Effect of fluvastatin on apoptosis in human CD4+ T cells

Statins are lipid-lowering agents with pleiotropic effects. We investigated the apoptotic effects of fluvastatin on peripheral CD4+ T cells from healthy subjects. Fluvastatin induced apoptosis in resting CD4+ T cells but not in CD4+ T cells strongly activated with a high concentration of PMA plus ionomycin (PMA/I) analyzed with annexin V and propidium iodide staining. However, CD4+ T cells activated with a low concentration of PMA/I or with anti-CD3 antibodies were apoptotic after treatment with fluvastatin. Activities of caspases-8, -9, and -3 were increased in resting CD4+ T cells treated with fluvastatin (10 microM). In strongly activated CD4+ T cells, fluvastatin inhibited the activation of caspase-8 induced by PMA/I and increased caspase-9 activity. The caspase-3 activity did not differ between untreated and fluvastatin-treated strongly activated CD4+ T cells. Treatment with fluvastatin (10 microM) enhanced cytochrome c release and increased the Bax/Bcl-2 ratio in both resting and strongly activated CD4+ T cells. Although the in vitro concentration of fluvastatin used in this study is higher than in vivo, other factors may sensitize apoptotic cell death of CD4+ T cells in vivo. In conclusion, fluvastatin induces apoptosis in resting T cells but not in strongly activated T cells, a difference that might be due to the interaction between caspase-8 and caspase-9.     

GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25⁻CD4⁺ effector (Teff) and CD25⁺CD4⁺ regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4⁺ T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4⁺ T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists.     

Seleno-short-chain chitosan induces apoptosis in human breast cancer cells through mitochondrial apoptosis pathway in vitro

Seleno-short-chain chitosan (SSCC) was a synthesized chitosan derivative with the molecular weight of 4826.986 Da. The study is aimed to investigate cytotoxicity of SSCC on human breast cancer MCF-7 and BT-20 cells and explore apoptosis-related mechanism in vitro. The MTT (3- [4,5-Dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide) assay showed that SSCC exhibited significantly cytotoxic effects on MCF-7 and BT-20 cells in a dose- and time-dependent manner, and the effective inhibitory concentration was 100 μg/ml and 200 μg/ml, respectively. Apoptosis assay of these two kinds of cells was determined by Hoechst 33,342/PI and Annexin V-FITC/PI double staining. The cell cycle assay showed that SSCC triggered S and G2/M phase cell cycle arrest in MCF-7 cells and S phase cell cycle arrest in BT-20 cells in a time-dependent manner. Further studies demonstrated that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) in these two kinds of cells. N- acetyl-L cysteine (NAC), as a radical scavenger, significantly inhibited the generation of ROS and decreased the apoptosis of MCF-7 and BT-20 cells. Moreover, the expression of mitochondrial apoptosis-related proteins was detected by western blot assay. SSCC up-regulated the expression of Bax, down-regulated the expression of Bcl-2, subsequently increased the release of cytochrome c from mitochondria to cytoplasm, and activated the cleavage of caspase-9 and ?3, which finally induced apoptosis in MCF-7 and BT-20 cells in vitro. Consequently, these data indicated that SSCC could induce apoptosis of MCF-7and BT-20 cells in vitro by mitochondrial pathway.     

STAT3 modulates CD4+ T mitochondrial dynamics and function in aging

Aging promotes numerous intracellular changes in T cells that impact their effector function. Our data show that aging promotes an increase in the localization of STAT3 to the mitochondria (mitoSTAT3), which promotes changes in mitochondrial dynamics and function and T-cell cytokine production. Mechanistically, mitoSTAT3 increased the activity of aging T-cell mitochondria by increasing complex II. Limiting mitoSTAT3 using a mitochondria-targeted STAT3 inhibitor, Mtcur-1 lowered complex II activity, prevented age-induced changes in mitochondrial dynamics and function, and reduced Th17 inflammation. Exogenous expression of a constitutively phosphorylated form of STAT3 in T cells from young adults mimicked changes in mitochondrial dynamics and function in T cells from older adults and partially recapitulated aging-related cytokine profiles. Our data show the mechanistic link among mitoSTAT3, mitochondrial dynamics, function, and T-cell cytokine production.     

