Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium

Cadmium (Cd), a human carcinogen, can induce apoptosis in various cell types. Three major mitogen-activated protein kinases (MAPKs), c-JUN N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), have been shown to regulate apoptosis. In this study we explore the ability of Cd to activate JNK, p38 and ERK, including their effects on Cd-mediated growth inhibition and apoptosis in a human non-small cell lung carcinoma cell line, CL3. The kinase activity of JNK was induced dose-dependently by 30-160 microM CdCl(2). High cytotoxic doses of Cd (130-160 microM) markedly activated p38, but low Cd doses did not. Conversely, the activities of ERK1 and ERK2 were decreased by low cytotoxic doses of Cd (相似文献   

Activation of JNK, p38 and ERK mitogen-activated protein kinases by chromium(VI) is mediated through oxidative stress but does not affect cytotoxicity

In this study we have explored the involvement of oxidative stress in Cr(VI)-induced JNK, p38 and ERK signaling pathways and their effects on Cr(VI) cytotoxicity in human non-small cell lung carcinoma CL3 cells. Exposure to K(2)Cr(2)O(7) markedly activated JNK and p38 and moderately activated ERK in a dose- (10-80 microM) and time-dependent (1-12 h) manner. The activated p38 decreased markedly and rapidly and the activated JNK decreased gradually when Cr(VI) was removed from the medium. Post-incubation of Cr(VI)-treated cells with H(2)O(2) increased the activities of JNK and p38, but not ERK. Co-administering Cr(VI) with 3-amino-1,2, 4-triazole (3AT), a catalase inhibitor, enhanced p38 activation, but did not influence JNK and ERK activation by Cr(VI). Conversely, co-administering Cr(VI) with mannitol, a hydroxyl radical scavenger and a Cr(V) chelator, reduced p38 activation and increased JNK and ERK activation by Cr(VI). These results indicate that p38 activation by Cr(VI) is positively correlated with oxidative stress, while JNK activity can be enhanced by either a quencher (mannitol) or activator (H(2)O(2)) of redox reactions in Cr(VI)-exposed CL3 cells. However, both 3AT and mannitol reduced the cytotoxicity of Cr(VI), but H(2)O(2) did not. The JNK activated by Cr(VI) was decreased (approximately 50%) by expression of a kinase-defective form of MKK7 (MKK7A) but not that of MKK4 (MKK4KR), suggesting that activation of JNK by Cr(VI) is mediated through MKK7. SB202190, a specific inhibitor of p38, markedly decreased JNK but did not change ERK activation by Cr(VI). PD98059, a specific inhibitor of ERK kinases MKK1/2, blocked ERK and p38 but did not alter JNK activation by Cr(VI). Neither the specific kinase inhibitors nor expression of MKK7A altered Cr(VI)-induced cytotoxicity. Together, these results suggest that activation of the JNK, p38 and ERK pathways by Cr(VI) is mediated through diverse redox mechanisms, yet their activation does not correlate with Cr(VI) cytotoxicity.     

Increased level of the p67phox subunit of NADPH oxidase by 4HPR in head and neck squamous carcinoma cells

N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has shown efficacy in cancer chemoprevention and therapy possibly due to its ability to induce growth inhibition and apoptosis in cancer cells. Reactive oxygen species (ROS) have been implicated in mediating 4HPR-induced apoptosis in various cancer cells, including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR increased the level of p67phox, which is a subunit of the NADPH oxidase complex that is involved in the generation of ROS. The increase in the level of p67phox protein may be a downstream effect of the activation of c-Jun N-terminal kinase (JNK) induced by 4HPR. Suppression of endogenous and 4HPR-induced levels of p67phox using small interfering RNA did not result in a change in ROS generation or apoptosis. These results suggest that p67phox is not a critical component or a limiting factor in the 4HPR-induced apoptosis pathway in HNSCC cells.     

p38 MAPK Signaling Mediates Mitochondrial Apoptosis in Cancer Cells Induced by Oleanolic Acid

