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.     

Role of oxidation-triggered activation of JNK and p38 MAPK in black tea polyphenols induced apoptotic death of A375 cells

Theaflavins (TF) and thearubigins (TR) are the major polyphenols of black tea. Our previous study revealed that TF- and TR-induced apoptosis of human malignant melanoma cells (A375) is executed via a mitochondria-mediated pathway. In our present study we observed the role of the three most important MAPK (ERK, JNK, and p38) in TF- and TR-induced apoptosis. TF and TR treatment of A375 cells led to sustained activation of JNK and p38 MAPK but not ERK, suggesting that JNK and p38 are the effector molecules in this polyphenol-induced cell death. This idea was further supported by subsequent studies in which JNK and p38 activation was inhibited by specific inhibitors. Significant inhibition was found in TF- and TR-treated A375 cell death pretreated with JNK- or p38-specific inhibitors only. Further, we have found that TF and TR treatment induces a time-dependent increase in intracellular reactive oxygen species generation in A375 cells. Interestingly, treatment with the antioxidant N -acetyl cystein inhibits TF- and TR-induced JNK and p38 activation as well as induction of cell death in A375 cells. We also provide evidence demonstrating the critical role of apoptosis signal-regulating kinase 1 in TF- and TR-induced apoptosis in A375 cells. Taken together our results strongly suggest that TF and TR induce apoptotic death of A375 cells through apoptosis signal-regulating kinase 1, MAPK kinase, and the JNK–p38 cascade, which is triggered by N -acetyl cystein intracellular oxidative stress. ( Cancer Sci  2009; 100: 1971–1978)     

ERK1/2 activation is required for resveratrol-induced apoptosis in MDA-MB-231 cells

Resveratrol (RSVL), a phytoalexin found in abundance in grapes and other grape-related products, has been shown to be antiproliferative and protective against various types of cancers, including breast cancer. However, the precise underlying mechanisms are not well understood. In this study, we show that treatment with RSVL induces growth inhibition and apoptosis in a highly invasive and metastatic breast cancer cell line MDA-MB-231. Cleavage of caspase-3 and PARP and fragmentation of DNA were observed following exposure to RSVL. Co-treatment with pan-caspase inhibitor completely prevents cell death induced by RSVL. We found that RSVL-induced apoptosis correlates with sustained activation of ERK1/2 and suppression of Bcl-2 expression. Inhibition of ERK1/2 activation by its specific inhibitor or small interfering RNA reverses the effect of RSVL on Bcl-2 suppression and inhibits apoptosis, while overexpression of MEK1, which is directly upstream of both ERK1 and ERK2, enhances apoptosis induced by RSVL. Moreover, ERK1/2 was found to act upstream of caspase-3 to induce apoptosis, while it was not directly involved in caspase-3 cleavage. The other closely related MAPK members, p38 and JNK are not involved in apoptosis induced by RSVL in MDA-MB-231 cells. These results suggest that activation of ERK1/2 is required for RSVL-induced apoptosis in MDA-MB-231 cells.     

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   

Guggulsterone-induced apoptosis in human prostate cancer cells is caused by reactive oxygen intermediate dependent activation of c-Jun NH2-terminal kinase

Guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, causes apoptosis in cancer cells but the sequence of events leading to cell death is poorly understood. We now show that guggulsterone-induced cell death in human prostate cancer cells is caused by reactive oxygen intermediate (ROI)-dependent activation of c-Jun NH(2)-terminal kinase (JNK). Exposure of PC-3 and LNCaP cells to apoptosis inducing concentrations of guggulsterone resulted in activation of JNK and p38 mitogen-activated protein kinase (p38 MAPK) in both cell lines and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LNCaP cells. The guggulsterone-induced apoptosis in PC-3/LNCaP cells was partially but statistically significantly attenuated by pharmacologic inhibition (SP600125) as well as genetic suppression of JNK activation. On the other hand, pharmacologic inhibition of p38 MAPK activation in PC-3 or LNCaP cells (SB202190) and ERK1/2 activation in LNCaP cells (PD98059) did not protect against guggulsterone-induced cell death. The guggulsterone treatment caused generation of ROI in prostate cancer cells but not in a normal prostate epithelial cell line (PrEC), which was also resistant to guggulsterone-mediated JNK activation. The guggulsterone-induced JNK activation as well as cell death in prostate cancer cells was significantly attenuated by overexpression of catalase and superoxide dismutase. In addition, guggulsterone treatment resulted in a decrease in protein level and promoter activity of androgen receptor in LNCaP cells. In conclusion, the present study reveals that the guggulsterone-induced cell death in human prostate cancer cells is regulated by ROI-dependent activation of JNK and guggulsterone inhibits promoter activity of androgen receptor.     

