Maternal protein restriction reduces perlecan at mid-metanephrogenesis in rats.

AIM: Maternal dietary protein restriction reduces nephron number in offspring and increases the risk of cardiovascular and chronic kidney diseases. Perlecan is the major basement membrane/extracellular matrix heparan sulfate proteoglycan (HSPG) that plays a crucial role in nephron formation. This study was to determine whether maternal dietary protein restriction during pregnancy leads to an abnormal perlecan expression pattern during kidney development and a correlation with aberrant cell proliferation and apoptosis. METHODS: Pregnant Sprague-Dawley rats were divided into two groups, maintained on either a low-protein diet (MLP group) or a normal-protein diet (MNP group). Kidneys were dissected from embryos of different kidney development stages. Real-time PCR and immunohistochemistry were performed to detect the transcript level of rHSPG2, the coding gene of perlecan, and its protein expression pattern. Apoptosis and proliferation cell were detected by TUNEL system and Ki67 marker. RESULTS: Embryonic weights and nephron number were significantly affected by maternal low protein diets. The transcript level of rHSPG2 in the MLP group was significantly lower at embryonic day 18 and the neonatal period. Immunohistochemistry study was consistent with the RT-PCR results. The proliferation level of the MLP group was significantly lower than the MNP group at E18 and more apoptotic cells was detected in MLP newborn. CONCLUSION: Maternal protein restriction reduced the expression of perlecan and lead aberrant cell proliferation and apoptosis during mid-metanephrogenesis in offspring. This data may provide new evidence to understand the mechanism of reduced nephron number due to maternal protein restriction and enlighten solution.

Angiopoietin-like 3 induces podocyte F-actin rearrangement through integrin α(V)ß3/FAK/PI3K pathway-mediated Rac1 activation.

Glomerular podocytes are highly differentiated cells whose foot processes, which are mainly maintained by the architecture of actin filaments, have a unique morphology. A rearrangement of F-actin in podocytes causes changes in their motility that involve foot process effacement and proteinuria in glomerular diseases. Members of the Rho family small GTPases, especially RhoA, Rac1, and Cdc42, are key molecules in the regulation of actin cytoskeleton rearrangement. Our previous study showed that angiopoietin-like 3 (Angptl3) can increase the motility of podocytes in vitro. In this study, we found that recombinant Angptl3 treatment, together with the activation of Rac1, could cause F-actin rearrangement in podocytes. We also found that these effects could be blocked by an integrin α(V)ß3 inhibitor, implicating integrin α(V)ß3 as the Angptl3 receptor in its effects on actin cytoskeleton rearrangement. In addition, we studied the molecular pathway for this process. Our results showed that in podocytes, Angptl3 could induce actin filament rearrangement, mainly in lamellipodia formation, and that this process was mediated by integrin α(V)ß3-mediated FAK and PI3K phosphorylation and Rac1 activation. Our results might provide a new explanation for the effect of Angptl3 on increasing podocyte motility.

254C>G: a TRPC6 promoter variation associated with enhanced transcription and steroid-resistant nephrotic syndrome in Chinese children.

BACKGROUND: Mutations in canonical transient receptor potential channel 6 (TRPC6) have been identified as responsible for the development of focal segmental glomerulosclerosis, a proteinuric disease with steroid resistance and poor prognosis. This study explores the prevalence of TRPC6 variants in Chinese children with idiopathic nephrotic syndrome (INS), the genotype/phenotype correlation of TRPC6 variants, the therapeutic response, and the underlying molecular mechanism. METHODS: Fifty-one children with sporadic INS were enrolled: 23 steroid-sensitive cases and 28 steroid-resistant cases Polymerase chain reaction was used to amplify 13 exons and the promoter sequences of TRPC6 before sequencing. The expression of TRPC6 in renal tissues was illustrated by immunohistochemistry staining. The transcriptional activity of variants in TRPC6 promoter was measured by the luciferase assay. RESULTS: Three variants (-254C>G, rs3824934; +43C/T, rs3802829; and 240 G>A, rs17096918) were identified. The allele frequency of the -254C>G single-nucleotide polymorphism (SNP) in the steroid-resistant nephrotic syndrome (SRNS) patients (40.5%) was higher than that in the steroid-sensitive nephrotic syndrome subjects (27.1%; P = 0.046). The -254C>G SNP enhanced transcription from TRPC6 promoter in vitro and was associated with increased TRPC6 expression in renal tissues of SRNS patients. CONCLUSION: -254C>G, a SNP underlying enhanced TRPC6 transcription and expression, may be correlated with the development of steroid resistance in Chinese children with INS.