Canstatin induces apoptosis in gastric cancer xenograft growth in mice through the mitochondrial apoptotic pathway

Canstatin, the non-collagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. In this study, the inhibitory effect of recombinant canstatin on tumour growth was investigated using a gastric cancer xenograft model. The volume and weight of tumours in mice treated with canstatin were lower than that in mice treated with PBS. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumours treated with PBS. Moreover, valuable insight into the mechanisms mediated by canstatin was obtained.     

Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells

Sarsasapogenin is a sapogenin from the Chinese medical herb Anemarrhena asphodeloides Bunge. In the present study, we revealed that sarsasapogenin exhibited antitumor activity by inducing apoptosis in vitro as determined by Hoechst staining analysis and double staining of Annexin V-FITC/PI. In addition, cell cycle arrest in G2/M phase was observed in sarsasapogenin-treated HeLa cells. Moreover, the results revealed that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio which led to the upregulation of cytochrome c, followed by activation of caspases. Meanwhile, treatment of sarsasapogenin also activated Unfolded Protein Response (UPR) signaling pathways and these changes were accompanied by increased expression of CHOP. Salubrinal (Sal), a selective inhibitor of endoplasmic reticulum (ER) stress, partially abrogated the sarsasapogenin-related cell death. Furthermore, sarsasapogenin provoked the generation of reactive oxygen species, while the antioxidant N-acetyl cysteine (NAC) effectively blocked the activation of ER stress and apoptosis, suggesting that sarsasapogenin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways. Taken together, the results demonstrate that sarsasapogenin exerts its antitumor activity through both reactive oxygen species (ROS)-mediate mitochondrial dysfunction and ER stress cell death.     

CD4+CD25+调节性T细胞

调节性T细胞(regulatory T cells,Treg)是机体维持自身耐受的重要组成部分。CD4^ CD25^ Treg细胞来源于胸腺,其主要功能是抑制自身反应性T细胞,并且其作用是通过直接的Treg-T效应细胞之间的相互接触方式来实现的。CD4^ CD25^ Treg细胞可分泌多种抑制性细胞因子,但与其抑制功能关系并不明确,目前有证据表明GITR和Foxp3与CD4^ CD25^ Treg细胞的抑制功能有关,并且Foxp3已作为CD4^ CD25^ Treg细胞的特异性标志。通过IL-10、TGF-β等抑制性细胞因子、imDC以及转基因技术可以产生具有免疫抑制功能的调节性T细胞。调节性T细胞在免疫相关性疾病、肿瘤免疫和抗感染免疫等方面具有重要意义。     

Antigen-specific CD8+ T cells induced by the ubiquitin fusion degradation pathway

We have developed a DNA vaccine encoding a fusion protein of ubiquitin (Ub) and target proteins at the N-terminus for effective induction of antigen-specific CD8⁺ T cells. A series of expression plasmids encoding a model antigen, ovalbumin (OVA), fused with mutated Ub, was constructed. Western blotting analyses using COS7 cells transfected with these plasmids revealed that there were three types of amino acid causing different binding capacities between Ub and OVA. Natural Ub with a C-terminal glycine readily dissociated from OVA; on the other hand, artificially mutated Ub, the C-terminal amino acid of which had been exchanged to valine or arginine, stably united with the polypeptide, while Ub with a C-terminal alanine partially dissociated. The ability of DNA vaccination to induce OVA-specific CD8⁺ T cells closely correlated with the stability of Ub fusion to OVA. Our strategy could be used to optimize the effect of genetic vaccines on the induction of CD8⁺ T cells.     

Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

Objectives

Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells.

Methods

Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.

Results

Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells.

Conclusions

Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis.

     

Inhibition of JAK2/STAT3 signalling induces colorectal cancer cell apoptosis via mitochondrial pathway

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.     

