Transforming growth factor-beta induces apoptosis in activated murine T cells through the activation of caspase 1-like protease

Transforming growth factor-beta (TGF-beta) has been known as a potent immunosuppressive cytokine that can induce apoptosis in lymphoid cells. We established an IL-2-independent cell line, CTLL-2A, from murine T cell line CTLL-2. CTLL-2A expressed higher levels of CD95, CD69, and CD18 molecules than CTLL-2 did, suggesting a more activated state in CTLL-2A than in the CTLL-2 by phenotype. Exposing both CTLL-2 and CTLL-2A to TGF-beta results in differential apoptosis patterns defined by DNA fragmentation and plasma membrane alteration. Among the bcl-2 family members, bcl-2, bcl-w, and bcl-x(L) were also differently expressed in these two cell lines. In CTLL-2A, bcl-x(L) was amplified as a major anti-apoptotic molecule, and TGF-beta-induced cell death was more enhanced than in the original cell line. Caspase 1-like protease was activated by TGF-beta treatment and consequently it cleaved bcl-x(L) in CTLL-2A. TGF-beta-induced DNA fragmentation and cleavage of bcl-x(L) were inhibited by pretreatment with tetra peptide caspase 1 inhibitor, YVAD.cmk. These findings suggest that TGF-beta induces cell death in activated murine T cells through cleavage of bcl-x(L) via activated caspase 1-like protease, which may act as an important executor in that process.     

Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.     

Hispolon induces apoptosis in oral squamous cell carcinoma cells through JNK/HO-1 pathway activation

Oral squamous cell carcinoma (OSCC) has a high recurrence rate and poor prognosis. Hispolon, a polyphenolic compound with antiviral, antioxidant, and anticancer activities, is a potential chemotherapy agent. However, few studies have investigated the anti-cancer mechanism of hispolon in oral cancer. This present study used the cell viability assay, clonogenic assay, fluorescent nuclear staining, and flow cytometry assay to analyse the apoptosis-inducing effects of hispolon in OSCC cells. After hispolon treatment, the apoptotic initiators, cleaved caspase-3, −8, and − 9, were upregulated, whereas the cellular inhibitor of apoptosis protein-1 (cIAP1) was downregulated. Furthermore, a proteome profile analysis using a human apoptosis array revealed the overexpression of heme oxygenase-1 (HO-1) by hispolon, which was determined to be involved in caspase-dependent apoptosis. Moreover, cotreatment with hispolon and mitogen-activated protein kinase (MAPK) inhibitors revealed that hispolon induces apoptosis in OSCC cells through activation of the c-Jun N-terminal kinase (JNK) pathway and not the extracellular signal-regulated kinase (ERK) or p38 pathway. These findings indicate that hispolon may exert an anticancer effect on oral cancer cells by upregulating HO-1 and inducing caspase-dependent apoptosis by activating the JNK pathway.     

Transforming growth factor-beta1 inhibits thrombin activation of endothelial cells

Transforming growth factor-beta1 (TGF-beta1) is reported to exert both pro- and anti-inflammatory effects on the chronic activation of endothelial cells (ECs) in vitro by cytokines such as tumour necrosis factor-alpha (TNF-alpha). However, the effects of TGF-beta1 on acute inflammatory responses of ECs in vitro (e.g. to thrombin) have not been characterised. Pretreatment with TGF-beta1 (10 ng/mL) effectively inhibited all the thrombin-stimulated responses in rat aortic endothelial cells (RAECs) examined: adhesion and migration of polymorphonuclear leukocytes, adhesion of platelets and lymphocytes. Substantial inhibition of thrombin stimulation occurred after 30 min of pretreatment with TGF-beta1 and maximal inhibition was obtained after 1-20 h of pretreatment. Inhibition by TGF-beta1 pretreatment for 30 min was not affected by cycloheximide and was therefore independent of protein synthesis. Treatment with TGF-beta1 for 20 h did not affect the total levels of P-selectin and von Willebrand factor (vWF) in RAECs, but reduced thrombin-stimulated recruitment of P-selectin and vWF to the cell surface. The data demonstrate that TGF-beta1 exerts a potent anti-thrombin effect on ECs, effective after long and short pretreatment times.     