Osteopontin and integrin are involved in cholesterol gallstone formation.

BACKGROUND: This study aimed to investigate the role of osteopontin and its receptor, integrin αv, in gallstone formation using human tissue specimens and a guinea pig lithogenic model. MATERIAL/METHODS: The nucleation role of osteopontin was determined in patients' and normal gallbladder bile samples in vitro. Normal gallbladder was the control, and gallstone gallbladders were divided into group I (with normal epithelia) and group II (with degenerated epithelia) based on pathology change. Immunostaining, mRNA and protein expressions of osteopontin and integrin αv were analyzed. The animals were randomly divided into a lithogenic diet group and a normal diet group; the osteopontin mRNA expression in gallbladder and liver and osteopontin concentrations were determined. RESULTS: Osteopontin prolonged nucleation time and inhibited the pro-nucleating role induced by calcium in human bile in vitro. Immunostaining for osteopontin and integrin αv in human gallbladder tissues showed a higher reactivity in Group I than control group and Group II. The immunostaining in Group II was weaker than control group; similar results were observed for mRNA and protein expression of osteopontin and integrin αv. In the animal assay, the mRNA expression and concentration of osteopontin in gallbladder and liver gradually increased at initial stages and decreased in later stages. The concentrations of osteopontin in bile and serum of guinea pig showed similar trends. CONCLUSIONS: Our results suggest that osteopontin is involved in cholesterol gallstone formation, and the role of osteopontin might correlate with integrin αv and calcium.

Ghrelin inhibits 5-fluorouracil-induced apoptosis in colonic cancer cells.

BACKGROUND AND AIM: The aim of the present study was to investigate if ghrelin inhibits apoptosis in colonic cancer cells. METHODS: Cell viability in HT-29 cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was measured using 4',6-diamidino-2-phenylindole staining and flow cytometry. The protein expression of Bcl-2, Bax, and caspase-3 activation was examined using Western blotting. RESULTS: Ghrelin dose dependently decreased the growth inhibition of HT-29 cells induced by 5-fluorouracil (5-FU). Cells treated with 5-FU displayed chromatin condensation and nuclear fragmentation, which are typical changes of apoptosis. However, co-treatment with ghrelin reduced these changes. Flow cytometry after staining with Annexin V and propidium iodide showed that ghrelin decreased the apoptotic rate of HT-29 cells induced by 5-FU. Caspase-3 activation was significantly lower in the co-treated group than in the group treated with 5-FU alone. In addition, ghrelin reversed the 5-FU-induced Bcl-2/Bax protein ratio. CONCLUSION: Ghrelin inhibits 5-FU-induced apoptosis in colon cancer cells through the regulation of the Bcl-2/Bax protein ratio.

Osteopontin plays an anti-nucleation role in cholesterol gallstone formation.

AIM: To investigate the role of osteopontin in cholesterol gallstone formation. METHODS: Nucleation time was determined in model and human gallbladder bile in vitro. Effect of osteopontin on vesicles of bile was investigated via transmission electron microscopy. The mRNA and protein expression of osteopontin were detected in human calculus and normal gallbladder tissues, and then lipid compositions of human bile were determined via commercial kits. RESULTS: Osteopontin could prolong the nucleation time in a dose-dependent manner, and inhibit the pro-nucleation effect induced by calcium ion. Cholesterol crystal growth was inhibited by osteopontin in a dose-dependent manner in model and human gallbladder bile, but not affected by calcium. Furthermore, the formation, aggregation and fusion of vesicles were suppressed by osteopontin in model and human bile as demonstrated by transmission electron microscopy. The mRNA and protein expression of osteopontin in calculus gallbladder tissues were lower than those in normal tissues. The concentrations of cholesterol, phospholipid and bile acids, and cholesterol saturated index were higher and the contents of osteopontin and calcium, nevertheless, were found to be lower in lithogenic bile than those in normal controls. CONCLUSION: These findings indicated that osteopontin could inhibit the cholesterol gallstone formation as an anti-nucleation factor, which may be involved in the pathogenesis of cholesterol gallstone formation.