Oleanolic acid (OA) is a nutritional component widely distributed in various vegetables. Although it has beenwell recognized for decades that OA exerts certain anti-tumor activity by inducing mitochondria-dependentapoptosis, it is still unclear that what molecular signaling is responsible for this effect. In this study, we employedcancer cell lines, A549, BXPC-3, PANC-1 and U2OS to elucidate the molecular mechanisms underlying OA antitumoractivity. We found that activation of MAPK pathways, including p-38 MAPK, JNK and ERK, was triggeredby OA in both a dose and time-dependent fashion in all the tested cancer cells. Activation was accompaniedby cleavage of caspases and PARP as well as cytochrome C release. SB203580 (p38 MAPK inhibitor), but notSP600125 (JNK inhibitor) and U0126 (ERK inhibitor), rescued the pro-apoptotic effect of OA on A549 and BXPC-3 cells. OA induced p38 MAPK activation promoted mitochondrial translocation of Bax and Bim, and inhibitedBcl-2 function by enhancing their phosphorylation. OA can induce reactive oxygen species (ROS)-dependentASK1 activation, and this event was indispensable for p38 MAPK-dependent apoptosis in cancer cells. In vivo,p38 MAPK knockdown A549 tumors proved resistant to the growth-inhibitory effect of OA. Collectively, weelucidated that activation of ROS/ASK1/p38 MAPK pathways is responsible for the apoptosis stimulated byOA in cancer cells. Our finding can contribute to a better understanding of molecular mechanisms underlyingthe antitumor activity of nutritional components.     

Regulation of hairy-cell survival through constitutive activation of mitogen-activated protein kinase pathways

The hairy cells (HCs) of hairy-cell leukemia are intrinsically activated mature clonal B cells. The aims of this study were to gain further insights into the nature of this activation and to assess its importance for the prolonged HC survival in this chronic disease. We show that HCs contain phosphorylated/activated p38 MAPK, JNK and ERK1/ERK2 (ERK1/2). PKC inhibitors increased the activation of p38 and JNK, but reduced the phosphorylation of ERK1/2. Moreover, PKC inhibition resulted in cell death; cell death was also observed when the activation of ERK1/2 in HCs was abrogated with an inhibitor of MEK1/2 activation. In addition to PKC, active Src kinase was also shown to be involved in the maintenance of Raf-independent ERK activation in HCs. During cell culture on a nonadherent surface, ERK phosphorylation was sustained, while phosphorylation of p38 and JNK decreased. This decrease was not observed in HCs cultured on vitronectin (VN), indicating that p38/JNK activation is probably a consequence of in vivo HC interaction with VN present in abundance in the red pulp of the spleen. Taken together, these results suggest that active p38/JNK make HCs susceptible to apoptosis, but the cells are effectively rescued by ERK activation involving constitutively active PKC and Src. These findings are relevant for the understanding of the prolonged cell survival of HCs and their selective sensitivity to some chemotherapeutic agents.     

Leukotriene B4 receptor inhibitor LY293111 induces cell cycle arrest and apoptosis in human anaplastic large-cell lymphoma cells via JNK phosphorylation.

Anaplastic large-cell lymphoma (ALCL) is a heterogeneous lymphoma category in which a subset of cases carry the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. In this study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in ALCL cell lines. LY293111 inhibited proliferation of both ALK(+) and ALK(-) ALCL cell in a dose-dependent fashion and induced complete G(1)-S cell cycle arrest, which was accompanied by upregulation of p27 and downregulation of cyclin E. Pretreatment with LY293111 for 4 h resulted in profound inhibition of serum-induced phosphorylation of extracellular-regulated kinases-1 and 2 and Akt and a concomitant increase in the phosphorylation of the stress-activated kinase c-jun N-terminal kinases (JNK). Simultaneously, LY293111 induced caspase-dependent apoptosis via activation of the intrinsic pathway, including early loss of mitochondrial inner transmembrane potential and the production of reactive oxygen species (ROS), cleavage of caspases-9, -3, poly ADP-ribose polymerase (PARP) and X-linked inhibitor of apoptosis. The phospho-JNK inhibitor SP600125 partially protected Sup-M2 cells from LY293111-induced apoptosis, PARP cleavage and ROS generation, suggesting a role for JNK in LY293111-induced cell death. These results warrant further studies of LY293111 in ALCL.     

