Hye Joung Choi

Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death.

Oxidative stress can induce cytotoxicity in neurons, which plays an important role in the etiology of neuronal damage and degeneration. This study sought to determine the cellular and biochemical mechanisms underlying resveratrols protective effect against oxidative neuronal death. Cultured HT22 cells, an immortalized mouse hippocampal neuronal cell line, were used as an in vitro model, and oxidative stress and neurotoxicity were induced in these neuronal cells by exposure to high concentrations of glutamate. Resveratrol strongly protected HT22 cells from glutamate-induced oxidative cell death. Resveratrols neuroprotective effect was independent of its direct radical scavenging property, but instead was dependent on its ability to selectively induce the expression of mitochondrial superoxide dismutase (SOD2) and, subsequently, reduce mitochondrial oxidative stress and damage. The induction of mitochondrial SOD2 by resveratrol was mediated through the activation of the PI3K/Akt and GSK-3beta/beta-catenin signaling pathways. Taken together, the results of this study show that up-regulation of mitochondrial SOD2 by resveratrol represents an important mechanism for its protection of neuronal cells against oxidative cytotoxicity resulting from mitochondrial oxidative stress.

Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells

Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time-dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8-12 h), glutamate induced mostly necrosis, whereas at late time points (16-24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.

Role of Cyclin B1/Cdc2 Up-Regulation in the Development of Mitotic Prometaphase Arrest in Human Breast Cancer Cells Treated with Nocodazole

Background During a normal cell cycle, the transition from G2 phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes. Methodology/Principal Findings It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment), and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest. Conclusions/Significance These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associated with exposure to microtubule inhibitors.

Critical role of the JNK-p53-GADD45α apoptotic cascade in mediating oxidative cytotoxicity in hippocampal neurons

BACKGROUND AND PURPOSE Glutamate‐induced oxidative stress plays a critical role in the induction of neuronal cell death in a number of disease states. We sought to determine the role of the c‐Jun NH2‐terminal kinase (JNK)‐p53‐growth arrest and DNA damage‐inducible gene (GADD) 45α apoptotic cascade in mediating glutamate‐induced oxidative cytotoxicity in hippocampal neuronal cells.

Induction of a reversible, non‐cytotoxic S‐phase delay by resveratrol: implications for a mechanism of lifespan prolongation and cancer protection

Background and purpose:  Resveratrol (RES) has been shown to prolong lifespan and prevent cancer formation. At present, the precise cellular mechanisms of RES actions are still not clearly understood, and this is the focus of this study.

Targeting interferon response genes sensitizes aromatase inhibitor resistant breast cancer cells to estrogen-induced cell death

IntroductionEstrogen deprivation using aromatase inhibitors (AIs) is currently the standard of care for postmenopausal women with hormone receptor-positive breast cancer. Unfortunately, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. The mechanism by which resistance occurs is still not completely known, however, recent studies suggest that impaired/defective interferon signaling might play a role. In the present study, we assessed the functional role of IFITM1 and PLSCR1; two well-known interferon response genes in AI resistance.MethodsReal-time PCR and Western blot analyses were used to assess mRNA and protein levels of IFITM1, PLSCR1, STAT1, STAT2, and IRF-7 in AI-resistant MCF-7:5C breast cancer cells and AI-sensitive MCF-7 and T47D cells. Immunohistochemistry (IHC) staining was performed on tissue microarrays consisting of normal breast tissues, primary breast tumors, and AI-resistant recurrence tumors. Enzyme-linked immunosorbent assay was used to quantitate intracellular IFNα level. Neutralizing antibody was used to block type 1 interferon receptor IFNAR1 signaling. Small interference RNA (siRNA) was used to knockdown IFITM1, PLSCR1, STAT1, STAT2, IRF-7, and IFNα expression.ResultsWe found that IFITM1 and PLSCR1 were constitutively overexpressed in AI-resistant MCF-7:5C breast cancer cells and AI-resistant tumors and that siRNA knockdown of IFITM1 significantly inhibited the ability of the resistant cells to proliferate, migrate, and invade. Interestingly, suppression of IFITM1 significantly enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells and markedly increased expression of p21, Bax, and Noxa in these cells. Significantly elevated level of IFNα was detected in AI-resistant MCF-7:5C cells compared to parental MCF-7 cells and suppression of IFNα dramatically reduced IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells. Lastly, neutralizing antibody against IFNAR1/2 and knockdown of STAT1/STAT2 completely suppressed IFITM1, PLSCR1, p-STAT1, and p-STAT2 expression in the resistant cells, thus confirming the involvement of the canonical IFNα signaling pathway in driving the overexpression of IFITM1 and other interferon-stimulated genes (ISGs) in the resistant cells.ConclusionOverall, these results demonstrate that constitutive overexpression of ISGs enhances the progression of AI-resistant breast cancer and that suppression of IFITM1 and other ISGs sensitizes AI-resistant cells to estrogen-induced cell death.

Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol

Earlier studies showed that 2-methoxyestradiol (2ME(2)), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G(2)/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME(2)-induced cell cycle arrest. We found that 2ME(2) can selectively induce mitotic prometaphase arrest, but not G(2) phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12-24h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME(2)-induced prometaphase arrest. The 2ME(2)-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME(2)-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME(2) causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.

Role of cyclin B1/Cdc2 in mediating Bcl-XL phosphorylation and apoptotic cell death following nocodazole-induced mitotic arrest.

