A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.Autophagy refers to an evolutionarily conserved process in which intracellular proteins and organelles are sequestered in autophagosomes and subsequently degraded by lysosomal enzymes for the purpose of recycling cellular components to sustain metabolism during nutrient deprivation and to prevent accumulation of damaged proteins and organelles (1, 2). Autophagy is a dynamic process, consisting of several sequential stages (initiation, nucleation, elongation, and maturation) controlled by a group of autophagy-related genes (Atg genes). So far, more than 30 Atg genes have been identified in yeast, and many of them have homologues in mammalian cells (3). Upstream of ATG proteins, mammalian target of rapamycin (mTOR) 4 has been well studied as the key regulatory molecule (4). mTOR is a serine/threonine protein kinase serving as the convergence point for many of the upstream stimuli and pathways to regulate cell growth, cell proliferation, cell motility, cell survival, protein synthesis, translation, and autophagy (5-7). Abundance of nutrients, including growth factors, glucose, and amino acids will activate mTOR and suppress autophagy, whereas nutrient deprivation will suppress mTOR, leading to activation of autophagy. At present, the molecular mechanisms downstream of mTOR responsible for its anti-autophagic function have not been fully understood. In yeast, TOR directly targets the ATG13-ATG1 complex and suppresses its function at the initiation stage of autophagy (8). In mammalian cells, the complex containing ULK1 (the ATG1 homologue), ATG13, and FIP200 is directly cont...

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

Rhubarb has been used as a traditional Chinese medicine since ancient times and today it is still present in various herbal preparations. In this review the toxicological and anti-neoplastic potentials of the main anthraquinones from Rhubarb, Rheum palmatum, will be highlighted. It is interesting to note that although the chemical structures of various anthraquinones in this plant are similar, their bioactivities are rather different. The most abundant anthraquinone of rhubarb, emodin, was capable of inhibiting cellular proliferation, induction of apoptosis, and prevention of metastasis. These capabilities are reported to act through tyrosine kinases, phosphoinositol 3-kinase (PI3K), protein kinase C (PKC), NF-kappa B (NF-kappaB), and mitogen-activated protein kinase (MAPK) signaling cascades. Aloe-emodin is another major component in rhubarb found to have anti-tumor properties. Its anti-proliferative property has been demonstrated to be through the p53 and its downstream p21 pathway. Our recent proteomic study also suggests that the molecular targets of these two anthraquinones are different. However, both components were found to be able to potentiate the anti-proliferation of various chemotherapeutic agents. Rhein is the other major rhubarb anthraquinone, although less well studied. This compound could effectively inhibit the uptake of glucose in tumor cells, caused changes in membrane-associated functions and led to cell death. Interestingly, all three major rhubarb anthraquinones were reported to have in vitro phototoxic. This re-evaluation of an old remedy suggests that several bioactive anthraquinones of rhubarb possess promising anti-cancer properties and could have a broad therapeutic potential.

It is intriguing that some pan-caspase inhibitors such as zVAD-fmk (zVAD) are capable of inducing necrotic cell death in a selected group of cells. As earlier reports from our laboratory have ruled out the original notion that zVAD-induced necrosis in mouse fibrosarcoma L929 cells was autophagic cell death, the main objective of this study was thus to determine the underlying mechanism of this form of cell death. In this study, we provided clear evidence that zVAD-induced necroptosis in L929 cells and such cell death is dependent on autocrine production of tumor necrosis factor-a (TNFa) at the transcriptional level. More importantly, we identified that activating protein-1 (AP-1), but not nuclear factor j-B, is the transcription factor controlling zVADinduced TNFa transcription. Moreover, zVAD is able to activate AP-1 through activation of two upstream mitogen-activated kinases (MAPKs), c-Jun N-terminal kinase and extracellular signal-regulated kinase. Finally, we found that protein kinase C is the important upstream signaling molecule in mediating zVAD-induced activation of MAPKs and AP-1, and subsequent autocrine production of TNFa and cell death. Data from this study reveal the molecular mechanisms underlying zVAD-induced necroptosis, an important form of programmed necrotic cell death with increasing understanding of its biological significance in health and diseases.

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVADinduced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.