Methylseleninic acid induces apoptosis of human bladder cancer cells through the ROS-mediated mitochondrial pathway

As the most common selenium derivative, methylseleninic acid (MSA) has attracted wide attention. Its apoptotic induction ability and the possible molecular mechanism in human bladder cancer (BC) J82 and T24 cells were investigated in the present study. We found that the survival of J82 and T24 cells were inhibited in a dose-dependent manner after MSA treatment. Propidium iodide (PI) staining and Annexin V-fluorescein isothiocyanate/PI double staining clarified that MSA stocked cells at G₂/M phase and caused apoptosis in J82 and T24 cells. Further, typical morphological features of apoptotic cells were also observed. Accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential were also detected by dichlorodihydrofluorescein diacetate and Rhodamin123 staining. Meanwhile, pretreatment with N-acetylcysteine, an ROS scavenging agent, found that the apoptosis of BC cells induced by MSA was related to the production of ROS. Western blot analysis results showed that MSA interrupted Bax/Bcl-2 balance, stimulated cytochrome c release into the cytoplasm, activated caspase-9 and caspase-3, and finally induced the apoptosis of the BC cells. These findings demonstrated that MSA was able to induce apoptosis in J82 and T24 cells through ROS-mediated mitochondrial apoptosis.     

CD4+CD25+ regulatory T cells control the progression from periinsulitis to destructive insulitis in murine autoimmune diabetes

Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells, leading to islet cell destruction and diabetes. Despite high genetic similarity, non-obese resistant (NOR) mice do not develop diabetes. We show here that spleen cells of both NOD and NOR mice respond to the islet cell antigen glutamic acid decarboxylase-65 in IFN-gamma-ELISPOT assays. Moreover, NOR-T cells induce periinsulitis in NOD SCID recipient mice. Thus, a potentially pathogenic islet cell-specific T-cell response arises in NOR and NOD mice alike; the mechanism that prevents the autoimmune progression of self-reactive T cells in NOR mice presumably acts at the level of effector function. Consistent with this hypothesis, CD4+CD25+ cell-depleted spleen cells from NOR mice mediated islet cell destruction and overt diabetes in NOD SCID mice. Therefore, islet cell-specific effector cells in NOR mice appear to be under the control of CD4+CD25+ regulatory T cells, confirming the importance of regulatory cells in the control of autoimmune diabetes.     

CD4+CD25+调节性T细胞的外周维持

CD4 CD25 调节性T细胞作为一种抑制性T细胞功能亚群,在维持机体的免疫自稳和免疫耐受方面发挥了关键作用。该作用的发挥与其外周细胞库的维持密切相关。新近的研究显示CD4 CD25 调节性T细胞主要通过两种机制来维持其外周细胞库,一些功能分子参与其中。     

Swainsonine activates mitochondria-mediated apoptotic pathway in human lung cancer A549 cells and retards the growth of lung cancer xenografts

Swainsonine (1, 2, 8-trihyroxyindolizidine, SW), a natural alkaloid, has been reported to exhibit anti-cancer activity on several mouse models of human cancer and human cancers in vivo. However, the mechanisms of SW-mediated tumor regression are not clear. In this study, we investigated the effects of SW on several human lung cancer cell lines in vitro. The results showed that SW significantly inhibited these cells growth through induction of apoptosis in different extent in vitro. Further studies showed that SW treatment up-regulated Bax, down-regulated Bcl-2 expression, promoted Bax translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, which in turn caused the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), resulting in A549 cell apoptosis. However, the expression of Fas, Fas ligand (FasL) or caspase-8 activity did not appear significant changes in the process of SW-induced apoptosis. Moreover, SW treatment inhibited Bcl-2 expression, promoted Bax translocation, cytochrome c release and caspase-3 activity in xenograft tumor cells, resulting in a significant decrease of tumor volume and tumor weight in the SW-treated xenograft mice groups in comparison to the control group. Taken together, this study demonstrated for the first time that SW inhibited A549 cancer cells growth through a mitochondria-mediated, caspase-dependent apoptotic pathway in vitro and in vivo.     

