Artesunate induces necrotic cell death in schwannoma cells

Established as a potent anti-malaria medicine, artemisinin-based drugs have been suggested to have anti-tumour activity in some cancers. Although the mechanism is poorly understood, it has been suggested that artemisinin induces apoptotic cell death. Here, we show that the artemisinin analogue artesunate (ART) effectively induces cell death in RT4 schwannoma cells and human primary schwannoma cells. Interestingly, our data indicate for first time that the cell death induced by ART is largely dependent on necroptosis. ART appears to inhibit autophagy, which may also contribute to the cell death. Our data in human schwannoma cells show that ART can be combined with the autophagy inhibitor chloroquine (CQ) to potentiate the cell death. Thus, this study suggests that artemisinin-based drugs may be used in certain tumours where cells are necroptosis competent, and the drugs may act in synergy with apoptosis inducers or autophagy inhibitors to enhance their anti-tumour activity.Artemisinin, a sesquiterpene lactone isolated from the Chinese herb Artemisia annua L., has profound activity against malaria.¹ Artemisinin contains an endoperoxide moiety that reacts with iron to produce toxic reactive oxygen species (ROS). When malaria parasite (Plasmodia) consumes iron-rich haemoglobin within its acidic food vacuole in erythrocytes, the exposure of artemisinin to haem-derived iron results in lethal ROS production that exerts fatal toxicity to the parasite.² Therefore, artemisinin, its water-soluble derivative artesunate (ART) and other analogues are potent in killing malarial parasites.^1,3Cancer cells contain substantial free iron, resulting from their higher-rate iron uptake via transferrin receptors compared with normal cells. Therefore, artemisinin-based drugs such as ART possess selective toxicity to cancer cells.^{4, 5, 6} Importantly, the pharmacokinetics and tolerance of ART as an anti-malarial drug have been well documented, with clinical studies showing excellent safety. Collectively, these properties make artemisinin-based compounds attractive drug candidates for cancer chemotherapy. Artemisinin and ART have been shown to induce cell death in multiple cancer cells, including colon, breast, ovarian, prostate,⁷ pancreatic⁸ and leukaemia⁹ cancer cells. Preliminary in vivo experiments also indicate the therapeutic potential for these drugs as anti-cancer treatments. In animal models, artemisinin or ART has shown promising results in Kaposi Sarcoma,¹⁰ pancreatic cancer¹¹ and hepatoma,¹² while compassionate use of ART in uveal melanoma patients fortifies standard chemotherapy potential for the patients.¹³ Currently, ART is on clinical trial for breast cancer treatment (ClinicalTrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT00764036","term_id":"NCT00764036"}}NCT00764036).Programmed cell death (PCD) is one of the critical terminal paths for the cells of metazoans. Among PCD, apoptosis has been well studied and it is known that caspase activation is essential in this process.¹⁴ In addition to apoptosis, necroptosis is another form of PCD. The RIP1-RIP3 complex highlights the signals that regulate necroptosis.^{15, 16, 17} Artemisinin derivatives, mostly ART, have been suggested to lead to apoptosis via ROS production in cancer cells. Efforts have been focused on ROS-mediated mitochondrial apoptosis,^9,18,19 and DNA damage²⁰ in cancer cells. Recent data suggest that artemisinin and its derivatives may induce cell death or inhibit proliferation through diverse mechanisms in different cell types. Artemisinin or its analogues were shown to inhibit cell proliferation in multiple cancer cells by regulating cell-cycle arrest^{21, 22, 23} or inducing apoptosis.^24,25 Nevertheless, the detailed molecular mechanisms underlying artemisinin or ART-induced cell death are poorly understood, thus need to be further addressed.Neurofibromatosis 2 (NF2) is caused by the loss of NF2 gene encoding Merlin protein. NF2 gene mutations cause the low grade tumour syndrome, composed of schwannomas, meningiomas and ependymomas.²⁶ All spontaneous schwannomas, the majority of meningiomas and a third of ependymomas are caused by NF2 gene mutations. Notably, approximately 10% of intracranial tumours are schwannomas.²⁷ Interestingly, NF2 gene mutations are also found in a variety of cancers, including breast cancer and mesothelioma.^{28, 29, 30} The low grade tumours caused by NF2 gene mutations do not respond well to current cancer drugs and therapy is restricted to surgery and radiosurgery.²⁶ Therefore, there is a need for drug treatment of the diseases. Here, we show that ART sufficiently induced schwannoma cell death in both RT4 cell line and human primary cells. Importantly, we show, for the first time, that ART-induced cell death is largely dependent on necroptosis. Our data suggest that ART has great potential in schwannoma chemotherapy, especially when used in synergy with an apoptosis-inducing drug and/or an autophagy-inhibitory drug.     

Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence.

Sugars are key regulatory molecules that affect diverse processes in higher plants. Hexokinase is the first enzyme in hexose metabolism and may be a sugar sensor that mediates sugar regulation. We present evidence that hexokinase is involved in sensing endogenous levels of sugars in photosynthetic tissues and that it participates in the regulation of senescence, photosynthesis, and growth in seedlings as well as in mature plants. Transgenic tomato plants overexpressing the Arabidopsis hexokinase-encoding gene AtHXK1 were produced. Independent transgenic plants carrying single copies of AtHXK1 were characterized by growth inhibition, the degree of which was found to correlate directly to the expression and activity of AtHXK1. Reciprocal grafting experiments suggested that the inhibitory effect occurred when AtHXK1 was expressed in photosynthetic tissues. Accordingly, plants with increased AtHXK1 activity had reduced chlorophyll content in their leaves, reduced photosynthesis rates, and reduced photochemical quantum efficiency of photosystem II reaction centers compared with plants without increased AtHXK1 activity. In addition, the transgenic plants underwent rapid senescence, suggesting that hexokinase is also involved in senescence regulation. Fruit weight, starch content in young fruits, and total soluble solids in mature fruits were also reduced in the transgenic plants. The results indicate that endogenous hexokinase activity is not rate limiting for growth; rather, they support the role of hexokinase as a regulatory enzyme in photosynthetic tissues, in which it regulates photosynthesis, growth, and senescence.     

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles’ heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.     

Overexpression of the disease resistance gene Pto in tomato induces gene expression changes similar to immune responses in human and fruitfly

The Pto gene encodes a serine/threonine protein kinase that confers resistance in tomato (Lycopersicon esculentum) to Pseudomonas syringae pv tomato strains that express the type III effector protein AvrPto. Constitutive overexpression of Pto in tomato, in the absence of AvrPto, activates defense responses and confers resistance to several diverse bacterial and fungal plant pathogens. We have used a series of gene discovery and expression profiling methods to examine the effect of Pto overexpression in tomato leaves. Analysis of the tomato expressed sequence tag database and suppression subtractive hybridization identified 600 genes that were potentially differentially expressed in Pto-overexpressing tomato plants compared with a sibling line lacking Pto. By using cDNA microarrays, we verified changes in expression of many of these genes at various time points after inoculation with P. syringae pv tomato (avrPto) of the resistant Pto-overexpressing line and the susceptible sibling line. The combination of these three approaches led to the identification of 223 POR (Pto overexpression responsive) genes. Strikingly, 40% of the genes induced in the Pto-overexpressing plants previously have been shown to be differentially expressed during the human (Homo sapiens) and/or fruitfly (Drosophila melanogaster) immune responses.     

Signal recognition and transduction mediated by the tomato Pto kinase: a paradigm of innate immunity in plants

Plant disease resistance is the result of an innate host defense mechanism, which relies on the ability of the plant to recognize pathogen invasion and to efficiently mount defense responses. In tomato, resistance to the pathogen Pseudomonas syringae is mediated by the specific interaction between the plant serine/threonine kinase Pto and the bacterial protein AvrPto. This article reviews molecular and biochemical properties that confer to Pto the capability to function as an intracellular receptor and to activate a signaling cascade leading to the induction of defense responses.     

Aquatic birnavirus induces necrotic cell death via the mitochondria-mediated caspase pathway

Aquatic birnavirus induces necrotic cell death by an ill-understood process. Presently, we demonstrate that infectious pancreatic necrosis virus (IPNV) induces post-apoptotic necrotic cell death through loss of mitochondrial membrane potential (MMP) followed by caspase-3 activation in CHSE-214 cells. Progressive phosphatidylserine externalization was observed at 6 h post-infection (p.i.). This was followed by the development of bulb-like vesicles (bleb formation) at 8 h p.i. Progressive loss of MMP was also observed in IPNV-infected CHSE-214 cells beginning at 6 h p.i. At 8 h and 12 h p.i., IPNV-infected cells demonstrated a dramatic increase in MMP loss, rapid entry into necrotic cell death, and activation of caspase-9 and -3. Additionally, treatment with an inhibitor of MMP loss, bongkrekic acid, an adenine nucleotide translocase inhibitor, blocked IPNV-induced PS exposure and MMP loss, as well as reduced the activation of caspase-3. Taken together, our results suggest that IPNV induces apoptotic cell death via loss of MMP, thereby triggering secondary necrosis and caspases-3 activation. Furthermore, this death-signaling pathway is disrupted by bongkrekic acid in fish cells, indicating that this drug may serve to modulate IPNV-induced pathogenesis.     

