Apoptosis induced by doxorubicin in neurotumor cells is divorced from drug effects on ceramide accumulation and may involve cell cycle-dependent caspase activation

Doxorubicin (0.5 microgram/ml) induced caspase-dependent apoptosis in SH-SY5Y neuroblastoma and CHP-100 neuroepithelioma cells. The apoptotic response started to be evident approximately 15 h after drug administration and, as monitored over a 48-h period, was more pronounced in CHP-100 than in SH-SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B(1) (25 microM); this compound, however, did not prevent drug-induced apoptosis. Untreated cells from both lines expressed negligible p53 levels; on the other hand, whereas p53 and p21(Cip1/Waf1) were rapidly up-regulated in doxorubicin-treated SH-SY5Y cells, such a response was not observed in CHP-100 cells. Doxorubicin induced a G(2)/M phase block in both cell lines, but whereas the G(1) phase was markedly depleted in CHP-100 cells, it was substantially retained in SH-SY5Y cells. In the latter system, double G(1) and G(2)/M block largely preceded cell death; however, as apoptosis underwent completion, it selectively targeted late S and G(2)/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G(1) cell population. These results support the notion that doxorubicin-induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G(1) cells appear to be refractory to doxorubicin-induced apoptosis, p53 up-regulation and p21(Cip1/Waf1) expression may provide an unfavorable setting for the apoptotic action of the drug.     

Apoptosis is induced by decline of mitochondrial ATP synthesis in erythroleukemia cells

Apoptosis is shown to occur in erythroleukemia cells after incubation with oligomycin, which specifically inactivates mitochondrial ATPsynthase. Energy charge and ATP content decline very early during the treatment. Mitochondrial respiration is dramatically decreased while lactate production results not modified. DNA fragmentation progressively increases starting one hour following oligomycin removal, while loss of plasma membrane integrity occurs with a much slower time-course. Similar effects are also shown in differentiation-induced erythroleukemia cells exposed to H(2)O(2). In this case, evidence is provided for the involvement of (*)OH generated by iron-catalyzed reactions in the mechanism by which H(2)O(2) impairs energy charge and induces apoptosis. We hypothesize a possible role played by interference with mitochondrial bioenergy through inactivation of mitochondrial ATPsynthase in the apoptosis triggered by oxidative stress under conditions in which cells undergo an iron overload-like status, as occurs in differentiation-induced erythroleukemia cells. These results point to the impairment of mitochondrial ATP synthesis and of energy charge as common early events critical for the execution of apoptosis, independently by the stimuli used for its induction: the specific inhibitor of mitochondrial ATPsynthase or H(2)O(2) exposure combined with the iron-enhancing differentiating treatment.     

Apoptosis induced by capsaicin in prostate PC-3 cells involves ceramide accumulation,neutral sphingomyelinase,and JNK activation

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89–99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.     

Apoptosis is induced in cells with cytolytic potential by L-leucyl-L-leucine methyl ester.

Previous studies have demonstrated that the selective toxicity of leucyl-leucine methyl ester (Leu-Leu-OMe) for cytotoxic lymphocytes and myeloid cells is dependent on intracellular conversion to membranolytic metabolites by the acyl transferase activity of the granule enzyme dipeptidyl peptidase I (DPPI) that is enriched in these cells. The mechanism of cell death remained unclear, however, and was the subject of the experiments reported here. When human U937, HL60, or THP-1 myeloid tumor cell lines or murine CTLL-2 cells were treated with Leu-Leu-OMe, early release of both cytosolic 51Cr and soluble [3H]TdR labeled DNA fragments was observed, whereas antibody + C treatment of these cells caused only 51Cr release. Killing of U937 or THP-1 cells by incubation with the lysosomotropic amino acid methyl ester, Phe-OMe also induced only 51Cr release without evidence of DNA fragmentation. Preincubation with Zn2+, a known inhibitor of endonuclease activity prevented Leu-Leu-OMe-induced 51Cr or [3H]TdR release from these cell lines, but had no effect on antibody + C or Phe-OMe-induced 51Cr release. Zn2+ also prevented Leu-Leu-OMe-mediated killing of normal human CD16+ NK cells. Zn2+ had no inhibitory effect on Leu-Leu-OMe uptake or intracellular conversion to (Leu-Leu)n-OMe metabolites by these cell lines. Moreover, Zn2+ did not inhibit 51Cr release from nonnucleated E or nucleated U937 targets induced by extracellular production of DPPI-generated metabolites of Leu-Leu-OMe. Thus, killing of cytotoxic lymphocytes and myeloid cells by Leu-Leu-OMe appears to be dependent on generation of metabolites with membranolytic properties, but cell death induced by this process does not involve simple lysis of the plasma membrane. Rather, intracellular production of DPPI generated (Leu-Leu)n-OMe metabolites appears to trigger, an additional Zn(2+)-sensitive process that is associated with induction of apoptosis in cells with cytolytic potential.     

