Background Neuroplasticity and neurorehabilitation have been extensively studied in animal models of stroke to guide clinical rehabilitation of stroke patients. Similar studies focused on traumatic brain injury (TBI) are lacking. Objective The current study was designed to examine the effects of individual and combined rehabilitative approaches, previously shown to be beneficial following stroke, in an animal model of moderate/severe TBI, the controlled cortical impact (CCI). Methods Rats received a unilateral CCI, followed by reach training, voluntary exercise, or unimpaired forelimb constraint, alone or in combination. Forelimb function was assessed at different time points post-CCI by tests of skilled reaching, motor coordination, and asymmetrical limb use. Results Following CCI, skilled reaching and motor coordination were significantly enhanced by combinations of rehabilitation strategies, not by individual approaches. The return of symmetrical limb use benefited from forelimb constraint alone. None of the rehabilitation strategies affected the size of injury, suggesting that enhanced behavioral function was not a result of neuroprotection. Conclusions The current study has provided evidence that individual rehabilitation strategies shown to be beneficial in animal models of stroke are not similarly sufficient to enhance behavioral outcome in a model of TBI. Motor rehabilitation strategies for TBI patients may need to be more intense and varied. Future basic science studies exploring the underlying mechanisms of combined rehabilitation approaches in TBI as well as clinical studies comparing rehabilitation approaches for stroke versus TBI would prove fruitful.
Summary The mechanisms by which cancer cell-intrinsic CYP monooxygenases promote tumor progression are largely unknown. CYP3A4 was unexpectedly associated with breast cancer mitochondria and synthesized arachidonic acid (AA)-derived epoxyeicosatrienoic acids (EETs), which promoted the electron transport chain/respiration and inhibited AMPKα. CYP3A4 knockdown activated AMPKα, promoted autophagy, and prevented mammary tumor formation. The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 AA epoxygenase activity. HBB also inhibited growth of established ER+ mammary tumors and suppressed intratumoral mTOR. CYP3A4 AA epoxygenase inhibition by biguanides thus demonstrates convergence between eicosanoid activity in mitochondria and biguanide action in cancer, opening a new avenue for cancer drug discovery.
#6019 Background: Literature reports demonstrate that genistein, a soy phytoestrogen, shows a biphasic response to cell proliferation in breast cancer cells. Genistein increases cell proliferation at low concentrations and decreases cell proliferation at high concentrations. Our previous studies suggest that at low concentrations of genistein the cell proliferative response depends on the type of ER. At high concentrations genistein is believed to act as a tyrosine kinase inhibitor. Currently, the mechanisms of genistein's effect on breast cancer cell proliferation are unclear. The aim of this study was to evaluate the mechanisms of the biphasic response of genistein on breast cancer cell proliferation, specifically by determining the effect of genistein on ER-related cell signaling molecules involved in cell proliferation, cell survival and apoptosis. Based on literature reports, the ER-related cell signaling molecules chosen for this study were ERK1/2, p90RSK, JNK, Akt and NFκB. ERK1/2 and, p90RSK are involved in cell proliferation; JNK, Akt and NFκB are involved in cell survival and/or apoptosis.
 Methods: The effect of genistein at 1 µM (low concentration) and 100 µM (high concentration) on cell signaling molecules was determined by a BioPlex Phosphoprotein detection kit. Results obtained from the BioPlex assay were confirmed by immunodetection. Cell proliferation was determined at 24, 48, and 72 hrs by the MTT (3-[4,5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium) assay. This study was carried out in T47D (ERα and ERβ) and MDA-MB-231 (ERβ) breast cancer cells.
