Barbara Sato

Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53.

Ischemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH](o)). Apoptosis did not occur when [pH](o) was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis-mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25-30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53.

Metallothionein Induction by Hypoxia Involves Cooperative Interactions between Metal-Responsive Transcription Factor-1 and Hypoxia-Inducible Transcription Factor-1α

Mammalian metallothionein (MT) genes are transcriptionally activated by the essential metal zinc as well as by environmental stresses, including toxic metal overload and redox fluctuations. In addition to playing a key role in zinc homeostasis, MT proteins can protect against metal- and oxidant-induced cellular damage, and may participate in other fundamental physiologic and pathologic processes such as cell survival, proliferation, and neoplasia. Previously, our group reported a requirement for metal-responsive transcription factor-1 (MTF-1) in hypoxia-induced transcription of mouse MT-I and human MT-IIA genes. Here, we provide evidence that the protumorigenic hypoxia-inducible transcription factor-1α (HIF-1α) is essential for induction of MT-1 by hypoxia, but not zinc. Chromatin immunoprecipitation assays revealed that MTF-1 and HIF-1α are both recruited to the mouse MT-I promoter in response to hypoxia, but not zinc. In the absence of HIF-1α, MTF-1 is recruited to the MT-I promoter but fails to activate MT-I gene expression in response to hypoxia. Thus, HIF-1α seems to function as a coactivator of MT-I gene transcription by interacting with MTF-1 during hypoxia. Coimmunoprecipitation studies suggest interaction between MTF-1 and HIF-1α, either directly or as mediated by other factors. It is proposed that association of these important transcription factors in a multiprotein complex represents a common strategy to control unique sets of hypoxia-inducible genes in both normal and diseased tissue. (Mol Cancer Res 2008;6(3):483–90)

Loss of metal transcription factor-1 suppresses tumor growth through enhanced matrix deposition

Metal transcription factor‐1 (MTF‐1) is a ubiquitous transcriptional regulator and chromatin in‐ sulator with roles in cellular stress responses and em‐ bryonic development. The studies described herein establish for the first time the involvement of MTF‐1 in tumor development. Genetically manipulated ras‐trans‐ formed mouse embryonic fibroblasts (MEFs), wild‐type (MTF‐1+/+), or nullizygous for MTF‐1 (MTF‐1‒/‒) were used to develop fibrosarcoma tumors. Loss of MTF‐1 resulted in delayed tumor growth associated with increased matrix collagen deposition and reduc‐ tions in vasculature density. Molecular consequences of MTF‐1 loss include increased expression and activation of the transforming growth factor–β1 (TGF‐β1) and tissue transglutaminase (tTG), two proteins with docu‐ mented roles in the production and stabilization of extracellular matrix (ECM). Our findings support the hypothesis that MTF‐1 enhances the ability of the developing tumor mass to evade fibrosis and scarring of the tumor, a critical step in tumor cell prolifera‐ tion.—Haroon, Z. A., Amin, K., Lichtlen, P., Sato, B., Huynh, N. T., Wang, Z., Schaffner, W., Murphy, B. J. Loss of metal transcription factor‐1 suppresses tumor growth through enhanced matrix deposition. FASEB J. 18, 1176 –1184 (2004)

A novel steroidal inhibitor of estrogen-related receptor α (ERRα)

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalphas transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.

A novel peroxisome proliferator-activated receptor delta antagonist, SR13904, has anti-proliferative activity in human cancer cells

The peroxisome proliferator-activated receptor delta (PPARδ) is a ligand-activated, nuclear receptor transcription factor that has a documented role in glucose and lipid homeostasis. Recent studies have implicated this nuclear receptor in numerous aspects of oncogenesis. We report herein the characterization of a novel small-molecule (SR13904) that inhibits PPARδ agonist- induced transactivation and functions as a PPARδ antagonist. SR13904 also antagonizes PPARγ transactivation, albeit with much weaker potency. SR13904 displays inhibitory effects on cellular proliferation and survival in several human carcinoma lines, including lung, breast, and liver. These inhibitory effects of SR13904 on tumor cells were linked to a G1/S cell cycle block and increased apoptosis. Molecular studies show that SR13904 treatment of a lung cancer cell line, A549, results in markedly reduced levels of a number of cell cycle proteins including cyclin A and D, and cyclin dependent kinase (CDK) 2 and 4. The inhibitory effects on CDK2 appear to be transcriptional. Several of these cell cycle-related genes are known to be upregulated by PPARδ. The anti-tumor activities of SR13904 suggest that antagonism of PPARδ-mediated transactivation may inhibit tumorigenesis and that pharmacological inhibition of PPARδ may be a potential strategy for treatment or prevention of cancer.