The role of CD4 T cell help for CD8 CTL activation

CD4 T cells play an important role in the initiation and persistence of CD8 T cells responses. In this review, we report on and evaluate the mechanisms by which CD4 T cells contribute to activation of CD8 T cells and the signal pathways of the down-streaming events after CD4 T cell help.     

Adoptive transfer of CD3+ T cells and CD4+CD44high memory T cells induces autoimmune pancreatitis in MRL/MpJ mice

The immunopathogenesis of autoimmune pancreatitis (AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3⁺ T cells both efficiently induced AIP, while CD4⁺ and CD8⁺ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4⁺CD44^high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (T_regs) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred T_regs on pancreatic histopathology. The results of our studies suggest a key role of T cell‐mediated processes in murine AIP. The effects of CD4⁺CD44^high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow‐up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.     

HCV-specific interleukin-21+CD4+ T cells responses associated with viral control through the modulation of HCV-specific CD8+ T cells function in chronic hepatitis C patients

Interleukin-21 (IL-21)+CD4+ T cells are involved in the immune response against hepatitis B virus (HBV) by secreting IL-21. However, the role of IL-21+CD4+ T cells in the immune response against chronic hepatitis C (CHC) virus infection is poorly understood. This study aimed to investigate the role of IL-21+CD4+ T cells in CHC patients and the potential mechanisms. The study subjects included nineteen CHC patients who were grouped by viral load (low, < 10⁶ RNA copies/ml, n = 8; high, > 10⁶ RNA copies/ml, n = 11). The peripheral frequency of HCV-specific IL-21+CD4+ T cells was higher in the low viral load group and was negatively correlated with the serum HCV RNA viral load in all CHC patients. Meanwhile, IL-21+ cells accumulated in the liver in the low viral load group. In vitro, IL-21 treatment increased the expression of proliferation markers and cytolytic molecules on HCV-specific CD8+ T cells. In summary, these findings suggest that HCV-specific IL-21+CD4+ T cells might contribute to HCV control by rescuing HCV-specific CD8+ T cells in CHC patients.     

Cross-primed CD8+ cytotoxic T cells induce severe Helicobacter-associated gastritis in the absence of CD4+ T cells

BACKGROUND: Although previous studies have reported important roles of CD4(+) type 1-helper T cells and regulatory T cells in Helicobacter-associated gastritis, the significance of CD8(+) cytotoxic T cells remains unknown. To study the roles of CD8(+) T cells, we examined the immune response in the gastric mucosa of Helicobacter felis-infected major histocompatibility complex (MHC) class II-deficient (II(-/-)) mice, which lack CD4(+) T cells. MATERIALS AND METHODS: Stomachs from H. felis-infected wild-type and infected MHC II(-/-) mice were examined histologically and immunohistochemically. Gastric acidity and serum levels of anti-H. felis antibodies were measured. The expression of pro-inflammatory and anti-inflammatory cytokine, Fas-ligand, perforin, and Foxp3 genes in the gastric mucosa was investigated. RESULTS: H. felis-infected MHC II(-/-) mice developed severe gastritis, accompanied by marked infiltration of CD8(+) cells. At 1 and 2 months after inoculation, mucosal inflammation and atrophy were more severe in MHC II(-/-) mice, although gastritis had reached similar advanced stages at 3 months after inoculation. There was little infiltration of CD4(+) cells, and no Foxp3-positive cells were detected in the gastric mucosa of the infected MHC II(-/-) mice. The expression of the interleukin-1beta and Fas-ligand genes was up regulated, but that of Foxp3 was down regulated in the infected MHC II(-/-) mice. Serum levels of anti-H. felis antibodies were lower in the infected MHC II(-/-) mice, despite severe gastritis. CONCLUSIONS: The present study suggests that cross-primed CD8(+) cytotoxic T cells can induce severe H.-associated gastritis in the absence of CD4(+) helper T cells and that Foxp3-positive cells may have an important role in the control of gastric inflammation.