The potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P), is produced by 2 sphingosine kinase isoenzymes, SphK1 and SphK2. Expression of SphK1 is up-regulated in cancers, including ...leukemia, and associated with cancer progression. A screen of sphingosine analogs identified (2R,3S,4E)-N-methyl-5-(4′-pentylphenyl)-2-aminopent-4-ene-1,3-diol, designated SK1-I (BML-258), as a potent, water-soluble, isoenzyme-specific inhibitor of SphK1. In contrast to pan-SphK inhibitors, SK1-I did not inhibit SphK2, PKC, or numerous other protein kinases. SK1-I decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of Bcl-2. Lethality of SK1-I was reversed by caspase inhibitors and by expression of Bcl-2. SK1-I not only decreased S1P levels but concomitantly increased levels of its proapoptotic precursor ceramide. Conversely, S1P protected against SK1-I–induced apoptosis. SK1-I also induced multiple perturbations in activation of signaling and survival-related proteins, including diminished phosphorylation of ERK1/2 and Akt. Expression of constitutively active Akt protected against SK1-I–induced apoptosis. Notably, SK1-I potently induced apoptosis in leukemic blasts isolated from patients with acute myelogenous leukemia but was relatively sparing of normal peripheral blood mononuclear leukocytes. Moreover, SK1-I markedly reduced growth of AML xenograft tumors. Our results suggest that specific inhibitors of SphK1 warrant attention as potential additions to the therapeutic armamentarium in leukemia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Sphingosine-1-phosphate is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 ...(SphK1), one of the two isoenzymes that produce sphingosine-1-phosphate, is up-regulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and nonestablished human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of extracellular signal-regulated kinase 1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the c-Jun-NH(2)-kinase pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced the tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization, and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease.
Interactions between the multikinase inhibitor sorafenib and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were examined in malignant hematopoietic cells. Pretreatment (24 h) of ...U937 leukemia cells with 7.5 micromol/L sorafenib dramatically increased apoptosis induced by sublethal concentrations of TRAIL/Apo2L (75 ng/mL). Similar interactions were observed in Raji, Jurkat, Karpas, K562, U266 cells, primary acute myelogenous leukemia blasts, but not in normal CD34+ bone marrow cells. Sorafenib/TRAIL-induced cell death was accompanied by mitochondrial injury and release of cytochrome c, Smac, and AIF into the cytosol and caspase-9, caspase-3, caspase-7, and caspase-8 activation. Sorafenib pretreatment down-regulated Bcl-xL and abrogated Mcl-1 expression, whereas addition of TRAIL sharply increased Bid activation, conformational change of Bak (ccBak) and Bax (ccBax), and Bax translocation. Ectopic Mcl-1 expression significantly attenuated sorafenib/TRAIL-mediated lethality and dramatically reduced ccBak while minimally affecting levels of ccBax. Similarly, inhibition of the receptor-mediated apoptotic cascade with a caspase-8 dominant-negative mutant significantly blocked sorafenib/TRAIL-induced lethality but not Mcl-1 down-regulation or Bak/Bax conformational change, indicating that TRAIL-mediated receptor pathway activation is required for maximal lethality. Sorafenib/TRAIL did not increase expression of DR4/DR5, or recruitment of procaspase-8 or FADD to the death-inducing signaling complex (DISC), but strikingly increased DISC-associated procaspase-8 activation. Sorafenib also down-regulated cFLIP(L), most likely through a translational mechanism, in association with diminished eIF4E phosphorylation, whereas ectopic expression of cFLIP(L) significantly reduced sorafenib/TRAIL lethality. Together, these results suggest that in human leukemia cells, sorafenib potentiates TRAIL-induced lethality by down-regulating Mcl-1 and cFLIP(L), events that cooperate to engage the intrinsic and extrinsic apoptotic cascades, culminating in pronounced mitochondrial injury and apoptosis.
Periostin is a modular glycoprotein frequently observed to be a major constituent of the extracellular milieu of mass-forming intrahepatic cholangiocarcinoma and other desmoplastic malignant tumors. ...In intrahepatic cholangiocarcinoma, as well as in desmoplastic pancreatic ductal adenocarcinoma, periostin is overexpressed and hypersecreted in large part, if not exclusively, by cancer-associated fibroblasts within the tumor stroma. Through its interaction with specific components of the extracellular tumor matrix, particularly collagen type I and tenascin-C, and with cell surface receptors, notably integrins leading to activation of the Akt and FAK signaling pathways, this TGF-β family-inducible matricellular protein appears to be functioning as a key extracellular matrix molecule regulating such critically important and diverse malignant tumor behaviors as tumor fibrogenesis and desmoplasia, invasive malignant cell growth, chemoresistance, and metastatic colonization. This review will discuss current evidence and basic molecular mechanisms implicating periostin as a mediator of intrahepatic cholangiocarcinoma invasive growth. In addition, its significance as a potential prognostic biomarker for intrahepatic cholangiocarcinoma patients, as well as future possibilities and challenges as a molecular target for cholangiocarcinoma therapy and/or prevention, will be critically evaluated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Calcineurin inhibitors are highly efficacious immunosuppressive agents used in pediatric kidney transplantation. However, calcineurin inhibitor nephrotoxicity (CNIT) has been associated with the ...development of chronic renal allograft dysfunction and decreased graft survival. This study evaluated 37 formalin-fixed paraffin-embedded biopsies from pediatric kidney transplant recipients using gene expression profiling. Normal allograft samples (
= 12) served as negative controls and were compared to biopsies exhibiting CNIT (
= 11). The remaining samples served as positive controls to validate CNIT marker specificity and were characterized by other common causes of graft failure such as acute rejection (
