Evaluation of Erastin as a Therapeutic Agent Under Hypoxic Conditions in Pancreatic Cancer Cells.

BACKGROUND/AIM: In pancreatic cancer tissues, hypoxic areas exist due to poor blood flow. Attenuation of the pharmacological efficacy of existing anticancer drugs in these hypoxic areas necessitates the search for novel anticancer compounds. We aimed to determine whether erastin exhibits anticancer effects in a hypoxic environment. MATERIALS AND METHODS: Pancreatic cancer cell lines were subjected to cobalt chloride, a hypoxia-mimicking agent. Cell viability assay, measurement of reactive oxygen species, and western blotting analysis were conducted to investigate the efficacy of erastin under hypoxic environments. RESULTS: Erastin exhibited remarkable cytotoxicity and induced apoptosis under hypoxic conditions. Furthermore, erastin triggered the intracellular accumulation of reactive oxygen species in a hypoxic environment. Subsequent treatment with N-acetylcysteine, an antioxidant, markedly attenuated cytotoxicity, and apoptosis. CONCLUSION: Erastin induces cell death by accumulation of intracellular reactive oxygen species and inducing apoptosis under hypoxic conditions, proving its potential for further development as a novel anticancer compound.

Metabolic reprogramming sustains cancer cell survival following extracellular matrix detachment.

Epithelial cells require attachment to a support, such as the extracellular matrix, for survival. During cancer progression and metastasis, cancerous epithelial cells must overcome their dependence on adhesion signals. Dependence on glucose metabolism is a hallmark of cancer cells, but the nutrient requirements of cancer cells under anchorage-deficient conditions remain uncharacterized. Here, we report that cancer cells prioritize glutamine-derived tricarboxylic acid cycle energy metabolism over glycolysis to sustain anchorage-independent survival. Moreover, glutamine-dependent metabolic reprogramming is required not only to maintain ATP levels but also to suppress excessive oxidative stress through interaction with cystine. Mechanistically, AMPK, a central regulator of cellular responses to metabolic stress, participates in the induction of the expression of ASCT2, a glutamine transporter, and enhances glutamine consumption. Most interestingly, AMPK activation induces Nrf2 and its target proteins, allowing cancer cells to maintain energy homeostasis and redox status through glutaminolysis. Treatment with an integrin inhibitor was used to mimic the alterations in cell morphology and metabolic reprogramming caused by detachment. Under these conditions, cells were vulnerable to glutamine starvation or glutamine metabolism inhibitors. The observed preference for glutamine over glucose was more pronounced in aggressive cancer cell lines, and treatment with the glutaminase inhibitor, CB839, and cystine transporter inhibitor, sulfasalazine, caused strong cytotoxicity. Our data clearly show that anchorage-independent survival of cancer cells is supported mainly by glutaminolysis via the AMPK-Nrf2 signal axis. The discovery of new vulnerabilities along this route could help slow or prevent cancer progression.

Setanaxib as a Potent Hypoxia-specific Therapeutic Agent Against Liver Cancer.

BACKGROUND/AIM: Liver cancer has extremely poor prognosis. The cancerous tissues contain hypoxic regions, and the available drugs are poorly effective in hypoxic environments. NADPH oxidase 4 (NOX4), producing reactive oxygen species (ROS), may contribute to cancer malignancy under hypoxic conditions. However, its role in liver cancer has not been examined in detail. Our aim was to explore the effects of setanaxib, a recently developed selective NOX4 inhibitor, in liver cancer cells under hypoxic conditions. MATERIALS AND METHODS: Liver cancer cell lines (HepG2, HLE and Alexander) were treated with hypoxia-mimetic agent cobalt chloride. Cytotoxicity assays, immunoblot analysis and ROS detection assay were performed to detect the effect of setanaxib under hypoxic conditions. RESULTS: Setanaxib exhibited hypoxia-selective cytotoxicity and triggered apoptosis in cancer cells. Moreover, setanaxib caused mitochondrial ROS accumulation under hypoxic conditions. Treatment with antioxidants markedly attenuated setanaxib-induced cytotoxicity and apoptosis under hypoxic conditions. CONCLUSION: Setanaxib caused mitochondrial ROS accumulation in a hypoxia-selective manner and evoked cancer cell cytotoxicity by inducing apoptosis. Thus, setanaxib has a great potential as a novel anticancer compound under hypoxic conditions.

