Ivana Stanic

Spermine causes caspase activation in leukaemia cells

Exposure of several leukaemia cell types to the polyamine spermine triggered caspase activation. In HL60 cells, the onset of caspase activity correlated with the accumulation of spermine, and was accompanied by the processing of the caspase‐3 precursor and the digestion of the substrate proteins PARP and gelsolin. Spermine also induced the accumulation of cytochrome c in the cytosol. Caspase activation triggered by spermine was not blocked by antioxidants or inhibition of polyamine oxidase. The deregulation of polyamine uptake strongly sensitised the cells to spermine‐induced caspase activation. These data show that an excessive intracellular level of spermine triggers caspase activation that is not mediated by oxidative mechanisms, and suggest a model where elevated free cytosolic polyamines may act as transducers of a death message.

Polyamines directly induce release of cytochrome c from heart mitochondria

Cytochrome c release from mitochondria to the cytosol represents a critical step in apoptosis, correlated to the activation of the caspase cascade. In this report, we show that addition of micromolar concentrations of polyamines to isolated rat heart mitochondria induces the release of cytochrome c. Spermine, which is effective at concentrations of 10-100 microM, is more potent than spermidine, whereas putrescine has no effect up to 1 mM. The release of cytochrome c caused by spermine is a rapid, saturable and selective process that is independent of mitochondria damage. Spermine, unlike polylysine, is able to release a discrete amount of cytochrome c from intact, functional mitochondria. The cytochrome c-releasing power of spermine is not affected by cyclosporin A, differently from the effect of permeability transition inducers. In a cardiac cell-free model of apoptosis, the latent caspase activity of cytosolic extracts from cardiomyocytes could be activated by cytochrome c released from spermine-treated heart mitochondria. These data indicate a novel mechanism of cytochrome c release from the mitochondrion, and suggest that prolonged and sustained elevation of polyamines, characteristic of some pathologies such as heart hypertrophy, could be involved in the development of apoptosis.

Caspase activation in etoposide-treated fibroblasts is correlated to ERK phosphorylation and both events are blocked by polyamine depletion.

Activation of the extracellular signal‐regulated kinases (ERKs) 1 and 2 is correlated to cell survival, but in some cases ERKs can act in signal transduction pathways leading to apoptosis. Treatment of mouse fibroblasts with 20 μM etoposide elicited a sustained phosphorylation of ERK 1/2, that increased until 24 h from the treatment in parallel with caspase activity. The inhibitor of ERK activation PD98059 abolished caspase activation, but caspase inhibition did not reduce ERK 1/2 phosphorylation, suggesting that ERK activation is placed upstream of caspases. Both ERK and caspase activation were blocked in cells depleted of polyamines by the ornithine decarboxylase inhibitor α‐difluoromethylornithine (DFMO). In etoposide‐treated cells, DFMO also abolished phosphorylation of c‐Jun NH2‐terminal kinases triggered by the drug. Polyamine replenishment with exogenous putrescine restored the ability of the cells to undergo caspase activation and ERK 1/2 phosphorylation in response to etoposide. Ornithine decarboxylase activity decreased after etoposide, indicating that DFMO exerts its effect by depleting cellular polyamines before induction of apoptosis. These results reveal a role for polyamines in the transduction of the death signal triggered by etoposide.

SPERMINE TRIGGERS THE ACTIVATION OF CASPASE-3 IN A CELL-FREE MODEL OF APOPTOSIS

Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post‐nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP‐dependent processing of pro‐caspase‐3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro‐apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.

Nitric oxide can function as either a killer molecule or an antiapoptotic effector in cardiomyocytes.

Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.

Effect of polyamine depletion on caspase activation: a study with spermine synthase-deficient cells.

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.

Polyamine Depletion Reduces TNFα/MG132‐Induced Apoptosis in Bone Marrow Stromal Cells

Polyamines are powerful modulators of both growth and survival in mammalian cells. In this study, we investigated the possibility of attenuating the process of apoptosis in bone marrow stromal cells (BMSCs), which comprise mesenchymal stem cells, by reducing the intracellular levels of polyamines. BMSCs were isolated from rat femurs and expanded for 12 days. At this time, BMSCs were CD34neg, CD45neg, and mostly CD90pos. BMSCs were grown for an additional 2 days in the presence of 1 mM α‐difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, which reduced the content of both putrescine and spermidine by nearly 90%. DFMO treatment progressively slowed down BMSC proliferation, as determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide (MTT) assay, without arresting their growth completely. The effect of polyamine depletion on caspase‐3 activity was evaluated in BMSCs after treatment with 500 U/ml tumor necrosis factor‐α (TNFα) and 5 μM MG132, an inhibitor of proteasome. Caspase‐3 activity increased linearly over a period of 24‐hour stimulation (p < .01), but this augmentation was blunted by 50% after DFMO administration (p < .05). The effect of DFMO on TNFα/MG132‐induced upregulation of caspase‐3 activity was reversed by the addition of 100 μM putrescine, confirming that polyamines were really involved in the apoptotic process. Also, the number of apoptotic BMSCs after TNFα/MG132 treatment, as determined by terminal transferase‐mediated dUTP nick end‐labeling (TUNEL) assay, were threefold reduced after polyamine depletion (p < .05). On the contrary, DFMO did not affect the MG132‐mediated increase in p53 abundance, nor its translocation to the nucleus. Thus, polyamine depletion can be considered a useful tool for counteracting programmed cell death in BMSCs without involving the p53 proapoptotic protein.

