Adaphostin

p210bcr/abl tyrosine kinase inhibitor (IC50 = 14 μM). Induces apoptosis in T-lymphoblastic human leukemia cell lines (IC50 values range from 16.8 to 216.3 nM) in vitro. Displays selectivity for chronic myelogenous leukemia (CML) myeloid progenitors in vit

Adaphostin toxicity in a sensitive non-small cell lung cancer model is mediated through Nrf2 signaling and heme oxygenase 1.[Pubmed:20618971]

J Exp Clin Cancer Res. 2010 Jul 9;29:91.

BACKGROUND: Preclinical toxicity of Adaphostin has been related to oxidative stress. This study investigated the regulatory mechanism underlying Adaphostin induction of heme oxygenase 1 (HMOX1) which plays a significant role in modulation of drug-induced toxicity in the non-small cell lung cancer cell line model, NCI-H522. METHODS: The transcriptional response of NCI-H522 to Adaphostin prominently involved oxidative stress genes, particularly HMOX1. Reactive oxygen species (ROS) involvement was additionally established by generation of ROS prior to modulation of Adaphostin-toxicity with antioxidants. To identify up-stream regulatory elements of HMOX1, immunofluorescence was used to evaluate nuclear translocation of the transcription factor, NF-E2-related factor 2 (Nrf2), in the presence of Adaphostin. The PI3-kinase inhibitor, wortmannin, was employed as a pharmacological inhibitor of this process. RESULTS: Generation of ROS provided a substantial foundation for the sensitivity of NCI-H522 to Adaphostin. However, in contrast to leukemia cell lines, transcriptional response to oxidative stress was associated with induction of HMOX1, which was dependent on nuclear translocation of the transcription factor, Nrf2. Pretreatment of cells with wortmannin inhibited translocation of Nrf2 and induction of HMOX1. Wortmannin pretreatment was also able to diminish Adaphostin induction of HMOX1, and as a consequence, enhance the toxicity of Adaphostin to NCI-H522. CONCLUSIONS: Adaphostin-induced oxidative stress in NCI-H522 was mediated through nuclear translocation of Nrf2 leading to upregulation of HMOX1. Inhibition of Nrf2 translocation by wortmannin inhibited this cytoprotective response, and enhanced the toxicity of Adaphostin, suggesting that inhibitors of the PI3K pathway, such as wortmannin, might augment the antiproliferative effects of Adaphostin in solid tumors that depend on the Nrf2/ARE pathway for protection against oxidative stress.

Proteomic analysis identifies oxidative stress induction by adaphostin.[Pubmed:17575232]

Clin Cancer Res. 2007 Jun 15;13(12):3667-81.

PURPOSE: Activities distinct from inhibition of Bcr/abl have led to Adaphostin (NSC 680410) being described as "a drug in search of a mechanism." In this study, proteomic analysis of Adaphostin-treated myeloid leukemia cell lines was used to further elucidate a mechanism of action. EXPERIMENTAL DESIGN: HL60 and K562 cells treated with Adaphostin for 6, 12, or 24 h were analyzed using two-dimensional PAGE. Differentially expressed spots were excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. The contribution of the redox-active hydroquinone group in Adaphostin was also examined by carrying out proteomic analysis of HL60 cells treated with a simple hydroquinone (1,4-dihydroxybenzene) or H(2)O(2). RESULTS: Analysis of Adaphostin-treated cells identified 49 differentially expressed proteins, the majority being implicated in the response to oxidative stress (e.g., CALM, ERP29, GSTP1, PDIA1) or induction of apoptosis (e.g., LAMA, FLNA, TPR, GDIS). Interestingly, modulation of these proteins was almost fully prevented by inclusion of an antioxidant, N-acetylcysteine. Validation of the proteomic data confirmed GSTP1 as an Adaphostin resistance gene. Subsequent analysis of HL60 cells treated with 1,4-dihydroxybenzene or H(2)O(2) showed similar increases in intracellular peroxides and an almost identical proteomic profiles to that of Adaphostin treatment. Western blotting of a panel of cell lines identified Cu/Zn superoxide dismutase (SOD) as correlating with Adaphostin resistance. The role of SOD as a second Adaphostin resistance gene was confirmed by demonstrating that inhibition of SOD using diethyldithiocarbamate increased Adaphostin sensitivity, whereas transfection of SOD I attenuated toxicity. Importantly, treatment with 1,4-dihydroxybenzene or H(2)O(2) replicated Adaphostin-induced Bcr/abl polypeptide degradation, suggesting that kinase inhibition is a ROS-dependent phenomenon. CONCLUSION: Adaphostin should be classified as a redox-active-substituted dihydroquinone.

