Secretory phospholipase A₂ induces apoptosis through TNF-α and cytochrome c-mediated caspase cascade in murine macrophage RAW 264.7 cells

Lee, ChuHee,Park, Dae-Weon,Lee, Jingu,Lee, Tae-Il,Kim, Young-Jo,Lee, Yun-Sik,Baek, Suk-Hwan Elsevier 2006 european journal of pharmacology Vol.536 No.1

<P><B>Abstract</B></P><P>Phospholipase A<SUB>2</SUB> (PLA<SUB>2</SUB>) is an esterase that cleaves the <I>sn</I>-2 ester bond in glycerophospholipids, thereby releasing free fatty acids and lysophospholipids. In addition to the apoptotic activity of cytosolic PLA<SUB>2</SUB> and Ca<SUP>2+</SUP>-independent PLA<SUB>2</SUB>, recent studies showed that secretory PLA<SUB>2</SUB> (sPLA<SUB>2</SUB>) also play a role in apoptosis. However, the details of molecular mechanism have not been fully elucidated. Our data demonstrated that group IB PLA (IB PLA<SUB>2</SUB>)-exposed murine macrophage 264.7 cells showed characteristic features of apoptosis such as morphological changes, DNA laddering, staining positive for propidium iodide (PI) as well as Annexin V and activation of caspases and subsequent cleavage of poly (ADP-ribose) polymerase (PARP) in dose- and time-dependent manner. Moreover, IB PLA<SUB>2</SUB> was found to elicit tumor necrosis factor (TNF)-α production and release of cytochrome c, suggesting that IB PLA<SUB>2</SUB> exerts its apoptotic activity via the induction of TNF-α production and cytochrome c release, which results in triggering the activation of caspase cascade and PARP cleavage.</P>

Overexpression of Tyro3 receptor tyrosine kinase leads to the acquisition of taxol resistance in ovarian cancer cells

SPANDIDOS PUBLICATIONS 2015 MOLECULAR MEDICINE REPORTS Vol.12 No.1

<P>The majority of patients with ovarian cancer are diagnosed at the advanced stages (III/IV) and their 5-year-survival rate is relatively low. One of the major causes of the poor prognosis of ovarian cancer is the development of resistance to first-line chemotherapy, including platinum and taxol. Therefore, improvements in current understanding of chemoresistance is required for the successful treatment of ovarian cancer. In the present study, taxol-resistant ovarian cancer cells, SKOV3/TR, were established by exposing parental SKOV3 cells to increasing concentrations of taxol. . Briefly, cells were treated with 1.5 nM (for 4 weeks), 3 nM (for 4 weeks), 6 nM (for 5 weeks), 12 nM (for 5 weeks) and 24 nM taxol (for 8 weeks) over 6 months. The SKOV3/TR cells were found to be smaller in size and rounder in shape compared with their parental cells. Cell viability and colony formation assays demonstrated an increase in the population doubling time of the SKOV3/TR cells, indicating a reduction in the proliferative capacity of these cells. Reverse transcription-polymerase chain reaction and western blot analysis revealed that, among the TAM receptor tyrosine kinases (RTKs), the mRNA and protein expression levels of Tyro3 RTK were increased, while those of Axl and Mer RTK were decreased in the SKOV3/TR cells. In addition, restoration of the level of Tyro3 by transfecting Tyro3-specific small interfering RNA into the SKOV3/TR cells reduced the proliferative capacity of the cells, indicating that upregulation of the expression of Tyro3 in SKOV3/TR cells may promote survival in the presence of taxol, which eventually resulted in the acquisition of resistance upon taxol treatment. The present study subsequently found that, in the SKOV3/TR cells, the level of intracellular reactive oxygen species (ROS) was elevated, and antioxidant treatment with N-acetyl cysteine (NAC) exerted more profound antiproliferative effects compared with the parental cells. The western blot analysis demonstrated that treatment of the SKOV3/TR cells with NAC reduced the protein expression of Tyro3, and the inhibitory effect of NAC on the phosphorylation of Akt was increased, which may have had a positive effect on the proliferation of the SKOV3/TR cells. The levels of phosphorylation and protein expression of signal transducers and activators of transcription 3 (STAT3) were not affected by NAC treatment, indicating that the phosphorylation of Akt, but not expression or phosphorylation of STAT3, was associated with the increased intracellular ROS level in the SKOV3/TR cells. Taken together, the results of the present study demonstrated that the acquired taxol resistance of ovarian cancer cells was associated with ROS-dependent upregulation in the expression of Tyro3 RTK and the subsequent activation of Akt.</P>

