Ki Mo Kim

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose.

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.

Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor

Cordycepin (3-deoxyadenosine), found in Cordyceps spp., has been known to have many therapeutic effects including immunomodulatory, anti-inflammatory, antimicrobial, and anti-aging effects. Moreover, anti-tumor and anti-metastatic effects of cordycepin have been reported, but the mechanism causing cancer cell death is poorly characterized. The present study was designed to investigate whether the mechanisms of cordycepin-induced cell death were associated with estrogen receptor in breast cancer cells. Exposure of both MDA-MB-231 and MCF-7 human breast cancer cells to cordycepin resulted in dose-responsive inhibition of cell growth and reduction in cell viability. The cordycepin-induced cell death in MDA-MB-231 cells was associated with several specific features of the mitochondria-mediated apoptotic pathway, which was confirmed by DNA fragmentation, TUNEL, and biochemical assays. Cordycepin also caused a dose-dependent increase in mitochondrial translocation of Bax, triggering cytosolic release of cytochrome c and activation of caspases-9 and -3. Interestingly, MCF-7 cells showed autophagy-associated cell death, as observed by the detection of an autophagosome-specific protein and large membranous vacuole ultrastructure morphology in the cytoplasm. Cordycepin-induced autophagic cell death has applications in treating MCF-7 cells with apoptotic defects, irrespective of the ER response. Although autophagy has a survival function in tumorigenesis of some cancer cells, autophagy may be important for cordycepin-induced MCF-7 cell death. In conclusion, the results of our study demonstrate that cordycepin effectively kills MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Hence, further studies should be conducted to determine whether cordycepin will be a clinically useful, ER-independent, chemotherapeutic agent for human breast cancer.

Puerarin suppresses AGEs-induced inflammation in mouse mesangial cells: A possible pathway through the induction of heme oxygenase-1 expression

Puerarin is a natural product isolated from Puerarin lobata and has various pharmacological effects, including anti-hyperglycemic and anti-allergic properties. In the present study, we investigated the effect of puerarin against advanced glycation end products (AGEs)-induced inflammation in mouse mesangial cells. Puerarin acts by inducing the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Puerarin was able to enhance phosphorylation of protein kinase C (PKC) delta, but not PKC alpha/beta II, in a time-dependent manner. Induction of HO-1 expression by puerarin was suppressed by GF109203X, a general inhibitor of PKC, and by rottlerin, a specific inhibitor of PKC delta. However, induction was not suppressed by Gö6976, a selective inhibitor for PKC alpha/beta II. Additionally, the knockdown of endogenous PKC delta by small interfering RNA (siRNA) resulted in the inhibition of HO-1 expression and Akt phosphorylation. Puerarin increased antioxidant response element (ARE)-Luciferase activity in a dose- and time-dependent manner in transfected mouse mesangial cells. Mutation of the ARE sequence abolished puerarin-induced HO-1 expression. Furthermore, puerarin treatments resulted in a marked increase in NF-E2 related factor-2 (Nrf-2) translocation, leading to up-regulation of HO-1 expression. However, transfection of Nrf-2 specific siRNA abolished HO-1 expression. Pretreatment with puerarin inhibited the expressions of COX-2, MMP-2 and MMP-9. But, these effects were reversed by ZnPP, an inhibitor of HO-1. Taken together, our results demonstrate that puerarin-induced expression of HO-1 is mediated by the PKC delta-Nrf-2-HO-1 pathway and inhibits N-carboxymethyllysine (CML)-induced inflammation in mouse mesangial cells.

Myricetin inhibits advanced glycation end product (AGE)-induced migration of retinal pericytes through phosphorylation of ERK1/2, FAK-1, and paxillin in vitro and in vivo.

Advanced glycation end products (AGE) have been implicated in the development of diabetic retinopathy. Characterization of the early stages of diabetic retinopathy is retinal pericytes loss, which is the result of pericytes migration. In this study, we investigated the pathological mechanisms of AGE on the migration of retinal pericytes and confirmed the inhibitory effect of myricetin on migration in vitro and in vivo. Migration assays of bovine retinal pericytes (BRP) were induced using AGE-BSA and phosphorylation of Src, ERK1/2, focal adhesion kinase (FAK-1) and paxillin were determined using immunoblot analysis. Sprague-Dawley rats (6 weeks old) were injected intravitreally with AGE-BSA and morphological and immunohistochemical analysis of p-FAK-1 and p-paxillin were performed in the rat retina. Immunoblot analysis and siRNA transfection were used to study the molecular mechanism of myricetin on BRP migration. AGE-BSA increased BRP migration in a dose-dependent manner via receptor for AGEs (RAGE)-dependent activation of the Src kinase-ERK1/2 pathway. AGE-BSA-induced migration was inhibited by an ERK1/2 specific inhibitor (PD98059), but not by p38 and Jun N-terminal kinase inhibitors. AGE-BSA increased FAK-1 and paxillin phosphorylation in a dose- and time-dependent manner. These increases were attenuated by PD98059 and ERK1/2 siRNA. Phosphorylation of FAK-1 and paxillin was increased in response to AGE-BSA-induced migration of rat retinal pericytes. Myricetin strongly inhibited ERK1/2 phosphorylation and significantly suppressed pericytes migration in AGE-BSA-injected rats. Our results demonstrate that AGE-BSA participated in the pathophysiology of retinal pericytes migration likely through the RAGE-Src-ERK1/2-FAK-1-paxillin signaling pathway. Furthermore, myricetin suppressed phosphorylation of ERK 1/2-FAK-1-paxillin and inhibited pericytes migration.

