Trifloxystrobin induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HaCaT, human keratinocyte cells.

As the outermost layer of the body, the skin plays an important role in exposure to pesticides, which could have negative impacts on human health. Trifloxystrobin is a widely used fungicide of the strobilurin class, however, there is little information regarding the skin contact-associated toxic mechanism. Therefore, the present study was performed in order to identify the skin toxicity mechanism of trifloxystrobin using HaCaT (keratinocyte of human skin) cells. Following 24 or 48 h treatment, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin. This could be proposed as a mechanism of skin toxicity by trifloxystrobin and considered in the management of pesticide exposure.

Cigarette Smoking Condensate Disrupts Endoplasmic Reticulum-Golgi Network Homeostasis Through GOLPH3 Expression in Normal Lung Epithelial Cells.

INTRODUCTION: Cigarette smoke (CS) is associated with a broad range of diseases including lung cancer. Many researchers have suggested that cigarette smoke condensate (CSC) may be more toxic compared to cigarette smoke extract (CSE) because CSC contains the lipid-soluble faction of smoke while CSE contains the hydrophilic or gas phase. The aim of this research is to investigate the effects of CSC on the disruption of endoplasmic reticulum (ER)-Golgi homeostasis in normal lung epithelial cells. METHODS: CS was generated according to the ISO 3308 method. To ascertain the mechanistic effects of CSC on lung toxicity, normal lung epithelial cells of the cell line 16HBE14o- were treated with CSC (0.1mg/mL) for 48 hours. The toxic effects of CSC on ER-Golgi homeostasis and GOLPH3 expression were observed through diverse molecular tools including transmission electron microscope analysis. RESULTS: Our results demonstrated that CSC treatment increased reactive oxygen species generation in lung cells and led to the alteration of ER-Golgi homeostasis in conjunction with increased autophagy. In particular, GOLPH3, known as an oncogene and a marker protein for the trans-Golgi network, was upregulated in CSC-treated cells. GOLPH3 protein overexpression was also confirmed in the lungs of human lung cancer patients as well as NNK-treated mice. CONCLUSION: Our study revealed that CSC caused lung damage through the disruption of ER-Golgi homeostasis and autophagy induction. The expression level of the trans-Golgi marker protein GOLPH3 could serve as a reliable bio-indicator for CS-related lung cancer. IMPLICATIONS: CS is a harmful factor in the development of many diseases including cancer. In this research, we demonstrated that CSC treatment led to malfunction of the ER-Golgi network, with the disrupted ER and Golgi causing GOLPH3 overexpression and abnormal autophagy accumulation. In addition, although the value of GOLPH3 as a predictor remains to be fully elucidated, our data suggest that GOLPH3 levels may be a novel prognostic biomarker of tobacco related lung disease.

Dihydroergotamine Tartrate Induces Lung Cancer Cell Death through Apoptosis and Mitophagy.

BACKGROUND: Mitochondria have emerged as a major target for anticancer therapy because of their critical role in cancer cell survival. Our preliminary works have suggested that dihydroergotamine tartrate (DHE), an antimigraine agent, may have effects on mitochondria. METHODS: We examined the effect of DHE on the survival of several lung cancer cells and confirmed that DHE suppressed diverse lung cancer cell growth effectively. To confirm whether such effects of DHE would be associated with mitochondria, A549 cells were employed for the evaluation of several important parameters, such as membrane potential, reactive oxygen species (ROS) generation, apoptosis, ATP production and autophagy. RESULTS: DHE decreased membrane permeability, increased ROS generation as well as apoptosis, and disturbed ATP production. Eventually, mitophagy was activated for damaged mitochondria. CONCLUSION: Taken together, our findings demonstrate that DHE induces lung cancer cell death by the induction of apoptosis and mitophagy, thus suggesting that DHE can be developed as an anti-lung cancer therapeutic agent.

Cancer-associated splicing variant of tumor suppressor AIMP2/p38: pathological implication in tumorigenesis.

Although ARS-interacting multifunctional protein 2 (AIMP2, also named as MSC p38) was first found as a component for a macromolecular tRNA synthetase complex, it was recently discovered to dissociate from the complex and work as a potent tumor suppressor. Upon DNA damage, AIMP2 promotes apoptosis through the protective interaction with p53. However, it was not demonstrated whether AIMP2 was indeed pathologically linked to human cancer. In this work, we found that a splicing variant of AIMP2 lacking exon 2 (AIMP2-DX2) is highly expressed by alternative splicing in human lung cancer cells and patient's tissues. AIMP2-DX2 compromised pro-apoptotic activity of normal AIMP2 through the competitive binding to p53. The cells with higher level of AIMP2-DX2 showed higher propensity to form anchorage-independent colonies and increased resistance to cell death. Mice constitutively expressing this variant showed increased susceptibility to carcinogen-induced lung tumorigenesis. The expression ratio of AIMP2-DX2 to normal AIMP2 was increased according to lung cancer stage and showed a positive correlation with the survival of patients. Thus, this work identified an oncogenic splicing variant of a tumor suppressor, AIMP2/p38, and suggests its potential for anti-cancer target.