Reversal of Cisplatin Resistance by Epigallocatechin Gallate Is Mediated by Downregulation of Axl and Tyro 3 Expression in Human Lung Cancer Cells

Kim, Kyung-Chan,Lee, ChuHee The Korean Society of Pharmacology 2014 The Korean Journal of Physiology & Pharmacology Vol.18 No.1

Lung cancer is still the number one cause of death from cancer worldwide. The clinical effect of platinum-based chemotherapy for non-small cell lung cancer is constrained by the resistance to drug. To overcome chemo-resistance, various modified treatment including combination therapy has been used, but overall survival has not been improved yet. In this study, chemo-resistant lung cancer cells, A549/Cis and H460/Cis, were developed by long-term exposure of cells to cisplatin and the proliferative capability of these resistant cells was verified to be reduced. We found cytotoxic effect of epigallocatechin gallate (EGCG), a major catechin derived from green tea, on both the parental lung cancer cells, A549 and H460, and their cisplatin resistant cells, A549/Cis and H460/Cis. ELISA and Western blot analysis revealed that EGCG was able to increase interlukine-6 (IL-6) production per cell, whereas its downstream effector Signal transducers and activators of transcription 3 (STAT3) phosphorylation was not changed by EGCG, indicating that IL-6/STAT3 axis is not the critical signaling to be inhibited by EGCG. We next found that EGCG suppresses the expression of both Axl and Tyro 3 receptor tyrosine kinases at mRNA and protein level, explaining the cytotoxic effect of EGCG on lung cancer cells, especially, regardless of cisplatin resistance. Taken together, these data suggest that EGCG impedes proliferation of lung cancer cells including their chemo-resistant variants through downregulation of Axl and Tyro 3 expression.

Curcumin Induces Downregulation of E2F4 Expression and Apoptotic Cell Death in HCT116 Human Colon Cancer Cells; Involvement of Reactive Oxygen Species

Kyung-Chan Kim,ChuHee Lee 대한생리학회-대한약리학회 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.6

E2F transcription factors and their target genes have been known to play an important role in cell growth control. We found that curcumin, a polyphenolic phytochemical isolated from the plant Curcuma longa, markedly suppressed E2F4 expression in HCT116 colon cancer cells. Hydrogen peroxide was also found to decrease E2F4 protein level, indicating the involvement of reactive oxygen species (ROS) in curucmin-induced downregulation of E2F4 expression. Involvement of ROS in E2F4 downregulation in response to curcumin was confirmed by the result that pretreatment of cells with N-acetylcystein (NAC) before exposure of curcumin almost completely blocked the reduction of E2F4 expression at the protein as well as mRNA level. Anti-proliferative effect of curcumin was also suppressed by NAC which is consistent to previous reports showing curcumin-superoxide production and induction of poly (ADP-ribose) polymerase (PARP) cleavage as well as apoptosis. Expression of several genes, cyclin A, p21, and p27, which has been shown to be regulated in E2F4-dependent manner and involved in the cell cycle progression was also affected by curcumin. Moreover, decreased (cyclin A) and increased (p21 and p27) expression of these E2F4 downstream genes by curcumin was restored by pretreatment of cells with NAC and E2F4 overexpression which is induced by doxycycline. In addition, E2F4 overexpression was observed to partially ameliorate curcumin-induced growth inhibition by cell viability assay. Taken together, we found curcumin-induced ROS down-regulation of E2F4 expression and modulation of E2F4 target genes which finally lead to the apoptotic cell death in HCT116 colon cancer cells, suggesting that E2F4 appears to be a novel determinant of curcumin-induced cytotoxicity.

