Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes

Growth hormone (GH) has been reported to be useful to treat heart failure. To elucidate whether GH has direct beneficial effects on the heart, we examined effects of GH on oxidative stress-induced apoptosis in cardiac myocytes. TUNEL staining and DNA ladder analysis revealed that hydrogen peroxide (H₂O₂)-induced apoptosis of cardiomyocytes was significantly suppressed by the pretreatment with GH. GH strongly activated extracellular signal-regulated kinases (ERKs) in cardiac myocytes and the cardioprotective effect of GH was abolished by inhibition of ERKs. Overexpression of dominant negative mutant Ras suppressed GH-stimulated ERK activation. Overexpression of Csk that inactivates Src family tyrosine kinases also inhibited ERK activation evoked by GH. A broad-spectrum inhibitor of protein tyrosine kinases (PTKs), genistein, strongly suppressed GH-induced ERK activation and the cardioprotective effect of GH against apoptotic cell death. GH induced tyrosine phosphorylation of EGF receptor and JAK2 in cardiac myocytes, and an EGF receptor inhibitor tyrphostin AG1478 and a JAK2 inhibitor tyrphostin B42 completely inhibited GH-induced ERK activation. Tyrphostin B42 also suppressed the phosphorylation of EGF receptor stimulated by GH. These findings suggest that GH has a direct protective effect on cardiac myocytes against apoptosis and that the effect of GH is attributed at least in part to the activation of ERKs through Ras and PTKs including JAK2, Src, and EGF receptor tyrosine kinase.     

Activation of ERK during DNA damage-induced apoptosis involves protein kinase Cdelta

We have previously shown that protein kinase C (PKC) acts upstream of caspases to regulate cisplatin-induced apoptosis. Since extracellular signal-regulated kinases (ERKs) have also been implicated in DNA damage-induced apoptosis, we have examined if ERK signaling pathway acts downstream of PKC in the regulation of cisplatin-induced apoptosis. PKC activator PDBu induced ERK1/2 phosphorylation which was inhibited by general PKC inhibitor bisindolylmaleimide and G? 6983 as well as the MEK inhibitor U0126 but not by the PKCdelta inhibitor rottlerin. Cisplatin caused a concentration-dependent activation of ERK1/2 in HeLa cells. The level of ERK2 was decreased in HeLa cells that acquired resistance to cisplatin (HeLa/CP). The MEK inhibitor U0126 inhibited cisplatin-induced ERK activation and attenuated cisplatin-induced cell death. Inhibition of PKCdelta by rottlerin or depletion of PKCdelta by siRNA inhibited cisplatin-induced ERK activation. These results suggest that cisplatin-induced DNA damage results in activation of ERK1/2 via PKCdelta.     

Both Gs and Gi proteins are critically involved in isoproterenol-induced cardiomyocyte hypertrophy

Activation of beta-adrenoreceptors induces cardiomyocyte hypertrophy. In the present study, we examined isoproterenol-evoked intracellular signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs) and cardiomyocyte hypertrophy. Inhibitors for cAMP and protein kinase A (PKA) abolished isoproterenol-evoked ERK activation, suggesting that Gs protein is involved in the activation. Inhibition of Gi protein by pertussis toxin, however, also suppressed isoproterenol-induced ERK activation. Overexpression of the Gbetagamma subunit binding domain of the beta-adrenoreceptor kinase 1 and of COOH-terminal Src kinase, which inhibit functions of Gbetagamma and the Src family tyrosine kinases, respectively, also inhibited isoproterenol-induced ERK activation. Overexpression of dominant-negative mutants of Ras and Raf-1 kinase and of the beta-adrenoreceptor mutant that lacks phosphorylation sites by PKA abolished isoproterenol-stimulated ERK activation. The isoproterenol-induced increase in protein synthesis was also suppressed by inhibitors for PKA, Gi, tyrosine kinases, or Ras. These results suggest that isoproterenol induces ERK activation and cardiomyocyte hypertrophy through two different G proteins, Gs and Gi. cAMP-dependent PKA activation through Gs may phosphorylate the beta-adrenoreceptor, leading to coupling of the receptor from Gs to Gi. Activation of Gi activates ERKs through Gbetagamma, Src family tyrosine kinases, Ras, and Raf-1 kinase.     

Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells.

We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines. Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP. Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h. Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells. In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis. Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly. Etoposide treatment of either cell line had no effect upon the activation of p38. Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation. These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways.     

Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.     