GOLGA2/GM130, cis-Golgi matrix protein, is a novel target of anticancer gene therapy.

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

Emerging role of LETM1/GRP78 axis in lung cancer.

The selective autophagy of damaged mitochondria is called mitophagy. Mitochondrial dysfunction, mitophagy, and apoptosis have been suggested to be interrelated in various human lung carcinomas. Leucine zipper EF-hand-containing transmembrane protein-1 (LETM1) was cloned in an attempt to identify candidate genes for Wolf-Hirschhorn syndrome. LETM1 plays a role in mitochondrial morphology, ion homeostasis, and cell viability. LETM1 has also been shown to be overexpressed in different human cancer tissues, including lung cancer. In the current study, we have provided clear evidence that LETM1 acts as an anchoring protein for the mitochondria-associated ER membrane (MAM). Fragmented mitochondria have been found in lung cancer cells with LETM1 overexpression. In addition, a reduction of mitochondrial membrane potential and significant accumulation of microtubule-associated protein 1 A/1B-light chain 3 punctate, which localizes with Red-Mito, was found in LETM1-overexpressed cells, suggesting that mitophagy is upregulated in these cells. Interestingly, glucose-regulated protein 78 kDa (GRP78; an ER chaperon protein) and glucose-regulated protein 75 kDa (GRP75) were posited to interact with LETM1 in the immunoprecipitated LETM1 of H460 cells. This interaction was enhanced in cells treated with carbonyl cyanide m-chlorophenylhydrazone, a chemical mitophagy inducer. Treatment of cells with honokiol (a GRP78 inhibitor) blocked LETM1-mediated mitophagy, and CRISPR/Cas9-mediated GRP75 knockout inhibited LETM1-induced autophagy. Thus, GRP78 interacts with LETM1. Taken together, these observations support the notion that the complex formation of LETM1/GRP75/GRP78 might be an important step in MAM formation and mitophagy, thus regulating mitochondrial quality control in lung cancer.

AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53.

AIMP2/p38 is a scaffolding protein required for the assembly of the macromolecular tRNA synthetase complex. Here, we describe a previously unknown function for AIMP2 as a positive regulator of p53 in response to genotoxic stresses. Depletion of AIMP2 increased resistance to DNA damage-induced apoptosis, and introduction of AIMP2 into AIMP2-deficient cells restored the susceptibility to apoptosis. Upon DNA damage, AIMP2 was phosphorylated, dissociated from the multi-tRNA synthetase complex, and translocated into the nuclei of cells. AIMP2 directly interacts with p53, thereby preventing MDM2-mediated ubiquitination and degradation of p53. Mutations in AIMP2, affecting its interaction with p53, hampered its ability to activate p53. Nutlin-3 recovered the level of p53 and the susceptibility to UV-induced cell death in AIMP2-deficient cells. This work demonstrates that AIMP2, a component of the translational machinery, functions as proapoptotic factor via p53 in response to DNA damage.

One-shot dual gene editing for drug-resistant pancreatic cancer therapy.

It is challenging to diagnose patients with pancreatic ductal adenocarcinoma (PDAC) early on, and their treatment is often complex. Gemcitabine (GEM) is the first-line treatment for PDAC, but its efficacy is limited in most patients due to the GEM resistance from KRAS and P53 gene mutations. We describe the correction of a double gene mutation and therapeutic effect for the GEM resistant PDAC. Bio-available nanoliposomes (NL) possessing Cas9-ribonucleoproteins and adenine-base editors were developed to conduct KRAS and P53 mutation gene editing directly. NLs were conjugated with EGFR antibodies to tumor-specific delivery, and the anti-cancer effect was verified in vitro and in vivo Model. Our GEM-combinatorial therapeutic strategies using double gene editing systems with one-shot may be a potent therapy for PDAC, overcoming chemoresistance.

High dietary inorganic phosphate increases lung tumorigenesis and alters Akt signaling.