The hexane fraction of Naematoloma sublateritium extract suppresses the TNF-α-induced metastatic potential of MDA-MB-231 breast cancer cells through modulation of the JNK and p38 pathways

Naematoloma sublateritium (Fr.) P. Karst is a basidiomycete that has been used as traditional medicine. N. sublateritium produces a triterpenoid antitumor compound, clavaric acid, but, in general, the effects of N. sublateritium constituents against tumor invasion and metastasis have been poorly studied. To investigate the inhibitory effect of N. sublateritium constituents on highly invasive and metastatic tumor cells, the TNF-α-stimulated human breast cancer cell line, MDA-MB‑231 was treated with the hexane fraction of an N. sublateritium extract (HFNS). Non-cytotoxic concentrations of HFNS markedly inhibited the invasion and migration of the MDA-MB‑231 cells in the Matrigel invasion assay and wound-healing analysis, respectively. Gelatin zymography showed that HFNS suppressed the activity of MMP-9, but not of MMP-2. Immunoblotting demonstrated that treatment with HFNS had decreased the level of MMP-9 and urokinase plasminogen activator-1 (uPA-1), but had upregulated expression of the endogenous inhibitor proteins, including TIMP-1,-2, and PAI-1, in a dose-dependent manner. Furthermore, HFNS suppressed the phosphorylation of p38 and JNK1/2, but not that of ERK1/2. This was confirmed by pretreatment of cells with specific inhibitors prior to stimulation with TNF-α. HFNS treatment also led to a dose-dependent inhibition of the DNA-binding activities of AP-1 and NFκB, which are downstream targets of JNK and p38. These data suggested that HFNS inhibits the metastatic potential of MDA-MB‑231 cells by inhibiting the phosphorylation of JNK/p38 and reducing AP-1 and NFκB DNA-binding activities. Therefore, HFNS may be a potential therapeutic agent against metastasis of breast cancer.

Therapeutic positioning of secretory acetylated APE1/Ref-1 requirement for suppression of tumor growth in triple-negative breast cancer in vivo

Triple-negative breast cancer (TNBC) represents a relatively small proportion of all BCs but a relatively large proportion of BC-related death. Thus, more effective therapeutic strategies are needed for the management of TNBC. We demonstrated that the stimulation of apoptosis by the binding of secreted acetylated-apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1) to the receptor for advanced glycation end products (RAGE) was essential for TNBC cell death in response to hyperacetylation. The aim of the present study was to assess the potential therapeutic efficacy of secretory Ac-APE1/Ref-1 in orthotopic TNBC xenografts in vivo. We found that hyperacetylation in xenografts caused secretion of Ac-APE1/Ref-1 into the blood, where the factor bound directly to RAGE in hyperacetylated tumor tissues. Hyperacetylation in the TNBC xenografts induced strong inhibition of tumor growth and development, leading to apoptotic cell death, accompanied by increased RAGE expression and generation of reactive oxygen species. Tissues exhibited markedly higher counts of apoptotic bodies, a reduced proliferation index, and reduced neovascularization compared with control tumors. Ac-APE1/Ref-1-stimulated apoptosis was markedly reduced in RAGE-knockdown tumors compared with RAGE-overexpressing tumors, even in the presence of hyperacetylation. The function of secreted Ac-APE1/Ref-1 was confirmed in other hyperacetylated TNBCs xenografts using BT-549 and MDA-MB-468 cells, demonstrating its relevance as an anti-cancer molecule.

Abstract 4422: Extracellularly secreted APE1/Ref-1 triggers apoptosis in triple-negative breast cancer cells via RAGE binding, which is mediated through acetylation

The present study evaluated the mechanism of apoptosis caused by posttranslational modification, hyperacetylation in triple-negative breast cancer (TNBC) cells. We previously showed that trichostatin A (TSA) induced secretion of acetylated apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1). This is the first report showing that Ac-APE1/Ref-1 initiates apoptosis in TNBC cells by binding to the receptor for advanced glycation end products (RAGE). The functional significance of secreted Ac-APE1/Ref-1 was studied by induction of intracellular hyperacetylation through co-treatment with acetylsalicylic acid and TSA in MDA-MB-231 cells. In response to hyperacetylation, secretion of Ac-APE1/Ref-1 in vesicles was observed, resulting in significantly decreased cell viability and induction of apoptosis with increased expression of RAGE. The hyperacetylation-induced apoptosis was similar in two other TNBC cell lines: BT-459 and MDA-MB-468. Therefore, hyperacetylation may be a therapeutic target for treatment of TNBCs. This study introduces a novel paradigm whereby posttranslational modification induces apoptotic cell death in breast cancer cells resistant to standard chemotherapeutic agents through secretion of auto- or paracrine molecules such as Ac-APE1/Ref-1. Citation Format: Yu Ran Lee, Hee Kyoung Joo, Ki Mo Kim, Byeong Hwa Jeon, Sunga Choi. Extracellularly secreted APE1/Ref-1 triggers apoptosis in triple-negative breast cancer cells via RAGE binding, which is mediated through acetylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4422.

