Song Iy Han

Cellular responses to mild heat stress

Abstract.Since its discovery in 1962 by Ritossa, the heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to heat stress. The most well characterized heat shock response is induction of the heat shock proteins that function as molecular chaperones and exert cell cycle regulatory and anti-apoptotic activities. While most investigators have focused their studies on the toxic effects of heat stress in organisms such as severe heat stress-induced cell cycle arrest and apoptosis, the cellular response to fever-ranged mild heat stress has been rather underestimated. However, the cellular response to mild heat stress is likely to be more important in a physiological sense than that to severe heat stress because the body temperature of homeothermic animals increases by only 1–2°C during febrile diseases. Here we provide information that mild heat stress does have some beneficial role in organisms via positively regulating cell proliferation and differentiation, and immune response in mammalian cells.

Wnt/Snail Signaling Regulates Cytochrome c Oxidase and Glucose Metabolism

Wnt signaling plays a critical role in embryonic development, and its deregulation is closely linked to the occurrence of a number of malignant tumors, including breast and colon cancer. The pathway also induces Snail-dependent epithelial-to-mesenchymal transition (EMT), which is responsible for tumor invasion and metastasis. In this study, we show that Wnt suppresses mitochondrial respiration and cytochrome C oxidase (COX) activity by inhibiting the expression of 3 COX subunits, namely, COXVIc, COXVIIa, and COXVIIc. We found that Wnt induced a glycolytic switch via increased glucose consumption and lactate production, with induction of pyruvate carboxylase (PC), a key enzyme of anaplerosis. In addition, Wnt-induced mitochondrial repression and glycolytic switching occurred through the canonical β-catenin/T-cell factor 4/Snail pathway. Short hairpin RNA-mediated knockdown of E-cadherin, a regulator of EMT, repressed mitochondrial respiration and induced a glycolytic switch via Snail activation, indicating that EMT may contribute to Wnt/Snail regulation of mitochondrial respiration and glucose metabolism. Together, our findings provide a new function for Wnt/Snail signaling in the regulation of mitochondrial respiration (via COX gene expression) and glucose metabolism (via PC gene expression) in tumor growth and progression.

Arachidonic acid induces the activation of the stress-activated protein kinase, membrane ruffling and H2O2 production via a small GTPase Rac1.

Arachidonic acid (AA) is generated via Rac‐mediated phospholipase A2 (PLA2) activation in response to growth factors and cytokines and is implicated in cell growth and gene expression. In this study, we show that AA activates the stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) in a time‐ and dose‐dependent manner. Indomethacin and nordihydroguaiaretic acid, potent inhibitors of cyclooxygenase and lipoxygenase, respectively, did not exert inhibitory effects on AA‐induced SAPK/JNK activation, thereby indicating that AA itself could activate SAPK/JNK. As Rac mediates SAPK/JNK activation in response to a variety of stressful stimuli, we examined whether the activation of SAPK/JNK by AA is mediated by Rac1. We observed that AA‐induced SAPK/JNK activation was significantly inhibited in Rat2‐Rac1N17 dominant‐negative mutant cells. Furthermore, treatment of AA induced membrane ruffling and production of hydrogen peroxide, which could be prevented by Rac1N17. These results suggest that AA acts as an upstream signal molecule of Rac, whose activation leads to SAPK/JNK activation, membrane ruffling and hydrogen peroxide production.

Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation

Radiation therapy is one of the major tools of cancer treatment, and is widely used for a variety of malignant tumours. Radiotherapy causes DNA damage directly by ionization or indirectly via the generation of reactive oxygen species (ROS), thereby destroying cancer cells. However, ionizing radiation (IR) paradoxically promotes metastasis and invasion of cancer cells by inducing the epithelial-mesenchymal transition (EMT). Metastasis is a major obstacle to successful cancer therapy, and is closely linked to the rates of morbidity and mortality of many cancers. ROS have been shown to play important roles in mediating the biological effects of IR. ROS have been implicated in IR-induced EMT, via activation of several EMT transcription factors—including Snail, HIF-1, ZEB1, and STAT3—that are activated by signalling pathways, including those of TGF-β, Wnt, Hedgehog, Notch, G-CSF, EGFR/PI3K/Akt, and MAPK. Cancer cells that undergo EMT have been shown to acquire stemness and undergo metabolic changes, although these points are debated. IR is known to induce cancer stem cell (CSC) properties, including dedifferentiation and self-renewal, and to promote oncogenic metabolism by activating these EMT-inducing pathways. Much accumulated evidence has shown that metabolic alterations in cancer cells are closely associated with the EMT and CSC phenotypes; specifically, the IR-induced oncogenic metabolism seems to be required for acquisition of the EMT and CSC phenotypes. IR can also elicit various changes in the tumour microenvironment (TME) that may affect invasion and metastasis. EMT, CSC, and oncogenic metabolism are involved in radioresistance; targeting them may improve the efficacy of radiotherapy, preventing tumour recurrence and metastasis. This study focuses on the molecular mechanisms of IR-induced EMT, CSCs, oncogenic metabolism, and alterations in the TME. We discuss how IR-induced EMT/CSC/oncogenic metabolism may promote resistance to radiotherapy; we also review efforts to develop therapeutic approaches to eliminate these IR-induced adverse effects.