Mitogen-activated protein kinase pathway is dispensable for microtubule-active drug-induced Raf-1/Bcl-2 phosphorylation and apoptosis in leukemia cells.

Raf-1 activation and Bcl-2 hyperphosphorylation following treatment with paclitaxel (Taxol) or other microtubule-active drugs is associated with mitotic arrest. Here we show that microtubule-active drugs do not activate the mitogen-activated protein kinase (MAPK) pathway in leukemia cells. PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, do not abrogate Bcl-2 phosphorylation nor apoptosis. Simultaneously with PARP cleavage, paclitaxel induces cleavage of Bcl-2 protein yielding a potentially pro-apoptotic 22 kDa product. In comparison, the stimulation of Raf-1 by phorbol ester (TPA) activates the MAPK pathway, causes MAPK-dependent p21WAF1/CIP1 induction, Rb dephosphorylation and growth arrest without Bcl-2 phosphorylation or apoptosis. Like TPA, cAMP induces p21WAF1/CIP1 but does not cause Bcl-2 phosphorylation. MEKK1 and Ras, upstream activators of JNK and ERK MAPK, also fail to induce Bcl-2 hyperphosphorylation. Although Lck tyrosine kinase has been recently implicated in Raf-1 activation during mitotic arrest, microtubule-active drugs induce Raf-1/Bcl-2 hyperphosphorylation and apoptosis in a Lck-deficient Jurkat cells. Therefore, microtubule-active drugs induce apoptosis which is associated with Raf-1 and Bcl-2 phosphorylation and Bcl-2 cleavage but is independent of the MAPK pathway. In contrast, TPA-activated MAPK pathway causes p21WAF1/CIP1-dependent growth arrest without apoptosis.     

All-trans retinoic acid potentiates Taxotere-induced cell death mediated by Jun N-terminal kinase in breast cancer cells

Taxotere is a cytotoxin effective in treating breast and prostate cancer. It stabilizes microtubules and causes catastrophic cell cycle arrest in G2/M. Taxanes also initiate apoptosis by activating signal pathways, such as the jun N-terminal kinase (JNK) pathway. Strategies aimed at potentiating cell death signaling may improve their efficacy while lessening the potential side effects. We reported that all-trans retinoic acid (ATRA) potentiated taxane-mediated cell death. Here we investigated whether ATRA potentiates cell death signaling through the JNK pathway. Activation of JNK by Taxotere 0.01, 0.1 and 1.0 microM was observed at 24 h in adherent cells and increased at 48 h. Taxotere 0.001 microM-induced JNK activation started after 48 h and increased at 72 h. The timing and intensity of PARP cleavage was similar to that of JNK activation. JNK activation and PARP cleavage induced by 30 nM Taxotere at 48 h were reversed by curcumin, PD169316 and SP600125, JNK inhibitors in order of progressive specificity. None of these inhibitors had an effect on p38 or ERK phosphorylation. All three inhibitors reversed Taxotere-induced phosphorylation of Bcl-2. ATRA induced JNK activation at 24, 48 and 72 h. Incubating cells with ATRA 0.01 microM for 3 days prior to Taxotere treatment potentiated Taxotere-induced JNK activation 24 and 48 h later, an effect sustained for 72 h. Cytotoxicities from 3-day ATRA 0.01 microM incubations were synergistic with subsequent 1-h Taxotere 0.01, 0.1 and 1.0 microM incubations in breast cancer cell lines MCF-7 and MDA-MB-231 and in prostate cancer cell lines LNCaP and PC-3, and additive in breast cancer cell line SK-Br-3. These data demonstrate the potentiation of Taxotere-induced cell death by ATRA pretreatment in breast and prostate cancer cells, and support a mechanism through accentuated and sustained JNK activation.     

