Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic

Mutations in genes that encode components of the phosphatidyl-inositol 3-kinase (PI3-kinase) signaling pathway are common in human cancer. The recent discovery of nonrandom somatic mutations in the PIK3CA gene of many human tumors suggests an oncogenic role for the mutated enzyme. We have determined the growth-regulatory and signaling properties of the three most frequently observed PI3-kinase mutations: E542K, E545K, and H1047R. Expressed in chicken embryo fibroblasts, all three mutants induce oncogenic transformation with high efficiency. This transforming ability is correlated with elevated catalytic activity in in vitro kinase assays. The mutant-transformed cells show constitutive phosphorylation of Akt, of p70 S6 kinase, and of the 4E-binding protein 1. Phosphorylation of S6 kinase and of 4E-binding protein 1 is regulated by the target of rapamycin (TOR) kinase and affects rates of protein synthesis. The inhibitor of TOR, rapamycin, strongly interferes with cellular transformation induced by the PI3-kinase mutants, suggesting that the TOR and its downstream targets are essential components of the transformation process. The oncogenic transforming activity makes the mutated PI3-kinase proteins promising targets for small molecule inhibitors that could be developed into effective and highly specific anticancer drugs.     

Construction, expression and characterization of human interferon α2b-（G4S）n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNc(2b and To(1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris.METHODS: Using PCR and molecular clone techniques,the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1(n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex^TM 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex^TM 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins‘ function in vivo.     

Expression of IL 1betaconverting enzyme in 5-FU induced apoptosis in esophageal carcinoma cells

AIM:To study the role of interleukin 1beta converting enzyme(ICE)in antitumor drug induced apoptosis in tumor cells.METHODS:Morphological changes in human esophageal carcinoma Eca-109 cells after treated with 5-fluorouracil (5-FU) were observed under light and electron microscope. Expression of ICE in the tumor cells exposed to 5-FU was examined by the immunocytochemical method.RESULTS:The cells treated with 5-FU displayed disappearance of nucleoli, chromatin gathering under nuclear envelope, karyorrhexis, budding and the formation of apoptotic bodies.The expression of ICE was negative in control cells, and 5-FU could induce the ICE expression in Eca-109 cells undergoing apoptosis. The number and the staining intensity of positive cells increased with the extension of action time.CONCLUSION: 5-FU may induce apoptosis in human esophageal carcinoma Eca-109 cells; ICE gene may be involved in the regulation of 5-FU induced apoptosis; and ICE protein may mediate apoptosis induced by 5-FU.     

IL-1β activates p44/42 and p38 mitogen-activated protein kinases via different pathways in cat esophageal smooth muscle cells

AIM: To examine the pathway related to the IL-1beta-induced activation of mitogen-activated protein (MAP) kinases in cat esophageal smooth muscle cells. METHODS: Culture of the esophageal smooth muscle cells from cat was prepared. Specific inhibitors were treated before applying the IL-1beta. Western blot analysis was performed to detect the expressions of COX, iNOS and MAP kinases. RESULTS: In the primary cultured cells, although IL-1beta failed to upregulate the COX and iNOS levels, the levels of the phosphorylated forms of p44/42 MAP kinase and p38 MAP kinase increased in both concentration- and time-dependent manner, of which the level of activation reached a maximum within 3 and 18 h, respectively. The pertussis toxin reduced the level of p44/42 MAP kinase phosphorylation. Tyrphostin 51 and genistein also inhibited this activation. Neomycin decreased the density of the p44/42 MAP kinase band to the basal level. Phosphokinase C (PKC) was found to play a mediating role in the IL-1beta-induced p44/42 MAP kinase activity. In contrast, the activation of p38 MAP kinase was inhibited only by a pretreatment with forskolin, and was unaffected by the other compounds. CONCLUSION: Based on these results, IL-1beta-induced p44/42 MAP kinase activation is mediated by the Gi protein, tyrosine kinase, phospholipase C (PLC) and PKC. The pathway for p38 MAP kinase phosphorylation is different from that of p44/42 MAP kinase, suggesting that it plays a different role in the cellular response to IL-1beta.     