Transforming growth factor-beta1 is responsible for maturation-dependent spontaneous apoptosis of cultured gastric pit cells

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-beta1 (TGF-beta1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-beta1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-beta1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-beta1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.     

Hispolon induces apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway

Severe side effects and complications such as gastrointestinal and hematological toxicities because of current anticancer drugs are major problems in the clinical management of gastric cancer, which highlights the urgent need for novel effective and less toxic therapeutic approaches. Hispolon, an active polyphenol compound, is known to possess potent antineoplastic and antiviral properties. In this study, we investigated the efficacy of hispolon in human gastric cancer cells and explored the cell death mechanism. Hispolon induced ROS-mediated apoptosis in gastric cancer cells and was more toxic toward gastric cancer cells than toward normal gastric cells, suggesting greater susceptibility of the malignant cells. The mechanism of hispolon-induced apoptosis was that hispolon abrogated the glutathione antioxidant system and caused massive ROS accumulation in gastric cancer cells. Excessive ROS caused oxidative damage to the mitochondrial membranes and impaired the membrane integrity, leading to cytochrome c release, caspase activation, and apoptosis. Furthermore, hispolon potentiated the cytotoxicity of chemotherapeutic agents used in the clinical management of gastric cancer. These results suggest that hispolon could be useful for the treatment of gastric cancer either as a single agent or in combination with other anticancer agents.     

Escherichia coli induces apoptosis in human monocytic U937 cells through the Fas/FasL signaling pathway

Apoptosis is a genetically regulated cellular suicide mechanism that plays an essential role in development and in defense of multicellular organism. Escherichia coli (E. coli) can induce monocyte apoptosis; however, the mechanism is not clear. This study determines if Fas/FasL regulates E. coli-induced human monocyte line U937 cell apoptosis. We found that infection of U937 cells with E. coli induced rapid cell death in a dose- and time-dependent manner displaying the characteristic features of apoptosis. Moreover, opsonized E. coli induced U937 apoptosis with a higher apoptotic rate (53.29 ± 5.83%) than non-opsonized E. coli (19.37 ± 2.56%). Studying the underlying mechanisms we found that the E. coli-induced apoptosis was associated with a more prominent induction expression of Fas/FasL in a time- and dose-dependent manner. Furthermore, E. coli treatment resulted in a significant increase in the levels of DR5, TRAIL, and FADD, but exerted no statistically significant effects on the levels of DR4. The activity of caspase-8 enzyme increased in infection groups, positively correlated with apoptosis rate. Taken together, these results clearly indicate that receptor-mediated phagocytosis of E. coli induces apoptosis. Moreover, our findings suggest a possible regulatory role of Fas/FasL in the pathway of E. coli infection.     

Transforming growth factor-beta1 induces the non-classical secretion of peroxiredoxin-I in A549 cells

Recent studies found that peroxiredoxin-I (Prx-I) is secreted from A549 cells although it does not contain a signal peptide and is known to be a cytosolic protein. Transforming growth factor-beta1 (TGF-beta1) treatment dramatically enhanced Prx-I secretion from A549 cells, and this effect was not inhibited by brefeldin A. Further investigation revealed that A549 cells constitutively secrete TGF-beta1. Furin, a TGF-beta1-converting enzyme, was also highly activated in A549 cells. Ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-PDX), a potent furin inhibitor, blocked both TGF-beta1 activation and Prx-I secretion. Our findings collectively suggest that non-classical secretion of Prx-I is induced by TGF-beta1, which is constitutively activated by furin in A549 cells.     