= 7) and interstitial fibrosis/tubular atrophy (
= 7). MiRNA profiles served as the platform for data integration. Oxidative phosphorylation and mitochondrial dysfunction were the top molecular pathways associated with overexpressed genes in CNIT samples. Decreased ATP synthesis was identified as a significant biological function in CNIT, while key toxicology pathways included NRF2-mediated oxidative stress response and increased permeability transition of mitochondria. An integrative analysis demonstrated a panel of 13 significant miRNAs and their 33 CNIT-specific gene targets involved with mitochondrial activity and function. We also identified a candidate panel of miRNAs/genes, which may serve as future molecular markers for CNIT diagnosis as well as potential therapeutic targets.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Interactions between histone deacetylase (HDAC) inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),
also known as Apo2 ligand, were examined in human leukemia cells ( e.g. ..., U937, Jurkat, and HL-60). Simultaneous exposure of cells to 100-ng/ml TRAIL with either 1-m m sodium butyrate or 2-μ m suberoylanilide hydroxamic acid resulted in a striking increase in leukemic cell mitochondrial damage, caspase activation,
and apoptosis. Lethal effects were significantly diminished in U937 cells ectopically expressing dominant-negative caspase-8,
dominant-negative Fas-associated death domain, CrmA (receptor pathway), or Bcl-2 or Bcl-X L (mitochondrial pathway). Analysis of mitochondrial events in U937 cells exposed to TRAIL/HDAC inhibitors revealed enhanced
Bid activation and Bax translocation, loss of mitochondrial membrane potential, and cytoplasmic release of cytochrome c , Smac/DIABLO, and apoptosis-inducing factor. No changes were observed in expression of FLICE-like inhibitory protein, TRAIL
receptors, or reactive oxygen species generation. TRAIL/HDAC inhibitor-induced apoptosis triggered caspase-dependent cleavage
of p21 WAF1/CIP1 ; moreover, enforced expression of a nuclear localization signal deletant form of p21 WAF1/CIP1 significantly diminished lethality. Lastly, p27 KIP1 , pRb, X-linked inhibitor of apoptosis, and Bcl-2 displayed extensive proteolysis. These findings indicate that coadministration
of TRAIL with HDAC inhibitors synergistically induces apoptosis in human myeloid leukemia cells and provide further evidence
that simultaneous activation of the extrinsic and intrinsic pathways in such cells leads to a dramatic increase in mitochondrial
injury and activation of the caspase cascade.
The role of the Ras/MEK/ERK pathway was examined in relation to DNA damage in human multiple myeloma (MM) cells exposed to Chk1 inhibitors in vitro and in vivo. Exposure of various MM cells to ...marginally toxic concentrations of the Chk1 inhibitors UCN-01 or Chk1i modestly induced DNA damage, accompanied by Ras and ERK1/2 activation. Interruption of these events by pharmacologic (eg, the farnesyltransferase inhibitor R115777 or the MEK1/2 inhibitor PD184352) or genetic (eg, transfection with dominant-negative Ras or MEK1 shRNA) means induced pronounced DNA damage, reflected by increased γH2A.X expression/foci formation and by comet assay. Increased DNA damage preceded extensive apoptosis. Notably, similar phenomena were observed in primary CD138+ MM cells. Enforced MEK1/2 activation by B-Raf transfection prevented R115777 but not PD184352 from inactivating ERK1/2 and promoting Chk1 inhibitor–induced γH2A.X expression. Finally, coadministration of R115777 diminished UCN-01–mediated ERK1/2 activation and markedly potentiated γH2A.X expression in a MM xenograft model, associated with a striking increase in tumor cell apoptosis and growth suppression. Such findings suggest that Ras/MEK/ERK activation opposes whereas its inhibition dramatically promotes Chk1 antagonist–mediated DNA damage. Together, these findings identify a novel mechanism by which agents targeting the Ras/MEK/ERK pathway potentiate Chk1 inhibitor lethality in MM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The role of reactive oxygen species (ROS) production on DNA damage and potentiation of fludarabine lethality by the histone
deacetylase inhibitor (HDACI) LAQ-824 was investigated in human leukemia ...cells. Preexposure (24 h) of U937, HL-60, Jurkat,
or K562 cells to LAQ-824 (40 nmol/L) followed by fludarabine (0.4 μmol/L) dramatically potentiated apoptosis (≥75%). LAQ-824
triggered an early ROS peak (30 min-3 h), which declined by 6 h, following LAQ-824-induced manganese superoxide dismutase
2 (Mn-SOD2) upregulation. LAQ-824/fludarabine lethality was significantly diminished by either ROS scavengers N -acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin or ectopic Mn-SOD2 expression and conversely increased
by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in γ-H2AX, which persisted (48
h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation,
diminished Ku70 and Ku86 DNA-binding activity, and down-regulated DNA repair genes BRCA1, CHEK1 , and RAD51 ). Addition of fludarabine further potentiated DNA damage, which was incompatible with cell survival, and triggered multiple
proapoptotic signals including activation of nuclear caspase-2 and release of histone H1.2 into the cytoplasm. The latter
event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a
mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes
in potentiation of nucleoside analogue-mediated DNA damage and lethality in leukemia. Moreover, they show for the first time
the link between HDACI-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents.
Mol Cancer Ther 2008;7(9):3285–97
Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138+ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and ...selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138+ primary samples, but spared normal CD138− and CD34+ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and increased apoptosis in all cell-cycle phases, including G0/G1. To determine whether this regimen is active against quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the G0/G1 population. G0/G1–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G0/G1–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst+), low pyronin Y (PY)–staining (2N Hst+/PY−) G0 population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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