Apoptosis induced by inhibition of intercellular contact

The LIM 1863 colon carcinoma cell line grows as structural organoids of goblet and columnar cells around a central lumen and provides a model for the development of stem cells in the normal colon. The organoid structure can be disrupted by removal of calcium from the medium, resulting in a suspension of single cells. Upon readdition of calcium, the cells reform the organoid structure over a period of 24 h, and ultrastructural examination of the reforming cells reveals that this involves a complex process that we have termed clutching. To determine the adhesion molecules involved in organoid formation we attempted to block this process by single cell suspensions of LIM 1863 reseeded in the presence of monoclonal antibodies. An anti-integrin antibody directed against a conformational epitope on the alpha v subunit totally inhibited organoid reformation. As a consequence of this inhibition of cell contact the colon carcinoma cells rapidly underwent apoptosis. Investigations of the apoptotic pathway involved suggested an induction mechanism since the onset of apoptosis in the contact- inhibited cells showed specific increased synthesis of 68- and 72-kD proteins. In addition, immunoblotting of cytosolic and nuclear extracts of the cells revealed the rapid translocation of the tumor suppressor gene product, p53 to the cell nucleus upon induction of apoptosis. These results suggest that cell-cell adhesion may be a vital regulator of colon development overcome in tumor cells by loss of adhesion molecules or of functional p53 protein.     

Apoptosis induction by MEK inhibition in human lung cancer cells is mediated by Bim

AZD6244 (ARRY-142886) is an inhibitor of MEK1/2 and can inhibit cell proliferation or induce apoptosis in a cell-type dependent manner. The precise molecular mechanism of AZD6244-induced apoptosis is not clear. To investigate mechanisms of AZD6244 induced apoptosis in human lung cancer, we determined the molecular changes of two subgroups of human lung cancer cell lines that are either sensitive or resistant to AZD6244 treatment. We found that AZD6244 elicited a large increase of Bim proteins and a smaller increase of PUMA and NOXA proteins, and induced cell death in sensitive lung cancer cell lines, but had no effect on other Bcl-2 related proteins in those cell lines. Knockdown of Bim by siRNA greatly increased the IC(50) and reduced apoptosis for AZD6244 treated cells. We also found that levels of endogenous p-Thr32-FOXO3a and p-Ser253-FOXO3a were lower in AZD6244-sensitive cells than in AZD6244-resistant cells. In the sensitive cells, AZD6244 induced FOXO3a nuclear translocation required for Bim activation. Moreover, the silencing of FOXO3a by siRNA abrogated AZD6244-induced cell apoptosis. In addition, we found that transfection of constitutively active AKT up-regulated p-Thr32-FOXO3a and p-Ser253-FOXO3a expression and inhibited AZD6244-induced Bim expression in sensitive cells. These results show that Bim plays an important role in AZD6244-induced apoptosis in lung cancer cells and that the PI3K/AKT/FOXO3a pathway is involved in Bim regulation and susceptibility of lung cancer cells to AZD6244. These results have implications in the development of strategies to overcome resistance to MEK inhibitors.     