Transforming growth factor-beta induces the expression of ANF and hypertrophic growth in cultured cardiomyoblast cells through ZAK

Transforming growth factor-beta (TGF-beta) has been associated with the onset of cardiac cell hypertrophy, but the mechanisms underlying this dissociation are not completely understood. By a previous study, we investigated the involvement of a MAP3K, ZAK, which in cultured H9c2 cardiac cells is a positive mediator of cell hypertrophy. Our results showed that expression of a dominant-negative form of ZAK inhibited the characteristic TGF-beta-induced features of cardiac hypertrophy, including increased cell size, elevated expression of atrial natriuretic factor (ANF), and increased organization of actin fibers. Furthermore, dominant-negative MKK7 effectively blocked both TGF-beta-and ZAK-induced ANF expression. In contrast, a JNK/SAPK specific inhibitor, sp600125, had little effect on TGF-beta- or ZAK-induced ANF expression. Our findings suggest that a ZAK mediates TGF-beta-induced cardiac hypertrophic growth via a novel TGF-beta signaling pathway that can be summarized as TGF-beta>ZAK>MKK7>ANF.     

Transforming growth factor-beta 1 induces expression of statin during differentiation of human promonocytic leukemia cells.

Transforming growth factor-Beta (TGF-beta) is a potent growth inhibitor for several cell types including epithelial cells and hematopoietic progenitor cells. Using a human promonocytic leukemia cell line, THP-1, we have shown that TGF-beta inhibits their proliferation and promotes differentiation into cells exhibiting macrophage-like properties. Therefore, a key question is whether TGF-beta influences the expression of genes associated with proliferation and/or growth inhibition. TGF-beta treatment of THP-1 cells results in downregulation of expression of c-myc. We also observe that TGF-beta 1-treated cells express reduced levels of the cell cycle regulated histone, H2B, but express elevated levels of an RNA splicing variant of this histone that has been observed to be upregulated in growth inhibited and terminally differentiated cells. In addition, a nuclear protein associated with senescence and withdrawal of cells from the cell cycle, statin, is also expressed by THP-1 cells in response to TGF-beta 1 treatment. These results suggest that TGF-beta 1 is capable of inducing expression of specific nuclear proteins associated with differentiation and/or cessation of proliferation that may result in changes in nuclear organization and altered gene expression. Such changes in nuclear organization may be incompatible with continued proliferation of the cells.     

Transforming growth factor-beta induces human T lymphocyte migration in vitro

In addition to its activities as a growth factor, recent studies suggest an immunoregulatory role for transforming growth factor-beta (TGF-beta). In this context we have demonstrated that TGF-beta is a potent chemotactic factor in vitro for human T lymphocytes at a concentration of 40 fM and for monocytes at a concentration of 0.4 fM but that it has no chemotactic activity for neutrophils. Furthermore, using an assay of lymphocyte subset chemotaxis we have been able to show that TGF-beta can induce migration of both CD4+ and CD8+ T lymphocytes in vitro. This study provides further evidence that TGF-beta acts as a cytokine, being able to attract T lymphocytes and monocytes to sites of inflammation. Its role in the pathogenesis of inflammatory reactions is likely to be complex.     

Transforming growth factor-beta 1 induces angiogenesis in vitro via VEGF production in human airway smooth muscle cells

