Roles of oxidative stress/JNK/ERK signals in paraquat-triggered hepatic apoptosis.

Paraquat (PQ), a toxic and nonselective bipyridyl herbicide, is one of the most extensively used pesticides in agricultural countries. In addition to pneumotoxicity, the liver is an important target organ for PQ poisoning in humans. However, the mechanism of PQ in hepatotoxicity remains unclear. In this study, we found that exposure of rat hepatic H4IIE cells to PQ (0.1-2 mM) induced significant cytotoxicity and apoptosis, which was accompanied by mitochondria-dependent apoptotic signals, including loss of mitochondrial membrane potential (MMP), cytosolic cytochrome c release, and changes in the Bcl-2/Bax mRNA ratio. Moreover, PQ (0.5 mM) exposure markedly induced JNK and ERK1/2 activation, but not p38-MAPK. Blockade of JNK and ERK1/2 signaling by pretreatment with the specific pharmacological inhibitors SP600125 and PD98059, respectively, effectively prevented PQ-induced cytotoxicity, mitochondrial dysfunction, and apoptotic events. Additionally, PQ exposure stimulated significant oxidative stress-related signals, including reactive oxygen species (ROS) generation and intracellular glutathione (GSH) depletion, which could be reversed by the antioxidant N-Acetylcysteine (NAC). Buffering the oxidative stress response with NAC also effectively abrogated PQ-induced hepatotoxicity, MMP loss, apoptosis, and phosphorylation of JNK and ERK1/2 protein, however, the JNK or ERK inhibitors did not suppress ROS generation in PQ-treated cells. Collectively, these results demonstrate that PQ exposure induces hepatic cell toxicity and death via an oxidative stress-dependent JNK/ERK activation-mediated downstream mitochondria-regulated apoptotic pathway.

Silica nanoparticles induce caspase-dependent apoptosis through reactive oxygen species-activated endoplasmic reticulum stress pathway in neuronal cells.

Human exposure to silica nanoparticles (SiNPs) has been widely applied as vehicles for drug delivery and cellular manipulations in nanoneuromedicine. SiNPs may cause adverse effects in the brain, but potential mechanisms underlying SiNPs-induced neurotoxicity are remained unclear. Here, we examined cytotoxic effects and the cellular mechanisms of SiNPs-induced neuronal cell death. In this study, the results showed that SiNPs significantly decreased cell viability and induced apoptosis in Neuro-2a cells as evidenced by the increase caspase-3 activity and the activation of caspase cascades and poly (ADP-ribose) polymerase (PARP). In addition, endoplasmic reticulum (ER) stress was triggered as indicated by several key molecules including glucose-regulated protein (GRP)78 and 94, C/EBP homologous protein (CHOP), activation transcription factor (ATF)-4, and caspase-12. Pretreatment of Neuro-2a cells with specific pharmacological inhibitor of ER stress (4-phenylbutyric acid (4-PBA)) effectively alleviated the SiNPs-induced ER stress and apoptotic related signals. Furthermore, 2',7'-Dichlorofluorescein fluorescence as an indicator of reactive oxygen species (ROS) formation after exposure of Neuro-2a cells to SiNPs significantly increased ROS levels. Antioxidant N-acetylcyseine (NAC) effectively reversed SiNPs-induced cellular responses. Taken together, these results suggest that SiNPs exposure exerts its neurotoxicity in cultured neuronal cells by inducing apoptosis via a ROS generation-activated downstream ER stress signaling pathway.

Methylmercury exposure induces ROS/Akt inactivation-triggered endoplasmic reticulum stress-regulated neuronal cell apoptosis.