RAF associates with phosphorylated nuclear BubR1 during endoreduplication induced by JAK inhibition

The role of JAK signaling in cell cycle transit and maintenance of genomic stability was determined in HL-60 human myeloblastic leukemia cells. We have previously reported that a pan-JAK inhibitor caused ERK-dependent endoreduplication. In the current study we find that JAK inhibition caused nuclear re-localization of RAF-1, which could be inhibited by RAF inhibitor GW5074. GW5074 also inhibited JAK inhibitor-induced appearance of nuclear phosphorylated RAF-1(pS621RAF) and MEK, and it inhibited the JAK inhibitor-induced co-immunoprecipitation of nuclear RAF-1 and MEK. JAK inhibition also increased nuclear BubR1 phosphorylation, which was diminished by RAF inhibitor GW5074. RAF-1 and BubR1 in the nucleus co-immunoprecipitated; and GW5074 eliminated this. Furthermore, inhibiting RAF with GW5074 blocked the pan-JAK inhibitor-induced endoreduplication. These data thus show that JAK inhibition causes nuclear re-localization and phosphorylation of RAF and MEK where RAF binds BubR1 with ensuing nuclear RAF-dependent BubR1 phosphorylation. Inhibiting RAF inhibited this and endoreduplication. The results suggest that there is a JAK/RAF/MEK/BubR1 axis that can regulate genomic stability. In this hypothetical model JAK suppresses RAF/MEK phosphorylation and nuclear re-localization, but JAK inhibition induces the phosphorylations and re-localization with association of RAF and phosphorylated BubR1 in the nucleus leading to endoreduplication.Key words: endoreduplication, JAK, genomic instability, MAPK, HL-60 cells     

Apoptosis induced by proteasome inhibition in human myoblast cultures

Dysfunction of the ubiquitin-proteasome system has recently been implicated in the pathogenesis of some untreatable myodegenerative diseases characterized by the formation of ubiquitinated inclusions in skeletal muscles. We have developed an in vitro model of proteasomal dysfunction by applying inhibitors of the proteasome to primary adult human skeletal muscle cultures. Our data show that proteasome inhibition causes both cytoplasmic accumulation of ubiquitinated inclusions and apoptotic death, the latter through accumulation of active caspase-3.     

Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage

Rose Bengal (RB) is a very efficient photosensitizer which undergoes inactivation of its photophysical and photochemical properties upon addition of a quencher group—i.e. acetate—to the xanthene rings. The resulting RB acetate (RB-Ac) derivative behaves as a fluorogenic substrate: it easily enters the cells where the native photoactive molecule is restored by esterase activities. It is known that the viability of RB-Ac-loaded cells is strongly reduced by light irradiation, attesting to the formation of intracellular RB. The aim of this study was to identify the organelles photodamaged by the intracellularly formed RB. RB-Ac preloaded rat C6 glioma cells and human HeLa cells were irradiated at 530 nm. Fluorescence confocal imaging and colocalization with specific dyes showed that the restored RB molecules redistribute dynamically through the cytoplasm, with the achievement of a dynamic equilibrium at 30 min after the administration, in the cell systems used; this accounted for a generalized damage to several organelles and cell structures (i.e. the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the cytoskeleton). The multiple organelle damage, furthermore, led preferentially to apoptosis as demonstrated by light and electron microscopy and by dual-fluorescence staining with FITC-labelled annexin V and propidium iodide.     

RAF associates with phosphorylated nuclear BubR1 during endoreduplication induced by JAK inhibition

The role of JAK signaling in cell cycle transit and maintenance of genomic stability was determined in HL-60 human myeloblastic leukemia cells. We have previously reported that a pan-JAK inhibitor caused ERK-dependent endoreduplication. In the current study we find that JAK inhibition caused nuclear re-localization of RAF-1, which could be inhibited by RAF inhibitor GW5074. GW5074 also inhibited JAK inhibitor-induced appearance of nuclear phosphorylated RAF-1(pS621RAF) and MEK, and it inhibited the JAK inhibitor-induced co-immunoprecipitation of nuclear RAF-1 and MEK. JAK inhibition also increased nuclear BubR1 phosphorylation, which was diminished by RAF inhibitor GW5074. RAF-1 and BubR1 in the nucleus co-immunoprecipitated; and GW5074 eliminated this. Furthermore, inhibiting RAF with GW5074 blocked the pan-JAK inhibitor-induced endoreduplication. These data thus show that JAK inhibition causes nuclear re-localization and phosphorylation of RAF and MEK where RAF binds BubR1 with ensuing nuclear RAF-dependent BubR1 phosphorylation. Inhibiting RAF inhibited this and endoreduplication. The results suggest that there is a JAK/RAF/MEK/BubR1 axis that can regulate genomic stability. In this hypothetical model JAK suppresses RAF/MEK phosphorylation and nuclear re-localization, but JAK inhibition induces the phosphorylations and re-localization with association of RAF and phosphorylated BubR1 in the nucleus leading to endoreduplication.     