Polyamine biosynthesis as a target to inhibit apoptosis of non-tumoral cells

Summary.Growing evidence suggests a role for polyamines in apoptosis, although the relationship appears to be complex. α-Difluoromethylornithine (DFMO), a largely used ornithine decarboxylase inhibitor, is cytostatic, hardly cytotoxic and may even increase the resistance of tumour cells to some apoptotic stimuli. This may represent a problem in cancer therapy, where the killing of tumoral cells would be a desired effect, but could be an advantage in other pathological contexts related to an excess of apoptosis, such as cardiovascular diseases, stem cell transplantation, arthritis and infections. In different cellular models, polyamine depletion following treatment with polyamine biosynthesis inhibitors appears to inhibit mitochondrial and death receptor pathways of apoptosis by affecting key proteins. These studies indicate that inhibition of polyamine biosynthesis may prevent or reduce the apoptotic response triggered by a variety of stimuli in non-tumoral cells, such as cardiac cells, stem cells, chondrocytes, macrophages and intestinal epithelial cells.

Polyamine depletion inhibits apoptosis following blocking of survival pathways in human chondrocytes stimulated by tumor necrosis factor‐α

Chondrocyte apoptosis can be an important contributor to cartilage degeneration, thereby making it a potential therapeutic target in articular diseases. To search for new approaches to limit chondrocytic cell death, we investigated the requirement of polyamines for apoptosis favored by tumor necrosis factor‐α (TNF), using specific polyamine biosynthesis inhibitors in human chondrocytes. The combined treatment of C‐28/I2 chondrocytes with TNF and cycloheximide (CHX) resulted in a prompt effector caspase activation and internucleosomal DNA fragmentation. Pre‐treatment of chondrocytes with α‐difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, markedly reduced putrescine and spermidine content as well as the caspase‐3 activation and DNA fragmentation induced by TNF and CHX. DFMO treatment also inhibited the increase in effector caspase activity provoked by TNF plus MG132, a proteasome inhibitor. DFMO decreased caspase‐8 activity and procaspase‐8 content, an apical caspase essential for TNF‐induced apoptosis. Although DFMO increased the amount of active, phosphorylated Akt, inhibitors of the Akt pathway failed to restore the TNF‐induced increase in caspase activity blunted by DFMO. DFMO also reduced the increase in caspase activity induced by staurosporine, but in this case Akt inhibition prevented the DFMO effect. Pre‐treatment with CGP 48664, an S‐adenosylmethionine decarboxylase (SAMDC) inhibitor markedly reduced spermidine and spermine levels, and provoked effects similar to those caused by DFMO. Finally DFMO was effective even in primary osteoarthritis (OA) chondrocyte cultures. These results suggest that the intracellular depletion of polyamines in chondrocytes can inhibit both the death receptor pathway by reducing the level of procaspase‐8, and the apoptotic mitochondrial pathway by activating Akt.

Sulforaphane protects human chondrocytes against cell death induced by various stimuli

Chondrocyte cell death can contribute to cartilage degeneration in articular diseases, such as osteoarthritis (OA). Sulforaphane (SFN), a natural compound derived from cruciferous aliment, is well known as an anti‐carcinogen, but according to recent evidence it also shows cytoprotective effects on a variety of non‐tumoral cells. Therefore we have tested the ability of SFN to protect chondrocytes from cell death in vitro. Treatment of growing monolayer cultures of human C‐28/I2 chondrocytes with SFN in the low micro‐molecular range for a few days, reduced cell growth without affecting cell survival or inducing apoptosis. However it decreased cell death in C‐28/I2 chondrocytes exposed to stimuli previously reported to promptly trigger apoptosis, that is, the cytokine tumor necrosis factor‐α (TNF) plus cycloheximide (CHX) or the polyamine analogue N1,N11‐diethylnorspermine (DENSPM) plus CHX. In particular pre‐treatment with SFN reduced effector and initiator caspase activities and the associated activation of JNK kinases. SFN exerted a cytoprotective action even versus H2O2, which differently from the previous stimuli induced cell death without producing an evident caspase activation. SFN pre‐treatment also prevented caspase activation in three‐dimensional micromass cultures of OA chondrocytes stimulated with growth‐related oncogene α (GROα), a pro‐apoptotic chemokine. The suppression of caspase activation in micromasses appeared to be related to the inhibition of p38 MAPK phosphorylation. In conclusion, the present work shows that low micro‐molecular SFN concentrations exert pro‐survival and anti‐apoptotic actions and influence signaling pathways in a variety of experimental conditions employing chondrocyte cell lines and OA chondrocytes treated with a range of death stimuli. J. Cell. Physiol. 226: 1771–1779, 2011.