Anti-obesity Effects of 3-hydroxychromone derivative, a Novel Small-Molecule Inhibitor of Glycogen Synthase Kinase-3

이수호 경희대학교 일반대학원 2012 국내박사

(A) novel role of AMP-activated protein kinase in chemotherapeutic effects in tumor

김학수 경희대학교 대학원 2010 국내박사

Part I Cisplatin is one of the most effective and widely used chemotherapeutic agents. However, one of the most salient limitations to the clinical application of cisplatin is the acquired or intrinsic drug resistance exhibited by some tumors. In the present study, we have assessed the potential of an intracellular energy balancing system as a target for augmentation of cisplatin sensitivity in tumors. AMP-activated protein kinase (AMPK) regulates the energy balance system by monitoring intracellular energy status. Here we demonstrate that AMPK is rapidly activated by cisplatin in AGS and HCT116 cancer cells. The inhibition of AMPK in those cells, and in xenograft of HCT116 resulted in a remarkable increase in cisplatin-induced apoptosis, which was associated with hyper-induction of a tumor suppressor p53. We further showed that ERK, but not ATM or ATR, was involved in the hyper-induction of p53 by the inhibition of cisplatin-induced AMPK. By way of contrast, cisplatin did not induce AMPK activation in HeLa cells, which appear to have a relatively high sensitivity to cisplatin-induced cytotoxicity, but expression of constitutive active form of AMPK in HeLa cells resulted in a significant increase of cell viability after cisplatin treatment. Collectively, our data suggest that AMPK performs a pivotal function for protection against the cytotoxic effect of cisplatin, thereby implying that AMPK is one of the cellular factors determining the cellular sensitivity to cisplatin. On the basis of these observations, we propose that a strategy combining cisplatin and AMPK inhibition could be developed into a novel chemotherapeutic modality. Part II Solid tumors are generally less well oxygenated than normal tissue. This leads to increased glycolysis for ATP generation (Warburg effect) and frequently associated with resistance to anticancer chemotherapy as well as predisposing to increased tumor metastasis. Therefore, tumor hypoxia is a major concern in therapeutic approaches. Nevertheless, mechanisms responsible for the hypoxia-induced anticancer drug resistance are somewhat poorly understood. Here, we report that quercetin enhanced hypoxia-induced apoptosis in HCT116 colon cancer cells. The activation of AMP-activated protein kinase (AMPK), as an energy sensor, by hypoxia-induced ATP depletion was dose dependently inhibited by quercetin treatment. Western blot and kinase assay data revealed that quercetin directly inhibited AMPK activity, and these events are likely to read to a reduction of hypoxia-induced HIF-1 transcriptional activity, major factor of hypoxia-induced drug resistance. Importantly, the inhibition of hypoxia-induced AMPK activity by quercetin also potently reduced hypoxia induced anticancer drugs resistance, such as cisplatin and etoposide. Here, we show that AMPK can protect cancer cells whose survival is threatened by hypoxia. However, inhibition of AMPK by chemopreventive agents, quercetin, can sensitize them to hypoxia. In conclusion, our results suggest that AMPK is a novel therapeutic target for the treatment of cancers.

AMP-activated protein kinase antagonizes pro-apoptotic extracellular signal-regulated kinase activation by inducing dual-specificity protein phosphatases in response to glucose deprivation in HCT116 carcinoma

김민정 경희대학교 대학원 2010 국내박사

Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular responses, including cell proliferation, differentiation, cell growth, and apoptosis. Because these responses are tightly related to cellular energy level, AMP-activated protein kinase (AMPK), which plays an essential role in energy homeostasis, has emerged as another key regulator. In the present study, we demonstrate a novel signal network between AMPK and MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK and three MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK. Under these conditions, inhibition of endogenous AMPK by expressing a dominant negative form significantly potentiated ERK activation, indicating that glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116 cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent expression of dual-specificity phosphatase (DUSP) 1 & 2, which are negative regulators of ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK, further implying that a signal network between AMPK and ERK is a critical regulatory point in coupling the energy status of the cell to the regulation of cell survival.

