SA Mice Research Articles

Mechanisms Mediating the Synergistic Anticancer Effects of Combined γ-Tocotrienol and Celecoxib Treatment

AIM: To characterize the intracellular signaling mechanisms mediating the synergistic anticancer effects of combined γ-tocotrienol and celecoxib treatment in neoplastic +SA mouse mammary epithelial cells in vitro. METHODS: +SA mammary tumor cells in different treatment groups were maintained in serum-free defined media containing 10ng/ml EGF as a mitogen and exposed to various doses of γ-tocotrienol and celecoxib alone or in combination. After a 96 hr culture period, cells were collected and whole cell lysates were subjected to Western blot analysis to determine treatment effects on intracellular signaling proteins associated with EGF-dependent mitogenesis and survival, and prostaglandin synthesis and responsiveness. RESULTS: Treatment with high doses of γ-tocotrienol or celecoxib alone inhibited Akt activation and downstream signaling and NFκB activation. Similar treatment with γ-tocotrienol also decreased concentration and activation of ErbB2-4 receptors, whereas celecoxib only inhibited ErbB2-4 receptor activation. In contrast, combined treatment with subeffective doses of γ-tocotrienol and celecoxib resulted in a large decrease ErbB2-4 receptor levels and activation, a decrease in PGE(2) levels, and a corresponding increase in prostaglandin EP2 and EP4 receptor levels. Combined treatment also induced an increase in the prostaglandin catabolizing enzyme, PGDH. CONCLUSION: The synergistic anticancer effects of combined low dose γ-tocotrienol and celecoxib treatment in +SA mammary tumor cells are mediated by COX-2-dependent mechanisms associated with a suppression in PGE(2) levels, as well as, COX-2-independent mechanisms associated with a reduction in ErbB2-4 receptor levels, activation, and subsequent reduction in downstream Akt and NFκB mitogenic signaling.

Abstract 5425: Characterization of synergistic antiproliferative effects of combined γ-tocotrienol and celecoxib treatment on neoplastic mouse mammary epithelial cells in vitro

Abstract In the present study, synergistic antiproliferative effects of combined treatment of γ-tocotrienol and celecoxib on +SA mouse mammary epithelial cells were evaluated and characterized for their mechanism(s) of action. Earlier studies have shown that these effects are mediated partly by inhibition of PGE2, which is a direct indicator of COX-2 activity in cells. Since 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD+-dependent oxidation of 15(S)-hydroxyl group of prostaglandins and has been considered a key enzyme in biological inactivation of prostaglandins, the effects of these drugs on intracellular levels of 15-PGDH were assessed. Consistent with earlier findings, western blot analysis showed increased levels of 15-PGDH by individual treatment of effective doses of both γ-tocotrienol (3.5 μM) and celecoxib (20 μM). Combined treatment with subeffective doses of γ-tocotrienol (0.25 μM) and celecoxib (2.5 μM) showed increased levels of 15-PGDH as compared to the same individual doses when treated alone. These studies indicate that the antiproliferative effects are mediated by inducing the expression of 15-PGDH, which leads to enhanced inactivation of PGE2. Similar to earlier studies which showed that these effects were associated with a suppression of Akt and NFκB activity, the present studies showed that treatment with 3.5 μM γ-tocotrienol alone induced a relatively large decrease in phospho-PDK-1 and phospho-Akt levels, but had no effect on PI3K p85, PTEN and phospho-PTEN levels. In contrast, treatment with 20 μM celecoxib alone was associated with a relatively large decrease in PI3K p85 levels. Combined treatment with subeffective doses of γ-tocotrienol and celecoxib caused a large decrease in PI3K, phospho-PDK-1, phospho-Akt and phospho-GSK-3β levels as compared to vehicle treated controls. In addition, a large decrease in phospho-IKK-α/β, phospho-IκB-α, and phospho-NFκB levels was observed in the combination treatment group relative to the same subeffective doses when given alone. In conclusion, these studies show that synergistic antiproliferative effects of combination treatment of γ-tocotrienol and celecoxib are associated with two different mechanisms; 1) a COX-2-dependent mechanism of induction of 15-PGDH leading to enhanced inactivation of PGE2; and 2) a COX-2-independent mechanism of attenuation of EGF-dependent PI3K/PDK-1/Akt and NFκB mitogenic signaling pathways. Further studies are required to evaluate the role of prostaglandin and EGF receptors in mediating these two independent mechanisms. This study was supported by grants from NIH (CA86833) and First Tech International Ltd. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5425.

