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Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is highly expressed in various kinds of tumors. In the present study, we investigated the cytotoxic mechanism of YM155, a unique small-molecule inhibitor of survivin, in human myelogenous leukemia cells. YM155 potently inhibited the cell growth of HL-60 and U937 cells with the half-maximal inhibitory concentration (IC50) value of 0.3 nM and 0.8 nM, respectively. YM155 significantly suppressed the levels of mRNA expression and protein of survivin in HL-60 and U937 cells. In addition, we also found that YM155 down-regulated the level of Mcl-1, another critical anti-apoptotic protein, in both HL-60 and U937 cells. Treatment of HL-60 and U937 cells with YM155 induced apoptosis concomitant with the activation of caspase-8 and caspase-3. Interestingly, we have found that caspase-8 inhibitor Z-IETD-FMK strongly inhibited YM155-induced apoptosis in HL-60 and U937 cells. When cells were pretreated with Z-IETD-FMK, the activation of caspase-3 was completely abolished, suggesting that caspase-8 may be involved in the activation of caspase-3 during YM155-induced apoptosis. We demonstrated for the first time that YM155 induces caspase-8 dependent apoptosis through downregulation of survivin and Mcl-1 in human leukemia cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Glutathione S-transferase mu (GSTM1) is mainly known as a detoxification enzyme but it has also been shown to be a negative regulator of apoptosis-related signaling cascades. Recently GSTM1 has been reported to be a significant risk factor for hematological relapse in childhood acute lymphoblastic leukemia, although the underlying mechanism remains largely unknown. Glucocorticoids play a crucial role in the treatment of childhood acute lymphoblastic leukemia, therefore we hypothesized that GSTM1 plays important roles in glucocorticoid-induced apoptotic pathways. To clarify the relationship between GSTM1 and drug resistance, GSTM1 was transfected into a T-acute lymphoblastic leukemia cell line, CCRF-CEM (CEM), and we established the GSTM1-expressing cell lines CEM/M1-4 and CEM/M1-9. Transduction of GSTM1 into CEM selectively decreased cellular sensitivity to dexamethasone in a manner that was independent of glutathione conjugation, but was due to apoptosis inhibition. Dexamethasone-induced p38-MAPK and Bim activation were concomitantly suppressed. Interestingly, nuclear factor kappa b (NF-kappaB) p50 activity was upregulated in GSTM1-expressing CEM. Inhibition of NF-kappaB by the pharmacological agent BAY11-7082 greatly enhanced the sensitivity of the GSTM1-expressing CEM to dexamethasone and was accompanied by an increase in Bim expression. Thus, we propose that GSTM1, a novel regulator of dexamethasone-induced apoptosis, causes dexamethasone resistance by suppression of Bim through dual mechanisms of both downregulation of p38-MAPK and upregulation of NF-kappaB p50.

We initially identified a nuclear protein, prothymosin-alpha1 (ProTalpha), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTalpha inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl(2)-family proteins. The apoptosis caused by ProTalpha was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTalpha-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCbeta(II) expression prevented this cell death mode switch. Collectively, these results suggest that ProTalpha is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors.

Topoisomerase I (Top1), an abundant nuclear enzyme expressed throughout the cell cycle, relaxes DNA supercoiling by forming transient covalent DNA cleavage complexes. We show here that staurosporine, a ubiquitous inducer of apoptosis in mammalian cells, stabilizes cellular Top1 cleavage complexes. These complexes are formed indirectly as staurosporine cannot induce Top1 cleavage complexes in normal DNA with recombinant Top1 or nuclear extract from normal cells. In treated cells, staurosporine produces oxidative DNA lesions and generates reactive oxygen species (ROS). Quenching of these ROS by the antioxidant N-acetyl-l-cysteine or inhibition of the mitochondrial dependent production of ROS by the caspase inhibitor benzyloxycarbonyl-VAD prevents staurosporine-induced Top1 cleavage complexes. Down-regulation of Top1 by small interfering RNA decreases staurosporine-induced apoptotic DNA fragmentation. We propose that Top1 cleavage complexes resulting from oxidative DNA lesions generated by ROS in staurosporine-treated cells contribute to the full apoptotic response.

Survivin, a new member of the inhibitor of apoptosis protein (IAP) family, has been reported to be expressed in many cancers but not in differentiated normal tissues. In the present study, we describe the identification of a novel alternatively spliced survivin transcript, designated as survivin-3B. It comprises 5 exons including novel exon 3B derived from a 165-bp long portion of intron 3. Acquisition of a new in-frame TGA stop codon within the novel exon 3B results in an open reading frame (ORF) of 363 nucleotides, predicting a truncated 120 amino acid protein. Expression of survivin-3B was detected in human colon and gastric adenocarcinoma cell lines as well as mononuclear cells prepared from patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Survivin-3B contains a single baculovirus IAP repeat (BIR), which is critical for apoptosis inhibition. However, it lacks a carboxyl-terminal coiled-coil domain, suggesting that survivin-3B may not be associated with G2/M phase. These data indicate that the function of survivin-3B may be different from that of regular survivin.

GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy)amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin, is a promising anticancer drug of podophyllotoxin class. The primary effect of GP7 is the anticancer activity on transplanted mouse tumors and cultured tumor cells. However, its molecular mechanism of action is still obscure. In this study, we investigated the activity of GP7 to induce apoptosis in human leukemia HL-60 and Jurkat cells. Apoptosis was determined by detection of DNA fragmentation in agarose gel electrophoresis. GP7 induced apoptotic DNA fragmentation of HL-60 and Jurkat cells in time- and dose-dependent manner. We further investigated the activity of caspase-3 in GP7-induced apoptotic DNA fragmentation of HL-60 and Jurkat cells. GP7 also induced time- and dose-dependent caspase-3 activation in both cell lines, and the kinetics of caspase-3 activation induced by GP7 was well correlated with that of apoptotic DNA fragmentation. To determine the role of caspase-3 in GP7-induced apoptotic DNA fragmentation, we examined the effect of specific caspase-3 inhibitor, Ac-DEVD-CHO, on GP7-induced apoptotic DNA fragmentation. Ac-DEVD-CHO prevented GP7-induced caspase-3 activation in both HL-60 and Jurkat cells, however, it only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. We then employed L-carnitine to investigate the role of caspase-3 in GP7-induced apoptotic DNA fragmentation. L-carnitine treatment prevented GP7-induced caspase-3 activation in both cell lines in a dose-dependent manner. Similar to Ac-DEVD-CHO, L-carnitine only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. These findings suggest that GP7 exerts an anti-leukemic effect by both caspase-3-dependent and -independent apoptotic signaling pathways.

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.

The cytolytic T lymphocyte protease granzyme A (GzmA) initiates a caspase-independent cell death pathway. Here we report that the rate-limiting enzyme of DNA base excision repair, apurinic endonuclease-1 (Ape1), which is also known as redox factor-1 (Ref-1), binds to GzmA and is contained in the SET complex, a macromolecular complex of 270-420 kDa that is associated with the endoplasmic reticulum and is targeted by GzmA during cell-mediated death. GzmA cleaves Ape1 after Lys31 and destroys its known oxidative repair functions. In so doing, GzmA may block cellular repair and force apoptosis. In support of this, cells with silenced Ape1 expression are more sensitive, whereas cells overexpressing noncleavable Ape1 are more resistant, to GzmA-mediated death.

Survivin is a member of the inhibitor of apoptosis protein (IAPs) family and considered to play a pivotal role in oncogenesis. We present the first report of survivin expression profile in myelodysplastic syndrome (MDS). Expression of survivin messenger RNA was evaluated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in patients with MDS and acute myeloid leukemia (AML). Eleven out of 12 patients with refractory anemia (RA) (91.6%), and all 3 patients with refractory anemia with excess blasts in transformation (RAEBt) (100%), were positive for survivin expression with the majority of cases showing abundant levels of the survivin transcript. On the other hand, expression of survivin was undetectable in the 4 patients with chronic myelomonocytic leukemia (CMMoL). The level and frequency of survivin expression in patients with refractory anemia were compared to those in patients with AML. Out of 12 patients with de novo AML, 5 patients (41.7%) showed detectable levels of survivin expression. Abundant survivin expression in RA was also confirmed by immunohistochemistry. In contrast, survivin was almost absent in two cases with aplastic anemia. We propose that high levels of survivin expression can serve as a reliable diagnostic marker of RA in MDS.

Mutations of the FMS-like tyrosine kinase 3 (FLT3) have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis. Thus, pharmacological inhibitors of FLT3 are of therapeutic interest for AML. Gö6976 is an indolocarbazole with a similar structural backbone to staurosporine. In the present study, we demonstrated that Gö6976 displays a potent inhibitory activity against recombinant FLT3 using an in vitro kinase assay, with an IC50 value of 0.7nM. Gö6976 markedly inhibited the proliferation of human leukemia cells having FLT3-ITD such as MV4-11 and MOLM13. We also observed that Gö6976 showed minimal toxicity for human normal CD34(+) cells. Gö6976 suppressed the phosphorylation of FLT3 and downstream signaling molecules such as STAT3/5, Erk1/2, and Akt in MV4-11 and MOLM13 cells. Interestingly, induction of apoptosis by Gö6976 was associated with rapid and pronounced down-regulation of the anti-apoptotic protein survivin and MCL-1. Suppression of survivin protein expression by Gö6976 was due to the inhibition of transcription via the suppression of STAT3/5. On the other hand, Gö6976 induced proteasome-mediated degradation of MCL-1. Previously described FLT3 inhibitors such as PKC412 are bound by the human plasma protein, α1-acid glycoprotein, resulting in diminished inhibitory activity against FLT3. In contrast, we found that Gö6976 potently inhibited phosphorylation of FLT3 and exerted cytotoxicity in the presence of human serum. In conclusion, Gö6976 is a potent FLT3 inhibitor that displays a significant antiproliferative activity against leukemia cells with FLT3-ITD through the profound down-regulation of survivin and MCL-1. [ABSTRACT FROM AUTHOR]