17-Allylamino-demethoxygeldanamycin (17-AAG), currently in phase I and II clinical trials as an anticancer agent, binds to the ATP pocket of heat shock protein (Hsp90). This binding induces a ...cellular stress response that up-regulates many proteins including Hsp27, a member of the small heat shock protein family that has cytoprotective roles, including chaperoning of cellular proteins, regulation of apoptotic signaling, and modulation of oxidative stress. Therefore, we hypothesized that Hsp27 expression may affect cancer cell sensitivity to 17-AAG. In colony-forming assays, overexpression of Hsp27 increased cell resistance to 17-AAG whereas down-regulation of Hsp27 by siRNA increased sensitivity. Because Hsp27 is known to modulate levels of glutathione (GSH), we examined cellular levels of GSH and found that it was decreased in cells transfected with Hsp27 siRNA when compared with control siRNA. Treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, also sensitized cells to 17-AAG. Conversely, treatment of Hsp27 siRNA-transfected cells with N-acetylcysteine, an antioxidant and GSH precursor, reversed their sensitivity to 17-AAG. A cell line selected for stable resistance to geldanamycin relative to parent cells showed increased Hsp27 expression. When these geldanamycin- and 17-AAG-resistant cells were transfected with Hsp27 siRNA, 17-AAG resistance was dramatically diminished. Our results suggest that Hsp27 up-regulation has a significant role in 17-AAG resistance, which may be mediated in part through GSH regulation. Clinical modulation of GSH may therefore enhance the efficacy of Hsp90-directed therapy.
To define the maximum tolerated dose, toxicities, pharmacokinetics, and pharmacodynamics of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17DMAG).
17DMAG was given intravenously over 1 hour ...daily for 5 days (schedule A) or daily for 3 days (schedule B) every 3 weeks. Plasma 17DMAG concentrations were measured by liquid chromatography/mass spectrometry. Heat-shock proteins (HSPs) and client proteins were evaluated at baseline and after treatment on day 1 in peripheral blood mononuclear cells (PBMCs) and in pre- and post-treatment (24 hours) biopsies done during cycle 1 at the recommended phase II dose (n = 7).
Fifty-six patients were entered: 26 on schedule A; 30 on schedule B. The recommended phase II doses for schedules A and B were 16 mg/m(2) and 25 mg/m(2), respectively. Grade 3/4 toxicities included liver function test elevation (14%), pneumonitis (9%), diarrhea (4%), nausea (4%), fatigue (4%) and thrombocytopenia (4%). There were no objective responses. Four patients had stable disease. 17DMAG half-life was 24 +/- 15 hours. 17DMAG area under the curve (range, 0.7 to 14.7 mg/mL x h) increased linearly with dose. The median HSP90, HSP70, and integrin-linked kinase levels were 87.5% (n = 14), 124% (n = 20), and 99.5% (n = 20) of baseline. Changes in HSPs and client proteins in tumor biopsies were not consistent between baseline and 24 hours nor did they change in the same direction as those in PBMCs collected at the time of biopsy.
The recommended phase II doses of 17DMAG (16 mg/m(2) x 5 days or 25 mg/m(2) x 3 days, every 3 weeks) are well tolerated and suitable for further evaluation.
Despite studies that show the antitumor activity of Hsp90 inhibitors, such as geldanamycin (GA) and its derivative 17-allylamino-demethoxygeldanamycin (17-AAG), recent reports indicate that these ...inhibitors lack significant single-agent clinical activity. Resistance to Hsp90 inhibitors has been previously linked to expression of P-glycoprotein (P-gp) and the multidrug resistant (MDR) phenotype. However, the stress response induced by GA treatment can also cause resistance to Hsp90-targeted therapy. Therefore, we chose to further investigate the relative importance of P-gp and the stress response in 17-AAG resistance. Colony-forming assays revealed that high expression of P-gp could increase the 17-AAG IC(50) 6-fold in cells transfected with P-gp compared with parent cells. A549 cells selected for resistance to GA overexpressed P-gp, but verapamil did not reverse the resistance. These cells also overexpressed Hsp27, and Hsp70 was induced with 17-AAG treatment. When the GA and 17-AAG resistant cells were transfected with Hsp27 and/or Hsp70 small interfering RNA (siRNA), the 17-AAG IC(50) decreased 10-fold compared with control transfected cells. Transfection with siRNA directed against Hsp27, Hsp70, or Hsp27 and Hsp70 also increased sensitivity to EC78, a purine scaffold-based Hsp90 inhibitor that is not a P-gp substrate. We conclude that P-gp may contribute, in part, to resistance to 17-AAG, but induction of stress response proteins, such as Hsp27 and Hsp70, by Hsp90-targeted therapy plays a larger role. Taken together, our results indicate that targeting of Hsp27 and Hsp70 should be exploited to increase the clinical efficacy of Hsp90-directed therapy.