(The) study of the relationship between the change of PTEN and antioxidant enzyme by antioxidant coenzymeQ10

오유림 경희대학교 대학원 2010 국내석사

Coenzyme Q10 is called to vitamin Q10, ubiquinone or ubidecarenone. This is a component of the mitochondrial electron transport chain and various cellular membranes [1]. It acts as an important in vivo antioxidant. Some Data suggest that consumption of CoQ10 can be protective against the development of prostate cancer. The CoQ10 may regulate proteins that control cellular division, such as the tumor suppressor PTEN(Phosphatase and tensin homologue). But the detailed mechanism of antioxidant effect of coenzyme Q10 on the cell functions has not been examined. In the present study, we investigated the relationship between the changes of PTEN and antioxidant enzyme by CoQ10 in prostate cancer cell (DU145). We explained thing that CoQ10 had antioxidant effect regarding oxygen free radical generation occurrend when an energy becomes a generation in mitochondria of a DU145 cell. PTEN protein level increased for the correspondence to CoQ10. CoQ10 increased PTEN, and we found it to affect to expression of antioxidant enzymes such as MnSOD, CuZnSOD and Catalase. This suggests thing at DU145 cell that we caused antioxidant enzymes increase by overexpression of PTEN by increase of concentration of CoQ10. We show that a cell decreases through cell viability measurement according to the concentration of CoQ10 at DU145 cell. These findings suggest an essential role for PTEN in prostate cancer cells against oxidative stress. It suggests that dietary changes may be adjunctive to traditional preventive and therapeutic strategies aginst prostate cancer.

(The) Differential Role of AMP-activated Protein Kinase Isoforms in Redox Balance

임세리 경희대학교 대학원 2010 국내석사

5’-AMP-activated protein kinase (AMPK) is a sensor of cellular status because of its capacity to detect in the concentration of AMP. This enzyme is a heterotrimetic serine/threonine kinase complex consisting of a catalytic α subunit and two regulatory β and γ subunits. Full activation of AMPK requires specific phosphorylation (Thr172) within the activation loop of the catalytic domain of the α-subunit by upstream kinases. In mammalian cells, two domain, have been identified (AMPK α1 and AMPK α2). The AMPK α1-isoform is widely distributed in different body tissues, whereas AMPK α2 is primarily expressed in skeletal muscle, heart, and liver. Interestingly, there is little mention of the possibility that reactive oxygen species (ROS) may play a role in regulated AMPK activation. Although Reactive Oxygen Species have been implicated in the regulation of hypoxic and non-hypoxic under various conditions, the role of ROS is quite controversial, and the mechanism underlying the ROS regulation by AMPK is not completely understood yet. We have been investigating the relationship between the AMPK α1-subunit, the expressed almost body tissue, and AMPK α2-subunit, the expressed a specific tissue, such as in skeletal muscle, heart, and liver for regulating ROS generation in AMPK modified MEFs (Mouse embryonic fibroblast). Here we report that AMPK α1-subunit induced by a direct application of ROS generator in MEFs. By way of contrast, AMPK α2-subunit induced by application of ROS scavenger in MEFs. Collectively, our findings identify AMPK as a key determinant of catalytic AMPKα1 and α2-subunit with different aspects in response to ROS.

(The) effects of anti cancer drug induced oxidative stress on the alpha 1 subunit degradation of AMPK complex