CaMKII Phosphorylation of RyR2 Mediates Heart Failure Development

Abnormal regulation of cardiac ryanodine receptor RyR2 by Ca2+/calmodulin‐dependent protein kinase 2 (CaMKII) has been identified as a potential cause of Ca2+ leakage and contractile dysfunction in heart failure. To test the hypothesis that CaMKII phosphorylation of RyR2 is crucial for heart failure development, we generated RyR2 knockin mice in which the CaMKII phosphorylation site S2814 is either mutated into aspartate (SD) to mimic CaMKII phosphorylation, or into alanine (SA) to prevent phosphorylation. Echocardiography revealed a decrease in cardiac function in SD mice at 12 months of age. Both WT and SA mice were subjected to transverse aortic constriction (TAC) to induce pressure overload. At 8 weeks after TAC, SA mice displayed a similar hypertrophic response as WT TAC mice. At 16 weeks after TAC, however, ejection fraction was significantly higher in SA mice compared to WT TAC mice, indicating an inhibition of heart failure development. This rescue effect was further verified by a lower lung‐weight‐to‐tibia‐length ratio and a decrease in expression levels of cardiac stress genes in SA mice. Furthermore, Ca2+ imaging in isolated cardiomyocytes demonstrated a decreased SR Ca2+ leak in SA TAC mice. Our results demonstrate that CaMKII phosphorylation of RyR2 is both sufficient and necessary for heart failure development.R.J.v.O. is recipient of the APS Postdoctoral Fellowship in Physiological Genomics

ACE2 over‐expression regulates oxidative stress gene expression in the brainstem

Angiotensin (Ang) I converting enzyme type 2 (ACE2) cleaves Ang II into the vasodilator Ang‐(1–7). We previously reported that ACE2 is expressed in the brain and involved in the prevention of hypertension via regulation of autonomic function. We also showed that ACE2 KO mice have increased levels of reactive oxygen species (ROS) in the brain. To clarify the role of ACE2 in the regulation of ROS levels, we performed an oxidative stress PCR array using brainstems from SA mice, with brain‐specific ACE2 overexpression, and control littermates (NT).In SA mice, the main ROS‐producing enzyme, NADPH oxidase, subunits genes Nox1 (gp91phox homolog), Nox4, p22phox and p67phox were downregulated by −3.1, −1.6, −13.4 and −11 folds respectively, compared to controls. The NADPH oxidase activator 1 (Noxa1) and the dual oxidase1 (Duox1), which generates hydrogen peroxide, were also decreased by −2.6 and −1 fold, respectively. Anti oxidant stress enzymes like glutathione peroxidase 1, 3, 6, 8 subunits were downregulated by −180, −3, −2.4, −3.1 folds and so were SOD 1 (−1.2 fold) and 3 (−1.8 fold). Moreover, there was a trend to a reduction in lipid peroxidation (Tbars assay) in the brainstem of SA mice. These data show that ACE2 is regulating the expression of multiple genes involved in the formation and removal of ROS in the brainstem, which may contribute to the prevention of hypertension in this model. NIH RR018766 and HL093178

Serines 13 and 16 Are Critical Determinants of Full-Length Human Mutant Huntingtin Induced Disease Pathogenesis in HD Mice