Inhibition of cell growth and invasion by epidermal growth factor-targeted phagemid particles carrying siRNA against focal adhesion kinase in the presence of hydroxycamptothecin.

BACKGROUND: Previous studies demonstrated the EGF-targeted phagemid particles carrying siRNA against Akt could be expressed efficiently in the presence of hydroxycamptothecin (HCPT). However, no significant cell growth inhibition was obtained. This study was to further investigate whether the EGF-targeted phagemid particles carrying siRNA would be a promising tool for anti-cancer siRNA delivery. RESULTS: We found that pSi4.1-siFAK phagemid particles could significantly inhibit the expression of focal adhesion kinase in the HCPT-treated cells. Moreover, we also observed that the particles could potently suppress cell growth and cell invasion. CONCLUSION: These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.

[Differential analysis of protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients].

OBJECTIVE: To identify the proteins which play key roles during the formation of cholesterol gallstone, differential analysis was carried out that the proteome of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients. METHODS: Vesicular and micellar phases were isolated by the density gradient ultracentrifugation method. Total proteins from the two phases were extracted, and the protein expressional profiles were established by two-dimensional electrophoresis respectively. The differentially expressed protein spots analyzed by ImageMaster two-dimensional electrophoresis analysis software were identified with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: The concentrations of proteins from vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were (1.5358 +/- 0.0682) mg/ml and (7.1222 +/- 0.2022) mg/ml (P < 0.01) respectively. The average matched protein spots were 120 +/- 24 and 198 +/- 37 in the two groups respectively. There were 72 +/- 16 matched spots in the two representative gels maps and the matched rate was 45.30%. Eight differentially expressed protein spots were identified from the two cholesterol-carrier phases. Among them, 6 were up-regulated with 2 down-regulated in vesicular phase compared with micellar phase. The abundance differentiation of RBP and HSA was confirmed by immunoblotting. CONCLUSIONS: The differential protein profiles of vesicular phase and micellar phase of gallbladder bile from cholesterol gallstone patients were established and 8 differential protein spots were identified successfully. The data may be a basis for further screening the key regulators of formation of cholesterol gallstone.

Localization of alpha-synuclein to mitochondria within midbrain of mice.

The interrelationship between alpha-synuclein (alpha-syn) and mitochondria is not clearly understood. Owing to the lack of the signal peptide and its predominant localization in the cytosol, alpha-syn is generally considered to affect mitochondrial function through some secondary effects. Contrary to this assumption, here, we show that a portion of alpha-syn is present in the membrane of mitochondria in normal dopaminergic neurons. The same profile is also found in other alpha-syn-positive neurons. Thus, binding to the membrane of mitochondria is the physiological nature of alpha-syn and might also contribute to the pathological role of this protein in the mitochondrial dysfunction in Parkinson's disease.

[Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1].

