Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli

Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c-mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.     

Mitochondria from TRAIL-resistant prostate cancer cells are capable of responding to apoptotic stimuli

TNFalpha-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells. However, some prostate cancer cells, such as LNCaP are resistant to TRAIL. In addition to the involvement of several pathways in the TRAIL-resistance of LNCaP, it has been shown that mitochondrial response to TRIAL is low in these cells. Therefore, in this study, using in vitro cell free and reconstitution models, we have demonstrated that mitochondria from these cells are capable of responding to apoptotic stimuli. Furthermore, experiments to determine the influence of cytochrome c on apoptotic response noted that incubation of cytosol with exogenous cytochrome c induced truncation of Bid. We have demonstrated that truncation of Bid by exogenous cytochrome c is mediated through the activation of caspases-9 and -3. Incubation of cytosol with recombinant caspases-9 and -3 in the absence or presence of inhibitors showed that activation of caspase-9, leading to the activation of caspase-3 was necessary for the truncation of Bid. Published results indicate that in apoptotic cells cytochrome c is released from the mitochondria in two installments, an early small amount and a late larger amount. Our results suggest that the initial release of cytochrome generates tBid that is capable of translocation into the mitochondria causing further release of cytochrome c. Thus, in addition to providing functional explanation for the biphasic release of cytochrome c from mitochondria, we demonstrate the presence of a feedback amplification of mitochondrial apoptotic signal.     

Differential susceptibility of subsarcolemmal and intermyofibrillar mitochondria to apoptotic stimuli

Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H₂O₂ (0, 25, 50, and 100 µM). However, IMF mitochondria were more sensitive to H₂O₂ and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, V_max) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H₂O₂-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn²⁺-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy. reactive oxygen species; skeletal muscle; mitochondrial permeability transition pore; cytochrome c; apoptosis     

Lowering the apoptotic threshold in colorectal cancer cells by targeting mitochondria

Background  

Colorectal cancer is the third most-common cancer and the second most-common cause of cancer related death in UK. Although chemotherapy plays significant role in the treatment of colorectal cancer, morbidity and mortality due to drug resistance and cancer metastasis are yet to be eliminated. Recently, doxycycline has been reported to have cytotoxic and anti-proliferating properties in various cancer cells. In this study, whether doxycycline was apoptosis threshold lowering agent in colorectal cancer cells by targeting mitochondria was answered.     

Giant mitochondria distinct from enlarged mitochondria in secretory and ciliated cells of gerbil trachea and bronchioles

Numerous mitochondria ranging from slightly larger than normal to several micrometers in diameter (giant) were found in about one-half the serous secretory cells in the surface epithelium of the normal gerbil trachea and proximal bronchi. Tracheal serous cells of mice also were found to contain numerous giant mitochondria. Clara cells of gerbil bronchioles contained abundant giant mitochondria in addition to normal tubular mitochondria and the second population of enlarged spherical mitochondria that have been described in Clara cells of several genera. In contrast, mouse Clara cells revealed the normal tubular and the enlarged spherical mitochondria but no giant mitochondria. A survey of a number of cell types in gerbils failed to disclose hypertrophied mitochondria outside tracheobronchial surface epithelium and bronchioles. The mitochondrial enlargement resulted from an increase of matrix but not cristae. The expansion of matrix displaced the relatively sparse cristae into small collections compressed against the outer membrane. The prevalence of giant mitochondria and of granular endoplasmic reticulum is similar among cells, and these two organelles are codistributed within cells. The megamitochondria and granular reticulum occupy a central stratum, whereas normal mitochondria occur in the apical and basal regions. The giant mitochondria are considered related to a normal biologic activity that is characteristic of respiratory tract epithelium of mice and gerbils selectively and is more prominent in secretory cells than in ciliated cells.     

Sorafenib sensitizes hepatocellular carcinoma cells to physiological apoptotic stimuli

Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib's main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic "BH3-domain only" PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knock-down, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of transforming growth factor-β (TGF-β) through the intrinsic pathway and to tumor necrosis factor-α (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-β or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines.     