Perturbation of transforming growth factor (TGF)-ß1 association with latent TGF-β binding protein yields inflammation and tumors

Transforming growth factor-β (TGF-β) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-β is often released as part of an inactive tripartite complex consisting of TGF-β, the TGF-β propeptide, and a molecule of latent TGF-β binding protein (LTBP). The interaction of TGF-β and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-β from this state is crucial for signaling. To examine the contribution of LTBP to TGF-β function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1^C33S/C33S mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-β1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1^−/− animals. Tgfb1^C33S/C33S mice exhibited decreased levels of active TGF-β1, decreased TGF-β signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-β complex is important for proper TGF-β1 function and that Tgfb1^C33S/C33S mice are hypomorphs for active TGF-β1. Moreover, although mechanisms exist to activate latent TGF-β1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.     

Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin

NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates the stability of the tumor suppressor WT p53. NQO1 binds and stabilizes WT p53, whereas NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. Here, we show that curcumin, a natural phenolic compound found in the spice turmeric, induced ubiquitin-independent degradation of WT p53 and inhibited p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Like dicoumarol, curcumin inhibited the activity of recombinant NQO1 in vitro, inhibited the activity of endogenous cellular NQO1 in vivo, and dissociated NQO1-WT p53 complexes. Neither dicoumarol nor curcumin dissociated the complexes of NQO1 and the human cancer hot-spot p53 R273H mutant and therefore did not induce degradation of this mutant. NQO1 knockdown by small-interfering RNA induced degradation of both WT p53 and the p53 R273H mutant. The results indicate that curcumin induces p53 degradation and inhibits p53-induced apoptosis by an NQO1-dependent pathway.     

Expression of Interleukin-11 and Interleukin-11 receptor α in human colorectal adenocarcinoma; lmmunohistochemical analyses and correlation with clinicopathological factors

AIM: There is strong evidence that interleukin-11 (IL-11) is involved in the regulation of tumor progression, cellular growth and differentiation. Recently, interleukin-11 receptor (IL-11R) has been detected on some cancer cells. In this study, we investigated the expression of IL-11 and IL-11R in colorectal adenocarcinoma. METHODS: To elucidate the involvement of IL-11 and IL-11Ra in human intestinal adenocarcinomas, we examined 115 cases of surgically resected human colonic adenocarcinoma and 11 cases of adenoma by immunohistochemistry and Western blotting. RESULTS: Among 115 cases of adenocarcinoma, 100 cases (87.0%) showed positive staining in the cytoplasm of carcinoma cells for the IL-11, and 87 cases (75.6%) were positive for the IL-11Ra. Six cases (54.5%) and four cases (36.4%) of 11 adenomas were positive for IL-11 and IL-11Ra, respectively. The expression of IL-11Ra correlated with the histological differentiation (P = 0.033503), the depth of tumor invasion (P = 0.006395), Dukes'classification (P = 0.015648) and lymphatic invasion (P = 0.003865). However, the expression of IL-11Ra was not correlated with the venous invasion and the presence of lymph node metastasis. The expression of IL-11 was not correlated with any clinicopathological factors. In Western blot analysis, two human colorectal carcinoma cell lines and four tissues of surgically resected human carcinoma expressed both IL-11 and IL-11Ra proteins. CONCLUSION: IL-11 and IL-11Ra are highly expressed in human colorectal adenocarcinoma and the IL-11Ra expression is correlated with clinicopathological factors. These findings suggest that the expression of IL-11Ra is an important factor for the invasion of human colorectal adenocarcinoma.     