A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death.

Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.     

A necrotic cell death model in a protist

While necrotic cell death is attracting considerable interest, its molecular bases are still poorly understood. Investigations in simple biological models, taken for instance outside the animal kingdom, may benefit from less interference from other cell death mechanisms and from better experimental accessibility, while providing phylogenetic information. Can necrotic cell death occur outside the animal kingdom? In the protist Dictyostelium, developmental stimuli induced in an autophagy mutant a stereotyped sequence of events characteristic of necrotic cell death. This sequence included swift mitochondrial uncoupling with mitochondrial 2',7'-dichlorofluorescein diacetate fluorescence, ATP depletion and increased oxygen consumption. This was followed by perinuclear clustering of dilated mitochondria. Rapid plasma membrane rupture then occurred, which was evidenced by time-lapse videos and quantified by FACS. Of additional interest, developmental stimuli and classical mitochondrial uncouplers triggered a similar sequence of events, and exogenous glucose delayed plasma membrane rupture in a nonglycolytic manner. The occurrence of necrotic cell death in the protist Dictyostelium (1) provides a very favorable model for further study of this type of cell death, and (2) strongly suggests that the mechanism underlying necrotic cell death was present in an ancestor common to the Amoebozoa protists and to animals and has been conserved in evolution.     

Overexpression of TTRAP inhibits cell growth and induces apoptosis in osteosarcoma cells

TTRAP is a multi-functional protein that is involved in multiple aspects of cellular functions including cell proliferation, apoptosis and the repair of DNA damage. Here, we demonstrated that the lentivirus-mediated overexpression of TTRAP significantly inhibited cell growth and induced apoptosis in osteosarcoma cells. The ectopic TTRAP suppressed the growth and colony formation capacity of two osteosarcoma cell lines, U2OS and Saos-2. Cell apoptosis was induced in U2OS cells and the cell cycle was arrested at G2/M phase in Saos-2 cells. Exogenous expression of TTRAP in serum-starved U2OS and Saos-2 cells induced an increase in caspase-3/-7 activity and a decrease in cyclin B1 expression. In comparison with wild-type TTRAP, mutations in the 5''-tyrosyl-DNA phosphodiesterase activity of TTRAP, in particular TTRAP^E152A, showed decreased inhibitory activity on cell growth. These results may aid in clarifying the physiological functions of TTRAP, especially its roles in the regulation of cell growth and tumorigenesis. [BMB Reports 2013; 46(2): 113-118]     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

avrPto enhances growth and necrosis caused by Pseudomonas syringae pv.tomato in tomato lines lacking either Pto or Prf

AvrPto was introduced into three tomato genotypes with two biotic agents to study its role in compatible interactions. avrPto enhanced the capacity of the Pseudomonas syringae pv. tomato strain T1 to induce necrotic symptoms on tomato plants that lacked either Pto or Prf genes. The enhanced necrosis correlated with a small increase in bacterial growth. In planta expression of avrPto in isolation did not elicit necrosis in the absence of a functional Prf gene.     

Genetic loci controlling lethal cell death in tomato caused by viral satellite RNA infection

Cucumber mosaic virus (CMV) associated with D satellite RNA (satRNA) causes lethal systemic necrosis (LSN) in tomato (Solanum lycopersicum), which involves programmed cell death. No resistance to this disease has been found in tomato. We obtained a line of wild tomato, S. habrochaitis, with a homogeneous non-lethal response (NLR) to the infection. This line of S. habrochaitis was crossed with tomato to generate F1 plants that survived the infection with NLR, indicating that NLR is a dominant trait. The NLR trait was successfully passed on to the next generation. The phenotype and genotype segregation was analyzed in the first backcross population. The analyses indicate that the NLR trait is determined by quantitative trait loci (QTL). Major QTL associated with the NLR trait were mapped to chromosomes 5 and 12. Results from Northern blot and in situ hybridization analyses revealed that the F1 and S. habrochaitis plants accumulated minus-strand satRNA more slowly than tomato, and fewer vascular cells were infected. In addition, D satRNA-induced LSN in tomato is correlated with higher accumulation of the minus-strand satRNA compared with the accumulation of the minus strand of a non-necrogenic mutant D satRNA.     

Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death

BACKGROUND: To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell-growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, resulting in part from the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. Here, we circumvent this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1. RESULTS: We find that overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild-type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. We find that cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, we demonstrate that Atg1-induced autophagy strongly inhibits cell growth and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, we show that Atg1 expression results in negative feedback on the activity of TOR itself. CONCLUSIONS: Our results reveal a central role for Atg1 in mounting a coordinated autophagic response and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

Activation-induced cell death in human T cells is a suicidal process regulated by cell density but superantigen induces T cell fratricide

Repeated ligation of the TCR results in apoptosis (activation-induced cell death; AICD). Superantigens such as Staphylococcal enterotoxin B (SEB) are particularly efficient at inducing AICD in T cells. We investigated whether apoptosis in human T cell subsets was due to fratricide (killing of neighboring cells) or suicide (cell autonomous death). AICD of Th1, Th2, Tc1, and Tc2 effector cells was dramatically enhanced at low cell densities and could be observed in single cell microcultures. AICD was unaffected by adhesion molecules or neighboring cells undergoing AICD, confirming the predominance of a suicidal mechanism. However, SEB was able to induce fratricidal apoptosis of type 1, but not type 2 cells. Fratricide was also observed when unstimulated T cells were exposed to activated Tc1 effector cells. Thus, AICD is tightly regulated to allow clonal T cell expansion and memory cell generation, but superantigens may subvert this process by allowing T cell fratricide.     

Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells

To investigate the effects of chloroquine diphosphate (CQ) on lung cancer cell growth, we treated A549 cells, a lung cancer cell line, with the drug at various concentrations (0.25-128 microM) for 24-72 h. The results showed that, at lower concentrations (from 0.25 to 32 microM), CQ inhibited the growth of A549 cells and, at the same time, it induced vacuolation with increased volume of acidic compartments (VAC). On the other hand, at higher concentrations (64-128 microM), CQ induced apoptosis at 24 h, while its effect of inducing vacuolation declined. The lactate dehydrogenase (LDH) assay showed that with the treatment of CQ 32-64 microM for 72 h or 128 microM for 48 h, CQ induced necrosis of A549 cells. To understand the possible mechanism by which CQ acts in A549 cells, we further incubated the cells with this drug at the concentrations of 32 or 128 microM in the presence of D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC). The results showed that D609 (50 microM) could inhibit the effects of CQ 32 microM on the viability and VAC, but it could not change the effects of CQ 128 microM on the same. Our data suggested that CQ inhibited A549 lung cancer cell growth at lower concentrations by increasing the volume of lysosomes and that PC-PLC might be involved in this process. The data also indicated that, at higher concentrations, CQ induced apoptosis and necrosis, but at this time its ability to increase the volume of lysosome gradually declined, and PC-PLC might not be implicated in the process.     

Selenium improves photosynthesis and induces ultrastructural changes but does not alleviate cadmium-stress damages in tomato plants

Protoplasma - The application of Se to plants growing under Cd contamination may become an alternative strategy to minimize Cd damage. However, there is no specific information available regarding...     

NF-kappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death

NF-kappaB downregulates tumor necrosis factor (TNF)-induced c-Jun N-terminal kinase (JNK) activation that promotes cell death, but the mechanism is not yet fully understood. By using murine embryonic fibroblasts (MEFs) that are deficient in TNF receptor-associated factor (TRAF) 2 and TRAF5 (DKO) or p65 NF-kappaB subunit (p65KO), we demonstrate here that TNF stimulation leads to accumulation of reactive oxygen species (ROS), which is essential for prolonged mitogen-activated protein kinase (MAPK) activation and cell death. Interestingly, dying cells show necrotic as well as apoptotic morphological changes as assessed by electron microscopy and flow cytometry, and necrotic, but not apoptotic, cell death is substantially inhibited by antioxidant. Importantly, TNF does not induce ROS accumulation or prolonged MAPK activation in wild-type MEFs, indicating that TRAF-mediated NF-kappaB activation normally suppresses the TNF-induced ROS accumulation that subsequently induces prolonged MAPK activation and necrotic cell death