Apoptosis is involved in the senescence of endothelial cells induced by angiotensin II

Vascular endothelial cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related cardiovascular disorders, e.g. atherosclerosis. In this study, induction of Angiotensin II (Ang II) promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence-associated beta-galactosidase (SA-beta-gal) positive staining cell, and depressed cell proliferation. Apoptotic changes were increased in senescent cells, with a small subset of the senescent cells showing aberrant morphology such as pronounced nuclear fragmentation or multiple micronuclei. The results suggest cell apoptosis is possibly an important factor in the process of pathologic and physiologic senescence of endothelial cells as well as vascular aging.     

Apoptosis induced by hydrogen peroxide under serum deprivation and its inhibition by antisense c-jun in F-MEL cells

Under serum deprivation F-MEL cells die by apoptosis. We previously showed that apoptosis induced by serum deprivation was suppressed by inhibition of c-jun expression using antisense c-jun transfected cell line, c-junAS. To elucidate the underlying mechanisms we examined the species which is responsible for apoptosis under serum deprivation. When catalase and N-acetyl-L-cysteine (NAC) were included in the medium, cell death under serum deprivation was effectively suppressed in F-MEL cells. Intracellular generation of hydrogen peroxide (H(2)O(2)) was also detected under serum deprivation in parental F-MEL cells, but it was suppressed in c-junAS (+) cells, in which antisense c-jun was expressed and c-Jun protein expression was inhibited as shown by Western blot. When H(2)O(2) was directly applied to F-MEL cells at 3 mM, apoptotic cell death was induced, whereas it was suppressed in c-junAS (+) cells. Induction of apoptosis by H(2)O(2) and its inhibition by antisense c-jun was confirmed by detection of internucleosomal fragmentation of DNA, TdT-mediated dUTP nick end labeling (TUNEL)-positive cells and morphological alteration of nuclei. These results indicate that apoptosis induced by serum deprivation in F-MEL cells is mediated by H(2)O(2) and c-jun expression is essential to apoptosis induced by H(2)O(2) in F-MEL cells.     

Stress-induced cell death is mediated by ceramide synthesis in Neurospora crassa

The combined stresses of moderate heat shock (45 degrees C) and analog-induced glucose deprivation constitute a lethal stress for Neurospora crassa. We found that this cell death requires fatty acid synthesis and the cofactor biotin. In the absence of the cofactor, the stressed cells are particularly sensitive to exogenous ceramide, which is lethal at low concentrations. When we extracted endogenous sphingolipids, we found that unique ceramides were induced (i) by the inhibitory glucose analog 2-deoxyglucose and (ii) by combined heat shock and 2-deoxyglucose. We determined that the former is a 2-deoxyglucose-modified ceramide. By structural analysis, we identified the latter, induced by dual stress, as C(18)(OH)-phytoceramide. We also identified C(24)(OH)-phytoceramide as a constitutive ceramide that continues to be produced during the combined stresses. The unusual C(18)(OH)-phytoceramide is not made by germinating asexual spores subjected to the same heat and carbon stress. Since these spores, unlike growing cells, do not die from the stresses, this suggests a possible connection between synthesis of the dual-stress-induced ceramide and cell death. This connection is supported by the finding that a (dihydro)ceramide synthase inhibitor, australifungin, renders cells resistant to death from these stresses. The OS-2 mitogen-activated protein kinase, homologous to mammalian p38, may be involved in the cell death signaling pathway. Strains lacking OS-2 survived the combined stresses better than the wild type, and phosphorylated OS-2 increased in wild-type cells in response to heat shock and combined heat and carbon stress.     