A nationwide cross-sectional survey on hepatitis B and C screening among workers in Japan.

In Japan, there is no publicly funded screening for hepatitis B virus (HBV) and hepatitis C virus (HCV) infections (using HBs antigen and HCV antibody, respectively) among workers, and workplace health programmes play a crucial role in reducing viral hepatitis-related deaths. The national number of hepatitis screening tests conducted in the workplace is unknown. To provide baseline data for policy formulation, we conducted a nationwide survey to estimate these parameters using data from approximately 10.5 million workers (6.8 million men and 3.8 million women) who underwent mandatory health examinations in their workplaces between April 2016 and March 2017. Among these workers, 494,303 (5.23%, 95% confidence interval [CI] 5.22%-5.24%) and 313, 193 (3.82%, 95% CI 3.81%-3.84%) were screened for HBV and HCV, respectively. Among those who were screened, 0.28% (95% CI 0.27-0.30%) and 0.35% (95% CI 0.33-0.37%) tested positive for HBs antigen and HCV antibody, respectively. According to the age-specific prevalence from the survey an estimated 0.30 and 0.14 million workers in Japan require treatment for HBV and HCV, respectively. To reduce viral hepatitis-related deaths by efficiently identifying workers who need treatment and promoting access to treatment, one-time hepatitis screening of all workers should be considered.

Glucose starvation induces LKB1-AMPK-mediated MMP-9 expression in cancer cells.

Cancer cells utilise the glycolytic pathway to support their rapid growth and proliferation. Since cells in most solid tumours are subjected to severe microenvironmental stresses including low nutrient and oxygen availability, such cancer cells must develop mechanisms to overcome these unfavourable growth conditions by metabolic adaptation. Although the liver kinase B1 (LKB1)-adenosine monophosphate-activated kinase (AMPK) signalling pathway plays a pivotal role in maintaining energy homeostasis under conditions of metabolic stress, the role of LKB1-AMPK signalling in aiding cancer cell survival and in malignant tumours has not yet been fully elucidated. We show that glucose starvation promotes cancer cell invasiveness and migration through LKB1-AMPK-regulated MMP-9 expression. Most intriguingly, triggering the LKB1-AMPK signalling pathway by glucose starvation-induced oxidative stress facilitates selective autophagy, which in turn enhances Keap1 degradation and the subsequent activation of Nrf2. Following this, Nrf2 regulates the transactivation of MMP-9 via Nrf2 binding sites in the promoter region of the MMP-9 gene. These mechanisms also contribute to the suppression of excessive oxidative stress under glucose starvation, and protect against cell death. Our data clearly shows that LKB1-AMPK signalling not only maintains energy and oxidative stress homeostasis, but could also promote cancer progression during metabolic stress conditions by MMP-9 induction.

Change in plasma lactate concentration during arctigenin administration in a phase I clinical trial in patients with gemcitabine-refractory pancreatic cancer.

Arctigenin is evaluated for antitumor efficacy in patients with pancreatic cancer. It has an inhibitory activity on mitochondrial complex I.Therefore, plasma lactate level of patients after arctigenin administration was evaluated for biomarker of clinical response and/or adverse effect. Plasma lactate level in 15 patients enrolled in a Phase I clinical trial of GBS-01 rich in arctigenin was analyzed by colorimetric assay. Statistical analyses for association of plasma lactate and clinical responses, pharmacokinetics of arctigenin, and background factors of each patient by multivariate and univariate analyses.In about half of the patients, transient increase of lactate was observed. Correlation between plasma lactate level and pharmacokinetic parameters of arctigenin and its glucuronide conjugate, and clinical outcome was not detected. Regarding to the determinant of lactate level, only slight association with liver function test was detected. Plasma lactate level is primary determined by reutilization rather than production for antitumor effect and dose not serve as a biomarker. Arctigenin, inhibition of mitochondrial complex I, plasma lactate concentration, phase I clinical trial of GBS-01, Cori cycle.