Protein kinase C-ERK1/2 signal pathway switches glucose depletion-induced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells

Cells typically die by either apoptosis or necrosis. However, the consequences of apoptosis and necrosis are quite different for a whole organism. In the case of apoptosis, the cell content remains packed in the apoptotic bodies that are removed by marcrophages, and thereby inflammation does not occur; during necrosis, the cell membrane is ruptured, and the cytosolic constituents are released into the extracellular space provoking inflammation. Recently, inflammation and necrosis have been suggested to promote tumor growth. We investigated the molecular mechanism underlying cell death in response to glucose depletion (GD), a common characteristic of the tumor microenvironment. GD induced necrosis through production of reactive oxygen species (ROS) in A549 lung carcinoma cells. Inhibition of ROS production by N‐acetyl‐L‐cysteine and catalase prevented necrosis and switched the cell death mode to apoptosis that depends on mitochondrial death pathway involving caspase‐9 and caspase‐3 activation, indicating a critical role of ROS in determination of GD‐induced cell death mode. We demonstrate that protein kinase C‐dependent extracellular regulated kinase 1/2 (ERK1/2) activation also switched GD‐induced necrosis to apoptosis through inhibition of ROS production possibly by inducing manganese superoxide dismutase (SOD) expression and by preventing GD‐induced degradation of cupper zinc SOD. Thus, these results suggest that GD‐induced cell death mode is determined by the protein kinase C/ERK1/2 signal pathway that regulates MnSOD and CuZnSOD and that these antioxidants may exert their known tumor suppressive activities by inducing necrosis‐to‐apoptosis switch. J. Cell. Physiol. 211: 371–385, 2007.

Lipid raft-dependent death receptor 5 (DR5) expression and activation are critical for ursodeoxycholic acid-induced apoptosis in gastric cancer cells

Ursodeoxycholic acid (UDCA) is known as a suppressor of cholestatic liver diseases and colorectal cancer development. Here, we demonstrate that UDCA induces apoptosis without necrotic features in SNU601, SNU638, SNU1 and SNU216 human gastric cancer cells, implying its possible use as an effective chemotherapeutic agent in treatment of gastric cancer. UDCA-induced apoptosis was dominantly mediated by an extrinsic pathway dependent on caspase-8, -6 and -3. UDCA increased expression of death receptor 5 [(DR5), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2], and this DR appeared to be responsible for UDCA-induced apoptosis, as evidenced by DR5 knockdown. UDCA triggered formation of lipid rafts that played crucial roles in UDCA-induced apoptotic actions. Lipid rafts were required not only for provision of a proper site for DR5 action but also for mediation of DR5 expression. In addition, reactive oxygen species (ROS) and protein kinase C (PKC) δ appeared to be implicated in UDCA-induced raft-dependent DR5 expression. Our results indicate that UDCA-induced apoptosis is mediated by DR5 expression, which is regulated by the raft formation/ROS production/PKCδ activation pathway and DR5 localization into lipid rafts in gastric cancer cells. Tumor-suppressive activity of UDCA was confirmed in an in vivo system: UDCA (120 mg/kg/day) significantly decreased tumor growth in gastric cancer xenograft mice. Taken together, our results demonstrate that UDCA can be used as a potent chemotherapeutic agent for treatment of gastric cancer.

Roles of cyclin-dependent kinase 8 and β-catenin in the oncogenesis and progression of gastric adenocarcinoma

Gastric adenocarcinoma is a common cause of cancer-related death. The Wnt/β-catenin pathway plays an important role in various cancers. However, relatively little is known about the regulatory mechanism of β-catenin in stomach cancer. To determine the patterns of cyclin-dependent kinase (CDK) 8 and β-catenin expression and the relationship between CDK8 and β-catenin, we conducted a study of immuno-histochemical staining of tumor tissues (12 adenomas, 24 early gastric carcinomas, 24 advanced gastric carcinomas and 21 metastatic lymph nodes), together with Western blot analysis and CDK8 interference studies using gastric cancer cell lines. Gastric adenocarcinomas with CDK8 expression had distinct clinical, prognostic and molecular attributes. CDK8 expression and the delocalization of β-catenin expression showed a significant positive correlation with carcinogenesis and tumor progression, especially lymph node metastasis. Immunohisto-chemically, CDK8 expression in gastric adenocarcinoma was independently associated with β-catenin activation (p<0.05). β-catenin expression was suppressed by CDK8 interference in the gastric adenocarcinoma cell lines, SNU-601 and SNU-638. These data support the potential link between CDK8 and β-catenin, and suggest that CDK8 detection and β-catenin delocalization could be related to a poor prognosis. Moreover, the interference of CDK8 could be a promising therapeutic modality for gastric adenocarcinoma.