 Results: At 100 µM genistein exposure we observed a decrease in phosphorylated p90RSK (40%) in T47D cells and an increase in phosphorylated JNK (45%) in MDA-MB-231 cells in a statistically significant (p<0.05) manner. At 100 µM genistein exposure, a statistically significant (p<0.001) decrease in cell proliferation was observed in T47D cells (20-40%) and MDA-MB-231 cells (30-60%) as compared to the control. At 1 µM genistein exposure, there was a statistically significant (p<0.05) decrease (15%) of phosphorylated JNK in MDA-MB-231 cells. MDA-MB-231 cells exposed to 1 µM genistein did not show any statistically significant difference in cell proliferation as compared to the control. T47D cells exposed to 1 µM of genistein for 24 – 72 hours showed a statistically significant (p<0.001) increase (25 to 70%) in cell proliferation.
 Conclusions: Our data suggests that in T47D cells which have both ERs, decreased cell proliferation due to high concentrations of genistein is probably due to a decrease in phosphorylated p90RSK, an ER-related cell proliferation protein. In MDA-MB-231 cells with only ERβ, the decrease in cell proliferation is probably due to an increase in phosphorylated JNK, an apoptotic protein. Our results suggest that at high concentrations of genistein, the cellular pathways of inhibition of breast cancer cell proliferation likely depend on ER status. This study reveals the importance of genistein as an effective chemotherapeutic agent in breast cancers that contain both ERs and only ERβ. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6019.
While cytochrome P450 enzymes (CYPs) are implicated in tumor angiogenesis through biosynthesis of epoxyeicosatrienoic acids (EETs), little is known about breast cancer cell-intrinsic CYPs that exhibit epoxygenase activity, such as CYP3A4. In an orthotopic breast cancer model, silencing of epithelial CYP3A4 suppressed angiogenesis-related escape of ER+ breast tumors from dormancy. While the diabetes drug metformin inhibits mitochondrial complex I and inhibits tumor growth, how it does so is unknown. Metformin inhibited CYP epoxygenase activity and co-crystallized in the active site of CYP3A4, hydrogen bonding with arginine 212, allowing the development of hexyl-benzyl-biguanide (HBB) as a CYP3A4 inhibitor using molecular modeling. HBB exhibited more than 10-fold greater potency than metformin for inhibition of ER+ mammary tumor growth and inhibited associated tumor angiogenesis. HBB inhibited EET biosynthesis ∼40-fold more potently than metformin and was ∼40-fold more potent for activation of AMPK phosphorylation. EETs suppressed and CYP silencing promoted AMPK phosphorylation, linking CYPs with AMPK regulation in breast cancer. HBB depolarized mitochondria, reduced oxygen consumption rates and suppressed the Warburg effect, while EETs restored the mitochondrial membrane potential. CYP3A4 silencing and HBB treatment increased reactive oxygen species (ROS) production, suggesting that CYPs suppress cancer cell death, in part, through suppression of ROS. CYP3A4 silencing sensitized breast cancer cells to hormonal therapy and chemotherapy, abrogated by EETs. Because EETs are autocrine, paracrine and endocrine, these results implicate CYPs in tumor growth, in part, through cell-cell mediation of mitochondrial homeostasis and demonstrate the potential of CYP3A4 as a therapeutic target in breast cancer. Citation Format: Zhijun Guo, Irina F. Sevrioukova, Eric Hanse, Ilia Denisov, Xia Zhang, Ting-Lan Chiu, Daniel Swedien, Justin Stamschror, Juan Alvarez, William Marerro Ortiz, Monique Morgan, Michael Maher, Kathryn J. Chavez, Dafydd Thomas, Young Kyung Bae, Jonathan Henriksen, Beverly Norris, Robert J. Schumacher, Henry Wang, Robin Bliss, Haitao Chu, Rebecca Cuellar, Thomas L. Poulos, Stephen G. Sligar, William Atkins, Stephen Schmechel, Jorge Capdevila, John Falck, Ian Blair, Jeffrey P. Jones, Gunda Georg, Kalpna Gupta, Ameeta Kelekar, Elizabeth Amin, David A. Potter. CYP3A4 epoxygenase activity mediates ER+ mammary tumor growth and angiogenesis, in part, through EET biosynthesis and is inhibited by biguanides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3568. doi:10.1158/1538-7445.AM2015-3568
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