RATIONALE: Phosphate (Pi) is an essential nutrient to living organisms. Recent surveys indicate that the intake of Pi has increased steadily. Our previous studies have indicated that elevated Pi activates the Akt signaling pathway. An increased knowledge of the response of lung cancer tissue to high dietary Pi may provide an important link between diet and lung tumorigenesis. OBJECTIVES: The current study was performed to elucidate the potential effects of high dietary Pi on lung cancer development. METHODS: Experiments were performed on 5-week-old male K-ras(LA1) lung cancer model mice and 6-week-old male urethane-induced lung cancer model mice. Mice were fed a diet containing 0.5% Pi (normal Pi) and 1.0% Pi (high Pi) for 4 weeks. At the end of the experiment, all mice were killed. Lung cancer development was evaluated by diverse methods. MEASUREMENT AND MAIN RESULTS: A diet high in Pi increased lung tumor progression and growth compared with normal diet. High dietary Pi increased the sodium-dependent inorganic phosphate transporter-2b protein levels in the lungs. High dietary consumption of Pi stimulated pulmonary Akt activity while suppressing the protein levels of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 as well as Akt binding partner carboxyl-terminal modulator protein, resulting in facilitated cap-dependent protein translation. In addition, high dietary Pi significantly stimulated cell proliferation in the lungs of K-ras(LA1) mice. CONCLUSIONS: Our results showed that high dietary Pi promoted tumorigenesis and altered Akt signaling, thus suggesting that careful regulation of dietary Pi may be critical for lung cancer prevention as well as treatment.

Poly(ester amine)-mediated, aerosol-delivered Akt1 small interfering RNA suppresses lung tumorigenesis.

RATIONALE: The low efficiency of conventional therapies in achieving long-term survival of patients with lung cancer calls for the development of novel therapeutic options. Recent advances in aerosol-mediated gene delivery have provided the possibility of an alternative for the safe and effective treatment of lung cancer. OBJECTIVES: To demonstrate the feasibility and emphasize the importance of noninvasive aerosol delivery of Akt1 small interfering RNA (siRNA) as an effective and selective option for lung cancer treatment. METHODS: Nanosized poly(ester amine) polymer was synthesized and used as a gene carrier. An aerosol of poly(ester amine)/Akt1 siRNA complex was delivered into K-ras(LA1) and urethane-induced lung cancer models through a nose-only inhalation system. The effects of Akt1 siRNA on lung cancer progression and Akt-related signals were evaluated. MEASUREMENTS AND MAIN RESULTS: The aerosol-delivered Akt1 siRNA suppressed lung tumor progression significantly through inhibiting Akt-related signals and cell cycle. CONCLUSIONS: The use of poly(ester amine) serves as an effective carrier, and aerosol delivery of Akt1 siRNA may be a promising approach for lung cancer treatment and prevention.

Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells.

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, is known to have anti-tumor activity in various malignant neoplasms. However, the precise mechanism by which TET inhibits tumor cell growth remains to be elucidated. The present studies were performed to characterize the potential effects of TET on phosphoinositide 3-kinase/Akt and extracellular signal-regulated kinase (ERK) pathways since these signaling pathways are known to be responsible for cell growth and survival. TET suppressed cell proliferation and induced apoptosis in A549 human lung carcinoma cells. TET treatment resulted in a down-regulation of Akt and ERK phosphorylation in both time-/concentration-dependent manners. The inhibition of ERK using PD98059 synergistically enhanced the TET-induced apoptosis of A549 cells whereas the inhibition of Akt using LY294002 had a less significant effect. Taken together, our results suggest that TET: i) selectively inhibits the proliferation of lung cancer cells by blocking Akt activation and ii) increases apoptosis by inhibiting ERK. The treatment of lung cancers with TET may enhance the efficacy of chemotherapy and radiotherapy and increase the apoptotic potential of lung cancer cells.

High dietary inorganic phosphate affects lung through altering protein translation, cell cycle, and angiogenesis in developing mice.

Inorganic phosphate (Pi) plays a key role in diverse physiological functions. Several studies indicate that Pi may affect lung cell development through Na/Pi cotransporter (NPT). Several NPT subtypes have been identified in mammalian lung, and considerable progress has been made in our understanding of their function and regulation. Therefore, current study was performed to elucidate the potential effects of high dietary Pi on lungs of developing mice. Our results clearly demonstrate that high dietary Pi may affect the lung of developing mice through Akt-related cap-dependent protein translation, cell cycle regulation, and angiogenesis. Our results support the hypothesis that Pi works as a critical signal molecule for normal lung growth and suggest that careful restriction of Pi consumption may be important in maintaining a normal development.