EGFR plays a pivotal role in the regulation of polyamine-dependent apoptosis in intestinal epithelial cells

Intracellular polyamine synthesis is regulated by the enzyme ornithine decarboxylase (ODC), and its inhibition by -difluromethylornithine (DFMO), confers resistance to apoptosis. We have previously shown that DFMO leads to the inhibition of de novo polyamine synthesis, which in turn rapidly activates Src, STAT3 and NF-κB via integrin β3 in intestinal epithelial cells. One mechanism to explain these effects involves the activation of upstream growth factor receptors, such as the epidermal growth factor receptor (EGFR). We therefore hypothesized that EGFR phosphorylation regulates the early response to polyamine depletion. DFMO increased EGFR phosphorylation on tyrosine residues 1173 (pY1173) and 845 (pY845) within 5 min. Phosphorylation declined after 10 min and was prevented by the addition of exogenous putrescine to DFMO containing medium. Phosphorylation of EGFR was concomitant with the activation of ERK1/2. Pretreatment with either DFMO or EGF for 1 h protected cells from TNF-/CHX-induced apoptosis. Exogenous addition of polyamines prevented the protective effect of DFMO. In addition, inhibition of integrin β3 activity (with RGDS), Src activity (with PP2), or EGFR kinase activity (with AG1478), increased basal apoptosis and prevented protection conferred by either DFMO or EGF. Polyamine depletion failed to protect B82L fibroblasts lacking the EGFR (PRN) and PRN cells expressing either a kinase dead EGFR (K721A) or an EGFR (Y845F) mutant lacking the Src phosphorylation site. Conversely, expression of WT-EGFR (WT) restored the protective effect of polyamine depletion. Fibronectin activated the EGFR, Src, ERKs and protected cells from apoptosis. Taken together, our data indicate an essential role of EGFR kinase activity in MEK/ERK-mediated protection, which synergizes with integrin β3 leading to Src-mediated protective responses in polyamine depleted cells.     

Platelet-derived growth factor stimulates phospholipase C-gamma 1, extracellular signal-regulated kinase, and arachidonic acid release in rat myometrial cells: contribution to cyclic 3',5'-adenosine monophosphate production and effect on cell proliferation

In the present study, we examined downstream signaling events that followed exposure of cultured rat myometrial cells to platelet-derived growth factor (PDGF) and their effect on cell proliferation. PDGF-BB induced tyrosine phosphorylation of PDGF-beta receptors and increased inositol trisphosphate production via the tyrosine phosphorylation of phospholipase (PL)C-gamma 1. PDGF-BB also increased cAMP synthesis. This increase was potentiated by forskolin and reduced by indomethacin, a cyclooxygenase inhibitor, reflecting a Gs protein-mediated process via prostaglandin biosynthesis. The prostaglandin produced by PDGF was characterized as prostacyclin (PGI(2)). PDGF-BB increased arachidonic acid (AA) release, which, similarly to cAMP accumulation, was abolished in the presence of AACOCF3, a cytosolic PLA(2) inhibitor, and in the absence of Ca(2+). U-73122, a potent inhibitor of PLC activity, blocked both the production of inositol phosphates and the AA release triggered by PDGF-BB. Extracellular signal-regulated kinases (ERKs) 1 and 2 are expressed in myometrial cells, and PDGF-BB selectively activated ERK2. PD98059, an inhibitor of the ERK-activating kinase, blocked PDGF-BB-mediated ERK2 activation, AA release, and cAMP production. The results demonstrate that PDGF-BB stimulated cAMP formation through both PLC activation and ERK-dependent AA release and PGI(2) biosynthesis. PDGF-BB also increased cell proliferation and [(3)H]thymidine incorporation. This was abolished by PD98059, demonstrating that the ERK cascade is required for the mitogenic effect of PDGF-BB. Forskolin, which potentiated the cAMP response to PDGF-BB, attenuated both DNA synthesis and ERK activation triggered by PDGF-BB, suggesting the presence of a negative feedback regulation.     

Activation of extracellular signal-regulated kinases ERK1 and ERK2 induces Bcl-xL up-regulation via inhibition of caspase activities in erythropoietin signaling

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.     

Mechanisms of ceramide‐induced COX‐2‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells partially overlapped with resveratrol

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular‐signal‐regulated kinases (ERK)1/2‐ and p38 kinase‐dependent apoptosis in human ovarian cancer OVCAR‐3 cells, concomitant with an increase in the expression of COX‐2 and p53 phosphorylation. Blockade of cyclooxygenase‐2 (COX‐2) activity by siRNA or NS398 correspondingly inhibited ceramide‐induced p53 Ser‐15 phosphorylation and apoptosis; thus COX‐2 appears at the apex of the p38 kinase‐mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide‐treated cells. Ceramide‐treated cells underwent a dose‐dependent reduction in trans‐membrane potential. Although both ceramide and resveratrol induced the expressions of caspase‐3 and ‐7, the effect of inducible COX‐2 was different in caspase‐7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis‐requiring, ERK1/2‐dependent signal transduction pathway and induction of COX‐expression as an essential molecular antecedent for subsequent p53‐dependent apoptosis. In addition, expressions of caspase‐3 and ‐7 are observed. However, a p38 kinase‐dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide‐induced apoptosis. J. Cell. Biochem. 114: 1940–1954, 2013. © 2013 Wiley Periodicals, Inc.     