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

Apoptosis induced by the SARS-associated coronavirus in Vero cells is replication-dependent and involves caspase

The pathogenesis of the severe acute respiratory syndrome (SARS), a newly emerging life-threatening disease in humans, remains unknown. It is believed that the modulation of apoptosis is relevant to diseases that are caused by various viruses. To examine potential apoptotic mechanisms related to SARS, we investigated features of apoptosis induced by the SARS-associated coronavirus (SARS-CoV) in host cells. The results indicated that the SARS-CoV-induced apoptosis in Vero cells in a virus replication-dependent manner. Additionally, the downregulation of Bcl-2, the activation of casapse 3, as well as the upregulation of Bax were detected, suggesting the involvement of the caspase family and the activation of the mitochondrial signaling pathway. Although there is a positive correlation between apoptosis and virus replication, the latter is not significantly blocked by treatment with the caspase inhibitor z-DEVD-FMK. These preliminary data provide important information on both the pathogenesis and potential antiviral targets of SARS-CoV.     

Selective phosphotyrosine phosphatase inhibition and increased ceramide formation is associated with B-cell death by apoptosis

Bis(maltolato)oxovanadium(IV) (BMOV), a protein phosphotyrosine phosphatase inhibitor, selectively induced apoptosis (as quantitated by TUNEL staining) in a B-cell line (Ramos) but not in a T-cell line (Jurkat). The pattern of BMOV-induced protein tyrosine phosphorylation was different in B-cells versus T-cells. Further, BMOV induced a 2-fold increase in ceramide levels in B-cells but not in T-cells and this resembled the ceramide increase following activation of the B-cell antigen receptor. A 2-fold increase in the ratio of ceramide to sphingomyelin in B-cells treated with BMOV suggested that sphingomyelinase activation was the result of the sustained tyrosine phosphorylation of specific proteins and activated the cell death pathway.     

Apoptosis in HeLa Hep2 cells is induced by low-dose,low-dose-rate radiation

Radioimmunotherapy with radiolabeled antibodies may cause inhibition of the growth of epithelial tumors, despite low total radiation doses and comparatively low radiosensitivity of epithelial tumor cells. The induction of apoptosis by low-dose radiation, such as delivered in radioimmunotherapy, was investigated in vitro in human HeLa Hep2 carcinoma cells. The cultured cells were exposed to defined radiation doses from a (60)Co radiation therapy source. The radiation source delivered 0.80 +/- 0.032 (mean +/- SD) Gy/min and the cells were given total doses of 1, 2, 5, 10 and 15 Gy. Using fluorescein-labeled Annexin V, followed by flow cytometry and DNA ladder analysis, apoptotic cells were detected and quantified. Radiation doses below 2 Gy did not cause any significant increase in apoptosis. Compared to control cells, apoptosis was pronounced after 5-10 Gy irradiation and was correlated to the radiation dose, with up to 42 +/- 3.5% of the cells examined displaying apoptosis. Clonogenic assays confirmed significantly decreased viability of the cells in the interval 2 to 10 Gy with low-dose-rate radiation, 60 +/- 2% compared to 2 +/- 2%. Lethal effects on the tumor cells were also evaluated by an assay of the cytotoxic effects of the release of (51)Cr. Significant cytotoxicity, with up to 64 +/- 6% dead cells, was observed at 5 Gy. Similar results were obtained when the dose rate was reduced to 0.072 +/- 0.003 Gy/min (mean +/- SD). In the case of the (137)Cs source, the dose rate could be reduced to 0.045 Gy/h, a level comparable to radioimmunotherapy, which induced significant apoptosis, and was most pronounced at 72-168 h postirradiation. It can be concluded that in vitro low-dose and low-dose-rate radiation induces apoptosis in epithelial HeLa Hep2 cells and thus may explain a mechanism by which pronounced inhibition of growth of HeLa Hep2 tumors at doses used in radioimmunotherapy has been obtained previously.