Vimentin intermediate filaments protect mitochondria from oxidative stress

The role of intermediate filaments (IFs) in eukaryotic cells is still unclear. The disturbance of mitochondria distribution and function, in particular the enhanced production of reactive oxygen species (ROS) and decreased membrane potential, is observed in cells devoid of IFs. The aim of this work was to study the dependence of mitochondria sensitivity to oxidative stress on the presence of vimentin IFs. It was found that mitochondria are less sensitive to ROS in cells containing vimentin than in cells devoid of vimentin. Besides, mitochondrial membrane potential was demonstrated to increase upon regeneration of vimentin IFs in the cells. Substitution of Pro-57 by Arg in N-terminal part of the vimentin molecule abandoned its protective ability and the effect on mitochondrial membrane potential.     

Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells

Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases.     

Dendritic cells crosspresent antigens from live B16 cells more efficiently than from apoptotic cells and protect from melanoma in a therapeutic model

Dendritic cells (DC) are able to elicit anti-tumoral CD8(+) T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8(+) T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8(+) T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy.     

Isotope-reinforced polyunsaturated fatty acids protect mitochondria from oxidative stress

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure–activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D₁-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D₂-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20–50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function.     

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.Apoptosis is an important regulatory mechanism of tissue homeostasis. It is triggered by the extrinsic pathway through the activation of proapoptotic receptors or by the intrinsic pathway through the destabilization of mitochondria in response to various forms of cell injury or stress.¹ Notably, stressed cells are also strongly influenced by intercellular communicative networks. This includes diffusible growth factors, cytokines and other small molecules secreted from neighbouring cells, which can modulate the fate of distressed cells. For example, stem cells release growth factors to protect dysfunctional neurons in the brain.² In tumour stroma, activated fibroblasts are thought to promote tumour progression by secreting growth factors that act in a paracrine manner.³ Moreover, contact-dependent signalling, for example, via adhesion molecules, can trigger contact inhibition or protection of endothelial cells.⁴ In addition, gap junctions have been shown to be involved in the transfer of death or survival molecules in different cell types.⁵ Therefore, the signals transferred from neighbouring cells influence the viability of target cells through different pathways.In 2004, our group described a previously unrecognized form of cell-to-cell interaction based on nanoscaled, F-actin-containing membrane tubes.^{6, 7} These tubes, referred to as membrane or tunneling nanotubes (TNTs), were subsequently found in numerous cell types in culture and in tissues.^{8, 9, 10, 11} Importantly, TNTs facilitate the intercellular exchange of diverse cellular signals and components ranging from electrical signalling to organelles.^{12, 13, 14, 15} Moreover, pathogens such as human immunodeficiency virus (HIV) and prions can spread between cells along TNTs.^{16, 17} Consistent with the model that TNTs are involved in cell-to-cell communication, apoptosis regulators may be transferred via TNTs between apoptotic and healthy cells to alter the fate of recipient cells. Indeed, it has been shown that TNTs can propagate the death signal Fas ligand between T lymphocytes to induce cell death.^{18, 19} TNTs have been also proposed to participate in the rescue of injured cardiomyoblasts or endothelial cells by mesenchymal stem cells (MSCs) through transferred mitochondria.^{20 ,21} However, the rescue mechanism by how and when this event was accomplished remains elusive.In this study, we found that PC12 cells stressed by ultraviolet (UV) radiation were rescued from apoptosis when cocultured with untreated, healthy PC12 cells. Single-cell analysis showed that stressed cells in the early stages of apoptosis form a new type of TNT to interact with untreated cells. These TNTs have a distinct cytoskeletal composition and biophysical properties when compared with TNTs interconnecting normal PC12 cells. We also observed the presence and transport of mitochondria in the TNTs formed by stressed cells. Notably, the rescue effect was inhibited when the formation of TNTs were impaired by incubating with an F-actin-depolymerizing drug, or when the mitochondria of rescuer cells were damaged. Our results suggest that the delivery of functional mitochondria via TNTs mediates the recovery of PC12 cells in the early stages of apoptosis.     

Smac/DIABLO is not released from mitochondria during apoptotic signalling in cells deficient in cytochrome c

We have characterised the apoptotic defects in cells null for cytochrome c (cyt c-/-). Such cells treated with staurosporine (STS) exhibited translocation to the mitochondria and activation of the proapoptotic signalling molecule Bax but failed to release Smac/DIABLO from these organelles, judged by both confocal microscopy and Western blotting. While reference cells expressing cytochrome c released both it and Smac/DIABLO under a variety of conditions of apoptotic induction, we have never observed release of Smac/DIABLO from cyt c-/- cells. We eliminate the possibility that proteasomal degradation of cytosolically localised Smac/DIABLO is responsible for our failure to visualise the protein outside the mitochondria. Our findings indicate an unanticipated nexus between release of cytochrome c and Smac/DIABLO from mitochondria, previously thought to be a more or less synchronised event early in apoptosis. We suggest that the failure of cyt c-/- cells to release Smac/DIABLO after recruitment of Bax to mitochondria represents an extreme manifestation of some inherent difference in the regulation of release of these two proteins from mitochondria.     