H2S-induced pancreatic acinar cell apoptosis is mediated via JNK and p38 MAP kinase

Treatment of pancreatic acinar cells by hydrogen sulphide has been shown to induce apoptosis. However, a potential role of mitogen-activated protein kinases (MAPKs) in this apoptotic pathway remains unknown. The present study examined the role of MAPKs in H₂S-induced apoptosis in mouse pancreatic acinar cells. Pancreatic acinar cells were treated with 10 μM NaHS (a donor of H₂S) for 3 hrs. For the evaluation of the role of MAPKs, PD98059, SP600125 and SB203580 were used as MAPKs inhibitors for ERK1/2, JNK1/2 and p38 MAPK, respectively. We observed activation of ERK1/2, JNK1/2 and p38 when pancreatic acini were exposed to H₂S. Moreover, H₂S-induced ERK1/2, JNK1/2 and p38 activation were blocked by pre-treatment with their corresponding inhibitor in a dose-dependent manner. H₂S-induced apoptosis led to an increase in caspase 3 activity and this activity was attenuated when caspase 3 inhibitor were used. Also, the cleavage of caspase 3 correlated with that of poly-(ADP-ribose)-polymerase (PARP) cleavage. H₂S treatment induced the release of cytochrome c , smac from mitochondria into the cytoplasm, translocation of Bax into mitochondria and decreased the protein level of Bcl-2. Inhibition of ERK1/2 using PD98059 caused further enhancement of apoptosis as evidenced by annexin V staining, while SP600125 and SB203580 abrogated H₂S-induced apoptosis. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to H₂S-induced apoptosis.     

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

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-alpha (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 alpha-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.     

Polyamine depletion inhibits etoposide-induced NF-κB activation in transformed mouse fibroblasts

Summary. In a previous research, we have shown that adequate levels of polyamines are required in transformed mouse fibroblasts for the correlated activations of MAPK subtypes (ERK and JNK) and caspases induced by etoposide and leading to apoptosis. We report now that the treatment of fibroblasts with etoposide also elicited a progressive and sustained increase of NF-B activation. The DNA binding activity of p65 NF-B subunit was increased up to approximately 4-fold and was accompanied by enhancement of p65 phosphorylation. A two days pre-treatment of fibroblasts with -difluoromethylornithine (DFMO), which caused polyamine depletion, provoked a slight activating effect when given alone, but markedly inhibited the etoposide-induced increases in p65 DNA binding and phosphorylation. The NF-B inhibiting effect of DFMO was prevented by the addition of exogenous putrescine, which restored the intracellular content of polyamines. Selective inhibitors of the etoposide-stimulated MAPK subtypes also reduced NF-B activation. Moreover, pharmacological NF-B inhibition reduced the increase in caspase activity and cell death elicited by etoposide, suggesting that NF-B is involved in signaling to apoptosis. The results of the present study, together with our previous findings, suggest that polyamines play a permissive role in the pathways triggered by etoposide and leading to cell death of fibroblasts, by supporting the activation of MAPKs, NF-B and caspases.     

TGF-beta1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production

Serum deprivation induces apoptosis in NIH3T3 cells, which is associated with increased intracellular ceramide generation and with the activation of p38 mitogen-activated protein (MAP) kinase. Treatment of cells with transforming growth factor-beta1 (TGF-beta1) activated the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2), inhibited the serum deprivation-induced p38 activation and the increase in intracellular ceramide formation, leading to the stimulation of cell proliferation and the suppression of apoptosis. Inhibition of p38 MAP kinase by SB203580 significantly reduced the serum-deprivation-induced apoptosis. Overexpression of p38 increased the cell apoptosis and reduced the antiapoptotic effect of TGF-beta1. Inhibition of ERK1/ERK2 by PD98059 completely inhibited the TGF-beta1-stimulated proliferation and partially inhibited the antiapoptotic effects of TGF-beta1. Neither SB203580 nor PD98059 has obvious effect on TGF-beta1-mediated inhibition of the increased ceramide generation. Serum-deprivation-induced apoptosis in NIH3T3 cells can also be blocked by broad-spectrum caspase inhibitor. TGF-beta1 treatment has an inhibitory effect on caspase activities. Our results indicate that ceramide, p38, and ERK1/ERK2 play critical but differential roles in cell proliferation and stress-induced apoptosis. TGF-beta1 suppresses the serum-deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 activation and increased ceramide generation.     

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.