Increase in size and number of bronchial blood vessels as well as hyperaemia are factors that contribute to airway wall remodelling in patients with chronic airway diseases, such as asthma and chronic obstructive pulmonary diseases (COPD). Expression of transforming growth factor beta 1 (TGF-beta 1), a multifunctional cytokine as well as vascular endothelial growth factor (VEGF), a key angiogenic molecule, has been shown in the inflammed airways in patients with chronic airway diseases. TGF-beta 1 has been implicated in the regulation of extracellular matrix, leading to airway remodelling in patients with chronic airway diseases. However, the role of TGF-beta 1 in regulating VEGF expression in patients with chronic airway diseases, as well as the underlying mechanisms are not yet well established. We investigated whether TGF-beta 1 stimulates VEGF expression in vitro and hence could influence vascular remodelling. Cultured human airway smooth muscle cells (HASMC) were serum deprived for 60 h before incubation with 5ng/ml of TGF-beta 1 for different time points. Control cells received serum-free culture medium. TGF-beta 1 treatment resulted in time dependent HASMC cell proliferation with maximal values for DNA biosynthesis at 24 h and cell number at 48 h. Northern blot analysis of VEGF mRNA expression showed increased levels in cells treated with TGF-beta 1 for 4 to 8 h. TGF-beta 1 also induced a time-dependent release of VEGF proteins in the conditioned medium after 48 h of treatment. Furthermore, the ability of HASMC-released VEGF proteins to induce human umbilical vein endothelial cells proliferation was inhibited by VEGF receptor antagonist, confirming that TGF-beta 1 induced VEGF was biologically active. We conclude that TGF-beta 1 in addition to an extracellular matrix regulator also could play a key role in bronchial angiogenesis and vascular remodelling via VEGF pathway in asthma.     

Sodium 4-phenylbutyrate induces apoptosis of human lung carcinoma cells through activating JNK pathway

Sodium 4-phenylbutyrate (PB) has been used in the therapy of urea cycle defects for many years. Recently, it has been shown to cause cellular differentiation, growth arrest, and apoptosis in certain malignancies. We have analyzed the effects of PB on human lung carcinoma cells. PB has distinct patterns of effects on different lung carcinoma cells, inducing apoptosis in NCI-H460 and NCI-H1792 cells, causing G1 arrest in A549 and SK-LU-1 cells, but having no effect on a non-transformed bronchial epithelial cell line HBE4-E6/E7. We investigated the role of MAP kinase family members, extracellular signal-regulated kinase (ERK), JNK, and p38 mitogen-activated protein kinase (MAPK), as well as other important cell survival signaling molecules in PB-induced apoptosis. We observed activation of JNK and ERK by PB in the lung cancer cells. JNK was activated only in the two apoptotic cells, whereas ERK was activated in both the apoptotic and the growth-arrested cells, demonstrating a correlation between apoptosis and activation of JNK in response to PB. Both JNK inhibitor and JNK RNA interference (RNAi) inhibited PB-induced apoptosis, whereas MEK inhibitor did not, supporting that apoptosis induced by PB is through activation of JNK. De novo protein synthesis is required for the PB-induced JNK activation and induction of apoptosis. However, the production of known upstream activators of JNK, namely Fas/Fas ligand, tumor necrosis factor (TNF)-alpha, TNF-beta, and TRAIL, are not altered by PB treatment. Therefore, PB activates JNK through an unidentified and cell type-specific mechanism. Understanding of this mechanism is of therapeutic value in treating cancer patients with PB.     

Ellipticine induces apoptosis through p53-dependent pathway in human hepatocellular carcinoma HepG2 cells

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), one of the simplest naturally occurring alkaloids, was isolated from the leaves of the evergreen tree Ochrosia elliptica Labill (Apocynaceae). Here, we reported that ellipticine inhibited the cell growth of human hepatocellular carcinoma cell line HepG2 and provided molecular understanding of this effect. The XTT assay results showed that ellipticine decreased the cell viability of HepG2 cells in a dose- and time-dependent manner, and the IC50 value was 4.1 microM. Furthermore, apoptosis induction by ellipticine in HepG2 cells was verified by the appearance of DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining assay. Ellipticine treatment was found to result in the upregulation of p53, Fas/APO-1 receptor and Fas ligand. Besides, ellipticine also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, alteration of mitochondrial membrane potential (DeltaPsim), and activation of caspase-9 and caspase-3. Taken together, ellipticine decreased the cell growth and induced apoptosis in HepG2 cell.