Functional activity and ultrastructure of mitochondria isolated from myocardial apoptotic tissue

Apoptosis in myocardial tissue slices was induced by extended incubation under anoxic conditions. Mitochondria were isolated from the studied tissue. A new method of isolation of mitochondria in special conditions by differential centrifugation at 1700, 10,000, and 17,000g resulted in three fractions of mitochondria. According to the data of electron microscopy the heavy mitochondrial fraction (1700g) consisted of mitochondrial clusters only, the middle mitochondrial fraction (10,000g) consisted of mitochondria with typical for isolated mitochondria ultrastructure, and the light fraction consisted of small mitochondria (2 or 3 cristae) of various preservation. The heavy fraction contained unusual structural elements that we detected earlier in apoptotic myocardial tissue—small electron-dense mitochondria incorporated in bigger mitochondria. The structure of small mitochondria from the light fraction corresponded to that of the small mitochondria from these unusual elements—mitochondrion in mitochondrion. The most important functions of isolated mitochondria are strongly inhibited when apoptosis is induced in our model. The detailed study of the activities of the two fractions of the apoptotic mitochondria showed that the system of malate oxidation is completely altered, the activity of cytochrome c as electron carrier is partly inhibited, while succinate oxidase activity is completely preserved (complexes II, III, and IV of the respiration chain). Succinate oxidase activity was accompanied by high permeability of the internal membrane for protons: the addition of uncoupler did not stimulate respiration. ATP synthesis in mitochondria was inhibited. We demonstrated that in our model of apoptosis cytochrome c remains in the intermembrane space, and, consequently, is not involved in the cascade of activation of effector caspases. The possible mechanisms of induction of apoptosis during anoxia are discussed.     

Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli

The proto-oncogene Akt is a potent inhibitor of apoptosis, and it is activated in many human cancers. A number of recent studies have highlighted the importance of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in mediating calcium (Ca²⁺) transfer from the endoplasmic reticulum (ER) to the mitochondria in several models of apoptosis. Akt is a serine-threonine kinase and recent data indicate the IP3R as a target of its phosphorylation activity.Here we show that HeLa cells, overexpressing the constitutively active myristoylated/palmitylated AKT1 (m/p-AKT1), were found to have a reduced Ca²⁺ release from ER after stimulation with agonist coupled to the generation of IP3. In turn, this affected cytosolic and mitochondria Ca²⁺ response after Ca²⁺ release from the ER induced either by agonist stimulation or by apoptotic stimuli releasing Ca²⁺ from intracellular stores.Most importantly, this alteration of ER Ca²⁺ content and release, reduces significantly cellular sensitivity to Ca²⁺ mediated proapoptotic stimulation. These results reveal a primary role of Akt in shaping intracellular Ca²⁺ homeostasis, that may underlie its protective role against some proapoptotic stimuli.     

Wild-type but not Parkinson's disease-related ala-53 --> Thr mutant alpha -synuclein protects neuronal cells from apoptotic stimuli

Recent works suggest that alpha-synuclein could play a central role in Parkinson's disease (PD). Thus, two mutations were reported to be associated with rare autosomal dominant forms of the disease. We examined whether alpha-synuclein could modulate the caspase-mediated response and vulnerability of murine neurons in response to various apoptotic stimuli. We established TSM1 neuronal cell lines overexpressing wild-type (wt) alpha-synuclein or the PD-related Ala-53 --> Thr mutant alpha-synuclein. Under basal conditions, acetyl-Asp-Glu-Val-Asp-aldehyde-sensitive caspase activity appears significantly lower in wt alpha-synuclein-expressing cells than in neurons expressing the mutant. Interestingly, wt alpha-synuclein drastically reduces the caspase activation of TSM1 neurons upon three distinct apoptotic stimuli including staurosporine, etoposide, and ceramide C(2) when compared with mock-transfected cells. This inhibitory control of the caspase response triggered by apoptotic agents was abolished by the PD-related pathogenic mutation. Comparison of wild-type and mutated alpha-synuclein-expressing cells also indicates that the former exhibits much less vulnerability in response to staurosporine and etoposide as measured by the sodium 3'-[1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid assay. Altogether, our study indicates that wild-type alpha-synuclein exerts an antiapoptotic effect in neurons that appears to be abolished by the Parkinson's disease-related mutation, thereby suggesting a possible mechanism underlying both sporadic and familial forms of this neurodegenerative disease.     

