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Tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), both related to persistent β-catenin activation, remain formidable challenges for chronic myeloid leukemia (CML). We observed overexpression of β-catenin in BC-CML stem/progenitor cells, particularly in granulocyte-macrophage progenitors, and highest among a novel CD34 + CD38 + CD123 hi Tim-3 hi subset as determined by CyTOF analysis. Co-culture with mesenchymal stromal cells (MSCs) induced the expression of β-catenin and its target CD44 in CML cells. A novel Wnt/β-catenin signaling modulator, C82, and nilotinib synergistically killed KBM5 T315I and TKI-resistant primary BC-CML cells with or without BCR-ABL kinase mutations even under leukemia/MSC co-culture conditions. Silencing of β-catenin by short interfering RNA restored sensitivity of primary BCR-ABL T315I/E255V BC-CML cells to nilotinib. Combining the C82 pro-drug, PRI-724, with nilotinib significantly prolonged the survival of NOD/SCID/IL2Rγ null mice injected with primary BCR-ABL T315I/E255V BC-CML cells. The combined treatment selectively targeted CML progenitors and inhibited CD44, c-Myc, survivin, p-CRKL and p-STAT5 expression. In addition, pretreating primary BC-CML cells with C82, or the combination, but not with nilotinib alone, significantly impaired their engraftment potential in NOD/SCID/IL2Rγ-null-3/GM/SF mice and significantly prolonged survival. Our data suggest potential benefit of concomitant β-catenin and Bcr-Abl inhibition to prevent or overcome Bcr-Abl kinase-dependent or -independent TKI resistance in BC-CML.

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Despite being highly effective for newly diagnosed chronic myeloid leukemia (CML), imatinib not only is inactive against quiescent CML stem cells, but also has limited activity against blast crisis (BC) CML. The relative activity of Bcr-Abl and the expression levels of antiapoptotic proteins in proliferating and quiescent CD34(+) BC CML progenitor cells and the effects of targeting antiapoptotic proteins in these cells are unknown. Here we report higher levels of p-CrkL in quiescent than in proliferating CD34(+) progenitor cells and comparable expression levels of Bcl-2, Bcl-xL, Mcl-1 and XIAP in the two populations in BC CML. Inhibition of Bcl-2/Bcl-xL by ABT-737 in cells from patients with tyrosine kinase inhibitor (TKI)-resistant BC CML promoted apoptosis in quiescent CD34(+) progenitor cells with an efficacy similar to that in proliferating cells. Combination of ABT-737 with imatinib (which decreases Mcl-1 levels) or triptolide (which decreases Mcl-1 and XIAP) synergistically induced death of both proliferating and quiescent CD34(+) progenitor cells obtained from TKI-resistant BC CML patients. These results suggest that antiapoptotic proteins are critical targets in BC CML and that activation of apoptosis signaling can eliminate both proliferating and quiescent CD34(+) progenitor cells in BC CML, independent of response to TKIs.

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently induce apoptosis in various malignant cells by inhibiting RNA synthesis and nuclear factor-κB activity. Previously, we showed that triptolide promotes apoptosis in acute myeloid leukemia (AML) cells via the mitochondria-mediated pathway, in part, by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a triptolide derivative, currently in preclinical development. Here we show that MRx102 potently promoted apoptosis in AML cell lines, with EC(50) values of 14.5±0.6 nM and 37.0±0.9 nM at 48 h for OCI-AML3 and MV4-11 cells, respectively. MRx102, at low nanomolar concentrations, also induced apoptosis in bulk, CD34(+) progenitor, and more importantly, CD34(+)CD38(-) stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. In vivo, MRx102 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Collectively, we demonstrated that MRx102 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.

Kinesin spindle protein (KSP), a microtubule-associated motor protein essential for cell cycle progression, is overexpressed in many cancers and is a potential anti-tumor target. We found that inhibition of KSP by a selective inhibitor, ARRY-520, blocked cell cycle progression, leading to apoptosis in acute myeloid leukemia cell lines that express high levels of KSP. Knockdown of p53, overexpression of XIAP and mutation in caspase-8 did not significantly affect sensitivity to ARRY-520, suggesting that the response is independent of p53, XIAP and the extrinsic apoptotic pathway. Although ARRY-520 induced mitotic arrest in both HL-60 and Bcl-2-overexpressing HL-60Bcl-2 cells, cell death was blunted in HL-60Bcl-2 cells, suggesting that the apoptotic program is executed through the mitochondrial pathway. Accordingly, inhibition of Bcl-2 by ABT-737 was synergistic with ARRY-520 in HL-60Bcl-2 cells. Furthermore, ARRY-520 increased Bim protein levels prior to caspase activation in HL-60 cells. ARRY-520 significantly inhibited tumor growth of xenografts in SCID mice and inhibited AML blast but not normal colony formation, supporting a critical role for KSP in proliferation of leukemic progenitor cells. These results demonstrate that ARRY-520 potently induces cell cycle block and subsequent death in leukemic cells via the mitochondrial pathway and has the potential to eradicate AML progenitor cells.