Naja nigricollis CMS-9 enhances the mitochondria-mediated death pathway in adaphostin-treated human leukaemia U937 cells.[Pubmed:21824174]

Clin Exp Pharmacol Physiol. 2011 Nov;38(11):755-63.

1. The aim of the present study was to explore the effect of the Naja nigricollis phospholipase A(2) CMS-9 on Adaphostin-induced death of human leukaemia U937 cells. 2. Leukaemia U937 cells (Bcr/Abl-negative cells) were treated with Adaphostin (0-10 mumol/L) and CMS-9 (0-1 mumol/L). The effects of CMS-9, Adaphostin and their combination on cell viability, the generation reactive oxygen species (ROS), [Ca(2+) ](i) , p38 mitogen-activated protein kinase (MAPK) activation, Akt and extracellular signal-regulated kinase (ERK) inactivation, mitochondrial membrane potential (DeltaPsi(m) ) and Bcl-2 family proteins were analysed. 3. Both Adaphostin and CMS-9 induced U937 cell apoptosis, characterized by dissipation of DeltaPsi(m) and ROS generation. Combined treatment further increased DeltaPsi(m) loss and reduced the viability of Adaphostin-treated cells. Unlike in CMS-9-treated cells, in Adaphostin-treated cells ROS-induced increases in [Ca(2+) ](i) were observed. CMS-9-induced ROS generation resulted in p38 MAPK activation, whereas Adaphostin treatment elicited ROS/Ca(2+) -mediated inactivation of Akt and ERK. Moreover, Akt was found to be involved in ERK phosphorylation. Suppression of p38 MAPK activation blocked CMS-9-induced DeltaPsi(m) loss and Bcl-xL downregulation. Overexpression of constitutively active Akt and mitogen-activated protein kinase kinase (MEK) 1 rescued Adaphostin-induced DeltaPsi(m) loss and Bcl-2 downregulation. Similarly, CMS-9 augmented Adaphostin toxicity in human leukaemia K562 cells via increased mitochondrial alterations. 4. The results suggest that two distinct pathways mediate Adaphostin- and CMS-9-induced mitochondrial damage (i.e. the ROS-Ca(2+) -Akt-ERK and ROS-p38 MAPK pathways, respectively). These distinct pathway explain the augmentation by CMS-9 of DeltaPsi(m) loss and apoptosis in Adaphostin-treated U937 cells.

Adaphostin promotes caffeine-evoked autocrine Fas-mediated death pathway activation in Bcr/Abl-positive leukaemia cells.[Pubmed:21745187]

Biochem J. 2011 Nov 1;439(3):453-67.