Epidemiological studies have positively linked mercury exposure and neurodegenerative diseases (ND). Methylmercury (MeHg), an organic form of mercury, is a ubiquitous and potent environmental neurotoxicant that easily crosses the blood-brain barrier and causes irreversible injury to the central nervous system (CNS). However, the molecular mechanisms underlying MeHg-induced neurotoxicity remain unclear. Here, the present study found that Neuro-2a cells underwent apoptosis in response to MeHg (1-5 µM), which was accompanied by increased phosphatidylserine (PS) exposure on the outer cellular membrane leaflets, caspase-3 activity, and the activation of caspase cascades and poly (ADP-ribose) polymerase (PARP). Exposure of Neuro-2a cells to MeHg also triggered endoplasmic reticulum (ER) stress, which was identified via several key molecules (including: glucose-regulated protein (GRP)78, GRP94, C/EBP homologous protein (CHOP) X-box binding protein(XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4, and ATF6. Transfection with GRP78-, GRP94-, CHOP-, and XBP-1-specific small interfering (si)RNA significantly suppressed the expression of these proteins, and attenuated cytotoxicity and caspase-12, -7, and -3 activation in MeHg-exposed cells. Furthermore, MeHg dramatically decreased Akt phosphorylation, and the overexpression of activation of Akt1 (myr-Akt1) could significantly prevent MeHg-induced Akt inactivation, as well as apoptotic and ER stress-related signals. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively prevented MeHg-induced neuronal cell reactive oxygen species (ROS) generation, apoptotic and ER stress-related signals, and Akt inactivation. Collectively, these results indicate that MeHg exerts its cytotoxicity in neurons by inducing ROS-mediated Akt inactivation up-regulated ER stress, which induces apoptosis and ultimately leads to cell death.

Cadmium exposure induces pancreatic ß-cell death via a Ca2+-triggered JNK/CHOP-related apoptotic signaling pathway.

Cadmium (Cd) is known to be ranked the 7th hazardous substance in the Substance Priority List by Agency for Toxic Substances and Disease Registry. The experimental and epidemiological data have suggested that Cd is linked to the development of diabetes mellitus (DM). The molecular mechanism of Cd on the pancreatic ß-cell cytotoxicity still remains unclear. Evidence has pointed toward that Ca2+ is an important regulator of toxic insult-induced ß-cell cytotoxicity. The role of Ca2+ in the Cd-induced ß-cell cytotoxicity is still unknown. In this study, we found that Cd exposure significantly inhibited insulin secretion and cell viability in the pancreatic ß-cell-derived RIN-m5F cells. Cd exposure induced apoptotic events, including the increased populations of apoptotic cells and sub-G1 hypodiploid cells, and caspase-3/-7/-9 and poly (ADP-ribose) polymerase (PARP) activation, which largely depended on the activation of c-Jun N-terminal kinase (JNK) and C/EBP homologous protein (CHOP). Transfection with siRNAs for JNK and CHOP or pretreatment with specific pharmacological inhibitor of JNK (SP600125) in ß-cells effectively prevented the Cd-induced insulin secretion dysfunction and apoptosis. JNK-specific siRNA dramatically suppressed Cd-induced JNK phosphorylation and CHOP protein expression, but JNK phosphorylation could not be inhibited by CHOP-specific siRNA. Furthermore, Cd exposure significantly increased the intracellular calcium ([Ca2+]i) levels. Buffering the Ca2+ response with BAPTA/AM effectively abrogated the Cd-induced [Ca2+]i elevation, insulin secretion dysfunction, apoptosis, and protein expression of JNK phosphorylation and CHOP activation. Taken together, these findings demonstrated that Cd exposure exerts ß-cell death via a [Ca2+]i-dependent JNK activation-activated downstream CHOP-related apoptotic signaling pathway.

Plasma Levels of Endothelial Cell-Specific Molecule-1 as a Potential Biomarker of Oral Cancer Progression.