In order to investigate the pharmacological basis of 'Yang-invigorating' action, the effect of oral treatment with the methanolic extract of 'Yang-invigorating' herbs on ATP-generation capacity was examined, using heart homogenates prepared from herb-pretreated mice. Tonifying (i.e., health-promoting) herbs of other functional categories were also included for comparison. The results indicated that 'Yang-invigorating' Chinese tonifying herbs could invariably enhance myocardial ATP-generation capacity, with the extent of stimulation varying among the herbs. In contrast, 'Yin-nourishing' herbs either did not stimulate or even decreased myocardial ATP-generation capacity. While 'Qi-invigorating' herbs produced variable effects on myocardial ATP-generation capacity, most of the 'blood-enriching' herbs did not cause any significant changes. The results obtained from studies using myocardial mitochondrial fractions isolated from herb-pretreated mice suggest that 'Yang-invigorating' herbs might speed up ATP generation by increasing mitochondrial electron transport. The ensemble of results has provided evidence for the first time to support the pharmacological basis of 'Yang invigoration' in Chinese medicine.

In the 16-week pilot study, the effect of a Yang-promoting Chinese herbal suppository preparation (VI-28) on the red cell antioxidant status was examined in 31 healthy male subjects aged 41-66 years old. VI-28 treatment for 12 weeks (one suppository (0.3 g) daily for week 1-4; one every 2 days for week 5-8; one every 3 days for week 9-12) produced a time/dose-dependent alteration in red cell antioxidant status. The VI-28-induced change is characterized by a slight depletion in cellular reduced glutathione (GSH) level and a decrease in susceptibility to peroxide-induced lipid peroxidation as well as increases in catalase (CAT) and Cu-Zn-superoxide dismutase (SOD) activities. While a reversal trend of change was observed in cellular GSH level, the susceptibility to lipid peroxidation as well as the CAT activity after the cessation of treatment for 4 weeks, the SOD activity exhibited a protracted increase. The results indicate that VI-28 treatment enhances red cell antioxidant status in male subjects. The beneficial effect of VI-28 treatment on red cells may re fl ect a corresponding change in antioxidant status of peripheral tissues.

The in vivo antioxidant action of a lignan-enriched extract of the fruit of Schisandra chinensis (FS) and an anthraquinone-containing extract of the root of Polygonum multiflorum (PME) was compared with their respective active constituents schisandrin B (Sch B) and emodin by examining their effect on hepatic mitochondrial glutathione antioxidant status in control and carbon tetrachloride (CCl 4 )-intoxicated mice. FS and PME pretreatments produced a dose-dependent protection against CCl 4 hepatotoxicity, with the effect of FS being more potent. Pretreatment with Sch B, emodin or alpha-tocopherol (alpha-Toc) also protected against CCl 4 hepatotoxicity, with the effect of Sch B being more potent. The extent of hepatoprotection afforded by FS/Sch B and PME/emodin pretreatment against CCl 4 toxicity was found to correlate well with the degree of enhancement in hepatic mitochondrial glutathione antioxidant status, as evidenced by increases in reduced glutathione level and activities of glutathione reductase, glutathione peroxidase as well as glutathione S-transferases, in both control and CCl 4 -intoxicated mice. alpha-Toc, which did not enhance mitochondrial glutathione antioxidant status, seemed to be less potent in protecting against CCl 4 hepatotoxicity. The ensemble of results indicates that FS/PME produced a more potent in vivo antioxidant action than alpha-Toc by virtue of their ability to enhance hepatic mitochondrial glutathione antioxidant status and that the differential potency of FS and PME can be attributed to the difference in in vivo antioxidant potential between Sch B and emodin. Abbreviations. ALT:alanine aminotransferases CCl 4 :carbon tetrachloride FS:lignan-enriched extract of Schisandra fruit GRD:glutathione reductase GSH:reduced glutathione GSH-Px: Se-glutathione peroxidase GST:glutathione S-transferases mt:mitochondrial MDA:malondialdehyde PME:anthraquinone-containing fraction of Polygonum root Sch B:schisandrin B SDH:sorbitol dehydrogenase alpha-Toc:alpha-tocopherol

Intragastric administration (100-200 micromol/kg) of tacrine (THA) or bis(7)-THA could cause an acute and dose-dependent increase in plasma alanine aminotransferases activity in mice at 6 h after the drug administration. The increase in plasma enzyme activity was associated with an increase in hepatic malondialdehyde level, an indirect index of oxidative tissue damage. Pretreating mice with schisandrin B (Sch B), an active dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, at a daily dose of 0.125-0.5 mmol/kg for 3 days protected against the THA/bis(7)-THA induced hepatic oxidative damage in a dose-dependent manner. Sch B treatment (0.025-0.5 mmol/kg/day x 5) also enhanced the passive avoidance-response in mice as assessed by the step-through task experiment. The ensemble of results suggests that Sch B may be useful for reducing the potential hepatotoxicity of THA/bis(7)-THA in anti-Alzheimer's therapy.

