Roles of Fas signaling pathway in vitamin E succinateinduced apoptosis in human gastric cancer SGC—7901 cells

AIM: To investigate the roles of Fas signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells. METHODS: Human gastric cancer SGC-7901 cells were treated with VES at 5, 10, 20 mg x L(-1), succinic acid and vitamin E as vehicle control and condition media only as untreated (UT) control. Apoptotic morphology was observed by DAPI staining. Western blot analysis was applied to measure the expression of Fas, FADD and caspase-8 proteins. After the cells were transiently transfected with Fas and FADD antisense oligonucleotides, respectively, caspase-8 activity was determined by flurometric method. RESULTS: The morphologically apoptotic changes were observed after VES treatment by DAPI staining. 23.7 % and 89.6 % apoptosis occurred after 24 h and 48 h of 20 mg x L(-1) VES treatment, respectively. The protein levels of Fas, FADD and caspase-8 were evidently increased in a dose-dependent manner after 24 h of VES treatment. The blockage of Fas by transfection with Fas antisense oligonucleotides obviously inhibited the expression of FADD protein. After SGC-7901 cells were transfected with Fas and FADD antisense oligonucleotides, caspase-8 activity was obviously decreased (P<0.01), whereas Fas blocked more than FADD. CONCLUSION: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves Fas signaling pathway including the interaction of Fas, FADD and caspase-8.     

TGF-β activation and lung fibrosis

Lung fibrosis can affect the parenchyma and the airways, classically giving rise to idiopathic pulmonary fibrosis (IPF) in the parenchyma or airway remodeling in asthma and chronic obstructive pulmonary disease. TGF-β activation has been implicated in the fibrosis of both IPF and airway remodeling. However, the mechanisms of TGF-β activation appear to differ depending on the cellular and anatomical compartments, with implications on disease pathogenesis. Although it appears that epithelial cell activation of TGF-β by the αvβ6 integrin is central in IPF, mesenchymal activation of TGF-β by the αvβ5 and αvβ8 integrins appears to predominate in airway remodeling. Interestingly, the mechanism of TGF-β by the integrins αvβ6 and αvβ5 is shared, relying on cytoskeletal changes, whereas activation of TGF-β by the αvβ8 integrin is distinct, relying on proteolytic cleavage of the latency-associated peptide of TGF-β by matrix metalloproteinase 14. This article describes the mechanisms through which epithelial cells activate TGF-β by the αvβ6 integrin and mesenchymal cells activate TGF-β by the αvβ5 integrin, and highlights their roles in lung fibrosis.     

Macrophage coculture enhanced invasion of gastric cancer cells via TGF-β and BMP pathways

Abstract

Objective. Transforming growth factor β (TGF-β) superfamily plays an important role in regulating gastric cancer progression. As previously demonstrated, tumor-associated macrophages (TAMs) promoted the invasion of gastric cancer cells in Matrigel. However, the role of TGF-β superfamily signaling between TAMs and gastric cancer remains unclear. Material and methods. Three-dimensional dynamic migration imaging system was used to detect gastric cancer invasion rate cocultured with macrophages in Matrigel before or after TGF-β receptor 1 or bone morphogenic protein (BMP) receptor 1A and 1B inhibition; real-time RT-PCR was used to quantitatively investigate gene expression (TGF-β1, TGF-β2, BMP4, and BMP7, ADAM9, MMP9, TIMP3, VEGF-A, and VEGF-C). Results. TGF-β1, TGF-β2, BMP4, and BMP7 expressions were increased significantly in macrophages grown with cancer cells as compared to macrophages grown alone. The invasion rate and invasion-related genes expressions of both AGS and Hs-746T gastric cancer cell lines were upregulated by macrophages, although the expression profile was different. Invasion rate and invasion-related genes' expressions of AGS cells cocultured with macrophages were downregulated significantly after TGF-βR1 and BMPR1 inhibition. Conclusions. Macrophages associated with tumor might promote gastric cancer cells invasion though enhancing TGF-β/BMPs signal pathway. Inhibiting TGF-β/BMPs signal between TAMs and gastric cancer cells might provide a new therapeutic method of gastric cancer.     