Reversal of Cisplatin Resistance by Epigallocatechin Gallate Is Mediated by Downregulation of Axl and Tyro 3 Expression in Human Lung Cancer Cells

Kyung-Chan Kim,ChuHee Lee 대한생리학회-대한약리학회 2014 The Korean Journal of Physiology & Pharmacology Vol.18 No.1

Lung cancer is still the number one cause of death from cancer worldwide. The clinical effect of platinum-based chemotherapy for non-small cell lung cancer is constrained by the resistance to drug. To overcome chemo-resistance, various modified treatment including combination therapy has been used, but overall survival has not been improved yet. In this study, chemo-resistant lung cancer cells, A549/Cis and H460/Cis, were developed by long-term exposure of cells to cisplatin and the proliferative capability of these resistant cells was verified to be reduced. We found cytotoxic effect of epigal-locatechin gallate (EGCG), a major catechin derived from green tea, on both the parental lung cancer cells, A549 and H460, and their cisplatin resistant cells, A549/Cis and H460/Cis. ELISA and Western blot analysis revealed that EGCG was able to increase interlukine-6 (IL-6) production per cell, whereas its downstream effector Signal transducers and activators of transcription 3 (STAT3) phosphorylation was not changed by EGCG, indicating that IL-6/STAT3 axis is not the critical signaling to be inhibited by EGCG. We next found that EGCG suppresses the expression of both Axl and Tyro 3 receptor tyrosine kinases at mRNA and protein level, explaining the cytotoxic effect of EGCG on lung cancer cells, especially, regardless of cisplatin resistance. Taken together, these data suggest that EGCG impedes proliferation of lung cancer cells including their chemo-resistant variants through downregulation of Axl and Tyro 3 expression.

Axl receptor tyrosine kinase is a novel target of apigenin for the inhibition of cell proliferation.

Kim, Kyung-Chan,Choi, Eun-Ha,Lee, Chuhee UNKNOWN 2014 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.34 No.2

<P>The Axl receptor tyrosine kinase (RTK), along with Tyro 3 and Mer, belongs to the TAM subfamily that promotes survival, stimulates proliferation and/or inhibits apoptosis. In various types of human cancer, including breast, lung and prostate cancer, Axl expression is increased and correlates with an advanced clinical stage. In this study, we examined whether apigenin has an effect on Axl expression, which in turn can affect cell proliferation. The treatment of the non?small cell lung cancer (NSCLC) cells, A549 and H460, with apigenin decreased Axl mRNA and protein expression in a dose?dependent manner. Axl promoter activity was also inhibited by apigenin, indicating that apigenin suppressed Axl expression at the transcriptional level. Upon treatment with apigenin, the viability of both the A549 and H460 cells was gradually decreased and the anti-proliferative effects were further confirmed by the dose?dependent decrease in the clonogenic ability of the apigenin?treated cells. Subsequently, we found that the viability and clonogenic ability of the cells treated with apigenin was less or more affected by transfection of the cells with a Axl-expressing plasmid or Axl targeting siRNA, compared to transfection with the empty vector or control siRNA, respectively. In addition, apigenin increased the expression of p21, a cyclin-dependent kinase inhibitor, but reduced the expression of X-linked inhibitor of apoptosis protein (XIAP). These cell cycle arrest and pro-apoptotic effects of apigenin were also attenuated or augmented by the up- or downregulation of Axl expression, respectively, which suggests that Axl is a novel target of apigenin through which it exerts its inhibitory effects on cell proliferation. Taken together, our data indicate that apigenin downregulates Axl expression, which subsequently results in the inhibition of NSCLC cell proliferation through the increase and decrease of p21 and XIAP expression, respectively.</P>

Exploration of Senescence-associated Genes by Differential Display Reverse Transcription Polymerase Chain Reaction: Prosaposin as a Novel Senescence-associated Gene

김남이,Anh-Myung Woo,김재룡,ChuHee Lee 대한약학회 2009 Archives of Pharmacal Research Vol.32 No.5

To understand the process and molecular mechanism of cellular aging, we explored novel senescence-associated genes using rat tissues with different age. Using total RNAs from 6 and 24-month old rat tissues, differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) were performed and 12 differentially expressed genes (DEGs) which were downor up-regulated in aged rat tissues were found. Among these DEGs, the level of prosaposin mRNA was elevated of in senescent human dermal fibroblast (HDF) and human umbilical vein endothelial cells (HUVECs). It was further confirmed that expression of prosaposin was up-regulated in prematurely senescent HUVECs induced by hydrogen peroxide or interferon- γ treatment. Taken together, we report the up-regulation of prosaposin in the senescent HDF and HUVECs as well as in hydrogen peroxide or interferon-γ induced prematurely senescent HUVECs, suggesting that prosaposin might be a novel senescence-associated gene.