The effects of short-term (2-week) diabetes on myocardial ischemia-reperfusion (I-R) injury and associated changes in myocardial non-enzymatic antioxidant level were examined. Isolated-perfused hearts prepared from control and diabetic rats were subjected to increasing periods of ischemia and reperfusion, and myocardial I-R injury was assessed by measuring the extent of lactate dehydrogenase (LDH) leakage and contractile force recovery. While a brief period (20 min) of post-ischemic reperfusion caused a smaller extent of LDH leakage, the prolonged period (40 min) of reperfusion produced a greater degree of I-R injury in diabetic hearts, as indicated by the impaired recovery of contractile force. The apparent protection against I-R injury in diabetic hearts during the early phase of post-ischemic reperfusion was associated with increases in myocardial reduced glutathione/ascorbic acid and a-tocopherol levels, with the effect on a-tocopherol being most prominent. Insulin treatment could reverse the diabetes-associated changes in susceptibility to myocardial I-R injury and antioxidant response. The ensemble of results indicates that the myocardium isolated from short-term diabetic rat can produce a beneficial antioxidant response to I-R challenge, which may, in turn, be attributable to the decreased susceptibility to I-R injury observable during the early phase of reperfusion.

'Dang-Gui Decoction for Enriching the Blood' (BE), a traditional Chinese formulation comprising Angelica sinensis and Astragalus membranaceus, is used for stimulating red blood cell production as well as enhancing cardiovascular function. In the present study, we have demonstrated the myocardial protection afforded by BE pretreatment against ischaemia-reperfusion (IR) injury in isolated-perfused rat hearts. A more complete and potent myocardial protection against IR injury was also shown by a Polygonum multiflorum extract supplemented BE preparation (BEA). The results suggest that the more potent cardioprotective action of BEA may be related to its ability to sustain the myocardial glutathione antioxidant status under conditions of IR-induced oxidative stress, which may possibly in turn result from the synergistic interaction between the BE and Polygonum extract., (Copyright 2000 John Wiley & Sons, Ltd.)

An ethyl acetate extract of Polygonum multiflorum Thunb. (PME) was fractionated into an anthraquinone-containing (PME-I) and a non-anthraquinone-containing (PME-II) fraction. The effects of PME and its related extracts pretreatment on myocardial ischemia-reperfusion (IR) injury in isolated perfused rat hearts were examined. Pretreatment with PME extract or its anthraquinone-containing fraction produced a dose-dependent protection against myocardial IR injury, as evidenced by a significant decrease in the extent of LDH leakage as well as an improvement in contractile force recovery. The myocardial protection was found to be associated with an enhancement in myocardial glutathione antioxidant status, as indicated by significant reductions in both the extent of IR-induced reduced glutathione (GSH) depletion and inhibition of Se-glutathione peroxidase (GPX) and glutathione reductase (GRD) activities. Both alpha-tocopherol acetate (VE) and emodin (EMD) pretreatments protected against IR-induced myocardial injury as assessed by the decrease in the extent of LDH leakage. But the contractile force recovery of the ischemic-reperfused hearts prepared from VE or EMD pretreated animals was not improved. The more complete myocardial protection afforded by the anthraquinone-containing fraction of PME extract may be related to its ability to sustain the glutathione antioxidant status under the condition of IR-induced oxidative stress.

The streptozotocin-induced short-term (2 week) diabetic rats showed an increase in susceptibility to carbon tetrachloride (CCl4)-induced hepatocellular damage. This diabetes-induced change was associated with a marked impairment in the hepatic glutathione antioxidant/detoxification response to CCl4 challenge, as indicated by the abrogation of the increases in hepatic reduced glutathione (GSH) level, glucose-6-phosphate dehydrogenase and microsomal glutathione S-transferases (GST) activities upon challenge with increasing doses of CCl4. While the hepatic GSH level was increased in diabetic rats, the hepatic mitochondrial GSH level and Se-glutathione peroxidase activity were significantly reduced. Insulin treatment could reverse most of the biochemical alterations induced by diabetes. Both insulin and schisandrin B (Sch B) pretreatments protected against the CCl4 hepatotoxicity in diabetic rats. The hepatoprotection was associated with improvement in hepatic glutathione redox status in both cytosolic and mitochondrial compartments, as well as the increases in hepatic ascorbic acid level and microsomal GST activity. The ensemble of results suggests that the diabetes-induced impairment in hepatic mitochondrial glutathione redox status may at least in part be attributed to the enhanced susceptibility to CCl4 hepatotoxicity. Sch B may be a useful hepatoprotective agent against xenobiotics-induced toxicity under the diabetic conditions.