Characterization of ANIT-Induced Toxicity using Precision-Cut Rat and Dog Liver Slices Cultured in a Dynamic Organ Roller System

This article describes the toxicity of α-naphthylisothiocyanate (ANIT), a compound known to induce dose-dependent hepatobiliary toxicity in vivo, using the slice model. Liver slices (200 μm thick) from male Sprague–Dawley rats and male beagle dogs were cultured for 7 days while exposed to a range of ANIT concentrations (1– 100 μM for rat and 4–320 μM for dog). Tissues (and medium for dog) were evaluated using a panel of clinically relevant biomarkers for liver and histological endpoints to assess viability and proliferation. ANIT increased slice levels of enzyme biomarkers corresponding to biliary markers. At high concentrations (80–100 μM for rat, 320 μM for dog) a diminution of tissue enzyme levels was observed, corresponding to severe hepatobiliary injury. By days 5 and 7, biochemical markers in the medium of dog slices indicated an elevation of hepatocellular and biliary markers. Histologically for both species, minimal hepatocellular injury was noted, but proliferation of biliary epithelial cells (BEC) was observed using 5-bromo-2-deoxyuridine (BrdU) immunostaining. In rat slices, ANIT increased the expression of inducible nitrous oxide synthase (iNOS) within 12 hrs of exposure. In summary, additional experimentation using slice culture may further demonstrate its value in screening compounds that cause hepatobiliary toxicity.

The metal-responsive transcription factor-1 protein is elevated in human tumors

We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis, and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung, and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.

Immunohematotoxicity studies with combinations of dapsone and zidovudine.

We investigated the immunohematoxicities of the antiparasitic drug dapsone (DDS) and the antiretroviral drug zidovudine (ZDV, AZT) given alone or in combination in BALB/c mice. DDS is used for prophylaxis and treatment of Pneumocystis carinii infection in AIDS patients. We examined the impact of concurrent administration of these drugs on the immune and hematopoietic systems because DDS causes hematotoxicity and ZDV therapy results in bone marrow toxicity. Daily oral administration of DDS at 25 and 50 mg/kg for 28 days caused a slight anemia, marked methemoglobinemia, reticulocytosis, and a moderate leukopenia (P < 0.01 for all parameters) but had no discernible effect on platelet count. In DDS-treated mice, the proliferative response of splenic T cells to concanavalin A was > or = 35% higher than that manifested by splenocytes from vehicle-treated control mice. ZDV at 240 and 480 mg/kg was not immunosuppressive but caused low-grade macrocytic anemia, thrombocytosis, and neutropenia; these effects were drug dose-dependent and statistically significant (P < 0.01). Concurrent administration of DDS and ZDV augmented the severity of ZDV-mediated macrocytic anemia, and 7 of 12 (58%) mice did not survive treatment with the high doses of DDS and ZDV (50 and 480 mg/kg, respectively). On the other hand, co-administration of ZDV mitigated DDS-induced methemoglobinemia and the DDS-associated elevation in lymphoproliferative response. These data suggest interaction between DDS and ZDV in mice and indicate a need for caution in using DDS as long-term therapy in AIDS patients receiving ZDV.

Abstract 3532: Antitumor activity of SR16388 on multiple myeloma targets factors needed for adaptation to bone marrow microenvironment