Benzyl isothiocyanate (BITC) induces apoptosis in ovarian cancer cells in vitro

Advanced ovarian cancer (OC) is not curable by surgery alone and chemotherapy is essential for its treatment. Isothiocyanates have been shown to inhibit carcinogen-induced tumorigenesis in animal models, yet no efforts have been made to determine their therapeutic potential in OC. In the present study, we investigated the mechanism of the anti-proliferative and apoptotic activity of benzyl isothiocyanate (BITC) in OC. BITC inhibited the proliferation of OC cells and induced apoptosis in OC cells. Apoptosis was induced by a strong activation of caspase-3 and -9, and cleavage of PARP-1. However, caspase-8 was not activated by BITC. Cytotoxic effects of BITC were reversed by the inhibition caspase-3 and -9 specific inhibitors. BITC showed a concentration dependent decrease in the levels of Bcl-2 with a concomitant increase in Bax levels. In addition, BITC activated proapoptotic signaling by phosphorylation JNK1/2 and p38 while simultaneously inhibiting survival signaling mediated by ERK1/2 and Akt phosphorylation in a dose-dependent manner. While JNK inhibitor SP600125 and p38 inhibitor SB203580, abolished the cytotoxic effect of BITC, MEK inhibitor, PD98059 and PI3 kinase inhibitor, LY294002 failed to show such reversal indicating a critical role played by JNK1/2 and p38 signaling in apoptosis induced by BITC. In summary, our studies demonstrate that BITC inhibits proliferation of OC cells and induces apoptosis via caspase-9 and -3 pathways. BITC inhibits ERK1/2 and Akt survival signaling while simultaneously activating pro-apoptotic p38 and JNK1/2. Therefore, BITC can be potentially developed as a therapeutic agent to treat OC.     

The inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in chronic myelogenous leukemic K562 cells

In this study, the downstream signaling of Bcr-Abl tyrosine kinase responsible for apoptosis resistance was investigated. DNA fragmentation, a hallmark of apoptosis, was observed after 2 days of herbimycin A treatment with a peak on 3 day. During the apoptosis induced by the treatment of herbimycin A, stress-activated protein kinase (SAPK) and p38 kinase were activated time- and dose-dependently, while extracellular signal-regulated kinase (ERK) was inhibited. However, apoptosis was induced by the treatment of PD98059, a specific inhibitor of MEK (MAPK or ERK kinase), not by the treatment of sorbitol, a strong activator of SAPK and p38 kinase. Although K562 cells were very resistant to sorbitol-induced apoptosis, DNA fragmentation was induced rapidly in Jurkat, HL-60 and U937 cells after exposure to sorbitol, despite that these apoptosis-sensitive cells have similar or lower activities of JNK/SAPK and p38 kinase compared with K562 cells after treatment of sorbitol. K562 cells had a much higher basal activity of ERK/MAPK than other apoptosis-sensitive cell lines, which were very susceptible to apoptosis induced by low dose of PD98059 compared with K562 cells. In HL-60 cells, sorbitol-induced apoptosis was prevented by the treatment of phorbol myristate 13-acetate (PMA), which activates the ERK/MAPK pathway, and this was blocked by PD98059. From these results, it could be suggested that the inhibition of ERK/MAPK not the activation of JNK/SAPK is primarily required to induce apoptosis in K562 cells.     

Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis through downregulation of ERK and Akt in lung adenocarcinoma A549 cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including A549 lung adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of sulforaphane (SFN) sensitizes TRAIL-resistant A549 cells to TRAIL-mediated apoptosis. Combined treatment with SFN and TRAIL induced chromatin condensation, DNA fragmentation, annexin V staining and sub-G(1) phase DNA content. These indicators of apoptosis correlate with the induction of caspase-3 activity that results in the cleavage of poly(ADP-ribose) polymerase and the release of lactate dehydrogenase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the observed cytotoxic effect. Combined treatment also triggered the activation of p38 MAPK and JNK, and downregulation of ERK and Akt. Inhibitors of ERK (PD98059) or Akt (LY294002), but not p38 MAPK, resulted in significantly decreased cell viability. Although the activation of JNK was increased in response to combined treatment, inhibition of the JNK pathway significantly attenuated cell viability. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined SFN and TRAIL in human lung adenocarcinoma A549 cells through downregulation of ERK and Akt.     

Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells

The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in apoptosis induction by phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived cancer chemopreventive agent, with DU145 and LNCaP human prostate cancer cells as a model. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. Exposure of DU145 and LNCaP cells to growth suppressive concentrations of PEITC resulted in activation of ERK1/2 and JNKs, but not p38 MAPK, in both cell lines. In DU145 cells, the apoptosis induction by PEITC was statistically significantly attenuated by pharmacological inhibition of JNKs with SP600125. Adenovirus-mediated overexpression of Flag-tagged JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), an inhibitor of JNK, also inhibited PEITC-induced apoptosis in DU145 cells. On the other hand, inhibition of ERK1/2 activation with MEK1 inhibitor PD98059 failed to offer protection against PEITC-induced apoptosis in DU145 cells. In LNCaP cells, the PEITC-induced cell death was not affected by either pretreatment with PD98059 or SP600125 or overexpression of JBD of JIP-1. These results indicate that involvement of MAPKs in apoptosis induction by PEITC in human prostate cancer cells is cell line-specific.     

Stomatin‐like protein 2 inhibits cisplatin‐induced apoptosis through MEK/ERK signaling and the mitochondrial apoptosis pathway in cervical cancer cells

Stomatin‐like protein 2 (STOML2 or SLP‐2) is an oncogenic anti‐apoptotic protein that is upregulated in several types of cancer, including cervical cancer. However, the mechanisms responsible for the SLP‐2 anti‐apoptotic function remain poorly understood. Here, we show that siRNA‐mediated SLP‐2 suppression decreases growth of human cervical cancer HELA and SIHA cells, and increases cisplatin‐induced apoptosis through activation of MEK/ERK signaling and suppression of the mitochondrial pathway. The inhibition of the mitochondrial pathway is mediated by increasing the mitochondrial Ca²⁺ concentration and mitochondrial membrane potential, thereby downregulating p‐MEK and p‐ERK levels, upregulating the Bax/Bcl‐2 ratio, increasing cytochrome C release from mitochondria into the cytosol, and upregulating levels of cleaved‐caspase 9, cleaved‐caspase 3, and cleaved poly ADP‐ribose polymerase (PARP). Overexpression of SLP‐2 using adenovirus‐STOML2 has the opposite effect: it upregulates p‐MEK and p‐ERK and downregulates the Bax/Bcl‐2 ratio and levels of cleaved‐caspase 9 to caspase 9, cleaved‐caspase 3 to caspase 3, and cleaved‐PARP to PARP in cisplatin‐treated cells. These data show that SLP‐2 inhibits cisplatin‐induced apoptosis by activating the MEK/ERK signaling and inhibiting the mitochondrial apoptosis pathway in cervical cancer cells.     

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.     

Involvement of JNK-mediated pathway in EGF-mediated protection against paclitaxel-induced apoptosis in SiHa human cervical cancer cells.

We investigated the signalling pathways by which epidermal growth factor (EGF) modulates paclitaxel-induced apoptosis in SiHa human cervical cancer cells. SiHa cells exposed to paclitaxel underwent apoptosis, which was strongly inhibited by EGF. This inhibition of apoptosis by EGF was not altered by pharmacological blockade of phosphatidylinositol 3'-OH kinase (PI-3K) with the PI-3K specific inhibitor LY294002 or blockade of the mitogen-activated protein kinase (MAPK) kinase (MEK) with the MEK specific inhibitor PD98059, or by transfection of the cells with PI-3K or MEK dominant-negative expression vectors. EGF did not stimulate PI-3K/Akt, MEK/MAPK, or p38 MAPK activity in SiHa cells but did transiently activate the c-Jun NH2-terminal kinase (JNK). Co-exposure of SiHa cells to SB202190 at concentrations that inhibit JNK abolished the protective effect of EGF on SiHa cells against paclitaxel-induced apoptosis. Our findings indicate that the JNK signaling pathway plays an important role in EGF-mediated protection from paclitaxel-induced apoptosis in SiHa cells.     

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.     

Involvement of ERK1/2 and p38 MAP kinase in doxorubicin-induced uPA expression in human RC-K8 lymphoma and NCI-H69 small cell lung carcinoma cells