Effects of Schisandrin B (Sch B) and alpha-tocopherol (alpha-TOC) on ferric chloride (Fe3+) induced oxidation of erythrocyte membrane lipids in vitro and carbon tetrachloride (CCl4) induced lipid peroxidation in vivo were examined. While alpha-TOC could produce prooxidant and antioxidant effect on Fe(3+)-induced lipid peroxidation, Sch B only inhibited the peroxidation reaction. Pretreatment with alpha-TOC (3 mmol/kg/day x 3) did not protect against CCl4-induced lipid peroxidation and hepatocellular damage in mice, whereas Sch B pretreatment (0.3 mmol/3.0 mmol/kg/day x 3) produced a dose-dependent protective effect on the CCl4-induced hepatotoxicity. The ensemble of results suggests that the ability of Sch B to inhibit lipid peroxidation, while in the absence of pro-oxidant activity, may at least in part contribute to its hepatoprotective action.

Changes in tissue glutathione antioxidant system in streptozotocin-induced diabetic rats for a period of 15 weeks were examined. Total glutathione level was significantly increased in kidney tissue, but were slightly decreased and increased in liver and heart tissues, respectively. The small changes in total glutathione level in the liver and heart, though not statistically significant, were associated with reciprocal alterations in the activity of gamma-glutamylcysteine synthetase (GCS). While the GCS activity was not changed in kidney tissue, the activity of gamma-glutathione peroxidase was significantly increased in kidney tissue. Insulin treatment could completely or partly normalize almost all of these changes induced by diabetes. However, the decrease in hepatic glutathione S-transferases activity in diabetic rats was not reversed by the insulin treatment. The ensemble of results suggests that the diabetes-induced alterations in tissue glutathione antioxidant system may possibly reflect an inter-organ antioxidant response to a generalized increase in tissue oxidative stress associated with diabetes.

Effects of Sheng Mai San (SMS), a traditional Chinese medicine used for the treatment of coronary heart disease, and the lignan-enriched extract of Fructus Schisandrae (FS), an antioxidant component of SMS, were examined in an in vivo model of myocardial infarction and an ex vivo model of myocardial ischemia-reperfusion injury in rats. Pretreatment with SMS (12 g/kg/day × 3, p. o.) or FS extract (0.8 g/kg/day × 3, p. o.) was found to protect against the isoproterenol-induced myocardial injury in rats and the ischemia-reperfusion injury in isolated perfused hearts prepared from the pretreated animals. Pretreatment with α-tocopherol (0.8 g/kg/day × 3, p. o.) produced similar beneficial effect on the myocardium. The myocardial protection afforded by SMS pretreatment is likely, at least in part, mediated by the FS-derived antioxidant activity., (Copyright © 1996 Gustav Fischer Verlag · Stuttgart · Jena · New York. Published by Elsevier GmbH.. All rights reserved.)

Treatment of rats with a lignan-enriched extract of the fruit of Schisandra chinensis could enhance hepatic antioxidant/detoxification system, as indicated by increases in hepatic reduced glutathione (GSH) level as well as hepatic glutathione reductase and glutathione S-transferase activities. The hepatoprotective action was evident after aflatoxin beta 1 or cadmium chloride (Cd) challenge. Schisandra chinensis pretreatment protected against aflatoxin B1-or Cd-induced hepatocellular damage in rats. However, pretreating rats with alpha-tocopherol acetate (vitamin E) did not protect against hepatic damage induced by both toxins. Results from the present as well as our previous studies demonstrate that the hepatoprotection afforded by Schisandra chinensis pretreatment is not hepatotoxin specific. Schisandra chinensis seems to be more effective than vitamin E in protecting against aflatoxin B1 and Cd toxicity. The mechanism of hepatoprotection afforded by Schisandra chinensis pretreatment may involve facilitation of both antioxidant and detoxification processes in the liver.

Exposure of liver homogenates to an in vitro tert-butyl hydroperoxide challenge can be used as a means for measuring the reduced glutathione regeneration capacity (GRC) of hepatic tissues. Pretreatment of rats with a lignan-enriched extract of Fructus Schisandrae (FS) caused a moderate enhancement of hepatic GRC in control rats, but the GRC enhancing effect of FS pretreatment on hepatic tissues was greatly exaggerated after CCl4 challenge. This in vitro bioassay can be used for investigating the hepatic GRC promoting action of orally active agents.