Myofibroblastic cell activation and neovascularization predict native liver survival and development of esophageal varices in biliary atresia

AIM:To study the relation between collagen 1,α-smooth muscle actin(α-SMA)and CD34 expression and the most essential portoenterostomy(PE)outcomes.METHODS:Liver specimens were obtained at PE from33 biliary atresia(BA)patients for immunohistochemical analysis of collagen 1,α-SMA and CD34.Liver biopsies from 35 organ donors were used as controls.Expression patterns were related to clinical data including age at PE,serum total and conjugated bilirubin concentration at the time of PE and during follow-up,incidence of esophageal varices in follow-up upper gastrointestinal endoscopies,and native liver survival as well as to detailed histopathological findings.RESULTS:Collagen 1(16.4%vs 4.5%,P<0.0001),α-SMA(17.9%vs 4.6%,P<0.0001)and CD34(4.9%vs 3.8%,P=0.017)were markedly overexpressed in BA patients compared with controls.Patients who underwent liver transplantation by age of two years had significantly higher expression of collagen 1(18.6%vs 13.7%,P=0.024),α-SMA(20.4%vs 15.4%,P=0.009)and CD34(5.9%vs 4.0%,P=0.029)at PE compared with native liver survivors.CD34-positive microvessels were identified in the centrizonal region close to central vein in every BA patient.In majority of BA cases(56%)neovascularization was frequent as CD34-positive microvessels were observed in over half of the hepatic lobules.In controls,the CD34-positive microvessels were rare as they were completely absent in 40%and were found in less than 5%of the hepatic lobules in the rest.The difference between BA patients and controls was significant(P<0.0001).Patients who developed esophageal varices by two years had significantly higher expression of CD34 at PE compared with patients without varices(5.6%vs 4.0%,P=0.019).Expression ofα-SMA(r=0.758,P<0.0001)and collagen 1(r=0.474,P=0.016),and the amount of CD34-positive microvessels(r=0.356,P=0.047)were related to patient age at PE.CONCLUSION:Hepatic myofibroblastic cell activation,fibrogenesis and neovascularization are enhanced in BA,progress with increasing PE age and relate to native liver survival and development of esophageal varices.     

Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after gammadelta T cell depletion and no effect of alphabeta T cell depletion

BACKGROUND AND AIM: Indirect evidence suggests that CD4+ T cells have a pathogenic while gammadelta T cells have a protective role in the initiation and perpetuation of inflammatory bowel disease. To define the role of T cell subsets in a rat colitis model (2,4,6-trinitrobenzene sulphonic acid (TNBS)) we analysed colitis severity after effective depletion of T helper cells, alphabeta T cells, or gammadelta T cells. METHODS: T helper cells, alphabeta T cells, or gammadelta T cells were depleted using previously described monoclonal antibodies directed at the CD4 molecule (OX38), the CD2 molecule (OX34, both depleting CD4+ T cells), the alphabeta T cell receptor (R73), and the gammadelta T cell receptor (V65). Depletion was verified by flow cytometry and/or immunohistology. Colitis was induced using intracolonic application of TNBS. RESULTS: Surprisingly, depletion of T helper cells or alphabeta T cells had no influence on survival, macroscopic or microscopic scores, or myeloperoxidase activity following colitis induction. In contrast, depletion of gammadelta T cells resulted in significantly increased mortality (V65: 73%, n=15) compared with controls (30%, n=13; p<0.03). In addition, colitis was histologically more severe in the gammadelta T cell depleted group compared with controls (p<0.05). CONCLUSIONS: T helper cells or alphabeta T cells did not influence the initiation or perpetuation of rat TNBS colitis. In contrast, gammadelta T cells had a protective role in rat TNBS colitis as depletion caused increased mortality.     

Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria

Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gram-negative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection.     

P120ctn overexpression enhances β-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.     

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53,proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation.METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry.Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0statistical package.RESULTS: TAp73α overexpressed in HCC tissues (36.2%)when compared with adjacent non-cancerous tissues(2.38%, P&lt;0.005) and normal liver tissues (0, P&lt;0.01).Mutant type p53 (rot-p53) overexpressed in HCC tissues(38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P&lt;0.05) and normal liver tissues (0,P&lt;0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%&#177;4.46% vs27.88%&#177;5.89%, t, P= 0.028).Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%&#177;2.28%vs7.38%&#177;2.61%, t, P = 0.019). Expression of TAp73α was associated with lymph node metastasis and rot-p53,with r = 0.407 and 0.265, respectively. Expression of rot-p53 was associated with Edmondson‘s stage and AFP,with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P= 0.039, 0.012, 0.002, 0.000,0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portalve in invasion, liver membrane invasion and age were independent factors of prognosis.CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.     