Abstract 3906: Magnolol suppresses vascular endothelial growth factor-induced angiogenesis by inhibiting Ras-dependent mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been reported to possess anti-cancer activity. Recent studies have also demonstrated that magnolol inhibits cell growth and induces the apoptosis of cancer cells. However, the effects of magnolol on vascular endothelial growth factor (VEGF)-induced angiogenesis in endothelial cells have not been studied. In the present study, we have used human umbilical vein endothelial cells (HUVECs) to investigate the anti-angiogenic effect and molecular mechanism of magnolol. Magnolol inhibited the VEGF-induced proliferation, chemotactic motility and tube formation of HUVECs in vitro as well as the vessel sprouting of the aorta ex vivo. Furthermore, magnolol inhibited VEGF-induced Ras activation and subsequently suppressed extracellular signal-regulated kinase (ERK), phosphatidylinositol-3-kinase (PI3K)/Akt and p38, but not Src and focal adhesion kinase (FAK). Interestingly, the knockdown of Ras by short interfering RNA produced inhibitory effects that were similar to the effects of magnolol on VEGF-induced angiogenic signaling events, such as ERK and Akt/eNOS activation, and resulted in the inhibition of proliferation, migration, and vessel sprouting in HUVECs. In combination, these results demonstrate that magnolol is an inhibitor of angiogenesis and suggest that this compound could be a potential candidate in the treatment of angiogenesis-related diseases. Citation Format: Ki Mo Kim, No Soo Kim, Jinhee Kim, Jong-Shik Park, Jin Mu Yi, Jun Lee, Ok-Sun Bang. Magnolol suppresses vascular endothelial growth factor-induced angiogenesis by inhibiting Ras-dependent mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3906. doi:10.1158/1538-7445.AM2013-3906

Abstract 2568: Hexane fraction of Naematoloma sublateritium extracts inhibits metastatic potential of MDA-MB-231 Breast cancer cells through modulation of the JNK and p38 pathway

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Despite years of therapeutic development and research, metastasis is still the major driver of mortality in cancer patients. Breast cancer is a second leading cause of cancer deaths in woman world-wide. The prevalence of breast cancer continues to increase by approximately 2% each year. Breast cancer also presents as metastatic disease, which has spread beyond the original organ, breast to bone, liver, lung and brain. Recently, many studies have reported that natural products- dietary phytochemicals including mushroom are known to exhibit a variety of anti-cancer, anti-invasive and anti-metastatic activities. Naematolma spp. is a basidiomycete of the fungus that is known to produce anti-tumor compound, clavaric acid. In our previous studies, HFNS has been shown to significantly inhibit cell proliferation and induce apoptosis of human estrogen-nonresponsive MDA-MB-231 breast cancer cells. In this present study, we report for the first time that hexane fraction of Naematoloma sublateritium (HFNS) regulates TNF-alpha induced invasion and migration in MDA-MB-231 cells. We observed that non-cytotoxic concentrations (10-50 μg/mL) of HFNS markedly inhibited the invasion and migration of highly metastatic MDA-MB-231 cells as shown by a Matrigel invasion assay and wound healing analysis, respectively. The results of a gelatin zymography showed that HFNS suppressed the activity of matrix metalloproteinase (MMP)-9 and urokinase plasminogen activator (uPA). Western blot results demonstrated that treatment with HFNS had decreased level of MMP-9, and uPA, while the expression of the endogenous inhibitor proteins including tissue inhibitors of MMP (TIMP-1 and TIMP-2), and plasminogen activator inhibitor (PAI)-1, was up-regulated in a dose-dependent manner. Furthermore, HFNS suppressed the phosphorylation of p38, and JNK1/2 in MDA-MB-231 cells, where extracellular signal-regulated kinase (ERK) 1/2 were not affected. It was confirmed by pretreatment with SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor), respectively, before stimulation with TNF-alpha. Interestingly, the HFNS treatment also led to a dose-dependent inhibition on NF-κB and activator protein-1(AP-1) binding activity which are downstream targets of JNK and p38. The gel-shift assay results supported that HFNS treatment caused down-regulation of MMP-9 and uPA gene in mRNA level. Taken together, these data suggest the inhibitory effect of HFNS on the metastatic potential of MDA-MB-231 cells through inhibiting the phosphorylation of JNK/p38 and reducing NFκB and AP-1 DNA binding activities. In conclusion, we propose that HFNS may be a safe and potential therapeutic agent against metastasis of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2568. doi:1538-7445.AM2012-2568