T via liposome reagent, followed by assessment of the activity and the protein expression of NF-kappaB by an electrophoretic gel mobility shift assay (EMSA) and Western blot analysis respectively. The alterations of heparanase expression were examined by RT-PCR and Western blot analysis, and heparanase activity in LS-174T cells was measured by specific enzymatic activity test using radiolabeled heparan sulfate as the substrate and gel filtration chromatography for the analysis of the degradation product. RESULTS: The 20-mer ASODN against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels, and the repression of eIF-4E gene expression was correlated with decreased expression levels and activity of NF-kappaB protein. Furthermore, this down regulation of the ubiquitous transcription factor NF-kappaB led to reduced transcription of the heparanase gene, and the transfected cells also showed a considerable decrease in heparanase protein and activity. CONCLUSION: These results suggest that eIF-4E play an important role in translational regulation of NF-kappaB expression in LS-174T cells.NF-kappaB is an essential factor in the regulatory mechanisms of heparanase gene transcription, and the suppressed NF-kappaB activity in LS-174T cells may significantly reduce the transcriptional expression of heparanase that consequently leads to decreased heparanase protein expression and enzymatic activity.

[The effects of bifidobacterium on the intestinal mucosa of the patients with ulcerative colitis].

OBJECTIVE: To investigate the effects of bifidobacterium (Bf) on the intestinal mucosa of the patients with ulcerative colitis (UC). METHODS: Thirty patients in clinical and endoscopic remission by sulphasalazine and glucocorticoid were randomized to receive either Bifid Triple Viable capsule (BIFICO), 420 mg/d, or an identical placebo (starch) for 8 weeks. Fecal samples were collected for stool culture before and after treatment. Patients were assessed clinically endoscopically and histologically after 2 months or in the case of a relapse. p65 and IkappaB expression was determined by Western blot analysis DNA-binding activity of NF-kappaB in colonic nuclear extracts was detected by electrophoretic mobility shift assay. The mRNA expression of cytokines were identified by a semi-quantitative assay, reverse transcription-polymerase chain reaction. RESULTS: Three patients in the BIFICO group had relapses within the 2-month follow-up period, compared with 14 in the placebo group (P < 0.01). Fecal concentration of lactobacilli, bifidobacteria, increased significantly from baseline levels only in the BIFICO-treated group (P < 0.01). The expression of NF-kappaB p65 and DNA binding activity of NF-kappaB were significantly attenuated in the treat group than that in control (P < 0.05). The mRNA expression of anti-inflammatory cytokines elevated obviously comparable of control group. CONCLUSIONS: The Bf maybe impede the activation of NF-kappaB, decrease the expression of TNF-alpha and IL-1beta and elevate the expression of IL-10. These results suggest that oral administration of this new probiotic preparation is effective in preventing flare-ups of chronic UC. It will become a prophylactic drug delaying the relapse of UC.

Contribution of eIF-4E inhibition to the expression and activity of heparanase in human colon adenocarcinoma cell line: LS-174T.

AIM: Heparanase degrades heparan sulfate proteoglycans (HSPGs) and is a critical mediator of tumor metastasis and angiogenesis. Recently, it has been cloned as a single gene family and found to be a potential target for antimetastasis drugs. However, the molecular basis for the regulation of heparanase expression is still not quite clear. The aim of this study was to determine whether the expression of eukaryotic initiation factor 4E (eIF-4E) correlated with the heparanase level in tumor cells and to explore the correlation between heparanase expression and metastatic potential of LS-174T cells. METHODS: A 20-mer antisense s-oligodeoxynucleotide (asODN) targeted against the translation start site of eIF-4E mRNA was introduced into LS-174T cells by lipid-mediated DNA-transfection. eIF-4E protein and mRNA levels were detected by Western blot analysis and RT-PCR, respectively. Heparanase activity was defined as the ability to degrade high molecular weight (40-100 kDa) radiolabeled HS (heparan sulfate) substrate into low molecular weight (5-15 kDa) HS fragments that could be differentiated by gel filtration chromatography. The invasive potential of tumor cell in vitro was observed by using a Matrigel invasion assay system. RESULTS: The 20-mer asODN against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels. As a result, the expression and activity of heparanase were effectively retarded and the decreased activity of heparanase resulted in the decreased invasive potential of LS-174T. CONCLUSION: eIF-4E is involved in the regulation of heparanase production in colon adenocarcinoma cell line LS-174T, and its critical function makes it a particularly interesting target for heparanase regulation. This targeting strategy in antisense chemistry may have practical applications in experimental or clinical anti-metastatic gene therapy of human colorectal carcinoma.

Effect of eIF-4E inhibition on heparanase mRNA and its expression in colon adenocarcinoma cell.

OBJECTIVE: To verify whether inhibition of the overexpressed eukaryotic initiation factor-4E (eIF-4E) in human colon adenocarcinoma cell line LS-174T may facilitate the degradation of heparanase mRNA and alter the translation and expression levels of heparanase protein. METHODS: A 20-mer antisense s-oligodeoxynucleotide (asODN) targeted against the translation start site of eIF-4E mRNA was introduced into LS-174T cells by means of lipid-mediated DNA-transfection, followed by Western blotting analysis and reverse transcription-PCR to determine eIF-4E protein and mRNA levels, respectively. Northern methods was applied to determine heparanase mRNA expression level, with the alterations of heparanase expression assessed by Western blotting analysis. RESULTS: The 20-mer asODN against eIF-4E specifically and significantly inhibited eIF-4E protein expression, and as a result, a significant reduction in heparanase mRNA level was observed by Northern blotting in conjunction with significantly decreased heparanase protein expression. CONCLUSION: The inhibition of eIF-4E strongly reduces the stability of heparanase mRNA in colon adenocarcinoma cell line LS-174T and results in an apparent reduction in the expression of heparanase protein.