OBJECTIVE: To illuminate the possible role of Prohibitin (PHB) in tubulointerstitial fibrosis. METHODS: (1) Forty-eight renal biopsy specimens were obtained from the patients with various primary glomerulonephritis, 26 male and 22 female, aged 7.5 +/- 5.5 (2.5 - 13 years), and nine kidney tissue specimens were obtained form the tissues far away from the tumor tissues and confirmed by pathological examination as normal tissues in the kidney dissected during operation as normal control. Immunohistochemistry was used to detect the protein expression of PHB and alpha-smooth muscle actin (alpha-SMA). The correlation between PHB and degree of tubulointerstitial lesion was compared. (2) Rat kidney fibroblastoma cells of the line NRK-49F were cultured, and laser scanning confocal microscopy was used to observe the subcellular location of PHB protein. The changes of PHB protein and mRNA expression in the NRK-49F cells upon TGF-beta1 stimulation were detected by Western blotting and RT-PCR analysis. (3) PHB expression plasmid was constructed and transfected into the NRK-49F cells. Then, cell cycle analysis was performed by flow cytometry, and Western blotting and RT-PCR were performed to detect the PHB and alpha-SMA protein and mRNA expression in the NRK-49F cells treated with or without TGF-beta1. RESULTS: (1) PHB protein expression was found in the normal renal tissues by immunohistochemistry, with a positive distribution in the interstitial cells and tubular epithelial cells. PHB was strongly down-regulated in the damaged interstitial and tubular epithelial cells, the higher the grade of damage, the lower the expression of PHB (all P < 0.01), and the PHB expression amount was negatively correlated with the degree of tubulointerstitial lesions (r = -0.802, P < 0.01). (2) Confocal microscopy showed that PHB was mainly located in the cytoplasm and weakly expressed in the nucleus of the NRK-49F cells. Treated with TGF-beta1, the PHB protein expression and mRNA expression in the NRK-49F cells were decreased both time-dependently and dose-dependently (all P < 0.01). (3) A recombinant pcDNA3.1 (-)/PHB plasmid was successfully constructed. PHB protein expression in the transfected NRK-49F cells was 2.54 times higher compared with the non-transfected cells. (4) The proportions of the cells in the S and G(2)/M phases were higher in the NRK-49F cells stimulated by TGF-beta1, however, more NRK-49F cells remained in the G(0)/G(1) phase after transfection of PHB (P < 0.01). (5) Both alpha-SMA protein and mRNA were not expressed in the control cells while de novo expression of alpha-SMA in the NRK-49F cells was increased after the treatment of TGF-beta1. Over-expression of PHB did not affect he basic alpha-SMA expression but dramatically repressed TGF-beta1-initiated alpha-SMA expression in the NRK-49F cells (P < 0.01). CONCLUSION: PHB protein is expressed in the normal renal tissues and adversely correlated with the degree of tubulointerstitial lesions. Extraneous PHB suppresses renal interstitial fibroblast proliferation and cell phenotypic change induced by TGF-beta1.

Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1.

Prohibitin (PHB), a potential tumor suppressor, has been shown to inhibit cell proliferation by repressing E2F-mediated transcription. But little is known about the role of PHB involved in tubulointerstitial fibrosis (TIF). Here, for the first time, we found PHB protein was positively expressed at normal renal tissues, strongly down-regulated in renal biopsy specimens, and negatively correlated with the expression of alpha-smooth-muscle actin (alpha-SMA) and with the degrees of tubulointerstitial lesions. Transforming growth factor-beta1 (TGF-beta1) is the most important profibrotic cytokine in the process of TIF and capable of inducing cell phenotypic change of interstitial fibroblasts characterized by the de novo expression of alpha-SMA. Confocal microscopy showed majority of PHB is located at cytoplasm as well as at nucleus in rat kidney fibroblasts cell (NRK-49F). As we found that PHB protein and mRNA expression were down-regulated in NRK-49F cells following TGF-beta1 stimulation. We used transient transfection to over-express PHB protein and found that cells with increased PHB levels had a significant reduction in the percentage entering cell cycle and abolished de novo expression of alpha-SMA following TGF-beta1 stimulation. Therefore, over-expression of PHB suppresses renal interstitial fibroblasts proliferation and cell phenotypic change induced by TGF-beta1, which indicates PHB as a potential therapeutic target to halt the progression of TIF.

Profilin 1 obtained by proteomic analysis in all-trans retinoic acid-treated hepatocarcinoma cell lines is involved in inhibition of cell proliferation and migration.