In Taiwan, oral cancer is the fourth most common cancer and the most common malignancy with a poor prognosis. Endothelial cell-specific molecule-1 (ESM-1) is secreted by vascular endothelial cells in the liver, lungs, kidneys, and gastrointestinal tract. ESM-1 expression is associated with tumor prognosis, metastasis, and angiogenesis in many cancers. However, few studies have examined the association of plasma ESM-1 levels with oral squamous cell carcinoma (OSCC) progression. We measured the plasma ESM-1 levels of 438 male OSCC patients through a commercial enzyme-linked immunosorbent assay. The Cancer Genome Atlas (TCGA) dataset was also used to analyze the ESM-1 levels in 328 OSCC patients and 33 normal tissues. Our results revealed that the plasma levels of ESM-1 in OSCC patients were significantly associated with the tumor (T) status but not with the lymph node status, metastasis, and cell differentiation. TCGA bioinformatics database analysis revealed that ESM-1 expression was significantly higher in OSCC patients than in normal individuals (p < 0.05). In addition, the examination revealed similar results for the ESM-1 expression levels and pathological stage in OSCC. In conclusion, plasma ESM-1 is a novel biomarker for predicting the T status in OSCC patients.

Cantharidin Induced Oral Squamous Cell Carcinoma Cell Apoptosis via the JNK-Regulated Mitochondria and Endoplasmic Reticulum Stress-Related Signaling Pathways.

Oral cancer is a subtype of head and neck cancer which represents 2.65% of all human malignancies. Most of oral cancer is histopathologically diagnosed as oral squamous cell carcinoma (OSCC). OSCC is characterized by a high degree of local invasion and a high rate of metastasis to the cervical lymph nodes. How to prevention and treatment of OSCC is important and imperative. Here, we investigated the therapeutic effect and molecular mechanism of cantharidin, an active compound isolated from blister beetles, on OSCC in vitro. Results showed that cantharidin significantly decreased cell viability in human tongue squamous carcinoma-derived SAS, CAL-27, and SCC-4 cell lines. The further mechanistic studies were carried out in SAS cells. Cantharidin also significantly increased apoptosis-related signals, including caspase-9, caspase-7 and caspase-3 proteins. Besides, cantharidin decreased mitochondrial transmembrane potential (MMP) and induced cytochrome c and apoptosis inducing factor (AIF) release. Cantharidin also increased Bax, Bid, and Bak protein expressions and decreased Bcl-2 protein expression. Cantharidin could also increase the endoplasmic reticulum (ER) stress signals, including the expressions of phosphorylated eIF-2α and CHOP, but not Grp78 and Grp94. Furthermore, cantharidin reduced pro-caspase-12 protein expression. In signals of mitogen-activated protein kinases, cantharidin increased the phosphorylation of JNK, but not ERK and p38. Transfection of shRNA-JNK to OSCC cells effectively reversed the cantharidin-induced cell apoptotic signals, including the mitochondrial and ER stress-related signaling molecules. Taken together, these findings suggest that cantharidin induces apoptosis in OSCC cells via the JNK-regulated mitochondria and ER stress-related signaling pathways.

Three-dimensional Doppler ultrasonography in assessing nodal metastases and staging head and neck cancer.

OBJECTIVES/HYPOTHESIS: This study built a simple prediction system by three-dimensional (3D) Doppler ultrasonography to evaluate the metastases of cervical lymph nodes and the preoperative initial stage of head and neck cancer. STUDY DESIGN: Retrospective review of cervical lymph node ultrasound features and prospective nodal staging of head and neck cancer. METHODS: One hundred thirty-nine suspicious cervical lymph nodes, receiving 3D Doppler ultrasonography, were used to establish a predictive model. Then nodal metastasis was initially staged from 27 patients with head and neck cancer by this model. RESULTS: The prediction system was constructed by major (internal matting, vascularity pattern) and minor (age ≥ 40 years, short/long ratio ≥ 0.5) sign categories. Cervical lymph node was regarded as metastasis with the presence of one major and any of the other factors. The predictive model resulted in sensitivity of 91.9%, specificity of 88.2%, and accuracy of 89.2%. Then we evaluated the initial staging of patients with head and neck cancer by this model, and the rate of correct N staging was 92.6%. CONCLUSIONS: According to this prediction system, 3D Doppler ultrasonography definitely provides a rapid and reliable method for initial staging of head and neck cancer.