Autophagy­mediated adaptation of hepatocellular carcinoma cells to hypoxia­mimicking conditions constitutes an attractive therapeutic target.

Hepatocellular carcinoma has extremely poor prognosis. In cancerous liver tissues, aberrant proliferation of cancer cells leads to the creation of an area where an immature vascular network is formed. Since oxygen is supplied to cancer tissues through the bloodstream, a part of the tumor is exposed to hypoxic conditions. As hypoxia is known to severely reduce the effectiveness of existing anticancer agents, novel valid therapeutic targets must be identified for the treatment of hepatocellular carcinoma. Generally, autophagy has been reported to play an important role in the adaptation of cancer cells to hypoxia. However, the exact role and significance of this process vary depending on the cancer type, requiring detailed analysis in individual primary tumors and cell lines. In the present study, we examined autophagy induced by cobalt chloride, a hypoxia­mimicking agent, in hepatocellular carcinoma cells with the aim to evaluate the validity of this process as a potential therapeutic target. We observed that treatment with cobalt chloride induced autophagy, including the intracellular quality control mechanism, in an AMPK­dependent manner. Furthermore, treatment with autophagy inhibitors (bafilomycin and LY294002) resulted in significant, highly­selective cytotoxicity and apoptosis activation under hypoxia­mimicking conditions. The knockdown of AMPK also revealed significant cytotoxicity in hypoxia­mimicking conditions. These results clearly demonstrated that autophagy, especially mitophagy, was induced by the AMPK pathway when hepatocellular carcinoma cells were subjected to hypoxic conditions and played an important role in the adaptation of these cells to such conditions. Thus, autophagy may constitute an attractive therapeutic target for the treatment of hepatocellular carcinoma.

An Adaptation System to Avoid Apoptosis via Autophagy Under Hypoxic Conditions in Pancreatic Cancer Cells.

BACKGROUND/AIM: Pancreatic cancer tissue is a hypoxic environment resistant to anticancer drugs. This study examined the role of autophagy as a response to hypoxic stress in pancreatic cancer. MATERIALS AND METHODS: Pancreatic cell lines (PANC-1, BxPC-3 and AsPC-1) were exposed to hypoxic conditions using cobalt chloride, a hypoxia-mimicking agent. Protein expression and cytotoxicity assays were performed to determine the effect of hypoxia on autophagy. RESULTS: When pancreatic cancer cells were exposed to hypoxia, autophagy was induced. The autophagy-inducing signal was dependent on the AMPK pathway. Inhibition of autophagy in a hypoxic state induced a remarkable cytotoxicity and enhanced apoptosis. When an AMPK inhibitor was added, cytotoxicity was observed in the hypoxic environment. CONCLUSION: The induced autophagy, dependent on the AMPK pathway, is a necessary survival strategy adopted by pancreatic cancer cells to adapt to hypoxic stress, and could be an attractive target for drug development.

[Therapeutic strategies targeting cancer--specific metabolism].

Basic, clinical and translational metabolic researches in cancer area have been extensively tried to discover and develop novel cancer metabolism drugs. Since tumor cells have metabolic dependencies that distinguish them from their normal counterparts, targeted inhibition of these metabolic dependencies is considered a promising therapeutic strategy against cancer. For example the representative cancer metabolism is that cancer cells exhibit profound metabolic alterations by choosing aerobic glycolysis to metabolize glucose to lactate regardless of the presence of adequate oxygen, although normal cells which mainly utilize glucose by using mitochondrial oxidative phosphorylation to generate ATP. In this review, we focus on several important oncogenes and enzymes, whose alterations have contributed extensively to the metabolic phenotype of cancer cells, with an emphasis on the therapeutic targets.

Epidermal growth factor receptor (EGFR) signaling regulates global metabolic pathways in EGFR-mutated lung adenocarcinoma.

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFRTKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.

Critical role of H2O2 generated by NOX4 during cellular response under glucose deprivation.

Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H2O2) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H2O2 levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H2O2 in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H2O2 could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.