The N-terminal 17 amino acids of huntingtin (NT17) can be phosphorylated on serines 13 and 16; however, the significance of these modifications in Huntington's disease pathogenesis remains unknown. In this study, we developed BAC transgenic mice expressing full-length mutant huntingtin (fl-mhtt) with serines 13 and 16 mutated to either aspartate (phosphomimetic or SD) or alanine (phosphoresistant or SA). Both mutant proteins preserve the essential function of huntingtin in rescuing knockout mouse phenotypes. However, fl-mhtt-induced disease pathogenesis, including motor and psychiatric-like behavioral deficits, mhtt aggregation, and selective neurodegeneration are abolished in SD but preserved in SA mice. Moreover, modification of these serines in expanded repeat huntingtin peptides modulates aggregation and amyloid fibril formation in vitro. Together, our findings demonstrate that serines 13 and 16 are critical determinants of fl-mhtt-induced disease pathogenesis in vivo, supporting the targeting of huntingtin NT17 domain and its modifications in HD therapy.

Biocatalysis of the Anticancer Sipholane Triterpenoids

The Red Sea sponge Callyspongia (= Siphonochalina) siphonella is a rich source of sipholane triterpenoids. Biocatalysis of the major sipholanes, sipholenol A (1) and sipholenone A (2), respectively, by Mucor ramannianus ATCC 9628 and Cunninghamella elegans ATCC 7929 afforded four new metabolites 3 - 6 along with sipholenol G and 28-hydroxysipholenol A. Major sipholanes along with their biocatalytic products were investigated for their antiproliferative activity against the highly malignant +SA mouse mammary epithelial cell line. Sipholenone A (2) was the most active sipholane inhibiting +SA cell proliferation with an IC(50) value of 20 - 30 microM. Sipholenone A, also, showed cytotoxicity against MCF-7 at a dose of 0.9 microM and antiangiogenic activity in the CAM (chorio-allantoic membrane) assay. This is the first report on anticancer activity of these triterpenoids.

γ‐Tocotrienol inhibits ErbB3‐dependent PI3K/Akt mitogenic signalling in neoplastic mammary epithelial cells

The antiproliferative effects of gamma-tocotrienol are associated with suppression in epidermal growth factor (EGF)-dependent phosphatidylinositol-3-kinase (PI3K)/PI3K-dependent kinase-1 (PDK-1)/Akt mitogenic signalling in neoplastic mammary epithelial cells. Studies were conducted to investigate the direct effects of gamma-tocotrienol treatment on specific components within the PI3K/PDK-1/Akt mitogenic pathway. +SA cells were grown in culture and maintained in serum-free media containing 10 ng/ml EGF as a mitogen. Treatment with 0-8 microm gamma-tocotrienol resulted in a dose-responsive decrease in the +SA cell growth and a corresponding decrease in phospho-Akt (active) levels. However, gamma-tocotrienol treatment had no direct inhibitory effect on Akt or PI3K enzymatic activity, suggesting that the inhibitory effects of gamma-tocotrienol occur upstream of PI3K, possibly at the level of the EGF-receptor (ErbB1). Additional studies were conducted to determine the effects of gamma-tocotrienol on ErbB receptor activation. Results showed that gamma-tocotrienol treatment had little or no effect on ErbB1 or ErbB2 receptor tyrosine phosphorylation, a prerequisite for substrate interaction and signal transduction, but did cause a significant and progressive decrease in the ErbB3 tyrosine phosphorylation. Because ErbB1 or ErbB2 receptors form heterodimers with the ErbB3 receptor, and ErbB3 heterodimers have been shown to be the most potent activators of PI3K, these findings strongly suggest that the antiproliferative effects of gamma-tocotrienol in neoplastic +SA mouse mammary epithelial cells are mediated by a suppression in ErbB3-receptor tyrosine phosphorylation and subsequent reduction in PI3K/PDK-1/Akt mitogenic signalling.