Treatment of cancer cells with microtubule inhibitors causes mitotic arrest, which subsequently leads to cell death via activation of the intrinsic apoptotic pathway. Mitotically arrested cells typically display increased phosphorylation (i.e., inactivation) of two key anti‐apoptotic proteins, Bcl‐2 and Bcl‐XL, but the mechanisms that regulate their phosphorylation as well as their role in apoptotic cell death following mitotic arrest are still poorly understood at present, which are the focus of this study. We recently showed that cyclin B1 and cell division cycle 2 (Cdc2) proteins are strongly up‐regulated in human breast cancer cells following treatment with nocodazole (a prototypical microtubule inhibitor), and their up‐regulation plays a critical role in the development of mitotic prometaphase arrest. In this study, we present evidence showing that the up‐regulated cyclin B1/Cdc2 complex in nocodazole‐treated human breast cancer cells is also responsible for the increased phosphorylation of Bcl‐2 and Bcl‐XL. However, only the increased phosphorylation of Bcl‐XL, but not the phosphorylation of Bcl‐2, contributes to subsequent activation of the intrinsic cell death pathway. In addition, evidence is presented to show that mitotic arrest deficient 2 (MAD2) is a key upstream mediator of the up‐regulation of cyclin B1/Cdc2 as well as the subsequent increase in phosphorylationof Bcl‐2 and Bcl‐XL in nocodazole‐treated cancer cells. Together, these results reveal that the up‐regulated cyclin B1/Cdc2 complex not only mediates prometaphase arrest in nocodazole‐treated cells, but also activates the subsequent intrinsic cell death pathway in these cells via increased phosphorylation of Bcl‐XL.

Abstract 25: Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Estrogen deprivation using aromatase inhibitors (AIs) is the standard of care for postmenopausal women with hormone receptor-positive breast cancer. However, the majority of patients treated with AIs eventually develop resistance, inevitably resulting in patient relapse and, ultimately, death. We have previously reported the development of an AI-resistant breast cancer cell line, MCF-7:5C, which was clonally derived from parental MCF-7 breast cancer cells following long term estrogen deprivation. A unique phenotype of AI-resistant MCF-7:5C cells is that they grow in the absence of estrogen; however, when they are treated with estradiol they undergo apoptosis/cell death through activation of the mitochondrial death pathway. More recently, we have also discovered that AI-resistant MCF-7:5C cells constitutively overexpress the interferon stimulated gene IFITM1. IFITM1 is a cell surface membrane protein that is most well-known for its ability to block viral replication, however, its role in breast cancer and AI-resistance is not known. We found that IFITM1 was constitutively overexpressed at the protein (> 20-fold) and mRNA (> 15-fold) level in AI-resistant MCF-7:5C breast cancer cells compared to parental MCF-7 cells and that its overexpression strongly correlated with enhanced growth and increased aggressiveness. Clinical data also indicated that IFITM1 was overexpressed in 36 out of 40 AI-resistant breast tumor samples compared to AI-sensitive tumors. In this study, we assessed the functional role of IFITM1 in AI-resistant MCF-7:5C cells. Notably, we found that knockdown of IFITM1 significantly induced cell death in MCF-7:5C cells and it reduced cell proliferation. The induction of cell death as a result of IFITM1 knockdown was associated with increases in p21, Bax, Noxa, and Puma expression; however, p53 was not altered in these cells. To validate the effect observed with IFITM1 knockdown in our resistant cells we used a second shRNA targeting IFITM1. We found that shRNA knockdown of IFITM1 induced PARP cleavage and p21, reduced cell proliferation, and induced cell death in AI-resistant MCF-7:5C cells. Moreover, we found that knockdown of IFITM1 dramatically enhanced estradiol-induced cell death in AI-resistant MCF-7:5C cells which was associated with induction of pro-apoptotic proteins and p21 via a p53-independent mechanism. Further analysis revealed that knockdown of STAT1 and STAT2, which are critical regulators of IFITM1, also significantly enhanced estradiol-induced apoptosis in MCF-7:5C cells. Taken together, these findings indicate that overexpression of IFITM1 plays a critical role in breast cancer and AI-resistance and that targeting this gene can sensitize AI-resistant cells to estrogen-induced death. Furthermore, these results suggest that IFITM1 can regulate the mitochondrial pathway through its interactions with anti-apoptotic proteins. Citation Format: Hye Joung Choi, Joan Lewis-Wambi. Loss of interferon-induced transmembrane protein 1 enhances estrogen-induced cell death in AI-resistant breast cancer cells. [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 25. doi:10.1158/1538-7445.AM2015-25

PDIp is a major intracellular oestrogen-storage protein that modulates tissue levels of oestrogen in the pancreas.

E(2) (17β-oestradiol), a female sex hormone, has important biological functions in a womans body. The pancreas, often considered a non-classical E(2)-targeting organ, is known to be functionally regulated by E(2), but little is known about how oestrogen actions are regulated in this organ. In the present study we report that PDIp (pancreas-specific protein disulfide isomerase), a protein-folding catalyst, can act as a major intracellular E(2) storage protein in a rat model to modulate the pancreatic tissue level, metabolism and action of E(2). The purified endogenous PDIp from both rat and human pancreatic tissues can bind E(2) with a K(d) value of approximately 150 nM. The endogenous PDIp-bound E(2) accounts for over 80% of the total protein-bound E(2) present in rat and human pancreatic tissues, and this binding protects E(2) from metabolic disposition and prolongs its duration of action. Importantly, we showed in ovariectomized female rats that the E(2) level in the pancreas reaches its highest level (9-fold increase over its basal level) at 24-48 h after a single injection of E(2), and even at 96 h its level is still approximately 5-fold higher. In contrast, the E(2) level in the uterus quickly returns to its basal level at 48 h after reaching its maximal level (approximately 2-fold increase) at 24 h. Taken together, these results show for the first time that PDIp is a predominant intracellular oestrogen storage protein in the pancreas, which offers novel mechanistic insights into the accumulation and action of oestrogen inside pancreatic cells.