Regulation of G1/S transition and induction of apoptosis in HL-60 leukemia cells by fenretinide (4HPR)

We previously reported that all-trans retinoic acid (RA) and fenretinide (4HPR) suppress HL-60 leukemia cell growth and cause partial cell arrest in the G₁-to-S phase. Moreover, 4HPR but not RA induces apoptosis in HL-60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40–75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR-treated cells. Evidence for 4HPR-induced apoptosis comes from (1) cleavage of the enzyme poly(ADP-ribose) polymerase (PARP) to an 89-kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3′-ends of DNA. Overnight pretreatment with 0.5–5.0 μM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid-derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2-fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR-treated HL-60 cells, the effects of fumonisin B₁, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25–100 μM fumonisin B₁ resulted in a dose-dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR-induced increase in ceramide. Int. J. Cancer 78:53–61, 1998.© 1998 Wiley-Liss, Inc.     

Critical role of the c-JunNH2-terminal kinase and p38 mitogen-activated protein kinase pathways on sodium butyrate-induced apoptosis in DU145 human prostate cancer cells.

Sodium butyrate (NaBu) is known to exhibit anti-cancer effects via the differentiation and apoptosis of various carcinoma cells. However, the mechanism by which NaBu induces apoptosis and the involvement of protein kinases during apoptosis is not completely understood. To investigate the underlying pathways, we performed cell culture experiments in androgen-independent human prostate cancer (DU145 cells) focusing on various protein kinases. NaBu causes concentration-dependent cell detachment and growth inhibition. Exposure of DU145 cells to NaBu for 24 h caused a strong apoptotic effect with 26% nuclear fragmentation and condensation. In addition, NaBu induced caspase-3 and poly-ADP ribose polymerase cleavage and up-regulation of bax, suggesting that mitochondrial damage is involved in NaBu-induced caspase-dependent apoptosis. Interestingly, NaBu stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) activation, but not extracellular signal-regulated kinase 1/2 activation during apoptosis. Furthermore, NaBu up-regulated total protein levels and phospho forms of MAPK kinase 3 (MKK3) and MAPK kinase 4 (MKK4) as the upstream kinases of p38 MAPK and JNK independently of oxidative stress. Taken together, it is suggested that NaBu can be a promising chemopreventive agent for prostate cancer and the p38 MAPK and JNK pathways have critical roles in NaBu-induced apoptosis in DU145 cells.     

Ex vivo study of MAPK profiles correlated with parameters of apoptosis during cervical carcinogenesis

Cervical cancer is a leading cause of death in developing countries and is the second highest occurring cancer in women all over the world. The progression of cancer is a multistep process affecting aspects of cellular function such as proliferation, differentiation and apoptosis. Mitogen activated protein kinases (MAPKs), which include p38-MAPK, c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinases (ERKs) are closely associated with cell proliferation and apoptosis and the balance between them could determine a cell's fate. Despite the expanding research effort in vitro, little is known about MAPK activation in clinical specimens of cervical cancer. Therefore, the aim of this ex vivo study was to correlate the phosphorylation status (activity) of MAPKs (p38-MAPK, JNK and ERK), as well as poly (ADP-ribose) polymerase (PARP) and caspase-3 (two cellular markers of apoptosis), during the different stages of cervical carcinogenesis, to observe whether correlations between MAPK activities and apoptosis during the disease process exist. Decreased p38-MAPK phosphorylation was found in the carcinoma (Ca) group) compared to the normal tissues, as well when the low grade squamous intraepithelial lesion--LSIL) group and high grade squamous intraepithelial lesion--HSIL) group were compared with the Ca group. Interestingly, a significant decrease in ERK44 phosphorylation was observed in Ca when compared to LSIL and HSIL. There was also a significant decrease in JNK phosphorylation in Ca when compared with normal tissue and HSIL. As expected, caspase-3 activation and PARP cleavage was significantly lower in Ca when compared with normal tissue. Our results present the first evidence of in vivo involvement of MAPKs in cervical cancer and indicate a possible correlation between MAPK activities and apoptosis in the disease process.     