The present study was conducted to verify whether caffeine is beneficial for improving leukaemia therapy. Co-treatment with Adaphostin (a Bcr/Abl inhibitor) was found to potentiate caffeine-induced Fas/FasL up-regulation. Although Adaphostin did not elicit ASK1 (apoptosis signal-regulating kinase 1)-mediated phosphorylation of p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase), co-treatment with Adaphostin notably increased p38 MAPK/JNK activation in caffeine-treated cells. Suppression of p38 MAPK and JNK abrogated Fas/FasL up-regulation in caffeine- and caffeine/Adaphostin-treated cells. Compared with caffeine, Adaphostin markedly suppressed Akt/ERK (extracellular-signal-regulated kinase)-mediated MKP-1 (MAPK phosphatase 1) protein expression in K562 cells. MKP-1 down-regulation eventually elucidated the enhanced effect of Adaphostin on p38 MAPK/JNK activation and subsequent Fas/FasL up-regulation in caffeine-treated cells. Knockdown of p38alpha MAPK and JNK1, ATF-2 (activating transcription factor 2) and c-Jun by siRNA (small interfering RNA) proved that p38alpha MAPK/ATF-2 and JNK1/c-Jun pathways were responsible for caffeine-evoked Fas/FasL up-regulation. Moreover, Ca2+ and ROS (reactive oxygen species) were demonstrated to be responsible for ASK1 activation and Akt/ERK inactivation respectively in caffeine- and caffeine/Adaphostin-treated cells. Likewise, Adaphostin functionally enhanced caffeine-induced Fas/FasL up-regulation in leukaemia cells that expressed Bcr/Abl. Taken together, the results of the present study suggest a therapeutic strategy in improving the efficacy of Adaphostin via Fas-mediated death pathway activation in Bcr/Abl-positive leukaemia.

In vitro and in vivo evaluations of the tyrosine kinase inhibitor NSC 680410 against human leukemia and glioblastoma cell lines.[Pubmed:12451475]

Cancer Chemother Pharmacol. 2002 Dec;50(6):479-89.

PURPOSE: NSC 680410, the novel adamantyl ester of AG957, an inhibitor of the p210bcr/abl tyrosine kinase (CML, Ph(+)) and possibly other kinases, was tested for antitumor activity in ten human leukemia and human glioblastoma cell lines. METHODS: CEM/0, seven ara-C- and/or ASNase-resistant clones, Jurkat/0, the myelomonocytic line U937 and U87 MG glioblastoma cell lines were used for these studies. The drug-resistant leukemic clones lack p53, express bcl-2 and VEGF-R1, and thus are refractory to apoptosis. Since tyrosine kinases drive many proliferative pathways and these activities are increased in many leukemic cells, we hypothesized that NSC 680410 may induce cytotoxicity in drug-resistant leukemia clones, independently of p210bcr/abl expression. RESULTS: NSC 680410 exhibited significant antileukemic activity in CEM/0, Jurkat E6-1, and in the drug-resistant leukemic cell lines. The IC(50) values in nine leukemia lines ranged from 17 to 216 n M. Western blot analyses after NSC 680410 treatment demonstrated caspase-3 cleavage and ELISAs showed a fivefold upregulation of its activity in cellular extracts. In addition, U87 MG human glioblastoma cells, which express VEGF-R1, were treated with the Flt-1/Fc chimera, a specific inhibitor of VEGF, and showed 30-43% cell kill in the MTT assay. Furthermore, the combination of NSC 680410 plus Flt-1/Fc chimera demonstrated an eightfold synergism against U87 MG cells in vitro. To verify this observation in vivo, athymic mice were inoculated orthotopically into the caudate putamen with 10(6) U87 MG cells. On day 3, five mice per group were treated i.p. with either 8.3 mg/kg NSC 680410 daily for three doses per week for 4 weeks alone or in combination with one dose of Flt-1/Fc chimera 100 mg/kg subcutaneously. Treatment with NSC 680410 alone produced no weight changes and increased the median survival to 133%, whereas treatment with NSC680410 plus Flt-1/Fc chimera increased survival to 142% over control. Control animals had large intra- and extracranial tumors while the NSC 680410-treated mice had small, only intracranial tumors with necrotic centers. The combination treatment resulted in small residual tumors around the needle track, indicating significant inhibition of tumor growth. CONCLUSIONS: These studies demonstrate that the tyrosine kinase inhibitor NSC 680410 has significant antileukemic activity in p53-null, drug-resistant human leukemia cell lines, as well as significant antitumor activity in combination with Flt-1/Fc chimera against U87 MG glioblastoma brain tumors implanted in situ in athymic mice.

Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro.[Pubmed:11781252]

Blood. 2002 Jan 15;99(2):664-71.

The adenosine triphosphate binding-site-directed agent STI571 and the tyrphostin Adaphostin are undergoing evaluation as bcr/abl kinase inhibitors. The current study compared the effects of these agents on the survival of K562 cells, bcr/abl-transduced FDC-P1 cells, and myeloid progenitors from patients with chronic myelogenous leukemia (CML) compared with healthy donors. Treatment of K562 cells with 10 microM Adaphostin resulted in decreased p210(bcr/abl) polypeptide levels in the first 6 hours, followed by caspase activation and accumulation of apoptotic cells in less than 12 hours. By 24 hours, 90% of the cells were apoptotic and unable to form colonies. In contrast, 20 microM STI571 caused rapid inhibition of bcr/abl autophosphorylation without p210(bcr/abl) degradation. Although this was followed by the inhibition of Stat5 phosphorylation and the down-regulation of Bcl-x(L) and Mcl-1, only 7% +/- 3% and 25% +/- 9% of cells were apoptotic at 16 and 24 hours, respectively. Instead, the cytotoxic effects of STI571 became more pronounced with prolonged exposure, with IC90 values greater than 20 microM and 1.0 +/- 0.6 microM after 24 and 48 hours, respectively. Consistent with these results, 24-hour Adaphostin exposure inhibited CML granulocyte colony-forming units (CFU-G) (median IC50, 12 microM) but not normal CFU-G (median IC50, greater than 20 microM), whereas 24-hour STI571 treatment had no effect on CML or normal CFU-G. Additional experiments revealed that STI571-resistant K562 cells remained sensitive to Adaphostin. Moreover, the combination of STI571 + Adaphostin induced more cytotoxicity in K562 cells and in CML CFU-G than either agent alone did. Collectively, these results identify Adaphostin as a mechanistically distinct CML-selective agent that retains activity in STI571-resistant cell lines.

Effects of the bcr/abl kinase inhibitors AG957 and NSC 680410 on chronic myelogenous leukemia cells in vitro.[Pubmed:10656455]

Clin Cancer Res. 2000 Jan;6(1):237-49.

The tyrphostin AG957 (NSC 654705) inhibits p210bcr/abl, the transforming kinase responsible for most cases of chronic myelogenous leukemia (CML). The present studies were performed to determine the fate of AG957-treated cells and assess the selectivity of AG957 for CML myeloid progenitors. When K562 cells (derived from a patient with blast crisis CML) were treated with AG957, dose- and time-dependent p210bc/abl down-regulation was followed by mitochondrial release of cytochrome c, activation of caspase-9 and caspase-3, and apoptotic morphological changes. These apoptotic changes were inhibited by transfection with cDNA encoding dominant negative caspase-9 but not dominant-negative FADD or blocking anti-Fas antibodies. In additional experiments, a 24-h AG957 exposure caused dose-dependent inhibition of K562 colony formation in soft agar. To extend these studies to clinical samples of CML, peripheral blood mononuclear cells from 10 chronic phase CML patients and normal controls were assayed for the growth of hematopoietic colonies in vitro in the presence of increasing concentrations of AG957. These assays demonstrated selectivity of AG957 for CML progenitors, with median IC50s (CML versus normal) of 7.3 versus >20 microM AG957 in granulocyte colony-forming cells (P < 0.001), 5.3 versus >20 microM in granulocyte/macrophage colony-forming cells (P < 0.05), and 15.5 versus > 20 microM in erythroid colony-forming cells (P > 0.05). The adamantyl ester of AG957 (NSC 680410) down-regulated p210bcr/abl in K562 cells and inhibited granulocyte colony formation in CML specimens at lower concentrations without enhanced toxicity in normal progenitors. These observations not only demonstrate that AG957-induced p210bcr/abl down-regulation is followed by activation of the cytochrome c/Apaf-1/caspase-9 pathway but also indicate that this class of kinase inhibitor exhibits selectivity worthy of further evaluation.