We previously demonstrated the doxorubicin-induced urokinase-type plasminogen activator (uPA) expression in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Western blotting analysis revealed phosphorylation/activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase and stress-activated protein kinase/c-jun N-terminal protein kinase (SAPK/JNK) in doxorubicin-treated RC-K8 and H69 cells, and, therefore, we attempted to identify the MAP kinases implicated in doxorubicin-induced uPA expression by the use of their specific inhibitors. U0126, SB202190 and JNKI-1, inhibitors for MAPK kinase, (MEK) 1/2, p38 MAP kinase and SAPK/JNK, respectively, specifically and clearly inhibited their corresponding kinases. U0126 and SB202190, but not JNKI-1, almost completely inhibited the doxorubicin-induced uPA expression in both RC-K8 and H69 cells. However, U0126 rather enhanced the doxorubicin-induced activation of caspase-3 and poly ADP-ribose polymerase (PARP), and U0126 itself activated caspase-3 and PARP. Interestingly, JNKI-1 inhibited the doxorubicin-induced activation of caspase-3 and PARP. Therefore, doxorubicin treatment activates the above three kinases, but different MAP kinase signaling is responsible in the doxorubicin-induced caspase activation and expression of uPA. Thus, we could possibly manipulate the direction of doxorubicin-induced MAP kinase activation and the effects of doxorubicin on the tumor cell biology by the use of MAP kinase inhibitors.     

Characterization of cell death induced by ethacrynic acid in a human colon cancer cell line DLD-1 and suppression by N-acetyl-L-cysteine

Since ethacrynic acid (EA), an SH modifier as well as glutathione S-transferase (GST) inhibitor, has been suggested to induce apoptosis in some cell lines, its effects on a human colon cancer cell line DLD-1 were examined. EA enhanced cell proliferation at 20–40 μ M , while it caused cell death at 60–100 μ M. Caspase inhibitors did not block cell death and DNA ladder formation was not detected. Poly(ADP-ribose) polymerase, however, was cleaved into an 82-kDa fragment, different from an 85-kDa fragment that is specific for apoptosisis. The 82-kDa fragment was not recognized by antibody against PARP fragment cleaved by caspase 3. N -Acetyl- l -cysteine (NAC) completely inhibited EA-induced cell death, but 3(2)- t -butyl-4-hydroxyanisole or pyrrolidinedithiocarbamate ammonium salt did not. Glutathione (GSH) levels were dose-dependently increased in cells treated with EA and this increase was hardly affected by NAC addition. Mitogen-activated protein kinase (MAPK) kinase (MEK) 1, extracellular signal-regulated kinase (ERK) 1 and GST P1-1 were increased in cells treated with 25–75 μ M EA, while c-Jun N-terminal kinase (JNK) 1 and p38 MAPK were markedly decreased by 100 μ M EA. NAC repressed EA-induced alterations in these MAPKs and GST P1-1. p38 MAPK inhibitors, SB203580 and FR167653, dose-dependently enhanced EA-induced cell death. An MEK inhibitor, U0126, did not affect EA-induced cell death. These studies revealed that EA induced cell death concomitantly with a novel PARP fragmentation, but without DNA fragmentation. p38 MAPK was suggested to play an inhibitory role in EA-induced cell death.     

Aplidin induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C delta

Aplidin, a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-delta) mediates the cytotoxic effect of Aplidin and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-delta show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-delta thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process.     

2-Chloro-2'-deoxyadenosine-induced apoptosis in T leukemia cells is mediated via a caspase-3-dependent mitochondrial feedback amplification loop

2-Chloro-2'-deoxyadenosine (CdA; cladribine) is a chemotherapeutic agent used in the treatment of certain leukemias. However, the signalling events that govern CdA-mediated cytotoxicity in leukemia cells remain unclear. We show here that CdA treatment caused Jurkat human T leukemia cells to die via apoptosis in a dose- and time-dependent fashion. Bcl-2 overexpression protected Jurkat T leukemia cells from CdA-induced apoptosis and loss of mitochondrial transmembrane potential (Delta Psi m). Furthermore, mitochondria that were isolated from Jurkat T leukemia cells and then exposed to CdA showed a loss of Delta Psi m, indicating that CdA directly compromised outer mitochondrial membrane integrity. CdA treatment of Jurkat T leukemia cells resulted in the activation of caspase-3, -8, and -9, while inhibition of these caspases prevented the CdA-induced loss of Delta Psi m, as well as DNA fragmentation. In addition, caspase-3 inhibition prevented caspase-8 activation while caspase-8 inhibition prevented caspase-9 activation. Death receptor signalling was not involved in CdA-induced apoptosis since cytotoxicity was not affected by FADD-deficiency or antibody neutralization of either Fas ligand or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Taken together, these data suggested that CdA-induced apoptosis in Jurkat T leukemia cells was mediated via a caspase-3-dependent mitochondrial feedback amplification loop. CdA treatment also increased p38 mitogen-activated protein (MAPK) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in Jurkat T leukemia cells. Although ERK1/2 inhibition did not affect CdA-mediated cytotoxicity, inhibition of p38 MAPK had an enhancing effect, which suggested a cytoprotective function for p38 MAPK. Agents that inhibit p38 MAPK might therefore increase the effectiveness of CdA-based chemotherapy.