Lack of p38 MAP kinase activation in TRAIL-resistant cells is not related to the resistance to TRAIL-mediated cell death

Activation of MAP kinases is involved in various cellular processes, including immunoregulation, inflammation, cell growth, cell differentiation, and cell death. To investigate the role of p38 MAP kinase activation in the signaling pathway of TRAIL-mediated apoptosis, we compared TRAIL-mediated MAP kinase activation in TRAIL-susceptible human colon cancer cell line DLD1 and TRAIL-resistant DLD1/TRAIL-R cells. TRAIL-mediated activation of ERK occurred in both cell lines. In contrast, both DLD1 and DLD1/TRAIL-R cells showed no obvious JNK activation after treatment with TRAIL. Interestingly, TRAIL-mediated activation of p38 MAP kinases was observed in DLD1 cells but not in DLD1/TRAIL-R cells. However, activation of p38 MAP kinases was observed in both DLD1 and DLD1/TRAIL-R cells after treatment with anisomycin. Furthermore, inhibiting activated p38 MAP kinases with known inhibitors or with an adenovector expressing dominant negative p38alpha did not block TRAIL-mediated cell death in DLD1 cells. Moreover, activation of p38 MAP kinases by adenovectors expressing constitutive MKK3 or MKK6 (Ad/MKK3bE or Ad/MKK6bE) did not induce cell death in either DLD1 or DLD1/TRAIL-R cell lines. Our results suggest that activation of p38 MAP kinases does not play a major role in TRAIL-mediated apoptosis in DLD1 cells and that lack of TRAIL-mediated p38 MAP kinase activation may not be the mechanism of TRAIL-resistance in DLD1/TRAIL-R cells.     

A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation.

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-kappaB, might determine the final cellular response.     

Inhibition of extracellular signal-regulated kinase (ERK) mediates cell cycle phase independent apoptosis in vinblastine-treated ML-1 cells

Chemotherapeutic agents induce alterations in intracellular signal transduction cascades that culminate in the initiation of the apoptotic program. Here, the relationship between the mitogen-activated protein kinase (MAPK) response and apoptosis in ML-1 cells treated with vinblastine and paclitaxel was investigated. We show that these compounds elicit different effects on MAPKs with vinblastine, but not paclitaxel, increasing both c-Jun-NH2-terminal kinase (JNK) and p38 activity. However, vinblastine and paclitaxel both induced apoptosis with similar kinetics, suggesting that increased JNK and p38 activity is not required for apoptosis that is induced by microtubule interfering agents. Strikingly, the abrogation of extracellular signal-regulated kinase (ERK)-signaling by the MAPK/ERK kinase (MEK)1/2 inhibitor PD098059 in combination with vinblastine robustly induced apoptosis in ML-1 cells at a rate much faster than treatment with vinblastine alone and occurred at all phases of the cell cycle. This apoptotic induction was attributed to JNK activation because: (a) non-JNK-activating concentrations of vinblastine failed to increase apoptosis in the presence of PD098059; (b) apoptosis induced by paclitaxel, which did not activate JNK, was not potentiated by PD098059; and (c) transduction of an inhibitor of JNK activity partially suppressed both JNK activity and apoptosis induced by vinblastine plus PD098059. Additionally, we found that the activation of JNK by vinblastine occurred upstream of effector caspase activation because treatment with a pan-specific caspase inhibitor (valine-alanine-aspartate-fluoromethylketone) resulted in complete abrogation of apoptosis with no effect on MAPK signaling. Taken together, these data suggest that inhibition of the MEK-->ERK signal transduction cascade alleviates cell cycle dependence for vinblastine-induced apoptosis by a mechanism that requires JNK activation.     

Mediation of N-(4-hydoxyphenyl)retinamide-induced apoptosis in human cancer cells by different mechanisms.