임동욱 경희대학교 대학원 2012 국내석사

The 5’-AMP- activated protein kinase (AMPK) functions as a metabolic fuel gauge that is activated in response to numerous environmental stressors to restore cellular and whole body energy balance. We show the regulation of AMPK α in the cell cycle by the increased ROS level in a cell damaged condition and we discovered that the total protein level of AMPK α is decreased by H2O2 treatment in the cell, but the activity of AMPK α is increased. Because the decreased of AMPK α is rescued by MG 132 treatment, we know that the AMPK α is degraded by proteasome pathway. Also, We attempted that the ubiquitination of AMPK α using immunoprecipitation assay. The poly-ubiquitination of AMPK α is dramatically increased in H2O2 treatment than non-treatment. Interestingly, the degradation rate of AMPK α1 is more decreased than the rate AMPK α2 in respond to H2O2. These facts are possible that the AMPK α2 escape from the proteasome target though the AMPK α2 increased activity is trans-localized to nucleus. Although increased activity of AMPK α by H2O2 is possible that is influence in cell cycle regulation thought directly phosphorylated p53. We don’t know the increased phosphor-p53 whether p53 dependant cell death or not the survival thought cell cycle arrest, yet. Particularly, high cell density in the physiologic condition of increased ROS level is similar result in H2O2 treatment condition. We show that total AMPK α1 is decreased by normal and cancer cell lines.

Resveratrol exerts anti-oxidant effect through regulation of AMP-activated protein kinase and FoxO1.

박설희 경희대학교 일반대학원 2015 국내석사

Redox-responsive signaling pathways control many physiological functions. Unbalance of redox status by oxidative stress is recognized as a main reason of many pathological conditions such as aging, cancer and diabetes, therefore it is important to investigate how antioxidants maintain redox homeostasis. AMP-activated protein kinase (AMPK) is activated in response to cellular conditions and plays a crucial role in regulation of energy homeostatasis, tumorigenesis, and longevity. Recently, activating AMPK by a variety of antioxidants have been reported, but the mechanisms how AMPK controls the levels of reactive oxygen species cellular are not fully understood. In this study, we investigated the molecular mechanisms and the role of AMPK in mediating resveratrol-induced antioxidant. We demonstrate that AMPK activity plays an indispensable role in the operation of the ROS defense system by inducing expression of the antioxidant enzymes, manganese superoxide dismutase (MnSOD) and catalase, in response to activated AMPK by resvertarol. Furthermore, we identified that AMPK directly phosphorylates human FoxO1 (forkhead box O1) and increases MnSOD and catalase through FoxO1, demonstrating the new mechanism involved in the antioxidant of AMPK. In conclusion, our result reveal a novel mechanism by which AMPK controls expression of antioxidant enzymes via FoxO1 and suggest that AMPK has major function in maintaining redox homeostasis. 산화-환원 반응 신호전달 경로는 많은 생리학적 기능을 조절한다. 산화 스트 레스로 인한 산화-환원의 불균형 상태는 노화, 암 그리고 당뇨와 같은 병리 적 상태의 주요 원인으로 인식되어 왔다. 따라서 항산화 효소의 의한 산화- 환원 항상성 유지는 생물학적으로 매우 중요한 기전에 해당된다. 아데노신 1 인산 의존 단백질 인산화 효소(AMP-activated protein kinase, 이하 AMPK)는 세포 내에서 에너지 항상성과 종양형성 그리고 수명 조절에 중요 한 역할을 수행한다. 최근 다양한 천연 항산화 제제가 AMPK의 활성을 증가 시킨다는 보고가 이루어 지고 있지만, AMPK의 세포 내 활성 산소 조절 기 작은 아직 규명되지 않았다. 본 연구에서 연구자는 항산화제인 레즈베라트롤 의 세포 내 항산화 효과에 대한 AMPK의 역할을 조사하였다. 인간 간 세포 인 CHANG 세포에 레즈베라트롤을 농도 의존적으로 처리하였을 때 AMPK 의 활성이 증가하였으며 AMPK 작용제와 길항제를 각각 처리하였을 때 다 음의 항산화 효소 즉, 망간 초산화 불균등화 효소(manganese superoxide dismutase, 이하 MnSOD), 과산화 수소 분해 효소(이하 catalase)의 전 사 조절 및 단백질 발현이 AMPK 활성 의존적으로 조절 되는 것을 밝혔다. 또한 AMPK에 의한 당 항산화 효소의 전사인자로 잘 알려져 있는 인간 포 크머리상자 단백질 O1 (ForkheadboxO1, 이하 FoxO1)을 인산화를 통하 여 이루어진다는 것을 증명하였다. 위 결과를 토대로 AMPK가 레즈베라트롤 항산화 기작에서 상위 조절인자임을 제안한다. 아울러 AMPK에 의한 항산화 기작 연구의 중요한 기초 연구 자료가 될 것으로 사료된다.