Antineoplastic effects of octreotide on human gallbladder cancer cells in vitro

AIM:To investigate whether octreotide can inhibit thegrowth of human gallbladder cancer cells in vitro and toelucidate the antineoplastic mechanism of octreotide ingallbladder cancer.METHODS:A human gallbladder cancer cell line,GBC-SD,was cultured in vitro.The antiproliferative effects of octreotidewere examined by means of an MTT assay and a colony formingability assay.Morphological variation was investigated underscanning electron microscopy and transmission electronmicroscopy.Cell cycle analysis and apoptosis rate wasevaluated by flow cytometry (FCM) after staining bypropidium iodide.DNA fragmentation was assayed byagarose gel electrophoresis.Immunohistochemical stainingwas performed to evaluate the expressions of mutant-typep53 and bc1-2.RESULTS:The growth curve and colony forming abilityassay showed significant inhibition of octreotide to theproliferation of GBC-SD cells in culture in a time-and dose-dependent manner.After exposure to octreotide,GBC-SDcells showed typically apoptotic characteristics,includingmorphological changes of chromatin condensation,vacuolardegeneration,nucleus fragmentation and apoptotic bodyformation.In FCM profile apoptotic cells showed increasedsub-G_1 peaks in the octreotide group,significantly higherthan the control group (P=0.013).There was also anaugmentation in the cell proportion of G_0/G_1 phase(P=0.015),while the proportion of S phase and G_2/M phaseremained unchanged (P=0.057 and P=0.280,respectively).DNA agarose gel electrophoresis displayed a ladder afterexposure to 1000 nmol/L octreotide.After being treatedwith octreotide,the expressions of both mutant-type p53and bc1-2 decreased considering the percentage of positivecells (P<0.05).CONCLUSION:Octreotide has a negative action to theproliferation of GBC-SD cells,and the mechanism may berelated to cytostatic and cytotoxic effects.The reduction ofmutant-type p53 and bc1-2 expressions may be associatedwith the apoptosis induced by octreotide.     

TGF-β IL-6 axis mediates selective and adaptive mechanisms of resistance to molecular targeted therapy in lung cancer

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N′-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-β–mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-β–dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-β are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.     

Over-expressing CYLD augments antitumor activity of TRAIL by inhibiting the NF-κB survival signaling in lung cancer cells

The death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in tumor cells. But studies have demonstrated that many tumor cells were resistant to TRAIL-induced apoptosis. CYLD is recognized as a negative regulator of nuclear factor-kappa B(NF-κB) activity. To explore a correlation between CYLD expression and responsiveness to TRAIL in lung cancer cell lines, we established lung cancer cell lines that stably express CYLD. Our data provided the first evidence that increased expression of CYLD directly blocks TRAIL-induced NF-κB activation, and consequently increases TRAIL-induced apoptosis in lung cancer cells. CYLD may act as a therapeutic target of lung cancer. Targeting CYLD, in combination with TRAIL, may be a new strategy to treat lung cancer with high NF-κB activity.     

Role of Tenascin-X in regulating TGF-β/Smad signaling pathway in pathogenesis of slow transit constipation

BACKGROUND Chronic constipation is a gastrointestinal functional disease that seriously harms physical and mental health and impacts the quality of life of patients.Its incidence rate is 2%-27%.Slow transit constipation(STC)is a common type of chronic functional constipation,accounting for 10.3%-45.5%of such cases.Scholars have performed many studies on the pathogenesis of STC.These studies have indicated that the occurrence of STC may be related to multiple factors,such as dysfunction of the enteric nervous system,interstitial cells of Cajal(ICC)damage,and changes in neurotransmitters regulating intestinal peristalsis.AIM To investigate the role of Tenascin-X(TNX)in regulating the TGF-β/Smad signaling pathway in the pathogenesis of STC.METHODS This study included an experimental group and a control group.The experimental group included 28 patients with severe colonic STC,and the control group included 18 patients with normal colon tissues.Immunohistochemistry(IHC)was used to detect c-Kit,a specific marker of the ICC.Western blot,immunofluorescence,and IHC were used to detect the localization and expression of TNX and TGF-β/Smad.RESULTS IHC showed that the number of ICC with positive c-Kit expression was significantly reduced in the colon of STC patients(22.17±3.28 vs 28.69±3.53,P<0.05)and that the distribution was abnormal.Western blot results showed that c-Kit and Smad7 levels were significantly decreased in the colon of STC patients(c-kit:0.462±0.099 vs 0.783±0.178,P<0.01;Smad7:0.626±0.058 vs 0.799±0.03,P<0.01)and that TNX and Smad2/3 levels were higher in the STC group(TNX:0.868±0.028 vs 0.482±0.032,P<0.01).There was no significant difference in TGF-βbetween the two groups(0.476±0.028 vs 0.511±0.044,P=0.272).Pearson correlation analysis showed that the TNX protein exhibited a strong correlation with Smad2/3 and Smad7(P<0.05,|R|>0.8)and TGF-β(P<0.05,|R|=0.7).CONCLUSION The extracellular matrix protein TNX may activate the TGF-β/Smad signaling pathway by upregulating the Smad 2/3 signaling protein and thereby induce slight or complete epithelial stromal cell transformation,leading to an abnormal distribution and dysfunction of ICC in the diseased colon,which promotes the occurrence and development of STC.     

Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.