Previous studies have shown that all-trans retinoic acid (ATRA) suppresses growth of hepatocarcinoma cell in vitro. To understand the underlying mechanisms, we investigated the protein expression profiles by 2-DE in hepatocarcinoma cell line SMMC-7721 treated with ATRA. Our results reveal that six proteins were differently expressed in response to ATRA. Using MS and database searching, they were identified as profilin 1, phosphoglycerate kinase 1, RuvB-like 1, alpha-enolase, pyridoxal kinase and F-actin capping protein. We selected the up-regulated protein, profilin 1 (PFN1), for further studies. The PFN1 expression was increased in response to ATRA in a dose- and time-dependent manner. The PFN1 expression was reduced dramatically in four hepatoma cell lines compared to L02 cell line of non-tumor origin. The PFN1 expression was also examined in 4 cases of primary hepatocarcinoma tissues by Western blot and 30 cases by tissues microarray. It was found that the protein level of PFN1 was lower in hepatocarcinoma tissues compared to that in the adjacent tissues. Similar to ATRA, overexpression of PFN1 led to inhibition of cell proliferation and migration. Furthermore, RNAi-based PFN1 knockdown could rescue the inhibitory effect of ATRA on cell proliferation and migration. In conclusion, ATRA inhibited cell proliferation and migration through up-regulation of PFN1.

Integrin beta(1A) upregulates p27 protein amount at the post-translational level in human hepatocellular carcinoma cell line SMMC-7721.

Integrins mediate many fundamental cellular processes by binding to components of the extracellular matrix. We showed previously that integrin beta(1A) could inhibit cell proliferation. Integrin beta(1A) stimulated the promoter activity of p21(cip1) and enhanced its transcription in SMMC-7721 cells. In this study, we demonstrated that integrin beta(1A) upregulated p27(kip1) at the post-translational level in SMMC-7721 cells. Our results showed that integrin beta(1A) increased the p27 protein amount, both in cytoplasm and nucleus, but did not affect the p27 mRNA amount. Cycloheximide treatment experiment revealed that the half-life of p27 protein was prolonged in integrin beta1A overexpressing cells, indicating that integrin beta(1A) inhibited the degradation of p27 protein. Our data also provided evidence that both the proteasome and calpain were involved in the degradation of p27 protein in SMMC-7721 cells. Integrin beta(1A) decreased the Skp2 expression and repressed the activity of calpain during G1 phase in SMMC-7721 cells. Taken together, these results indicated that integrin beta(1A) might upregulate the protein amount of p27 through repressing Skp2-dependent proteasome degradation and calpain-mediated proteolysis in SMMC-7721 cells.

The membrane-cytoskeleton organizer ezrin is necessary for hepatocellular carcinoma cell growth and invasiveness.

PURPOSE: The change of cell mobility is one of the preconditions of tumor metastasis. Cell skeleton alteration and rearrangement of F-actin was closely related to cell mobility. Ezrin is a membrane-cytoskeleton organizer that can mediate the rearrangement and the function of F-actin. In this paper, we investigated the effect of ezrin on hepatocellular carcinoma cell growth and invasiveness. METHODS: Hepatocellular carcinoma cell lines such as MHCC-1, MHCC97-H, SF7721, SMMC7721, Hep3B, and HepG2 were chosen in this study. We first examined the expression and the distribution of ezrin and F-actin in these cell lines using immunofluorescence, RT-PCR, and the western blot. Next we used small interfering RNA (siRNA) to down-regulate ezrin expression in MHCC-1, MHCC97-H, SF7721, and HepG2 to investigate the role of ezrin in tumor cell growth and invasiveness. RESULTS: Our preliminary results showed that the expression of ezrin and gamma-actin in MHCC-1, MHCC97-H, and SF7721 with higher metastatic potential were obviously up-regulated than those in SMMC7721, Hep3B, and HepG2 with lower potential. No different expression of beta-actin was found in the above tumor cell lines. The outcome of RNAi indicated that decreasing ezrin expression can notably inhibit the proliferation of the four hepatocellular carcinoma cell lines (p < 0.01, n = 10). The proportion of cells in G2-M phase also decreased after RNAi. The number of pseudopods decreased as well after RNAi treatment (p < 0.01, n = 5). The mobility and invasiveness of cancer cells decreased with decreasing ezrin expression tested by transwell assay (p < 0.01, n = 8). CONCLUSION: Ezrin plays an important role in the process of hepatocellular carcinoma cell proliferation, migration, and invasiveness.

PPARgamma activator rosiglitazone inhibits cell migration via upregulation of PTEN in human hepatocarcinoma cell line BEL-7404.