We have reported previously the activity of the insulin-like growth factor-I (IGF-IR)/insulin receptor (InsR) inhibitor, BMS-554417,
in breast and ovarian cancer cell lines. Further studies indicated ...treatment of OV202 ovarian cancer cells with BMS-554417
increased phosphorylation of HER-2. In addition, treatment with the pan-HER inhibitor, BMS-599626, resulted in increased phosphorylation
of IGF-IR, suggesting a reciprocal cross-talk mechanism. In a panel of five ovarian cancer cell lines, simultaneous treatment
with the IGF-IR/InsR inhibitor, BMS-536924 and BMS-599626, resulted in a synergistic antiproliferative effect. Furthermore,
combination therapy decreased AKT and extracellular signal-regulated kinase activation and increased biochemical and nuclear
morphologic changes consistent with apoptosis compared with either agent alone. In response to treatment with BMS-536924,
increased expression and activation of various members of the HER family of receptors were seen in all five ovarian cancer
cell lines, suggesting that inhibition of IGF-IR/InsR results in adaptive up-regulation of the HER pathway. Using MCF-7 breast
cancer cell variants that overexpressed HER-1 or HER-2, we then tested the hypothesis that HER receptor expression is sufficient
to confer resistance to IGF-IR-targeted therapy. In the presence of activating ligands epidermal growth factor or heregulin,
respectively, MCF-7 cells expressing HER-1 or HER-2 were resistant to BMS-536924 as determined in a proliferation and clonogenic
assay. These data suggested that simultaneous treatment with inhibitors of the IGF-I and HER family of receptors may be an
effective strategy for clinical investigations of IGF-IR inhibitors in breast and ovarian cancer and that targeting HER-1
and HER-2 may overcome clinical resistance to IGF-IR inhibitors. Mol Cancer Ther 2008;7(9):2589–98
Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the NH 2 -terminal ATP-binding domain of heat shock protein (Hsp) 90 and inhibit its chaperone functions. Despite in vitro and in ...vivo studies indicating promising antitumor activity, derivatives of GA, including 17-allylaminogeldanamycin (17-AAG), have shown
little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including
cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic and caused
increased apoptosis compared with each agent alone. One measurable response that results from treatment with Hsp90-targeted
agents is the induction of a heat shock factor-1 (HSF-1) heat shock response. Treatment with GA + CDDP revealed that CDDP
suppresses up-regulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70.