Metformin targets Axl and Tyro3 receptor tyrosine kinases to inhibit cell proliferation and overcome chemoresistance in ovarian cancer cells

KIM, NAM-YI,LEE, HWA-YOUNG,LEE, CHUHEE Spandidos Publications 2015 International journal of oncology Vol.47 No.1

<P>Metformin, the most frequently prescribed anti-diabetic drug, has recently been paid attention as a chemotherapeutic agent. In this study, we demonstrated that metformin decreased the viability of parental as well as cisplatin/taxol-resistant ovarian cancer cells. Its anti-proliferative effect was further demonstrated by dose?dependent reduction of the clonogenic ability of the metformin?treated cells. We next observed the effect of metformin on expression of Axl and Tyro3 receptor tyrosine kinases (RTKs) which belong to the TAM subfamily of RTKs transducing pro-survival and anti-apoptotic signals. Metformin treatment of ovarian cancer cells decreased both mRNA and protein levels of Axl and Tyro3 in a dose?dependent manner. Axl promoter activity was also inhibited by metformin, indicating that metformin suppresses Axl and Tyro3 expression at the transcriptional level. Metformin treatment was also found to augment its anti?proliferative effect in SKOV3 and taxol-resistant SKOV3/TR cells transfected with Axl and Tyro3 specific siRNAs, siAxl and siTyro3, respectively, suggesting that metformin might target Axl and Tyro3 RTKs to restrain cell proliferation. In parallel, the level of X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptotic molecule, was reduced in the metformin-treated cells. Collectively, our data showed that metformin caused reduction of Axl and Tyro3 RTKs' expression, inactivation of downstream effectors, and decrease of anti-apoptotic protein level, forming a potent therapeutic strategy to facilitate its anticancer activity as well as to overcome chemoresistance in human ovarian cancer cells.</P>

Preparation of Gold Nanoisland Arrays from Layer-by-Layer Assembled Nanoparticle Multilayer Films

Hyung Y. Choi,Michael S. Guerrero,Michael Aquino,Chuhee Kwon,Young-Seok Shon 대한화학회 2010 Bulletin of the Korean Chemical Society Vol.31 No.2

This article introduces a facile nanoparticle self-assembly/annealing method for the preparation of nanoisland films. First, nanoparticle-polymer multilayer films are prepared with layer-by-layer assembly. Nanoparticle multilayer films are then annealed at ~ 500℃in air to evaporate organic matters from the films. During the annealing process, the nanoparticles on the solid surface undergo nucleation and coalescence, resulting in the formation of nanostructured gold island arrays. By controlling the overall thickness (number of layers) of nanoparticle multilayer films, nanoisland films with various island density and different average sizes are obtained. The surface property of gold nanoisland films is further controlled by the self-assembly of alkanethiols, which results in an increased surface hydrophobicity of the films. The structure and characteristics of these nanoisland film arrays are found to be quite comparable to those of nanoisland films prepared by vacuum evaporation method. However, this self-assembly/annealing protocol is simple and requires only common laboratory supplies and equipment for the entire preparation process.

Involvement of ROS in Curcumin-induced Autophagic Cell Death

Youn Ju Lee,Nam-Yi Kim,Young-Ah Suh,ChuHee Lee 대한생리학회-대한약리학회 2011 The Korean Journal of Physiology & Pharmacology Vol.15 No.1

Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant <i>Curcuma longa</i>, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome- lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin- induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell. However, ROS-dependent activation of ERK1/2 and p38 MAPK, but not JNK, might not be involved in the curcumin-induced autophagy.