PPARgamma agonists were reported to be implicated in many biological functions in certain kinds of cells, however, little is known about the effects of PPARgamma on hepatocarcinoma cell. We explored the effects of rosiglitazone, a PPARgamma activator, on human hepatocarcinoma cell line BEL-7404 and its mechanism. After BEL-7404 was exposed to rosiglitazone, its migration was significantly inhibited, which associated with downregulation of the phosphorylation of Akt and FAK, while no significant change was detected in the phosphorylation of ERK after rosiglitazone treatment. It is now known that phosphorylated FAK is a substrate of PTEN and Akt phosphorylation can be regulated by PTEN via the PIP(3) level. We found rosiglitazone upregulated PTEN expression in a dose- and time-dependent manner, which was mediated by PPARgamma. Furthermore, PTEN overexpression resulted in inhibition of cell migration and PTEN knock-down blocked the effect of rosiglitazone on cell migration. It suggested that PTEN was required for rosiglitazone-induced inhibition of BEL-7404 cells migration. In conclusion, our results demonstrated that PTEN played a critical role in rosiglitazone inhibiting cell migration in BEL-7404.

[The influence of downregulation of ezrin expression by RNA interference on the growth and metastasis of hepatocellular carcinoma: experiment in vitro].

OBJECTIVE: To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of human hepatocellular carcinoma cell (HCC) lines. METHODS: Human HCC cells of the lines SF/SMMC7721, MHCC97-H, MHCC-1, and HepG2 were cultured. Four pairs of small interfering RNA (siRNA) targeting erzin were designed and transfected into the HCC cells. 48 h after transfection the cell total RNA was extracted and 72 h later the total cell protein was extracted. RT-PCR and Western blotting were used to detect the transfection rates so as to screen the most effective siRNA to be transfected into the HCC cells. HCC cells were collected every day for 7 days to extract the total RNA and protein. Real-time PCR and Western blotting were used to detect the downregulation rate of erzin at different times. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell cycle and apoptosis. Scanning electron microscopy was used to observe the cell pseudopods. Transwell test was used to detect the invasion ability of the transfected HCC cells. RESULTS: Real-time PCR and western-blotting revealed that ezrin siRNA notably down-regulated ezrin expression at both mRNA and protein levels. Down-regulation of ezrin expression distinctly decreased the proliferation rates of these 4 kinds of HCC line. After RNAi treatment the cell proportion in G(2)-M phase decreased from 28.07% to 21.53% in the SF/SMMC7721 cells, from 24.94% to 13.92% in the MHCC97-H cells, from 19.30% to 13.2% in the MHCC-1cells, and from 7.73% to 4.24% in the HepG2 cells. After RNAi treatment, the number of pseudopods decreased from 20.8 +/- 3.0 to 13.2 +/- 2.4 in the SF/SMMC7721: cells (P < 0.05), from 18.4 +/- 2.7 to 14.0 +/- 2.9 in the MHCC97-H cells (P < 0.01), from 22.6 +/- 3.5 to 13.3 +/- 1.9 in the MHCC-1: cells (P < 0.01), and from 31.0 +/- 2.9 to 17.8 +/- 2.3 in the HepG2 cells (P < 0.01); and the motility and invasiveness decreased from 49.9 +/- 7.7 to 31.9 +/- 5.2 in the SF/SMMC7721 cells (P < 0.05), from 58.5 +/- 4.2 to 33.0 +/- 3.3 in the MHCC97-H cells (P < 0.01), from 57.6 +/- 6.1 to 28.3 +/- 3.4 in the MHCC-1 cells (P < 0.01), and from 37.3 +/- 3.0 to 25.3 +/- 2.3 in the HepG2 cells (P < 0.01). The pseudopods of the HCC cells remarkably shortened and decreased in number (for the SF/SMMC7721 cells: t = 4.95, P < 0.05, for the MHCC97-H cells: t = 5.88, P < 0.01, for the MHCC-1 cells: t = 5.56, P < 0.01, and for the HepG2 cells: t = 5.71, P < 0.01) after siRNA interference. CONCLUSION: Ezrin is necessary for HCC proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis.

P120ctn overexpression enhances beta-catenin-E-cadherin binding and down regulates expression of survivin and cyclin D1 in BEL-7404 hepatoma cells.