The induction of apoptosis by the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been documented in vitro in various cancer types. A role for reactive oxygen species (ROS) in apoptosis induced by 4HPR in some cancer cells has been demonstrated recently. We studied five different human head and neck and five lung cancer cell lines to determine whether the ROS play a general role in 4HPR-induced apoptosis. We found that 4HPR induced apoptosis in all of the cell lines; however, this effect was blocked by antioxidants in only 2 of the 10 cell lines. 4HPR induced a greater than 4-fold increase in the generation of intracellular ROS in these two cell lines compared with a much lower effect in other cell lines. Furthermore, these two cell lines were most sensitive to the induction of apoptosis by 4HPR. The level of the cellular antioxidant thiol and superoxide dismutase activity were relatively lower in cells, which responded to 4HPR with a high level of ROS generation. These results indicate that although ROS can mediate 4HPR-induced apoptosis in some cells, which may have a low endogenous cellular antioxidant levels, other mechanisms exist for 4HPR-induced apoptosis. One such mechanism may involve retinoic acid receptors (RARs) because an RAR antagonist was able to block partially 4HPR-induced apoptosis. In conclusion, 4HPR-induced apoptosis involves at least three different mechanisms, which are complex and can overlap in the same cell line: (a) one mechanism involving 4HPR-induced ROS; (b) one involving RARs; and (c) at least one that does not involve ROS or RARs and remains unclear.     

Mechanism of 2-methoxyestradiol-induced apoptosis and growth arrest in human breast cancer cells

2-Methoxyestradiol, a well-known nonpolar endogenous metabolite of 17beta-estradiol, has been shown to selectively induce apoptosis in a number of cancer cell lines, but not in normal cells. The mechanism of 2-methoxyestradiol-induced apoptosis appears to vary considerably in different cell lines examined. In the present study, we systematically analyzed the mechanisms of 2-methoxyestradiol-induced apoptosis in the estrogen receptor-negative MDA-MB-435s human breast cancer cells. We found that 2-methoxyestradiol induced the activation of JNK, ERK, and p38 MAPKs. 2-methoxyestradiol-induced JNK activation was associated with the induction of apoptosis through the mitochondrial pathways as a result of increased phosphorylation (inactivation) of the anti-apoptotic Bcl-2 and Bcl-xL proteins. In comparison, 2-methoxyestradiol-induced activation of ERK and p38 in these cells was found to have a protective effect against 2-MeO-E(2)-induced apoptosis. Consistent with this observation, the presence of pharmacological inhibitor of ERK or p38 enhanced 2-methoxyestradiol-induced apoptosis. Mechanistically, inhibition of ERK and p38 activity was associated with activation of various caspases and PARP cleavage, and it also stabilized the pro-apoptotic proteins Bax and Bim, thereby preventing them from degradation during 2-methoxyestradiol treatment. These results suggest that ERK and p38 MAPKs may serve as viable targets for the sensitization of human breast cancer cells to 2-methoxyestradiol-induced apoptosis.     

Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells.

The mechanism of Taxol-induced apoptosis was investigated in MCF-7 human breast carcinoma cells. Taxol-induced apoptosis was associated with phosphorylation of both c-Raf-1 and Bcl-2 and activation of ERK and JNK MAP kinases. The serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) effectively blocked apoptosis, but N-p-tosyl-L-lysine chloromethyl ketone (TLCK), another serine protease inhibitor, was without effect. TPCK treatment also prevented phosphorylation of c-Raf-1 and Bcl-2 in response to Taxol treatment. The serine protease inhibitor did not alter JNK activity, but it enhanced Taxol-induced activation of ERK1/2. Treatment of cells with the inhibitor of MEK activation, PD98059, prevented Taxol-induced ERK activation both in the presence and absence of TPCK, but did not influence survival of either Taxol- or Taxol plus TPCK-treated cells. In addition, PD98059 had no effect on c-Raf-1 or Bcl-2 phosphorylation. Thus, while the Taxol-induced phosphorylations of c-Raf-1 and Bcl-2 proteins appear to be coupled, these events can be disassociated from ERK1/2 activation. In summary, these findings suggest that phosphorylation of c-Raf-1 and Bcl-2, but not ERK1/2, are important signaling events in Taxol-induced apoptosis of MCF-7 breast cancer cells and that a TPCK inhibitable protease(s) is required for these processes.     

Ajoene-induced cell death in human promyeloleukemic cells does not require JNK but is amplified by the inhibition of ERK

Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-x(L), and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards ajoene suggesting that the activation of JNK is not necessary for ajoene-induced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced ajoene-induced cell death. The survival kinase Akt, in contrast, did not participate in ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, ajoene-mediated activation of JNK and p38 has no impact on ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards ajoene.