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells.

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.

Role of Caspase-8 Activation in Mediating Vitamin E-Induced Apoptosis in Murine Mammary Cancer Cells

The vitamin E family of compounds is divided into two subgroups, tocopherols and tocotrienols. However, tocotrienols display more potent apoptotic activity in mammary cancer cells. Although the mechanism(s) mediating tocotrienol-induced apoptosis is presently unknown, apoptosis is carried out by activation of initiator caspases (caspase-8 or -9) that subsequently activate effector caspases (caspase-3, -6, or -7). Studies were conducted to determine whether tocotrienol-induced apoptosis is mediated by activation of the caspase-8 and/or caspase-9 pathway. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained on serum-free media. Treatment with tocotrienol-rich-fraction of palm oil (TRF) and γ-tocotrienol, but not α-tocopherol, induced a dose-dependent decrease in +SA cell viability. TRF- and γ-tocotrienol-induced cell death resulted from apoptosis, as determined by DNA fragmentation and positive TUNEL assay staining. Additional studies showed that treatment with 50 μM TRF or 20 μM γ-tocotrienol increased intracellular activity and levels of processed caspase-8 and -3 but not caspase-9. Furthermore, treatment with specific caspase-8 or -3 inhibitors, but not caspase-9 inhibitor, completely blocked the tocotrienol-induced apoptosis in +SA cells. These findings demonstrate that tocotrienol-induced apoptosis in +SA mammary cancer cells is mediated through activation of the caspase-8 signaling pathway and is independent of caspase-9 activation.

Mouse brain gene expression changes after acute and chronic amphetamine.

Gene expression changes are candidate mechanisms to contribute to long-term consequences of psychostimulant use. We use microarrays to examine the expression of 6340 genes in brains of mice killed 5 or 20 h following 14 day, twice-daily treatments with saline (SS), saline followed by a single 7.5 mg/kg amphetamine dose (SA), or repeated 7.5 mg/kg amphetamine doses (AA) that produce sensitization but no clear-cut neuronal toxicities. Arrays display robust hybridization for about 3600 transcripts. One hundred and seventeen of these expressed transcripts are candidate positives for drug-related changes, displaying > 1.8-fold differences from SS control values in whole brains of either SA or AA mice. Five transcripts reveal altered expression in both AA and SA mice. SA mostly enhances expression while AA treatments largely reduce expression. Fourteen SA and four AA changes in whole brain mRNA were replicated by > 1.8-fold changes in independent microarray assessments of either cerebral cortical or brainstem mRNAs, with more changes identified in frontal than in entorhinal/parietal cortical samples. About one-quarter of these changes persist in initial studies of mice killed 20 h after the last amphetamine injection. Each of these genes, including transcription factor, cellular regulatory, structural and other gene family members, are candidates to contribute to brain adaptations to psychostimulants.

Diabetic microangiopathy in KK mice: V. Effect of early glyburide treatment on kidney glucosyltransferase activity

KK mice with spontaneous hereditary diabetes were treated with 0.5 mg/kg of glyburide once a day by intubation for 5 days a week from 20 days of age. A representative number of KK mice received 0.9% saline (0.1 ml/10 g) and served as controls for glyburide treated mice. The SA mice were not treated with saline. After an 18 hr fast, both saline and glyburide-treated mice were sacrificed at 40, 50, 70, and 100 days of age. Glyburide-treated mice showed a decreased trend in blood sugar levels, and this was significant in 50- and 70-day-old mice. The serum IRI levels increased significantly in the 70-day-old glyburide-treated mice. Glyburide caused a significant decrease in glucosyltransferase activity in the renal cortex of the KK mice at 40, 50, and 70 days of age. Early glyburide treatment is effective not only in preventing the progression of glomerulosclerosis but also in normalizing biochemical changes contributory to genetic diabetic glomerulosclerosis in KK mice.