AMP-activated protein kinase mediates antioxidant effects of hydrogen-rich media

이지현 경희대학교 대학원 2017 국내박사

Persistent oxidative stress has been recognized as a major cause of many pathological conditions or ageing. However, the most clinical trials with dietary antioxidants failed to show successful outcomes in preventing oxidative stress-related diseases. Recently, molecular hydrogen (H2) has received a great deal of attention as a therapeutic agent due to its novel antioxidant property, but its underlying mechanisms remain very elusive. H2 was initially reported to selectively scavenge hydroxyl and peroxynitrite radicals. In the present study, we report a novel mechanism for the antioxidant properties of H2. Hydrogen-rich media activated AMP-activated protein kinase (AMPK), a central regulator of energy hemostasis, which in turn induced FoxO1-dependent transcription of manganese superoxide dismutase and catalase in mouse embryonic fibroblasts. Moreover, hydrogen-rich media effectively reduced the level of reactive oxygen species in cells treated with hydrogen peroxide and protected the cells from apoptosis. These results suggest that AMPK signal pathway is a novel component for the antioxidant properties of H2 in addition to its direct reactive oxygen species scavenging activity.

Melatonin suppresses cellular senescence by activating p62-Nrf2 mediated antioxidant response in keratinocyte

양구원 경희대학교 대학원 2019 국내박사

Melatonin, a natural hormone primarily secreted by the pineal gland, not only regulates circadian rhythms, but also shows a number of beneficial effects on age-related human physiology. Due to the strong anti-oxidant properties and low side effects, it has been hypothesized that melatonin is an effective and safe remedy to slow down aging and prolong the life span, but the underlying mechanisms are far from complete understanding. Here, we explored mechanisms by which melatonin inhibits the replicative senescence of HaCaT keratinocytes and the aging of mouse skin tissue, and identified a novel signaling complex consisting of p62/SQSTM1, p38 MAPK and Nrf2, which plays a central role in inhibiting senescence. As the senescence progressed, the activity of this complex gradually decreased, and the level of reactive oxygen species increased, but in response to melatonin, the complex was highly activated by forming an interactive positive feedback loop between three components, preventing senescence as a result of relaying the melatonin signal to the expression of antioxidant genes from MT2 receptor. Melatonin also blocked aging of mouse skin through this complex, highlighting the central role of the complex in aging and signal transduction of melatonin.

(The) effect of curcumin on AMP-activated protein kinase (AMPK) in human breast cancer MCF7 cell and cell death

노경진 경희대학교 대학원 2009 국내석사

Pharmacologically safe compounds that can inhibit the proliferation of tumor cells have potential as anticancer agents. Curcumin, a diferuloylmethane, is a major active component of the food flavor turmeric (Curcuma longa) that has been shown to inhibit the proliferation of a wide variety of tumor cells. From quantitative image analysis data showing an increase in the percentage of cells with a sub-G1 DNA content. Treatment of MCF-7 cell with 25uM curcumin resulted in a PARP cleavage and inhibited expression of bcl-2 family. We demonstrated curcumin-induced apoptosis in the breast cancer cell line MCF-7. AMP-activated protein kinase (AMPK) regulates the energy balance system by monitoring intracellular energy status. Here we demonstrate that AMPK is rapidly activated by curcumin in MCF-7 cancer cell. The involvement of AMPK signaling cascade in curcumin-based cancer therapy was clearly shown by comparing the conditions of AMPK activated states and inactivated states. We have identified ROS as an upstream regulator of AMPK. Collectively, our data suggest that AMPK performs a pivotal function for protection against the cytotoxic effect of curcumin, thereby implying that AMPK is one of the cellular factors determining the cellular sensitivity to curcumin. Further investigation warrants to elucidate the mechanism by which curcumin generates ROS and AMPK activation.