AIM: To understand the role of P120ctn in E-cadherin-mediated cell-cell adhesion and signaling as well as in hepatoma cell biological function. METHODS: We stably overexpressed p120ctn isoform 3A in BEL-7404 human hepatoma cells and studied the effect of p120ctn on beta-catenin and E-cadherin binding as well as p120ctn and beta-catenin subcellular localization using immunoprecipitation, Western blotting and confocal microscopy. We also investigated the inhibitory effect of p120ctn transfection on the expression of apoptotic protein survivin survivin and cell cycle regulator cyclin D1 in the cells. RESULTS: Western blotting indicated that p120ctn expression increased after cells were transfected with p120ctn isoform 3A. The protein was located mainly at membrane under immunofluorescent microscope. Beta-catenin nuclear expression was reduced after overexpression of p120ctn isoform 3A. The p120ctn-E-cadherin binding increased after transfection of p120ctn isoform 3A. Furthermore, overexpression of p120ctn down regulated the expression of apoptotic protein survivin and cell cycle regulator cyclin D1. These effects led to reduction of cell proliferation. CONCLUSION: Our results indicate that p120ctn plays an important role in regulating the formation of E-cadherin and -catenin complex, cell apoptosis, cell cycle and cancer cell biological function.

[Molecular cloning and expression of anti-tumor adhesion peptide (beta3)].

To block tumor cell adhesion, inhibit tumor metastasis and recurrence, the anti-adhesion peptide-trimeric beta peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK, beta3) was designed. The DNA fragment of beta3 was cloned into expression vector pET-His and the fusion protein His-beta3 was expressed in E. coli. BL21(DE3)plysS. After 1.5 hours' induction with IPTG, His-beta3 peptide was expressed significantly amounting to 10% of the insoluble proteins and 4% of the total proteins. 20mg of beta3 peptide was obtained from one litter culture medium after purification by using metal-chelating sepharose 6B FF. The purity of beta3 is 92.2% according to Gel-Pro analysis. The anti-adhesion effects of beta3 peptide, beta1 peptide (DLYYLMDLSYSMK) and GRGDS on the hepatocellular carcinoma cell line SMMC-7721 and the high metastasis hepatocellular carcinoma cell line HCCLM6 were studied. The result showed the beta3 blocked the adhesion of HCCLM6 cells and SMMC-7721 cells to fibronectin (FN) specifically. The inhibition effect was dose-dependent and time-dependent and the inhibition rate of beta3 was higher than three times concentration of beta1 and GRGDS. This suggested that pET-His-beta3/BL21(DE3)plysS was a suitable expression system for beta3, and the expressed beta3 specially inhibited the adhesion of cancer cells.

Role of cell adhesion signal molecules in hepatocellular carcinoma cell apoptosis.

AIM: Cell adhesion molecules and their signal molecules play a very important role in carcinogenesis. The aim of this study is to elucidate the role of these molecules and the signal molecules of integrins and E-cadherins, such as (focal adhesion kinase) FAK, (integrin linked kinase) ILK, and beta-catenin in hepatocellular carcinoma cell apoptosis. METHODS: We first synthesized the small molecular compound, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and identified it, by element analysis and (1)H NMR. To establish the apoptosis model of the SMMC-7721 hepatocellular carcinoma cell, we treated cells with DCVC in EBSS for different concentrations or for various length times in the presence of 20 micromol/L N,N'-diphenyl-p-phenylenediamine, which blocks necrotic cell death and identified this model by flow cytometry and DNA ladder. Then we studied the changes of FAK, ILK, beta-catenin, and PKB in this apoptotic model by Western blot. RESULTS: We found that the loss or decrease of cell adhesion signal molecules is an important reason in apoptosis of SMMC-7721 hepatocellular carcinoma cell and the apoptosis of SMMC-7721 cell was preceded by the loss or decrease of FAK, ILK, PKB, and beta-catenin or the damage of cell-matrix and cell-cell adhesion. CONCLUSION: Our results suggested that the decrease of adhesion signal molecules, FAK, ILK, PKB, and beta-catenin, could induce hepatocellular carcinoma cell apoptosis.