Mild heat shock induces cyclin D1 synthesis through multiple Ras signal pathways

Hyperthermia such as that occurring during fever may improve cell survival during infection, although its mechanism of action is largely unknown. Here we show that acute exposure to mild, but not severe, heat shock induces the synthesis of cyclin D1 that plays a critical role(s) in G1 progression of the cell cycle. This induction seemed to be regulated through multiple Ras signal pathways involving extracellular signal‐regulated kinase, phosphatidylinositol 3‐kinase, and Rac1/NADPH oxidase, all of which have well been documented to be responsible for growth factor‐induced cyclin D1 expression. In a physiological sense, mild heat shock may regulate cell proliferation through inducing cyclin D1 along with growth factors.

Homeobox gene Dlx-2 is implicated in metabolic stress-induced necrosis

BackgroundIn contrast to tumor-suppressive apoptosis and autophagic cell death, necrosis promotes tumor progression by releasing the pro-inflammatory and tumor-promoting cytokine high mobility group box 1 (HMGB1), and its presence in tumor patients is associated with poor prognosis. Thus, necrosis has important clinical implications in tumor development; however, its molecular mechanism remains poorly understood.ResultsIn the present study, we show that Distal-less 2 (Dlx-2), a homeobox gene of the Dlx family that is involved in embryonic development, is induced in cancer cell lines dependently of reactive oxygen species (ROS) in response to glucose deprivation (GD), one of the metabolic stresses occurring in solid tumors. Increased Dlx-2 expression was also detected in the inner regions, which experience metabolic stress, of human tumors and of a multicellular tumor spheroid, an in vitro model of solid tumors. Dlx-2 short hairpin RNA (shRNA) inhibited metabolic stress-induced increase in propidium iodide-positive cell population and HMGB1 and lactate dehydrogenase (LDH) release, indicating the important role(s) of Dlx-2 in metabolic stress-induced necrosis. Dlx-2 shRNA appeared to exert its anti-necrotic effects by preventing metabolic stress-induced increases in mitochondrial ROS, which are responsible for triggering necrosis.ConclusionsThese results suggest that Dlx-2 may be involved in tumor progression via the regulation of metabolic stress-induced necrosis.

HEAT SHOCK-INDUCED ACTIN POLYMERIZATION, SAPK/JNK ACTIVATION, AND HEAT-SHOCK PROTEIN EXPRESSION ARE MEDIATED BY GENISTEIN-SENSITIVE TYROSINE KINASE(S) IN K562 CELLS

Upon exposure to elevated growth temperatures, mammalian cells exhibit a variety of cellular responses, such as the expression of heat‐shock proteins (HSPs) and the activation of stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK). In this study, we show that heat shock transiently induces morphological change (cell elongation) and polymerization of actin, but not of microtubules, in human erythroleukaemic K562 cells. Pretreatment with actinomycin D or cycloheximide did not prevent the heat shock‐induced cell elongation and actin reorganization, indicating that gene transcription and protein synthesis are not required for this phenomenon. The alterations in cell morphology and actin structure in response to heat shock were specifically inhibited by genistein, a tyrosine kinase inhibitor, but not by other kinase inhibitors, including tyrosine kinase inhibitors (herbimycin and tyrphostin) and protein kinase C inhibitors (staurosporine and H7). The activities of genistein‐sensitive tyrosine kinase (GTK) and c‐Src were enhanced by heat‐shock treatment. In addition, a 75kDa protein was highly phosphorylated in its tyrosine residues(s) by heat shock, and the phosphorylation was prevented by genistein pretreatment. Genistein also inhibited the heat‐shock‐induced SAPK/JNK activation and HSP expression. In contrast, while colchicine, a microtubule‐disrupting agent, was able to induce actin polymerization and SAPK/JNK activation, these events were not inhibited by genistein. These results suggest that the heat‐shock‐induced actin polymerization, HSP expression, and SAPK/JNK activation may be mediated by the specific signal pathway involving GTK(s), while colchicine‐induced actin polymerization and SAPK/JNK activation is regulated in a different manner.