However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1 but inhibited DNA binding of HSF-1
as shown by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1–mediated
Hsp70 up-regulation. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell survival
assays revealed that deletion of Hsp70 caused increased sensitivity to GA ( Hsp70 +/+ IC 50 = 63.7 ± 14.9 nmol/L and Hsp70 −/− IC 50 = 4.3 ± 2.9 nmol/L), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results
suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition
of HSF-1 activity. Clinical modulation of the HSF-1–mediated heat shock response may enhance the efficacy of Hsp90-directed
therapy. Mol Cancer Ther 2008;7(10):3256–64
Despite the most aggressive medical and surgical treatments, glioblastoma multiforme remains incurable with a median survival of <1 year. We investigated the antitumor potential of a novel viral ...agent, an attenuated strain of measles virus (MV), derived from the Edmonston vaccine lineage, genetically engineered to produce carcinoembryonic antigen (CEA). CEA production as the virus replicates can serve as a marker of viral gene expression. Infection of a variety of glioblastoma cell lines including U87, U118, and U251 at MOIs 0.1, 1, and 10 resulted in significant cytopathic effect consisting of excessive syncycial formation and massive cell death at 72-96 h from infection. terminal deoxynucleotidyltransferase-mediated nick end labeling assays demonstrated the mechanism of cell death to be predominantly apoptotic. The efficacy of this approach in vivo was examined in BALB/c nude mice by using both s.c. and intracranial orthotopic U87 tumor models. In the s.c. U87 model, mice with established xenografts were treated with a total dose of 8 x 10(7) plaque forming units of MV-CEA, administered i.v. Mice treated with UV light inactivated MV, and untreated mice with established U87 tumors were used as controls. There was statistically significant regression of s.c. tumors (P < 0.001) and prolongation of survival (P = 0.007) in MV-CEA treated animals compared with the two control groups. In the intracranial orthotopic U87 model, there was significant regression of intracranial U87 tumors treated with intratumoral administration of MV-CEA at a total dose of 1.8 x 10(6) plaque forming units as assessed by magnetic resonance image (P = 0.002), and statistically significant prolongation of survival as compared with mice that received UV-inactivated virus and untreated mice (P = 0.02). Histological examination of brains of MV-CEA-treated animals revealed complete regression of the tumor with the presence of a residual glial scar and reactive changes, mainly presence of hemosiderin-laden macrophages. In addition, CEA levels in the peripheral blood in both the s.c. and orthotopic models increased before tumor regression, indicating viral gene expression, and returned to normal when the tumors regressed. Ifnar(ko) CD46 Ge transgenic mice, susceptible to MV infection, were used to assess central nervous system toxicity of MV-CEA. Intracranial administration of MV-CEA into the caudate nucleus of Ifnar(ko) CD46 Ge did not result in clinical neurotoxicity. Pathologic examination demonstrated limited microglial infiltration surrounding the injection site. In summary, MV-CEA has potent antitumor activity against gliomas in vitro, as well as in both s.c. and orthotopic U87 animal models. Monitoring CEA levels in the serum can serve as a low-risk method of detecting viral gene expression during treatment, and could allow dose optimization and individualization of treatment.
The use of replicating viruses for cancer therapy (virotherapy) holds much promise. We reported previously that the live attenuated Edmonston B vaccine strain of measles virus (MV-Edm) had ...antineoplastic efficacy against hematological malignancies. In this study, we demonstrate that a recombinant MV-Edm, genetically engineered to express an inert soluble marker peptide (MV-hCEA), is potent against human epithelial ovarian cancer cells in vitro and in vivo. The virus was selectively oncolytic for ovarian tumor cells but caused minimal cytopathic damage on nontransformed ovarian surface epithelium and mesothelium. In contrast to nontransformed cells, the ovarian tumor cells expressed high levels of the measles virus receptor CD46. When injected directly into large established s.c. SKOV3ip.1 human epithelial ovarian xenografts in athymic mice, the virus induced complete regression of 80% of the tumors. i.p. administration of virus enhanced the median survival of mice with advanced i.p. SKOV3ip.1 tumors by >50 days. In addition, we could easily follow the kinetic profile of viral gene expression in the treated mice by determining serum levels of the virally encoded marker peptide (soluble human carcinoembryonic antigen). Trackable recombinant measles viruses warrant further investigation for therapy of ovarian cancer.