Local injection of botulinum toxin type A (BTX-A) prevents scarring esophageal stricture caused by electrocautery in rabbit models

BackgroundBenign esophageal strictures are common in clinical practice. The commonly used methods for preventing benign esophageal strictures still have many shortcomings. In this study, we investigated the preventive effect and possible mechanism of endoscopic local injection of botulinum toxin type A (BTX-A) on scarring esophageal stricture caused by electrocautery in rabbit models, with an attempt to provide a theoretical basis for the clinical application of BTX-A in the prevention of benign esophageal stricture.MethodsAdult male New Zealand rabbits were randomly divided into 4 groups: cautery group (cauterized with 30 W electrocoagulation power without other intervention), saline group (injected with normal saline at 4 spots in the local esophagus after modeling), BTX-A I group (injected with 10 U of BTX-A after modeling), and BTX-A II group (injected with 20 U of BTX-A after modeling). Body weight was measured at postoperative weeks 1, 2, and 4. Esophagography was performed, and the internal diameter of the esophagus was measured. The esophageal tissues were harvested for hematoxylin and eosin (HE) staining and Masson staining. Type I, type III collagen levels and the mRNA expression of transforming growth factor β1 (TGF-β1) in esophageal tissues were detected.ResultsCompared with the cautery and saline groups, the BTX-A I and BTX-A II groups had significantly higher body weight, larger esophageal internal diameter, lower type I and type III collagen levels, and lower TGF-β1 mRNA expression levels in esophageal tissues at postoperative week 4. Comparisons between the BTX-A I and BTX-A II groups showed no significant differences in terms of body weight, esophageal internal diameter, and type I collagen level at postoperative week 4. However, the BTX-A II group had a significantly lower type III collagen level and TGF-β1 mRNA expression level than the BTX-A I group.ConclusionsLocal injection of BTX-A can alleviate esophageal stricture after electrocautery and has a preventive effect on benign esophageal stricture caused by electrocautery in rabbits. The mechanism may be that BTX-A down-regulates the expression of TGF-β1 in the esophageal tissue at the burn site and reduces the deposition of collagen.     

Role of checkpoint kinase 1 (Chk1) in the mechanisms of resistance to histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACi) are a new group of anticancer drugs with tumor selective toxicity. Normal cells are relatively resistant to HDACi-induced cell death compared with cancer cells. Previously, we found that vorinostat induces DNA breaks in normal and transformed cells, which normal but not cancer cells can repair. In this study, we found that checkpoint kinase 1 (Chk1), a component of the G2 DNA damage checkpoint, is important in the resistance of normal cells to HDACi in vitro and in vivo. Inhibition of Chk1 activity with Chk1 inhibitor (UCN-01, AZD7762, or CHIR-124) in normal cells increases their sensitivity to HDACi (vorinostat, romidepsin, or entinostat) induced cell death, associated with extensive mitotic disruption. Mitotic abnormalities included loss of sister chromatid cohesion and chromosomal disruption. Inhibition of Chk1 did increase HDACi-induced cell death of transformed cells. Thus, Chk1 is an important factor in the resistance of normal cells, and some transformed cells, to HDACi-induced cell death. Use of Chk1 inhibitors in combination with anticancer agents to treat cancers may be associated with substantial toxicity.     

Upregulation of 25-hydroxyvitamin D3-1α-hydroxylase by butyrate in Caco-2 cells

AIM: To investigate the possible involvement of 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase [1alpha-25(OH) (2) D(3)] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells. METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 micromol/L 25(OH) (2) D(3) or with 1 micromol/L 1alpha-25(OH) (2) D(3) for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH) (2) D(3) or 1alpha-25(OH) (2) D(3). 1alpha-25(OH) (2) D(3) mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1alpha-25(OH) (2) D(3) protein. Finally, enzymatic activity was measured by ELISA. RESULTS: Both butyrate and 1alpha-25(OH) (2) D(3) stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH) (2) D(3) had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1alpha-25(OH) (2) D(3) mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites. CONCLUSION: Our experimental data provide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1alpha-25(OH) (2) D(3) followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH) (2) D(3) in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.