Effects of 7-hydroxy-3-methoxycadalene on cell cycle, apoptosis and protein translation in A549 lung cancer cells.

Previously we reported that cadalene extracted from Zelkova serrata inhibited lung tumorigenesis in mice. However, the precise mechanism has not yet investigated. Here, we examined the effects of cadalene on signal pathways important for apoptosis, cell cycle, and protein translation in lung cancer cells. Our results showed that cadalene suppressed the expression of Akt and its phosphor-forms through controlling PI3K and PTEN. Cadalene also induced apoptosis through facilitating pro-apoptotic protein expression. In addition, cadalene caused cell cycle arrest and decreased mTOR-mediated protein translation. Taken together, cadalene may be developed as a lung cancer therapeutic agent in the future.

Aerosol delivery of urocanic acid-modified chitosan/programmed cell death 4 complex regulated apoptosis, cell cycle, and angiogenesis in lungs of K-ras null mice.

The low efficiency of conventional therapies in achieving long-term survival of patients with lung cancer calls for development of novel treatment options. Although several genes have been investigated for their antitumor activities through gene delivery, problems surrounding the methods used, such as efficiency, specificity, and toxicity, hinder application of such therapies in clinical settings. Aerosol gene delivery as nonviral and noninvasive method for gene therapy may provide an alternative for a safer and more effective treatment for lung cancer. In this study, imidazole ring-containing urocanic acid-modified chitosan (UAC) designed in previous study was used as a gene carrier. The efficiency of UAC carrier in lungs was confirmed, and the potential effects of the programmed cell death protein 4 (PDCD4) tumor suppressor gene on three major pathways (apoptosis, cell cycle, and angiogenesis) were evaluated. Aerosol containing UAC/PDCD4 complexes was delivered into K-ras null lung cancer model mice through the nose-only inhalation system developed by our group. Delivered UAC/PDCD4 complex facilitated apoptosis, inhibited pathways important for cell proliferation, and efficiently suppressed pathways important for tumor angiogenesis. In summary, results obtained by Western blot analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay suggest that our aerosol gene delivery technique is compatible with in vivo gene delivery and can be applied as a noninvasive gene therapy.

Ephedrine-induced mitophagy via oxidative stress in human hepatic stellate cells.

The herb Ephedra sinica (also known as Chinese ephedra or Ma Huang), used in traditional Chinese medicine, contains alkaloids identical to ephedrine and pseudoephedrine as its principal active constituents. Recent studies have reported that ephedrine has various side effects in the cardiovascular and nervous systems. In addition, herbal Ephedra, a plant containing many pharmacologically active alkaloids, principally ephedrine, has been reported to cause acute hepatitis. Many studies reported clinical cases, however, the cellular mechanism of liver toxicity by ephedrine remains unknown. In this study, we investigated hepatotoxicity and key regulation of mitophagy in ephedrine-treated LX-2 cells. Ephedrine triggered mitochondrial oxidative stress and depolarization. Mitochondrial swelling and autolysosome were observed in ephedrine-treated cells. Ephedrine also inhibited mitochondrial biogenesis, and the mitochondrial copy number was decreased. Parkin siRNA recovered the ephedrine-induced mitochondrial damage. Excessive mitophagy lead to cell death through imbalance of autophagic flux. Moreover, antioxidants and reducing Parkin level could serve as therapeutic targets for ephedrine-induced hepatotoxicity.

Effects of feeding a diet containing Gymnema sylvestre extract: Attenuating progression of obesity in C57BL/6J mice.

OBJECTIVE: To investigate the effect of Gymnema sylvestre extract (GS) on initial anti-obesity, liver injury, and glucose homeostasis induced by a high-fat diet (HFD). METHODS: The dry powder of GS was extracted with methanol, and gymnemic acid was identified by high performance liquid chromatography as deacyl gymnemic acid. Male C57BL/6J mice that fed on either a normal diet, normal diet containing 1 g/kg GS (CON+GS), HFD, or HFD containing 1.0 g/kg GS (HFD + GS) for 4 weeks were used to test the initial anti-obesity effect of GS. Body weight gain and food intake, and serum levels about lipid and liver injury markers were measured. Histopathology of adipose tissue and liver stained with hematoxylin and eosin (H&E) and oil-red O were analyzed. After 4 weeks of GS extract feeding, intraperitoneal glucose tolerance test (IPGTT) was performed. RESULTS: The methanol extracts of GS exerted significant anti-obesity effects in HFD + GS group. They decreased body weight gain, a lower food and energy efficiency ratio, and showed lower serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL)-cholesterol, very-low density lipoprotein (VLDL)-cholesterol and leptin compared with the HFD group. The decreases of abdominal as well as epididymal fat weight and adipocyte hypertrophy, lipid droplets in liver, and serum levels of aspartate aminotransferase (AST) and alanine transaminase (ALT) were also observed. The CON + GS group showed an effect of glucose homeostasis compared to the CON group. CONCLUSIONS: This study shows that GS provide the possibility as a key role in an initial anti-obesity effects feeding with a HFD.