SG2NA recruits DJ-1 and Akt into the mitochondria and membrane to protect cells from oxidative damage

SG2NA is a WD-40 repeat protein with multiple protein–protein interaction domains of unknown functions. We demonstrate that it associates with the antioxidant protein DJ-1 and the survival kinase Akt. The C-terminal WD-40 repeat domain of SG2NA is required for its interaction with Akt, while DJ-1 binds it further upstream. No interaction between DJ-1 and Akt occurs in the absence of SG2NA. SG2NA, DJ-1, and Akt colocalize in mitochondria and plasma membrane. Their association is enhanced by increasing levels of reactive oxygen species up to a threshold level but falters thereafter with further increase in oxidants. Mutants of DJ-1 found in patients with familial parkinsonism are not recruited by SG2NA, suggesting its role in neuroprotection. Cells depleted of SG2NA are susceptible, while those overexpressing it are resistant to apoptosis induced by oxidative stress. Our study thus unravels a novel pathway of recruitment of Akt and DJ-1 that provides protection against oxidative stress, especially in neurons.     

Tyrphostins protect neuronal cells from oxidative stress

Tyrphostins are a family of tyrosine kinase inhibitors originally synthesized as potential anticarcinogenic compounds. Because tyrphostins have chemical structures similar to those of the phenolic antioxidants, we decided to test the protective efficacy of tyrphostins against oxidative stress-induced nerve cell death (oxytosis). Many commercially available tyrphostins, at concentrations ranging from 0.5 to 200 microm, protect both HT-22 hippocampal cells and rat primary neurons from oxytosis brought about by treatment with glutamate, as well as by treatment with homocysteic acid and buthionine sulfoximine. The tyrphostins protect nerve cells by three distinct mechanisms. Some tyrphostins, such as A25, act as antioxidants and eliminate the reactive oxygen species that accumulate as a result of glutamate treatment. These tyrphostins also protect cells from hydrogen peroxide and act as antioxidants in an in vitro assay. In contrast, tyrphostins A9 and AG126 act as mitochondrial uncouplers, collapsing the mitochondrial membrane potential and thereby reducing the generation of reactive oxygen species from mitochondria during glutamate toxicity. Finally, the third group of tyrphostins does not appear to be effective as antioxidants but rather protects cells by increasing the basal level of cellular glutathione. Therefore, the effects of tyrphostins on cells are not limited to their ability to inhibit tyrosine kinases.     

Microenvironment mesenchymal cells protect ovarian cancer cell lines from apoptosis by inhibiting XIAP inactivation

Epithelial ovarian carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. Carcinoma-associated mesenchymal stem cells (CA-MSC) have been shown to induce ovarian cancer chemoresistance through trogocytosis. Here we examined CA-MSC properties to protect ovarian cancer cells from carboplatin-induced apoptosis. Apoptosis was determined by Propidium Iodide and Annexin-V-FITC labelling and poly-ADP-ribose polymerase cleavage analysis. We showed a significant increase of inhibitory concentration 50 and a 30% decrease of carboplatin-induced apoptosis in ovarian cancer cells incubated in the presence of CA-MSC-conditioned medium (CM). A molecular analysis of apoptosis signalling pathway in response to carboplatin revealed that the presence of CA-MSC CM induced a 30% decrease of effector caspases-3 and -7 activation and proteolysis activity. CA-MSC secretions promoted Akt and X-linked inhibitor of apoptosis protein (XIAP; caspase inhibitor from inhibitor of apoptosis protein (IAP) family) phosphorylation. XIAP depletion by siRNA strategy permitted to restore apoptosis in ovarian cancer cells stimulated by CA-MSC CM. The factors secreted by CA-MSC are able to confer chemoresistance to carboplatin in ovarian cancer cells through the inhibition of effector caspases activation and apoptosis blockade. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and the phosphorylation of its downstream target XIAP underlined the implication of this signalling pathway in ovarian cancer chemoresistance. This study reveals the potentialities of targeting XIAP in ovarian cancer therapy.