Multiple myeloma (MM) is a B-cell malignancy that is characterized by accumulation of clonal plasma cells in the bone marrow. The bone marrow microenvironment plays a critical role in MM cell pathogenesis and progression. In the bone marrow milieu, MM cells adhere to the bone marrow stromal cells and trigger the secretion of growth factors such as vascular endothelial growth factor (VEGF). VEGF in turn upregulates the secretion of interleukin-6 (IL-6), the major cytokine that mediates MM cell growth and survival in part through the activation of Janus kinase (Jak)/signal transducers and activators of transcription 3 (STAT3). VEGF also stimulates the proliferation of endothelial and stromal cells and induces angiogenesis via its receptors. Thus, targeting both myeloma cells and bone marrow microenvironment is a valid approach for the development of therapeutic agents for MM. At SRI International, we have developed an antiestrogen, SR16388, that binds to ER-α, ER-β and ERR-α with potent antitumor and antiangiogenic properties. We have previously reported that SR16388 inhibited MM cell proliferation in vitro and MM tumors in vivo. In the current study, we are presenting data to show that the inhibitory effect of SR16388 on MM cell proliferation was achieved by inducing apoptosis, and arresting cells at the G2 phase. Furthermore, at nanomolar concentrations SR16388 inhibited IL-6-induced cell proliferation and blocked the activation of STAT3 induced by IL-6 in RPMI-8226 human MM cells. In human endothelial cells, SR16388 inhibited VEGF-induced cell proliferation and VEGF-induced phosphorylation of STAT3. In a RPIM-8226 tumor xenograft model, the microvessel density in the tumor tissue was markedly reduced by treatment with SR16388. We are currently investigating the effect of SR16388 in combination with known therapeutic agents on the growth of MM tumors in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3532. doi:10.1158/1538-7445.AM2011-3532

Abstract 731: SRI-28731, a highly potent and selective MAP4K4 (HGK) inhibitor for cancer therapy

MAP4K4, a Ser/Thr kinase, was identified as an important pro-migratory kinase in an siRNA screen, targeting 5,234 human genes for modulators of tumor cell motility. MAP4K4 siRNA potently suppressed cell invasion and migration of multiple cancer cell lines, indicating a broad role in cell motility. There are no drugs in the clinic that are known to specifically target MAP4K4 for cancer therapy. We have successfully developed an orally active, highly effective and selective MAP4K4 inhibitor (SRI-28731) with potent in vitro and in vivo anticancer activity. SRI-28731 is more potent than Paclitaxel (Taxol) against most of the breast cancer cell lines tested. SRI-28731 exhibits more potent activities against triple negative (MDA-MB-231, BT549 and Hs578T) than estrogen-dependent (T47D and MCF-7) breast cancer cell lines, and its potency is positively correlated with MAP4K4 expression in cancer cell lines. SRI-28731 is also more potent than Docetaxel against both androgen-dependent (LNCaP) and -independent (PC-3 and DU-145) prostate cancer cell lines. In vitro mechanistic studies showed that SRI-28731 induced apoptosis and a time-dependent M phase arrest. Treatment with SRI-28731 (12.5, 25 and 50 mg/kg/day) caused a significant dose-dependent growth reduction of PC-3 tumors (30%, 61% and 88% growth inhibition, respectively), while Docetaxel at its MTD (7.5 mg/kg; Q3Dx2) produced only 10-15% growth inhibition. At the end of PC-3 tumor xenograft studies, we conducted an ex vivo invasion assay using PC-3 tumor cells isolated from tumor-bearing mice. SRI-28731 significantly reduced ex vivo tumor cell invasion by ∼80%. Pharmacokinetic studies showed that SRI-28731 could be detected in plasma up to 8 hours after oral dosing, and drug plasma concentrations remained above the IC50 values needed to inhibit prostate or breast cancer proliferation. To quantitatively define the kinase selectivity of SRI-28731, we tested the interaction of SRI-28731 with 456 kinase protein kinases (KINOMEscan), followed by in vitro pharmacology studies. Our data indicated that SRI-28731 is a highly selective Type-II MAP4K4 inhibitor. Type II kinase inhibitors bind to both the ATP site and an adjacent hydrophobic site exposed in the non-activated kinase state. Generally, type II inhibitors show higher selectivity for targets, and act primarily by locking the equilibrium switch between conformational states in a way that prevents kinase activation, rather than directly inhibiting it. Elevated MAP4K4 expression is strongly associated with higher rate of metastasis, and is regarded as an independent predictor of overall survival in cancer patients. Since MAP4K4 is overexpressed in many human cancer cell lines but is undetectable in non-transformed epithelial cells, targeting MAP4K4 may provide effective anti-metastatic therapy with limited side effects on normal tissues. Citation Format: Chih-Tsung Chang, Jaehyeon Park, Wei Zhou, Xiaohe Liu, Barbara Sato, Dominic Dinh, Anna Furimsky, Lucia Beviglia, Lidia Sambucetti, Ling Jong. SRI-28731, a highly potent and selective MAP4K4 (HGK) inhibitor for cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 731. doi:10.1158/1538-7445.AM2014-731