Numerous metabolic adaptations occur in the heart after birth. Important transcription factors that regulate expression of the glycolytic and mitochondrial oxidative genes are hypoxia-inducible ...factors (HIF-1alpha and -2alpha) and nuclear respiratory factor-1 (NRF-1). The goal of this study was to examine expression of HIF-1alpha, HIF-2alpha, and NRF-1 and the genes they regulate in pre- and postnatal myocardium. Ovine right and left ventricular myocardium was obtained at four time points: 95 and 140 d gestation (term = 145 d) and 7 d and 8 wk postnatally. Steady-state mRNA and protein levels of HIF-1alpha and NRF-1 and protein levels of HIF-2alpha were measured along with mRNA of HIF-1alpha-regulated genes (aldolase A, alpha- and beta-enolase, lactate dehydrogenase A, liver and muscle phosphofructokinase) and NRF-1-regulated genes (cytochrome c, Va subunit of cytochrome oxidase, and carnitine palmitoyltransferase I ). HIF-1alpha protein was present in fetal myocardium but dropped below detectable levels at 7 d postnatally. HIF-2alpha protein levels were similar at the four time points. Steady-state mRNA levels of alpha-enolase, lactate dehydrogenase A, and liver phosphofructokinase declined significantly postnatally. Aldolase A, beta-enolase, and muscle phosphofructokinase mRNA levels increased postnatally. Steady-state mRNA and protein levels of NRF-1 decreased postnatally in contrast to the postnatal increases in cytochrome c, subunit Va of cytochrome oxidase, and carnitine palmitoyltransferase I mRNA levels. The in vivo postnatal regulation of enzymes encoding glycolytic and mitochondrial enzymes is complex. As transactivation response elements for the genes encoding metabolic enzymes continue to be characterized, studies using the fetal-to-postnatal metabolic transition of the heart will continue to help define the in vivo role of these transcription factors.
T. D. Scholz, C. J. TenEyck and B. C. Schutte. Thyroid Hormone Regulation of the NADH Shuttles in Liver and Cardiac Mitochondria. Journal of Molecular and Cellular Cardiology (2000) 32, 1–10. Thyroid ...hormone can potentially regulate the malate/aspartate and α -glycerophosphate shuttle pathways in cardiac mitochondria either directly, by altering gene expression, or indirectly, by increasing myocardial workload. The goal of the current study was to determine the influence of thyroid hormone on the NADH shuttles in cardiac and liver mitochondria. Malate/aspartate andα -glycerophosphate shuttle capacities were significantly increased in cardiac mitochondria from adult rats treated for 9 days with T3 compared to saline-treated controls. Liver mitochondria demonstrated a significant increase in α -glycerophosphate and no change in malate/aspartate shuttle capacity. T3 increased steady-state mRNA levels and activity of mitochondrial α -glycerophosphate dehydrogenase in both myocardium and liver. Quantitative immunoblot studies demonstrated a significant increase in aspartate-glutamate carrier levels in T3-treated myocardium suggesting a regulatory role of the aspartate/glutamate carrier in T3-treated hearts. Thyroid hormone effects on the NADH shuttles are tissue-specific. Changes in the NADH shuttles in the presence of thyroid hormone excess occur both directly at the gene level and indirectly as an adaptive response.
Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242
Developmental downregulation of the malate-aspartate shuttle has
been observed in cardiac mitochondria. The goals of this study ...were to
determine the time course of the postnatal decline and to identify
potential regulatory sites by measuring steady-state myocardial mRNA
and protein levels of the mitochondrial proteins involved in the
shuttle. By use of isolated porcine cardiac mitochondria incubated with
saturating concentrations of the cytosolic components of the
malate-aspartate shuttle, shuttle capacity was found to decline by
~50% during the first 5 wk of life (from 921 ± 48 to 531 ± 53 nmol · min 1 · mg
protein 1 ). Mitochondrial
aspartate aminotransferase mRNA levels were greater in adult than in
newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase
in adult cardiac tissue were 224% of levels in newborn tissue, whereas
protein levels were 54% greater in adult myocardium.
Aspartate/glutamate carrier protein levels were also greater in adult
than in newborn tissue. mRNA and protein levels of the
oxoglutarate/malate carrier were increased in newborn myocardium. It
was concluded that 1 ) myocardial
malate-aspartate shuttle capacity declines rapidly after birth,
2 ) divergence of mitochondrial
malate dehydrogenase mRNA and protein levels during development
suggests posttranscriptional regulation of this protein, and
3 ) the developmental decline in
malate-aspartate shuttle capacity is regulated by decreased
oxoglutarate/malate carrier gene expression.
development; energy metabolism; gene expression; oxoglutarate/malate carrier; malate dehydrogenase
PDF