Endoplasmic reticulum-Golgi intermediate compartment protein 3 knockdown suppresses lung cancer through endoplasmic reticulum stress-induced autophagy.

Trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus is elevated in cancer cells. Therefore, proteins of the ER-Golgi intermediate compartment (ERGIC) attract significant attention as targets for cancer treatment. Enhanced cancer cell growth and epithelial-mesenchymal transition by ERGICs correlates with poor-prognosis of lung cancer. This prompted us to assess whether knockdown of ERGIC3 may decrease lung cancer growth. To test the hypothesis, the effects of ERGIC3 short hairpin RNA (shERGIC3) on ER stress-induced cell death and lung tumorigenesis were investigated both in vitro and in vivo. Knockdown of ERGIC3 led to ER stress-induced autophagic cell death and suppression of proliferation in the A549 human lung cancer cell-line. Moreover, non-invasive aerosol-delivery of shERGIC3 using the biocompatible carrier glycerol propoxylate triacrylate and spermine (GPT-SPE) inhibited lung tumorigenesis in the K-rasLA1 murine model of lung cancer. Our data suggest that suppression of ERGIC3 could provide a framework for the development of effective lung cancer therapies.

Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells.

Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO2-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO2-NP in cell organelles. Human bronchial epithelial cells (16HBE14o-) were exposed to 50 and 100 µg/mL TiO2-NP for 24 and 48 h. Our results showed that TiO2-NP induced endoplasmic reticulum (ER) stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs) and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA), mitigated the cellular toxic response, suggesting that TiO2-NP promoted toxicity via ER stress. This novel mechanism of TiO2-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application.

B6C3F1 mice exposed to ozone with 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and/or dibutyl phthalate showed toxicities through alterations of NF-kappaB, AP-1, Nrf2, and osteopontin.

Toxic effects of ozone, 4-(N-methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK), and/or dibutyl phthalate (DBP) were examined through NF-kappaB, AP-1, Nrf2, and osteopontin (OPN) in lungs and livers of B6C3F1 mice. Electrophoretic mobility shift assay (EMSA) indicated that mice treated with combination of toxicants induced high NF-kappaB activities. Expression levels of p105, p65, and p50 proteins increased in all treated mice, whereas IkB activity was inhibited in NNK-, DBP-, and combination-treated ones. All treated mice except ozone-treated one showed high AP-1 binding activities. Expression levels of c-fos, c-jun, junB, jun D, Nrf2, and OPN proteins increased in all treated mice. Additive interactions were frequently noted from two-toxicant combination mice compared to ozone-treated one. These results indicate treatment of mixture of toxicants increased toxicity through NF-kappaB, AP-1, Nrf2, and OPN. Our data could be applied to the elucidation of mechanism as well as the risk assessment of mixture-induced toxicity.

Role of p53 in the cellular response following oleic acid accumulation in Chang liver cells.

Abnormal accumulation of fatty acids triggers the harmful cellular response called lipotoxicity. In this study, we investigated the cellular response following accumulation of oleic acid (OA), a monounsaturated fatty acid, in human Chang liver cells. OA droplets were distributed freely in the cytoplasm and/or degraded within lysosomes. OA exposure increased ATP production and concomitantly dilated mitochondria. At 24h after OA exposure, cell viability decreased slightly and was coupled with a reduction in mitochondrial Ca(2+) concentration, the alteration in cell viability was also associated with the generation of reactive oxygen species and changes in the cell cycle. Moreover, OA treatment increased the expression of autophagy- and apoptotic cell death-related proteins in a dose-dependent manner. Furthermore, we investigated the role of p53, a tumor suppressor protein, in the cellular response elicited by OA accumulation. OA-induced changes in cell viability and ATP production were rescued to control levels when cells were pretreated with pifithrin-alpha (PTA), a p53 inhibitor. By contrast, the expressions of LC3-II and perilipin, proteins required for lipophagy, were down-regulated by PTA pretreatment. Taken together, our results suggest that p53 plays a key role in the cellular response elicited by OA accumulation in Chang liver cells.