DHA Induces Cell Death through the Production of ROS and the Upregulation of CHOP in Fibroblast-like Synovial Cells from Human Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is an inflammatory disease marked by a massive proliferation of synovial cells in the joints. In this study, we investigated the pro-apoptotic effects of docosahexaenoic acid (DHA) in human fibroblast-like synovial cells from RA patients (RA-FLS). An in vitro study using MH7A cells showed that DHA treatment induced caspase-8-dependent apoptosis in a dose-dependent manner and reduced the TNF-α-mediated induction of MMP-9 and IL-1ß. DHA also induced the phosphorylation of eIF2α, the expression of the ER stress markers ATF4 and C/EBP homologous protein (CHOP), and death receptor 5 (DR5). The knockdown of CHOP or DR5 increased cell viability and reduced apoptosis in DHA-treated cells. Furthermore, the knockdown of CHOP reduced DHA-mediated DR5 expression, while the overexpression of CHOP increased DR5 expression. We also found that DHA treatment induced the accumulation of reactive oxygen species (ROS), and pretreatment with the anti-oxidant Tiron effectively abrogated not only the expression of CHOP and DR5, but also DHA-induced apoptosis. Under this condition, cell viability was increased, while PARP-1 cleavage and caspase-8 activation were reduced. All the findings were reproduced in human primary synovial cells obtained from RA patients. These results suggest that the DHA-mediated induction of ROS and CHOP induced apoptosis through the upregulation of DR5 in RA-FLSs, and that CHOP could be used as a therapy for RA.

PKR-Mediated Phosphorylation of eIF2a and CHK1 Is Associated with Doxorubicin-Mediated Apoptosis in HCC1143 Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer is more aggressive than other types of breast cancer. Protein kinase R (PKR), which is activated by dsRNA, is known to play a role in doxorubicin-mediated apoptosis; however, its role in DNA damage-mediated apoptosis is not well understood. In this study, we investigated the roles of PKR and its downstream players in doxorubicin-treated HCC1143 triple-negative breast cancer cells. Doxorubicin treatment induces DNA damage and apoptosis. Interestingly, doxorubicin treatment induced the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) via PKR, whereas the inhibition of PKR with inhibitor C16 reduced eIF2α phosphorylation. Under these conditions, doxorubicin-mediated DNA fragmentation, cell death, and poly(ADP ribose) polymerase and caspase 7 levels were recovered. In addition, phosphorylation of checkpoint kinase 1 (CHK1), which is known to be involved in doxorubicin-mediated DNA damage, was increased by doxorubicin treatment, but blocked by PKR inhibition. Protein translation was downregulated by doxorubicin treatment and upregulated by blocking PKR phosphorylation. These results suggest that PKR activation induces apoptosis by increasing the phosphorylation of eIF2α and CHK1 and decreasing the global protein translation in doxorubicin-treated HCC1143 triple-negative breast cancer cells.

G-Protein-Coupled Receptor 120 Mediates DHA-Induced Apoptosis by Regulating IP3R, ROS and, ER Stress Levels in Cisplatin-Resistant Cancer Cells.

The omega-3 fatty acid docosahexaenoic acid (DHA) is known to induce apoptosis and cell cycle arrest via the induction of reactive oxygen species (ROS) production and endoplasmic reticulum (ER) stress in many types of cancers. However, the roles of DHA in drug-resistant cancer cells have not been elucidated. In this study, we investigated the effects of DHA in cisplatin-resistant gastric cancer SNU-601/cis2 cells. DHA was found to induce ROS-dependent apoptosis in these cells. The inositol 1,4,5-triphosphate receptor (IP3R) blocker 2-aminoethyl diphenylboninate (2-APB) reduced DHA-induced ROS production, consequently reducing apoptosis. We also found that G-protein-coupled receptor 120 (GPR120), a receptor of long-chain fatty acids, is expressed in SNU-601/cis2 cells, and the knockdown of GPR120 using specific shRNAs alleviated DHA-mediated ROS production and apoptosis. GPR120 knockdown reduced the expression of ER stress response genes, similar to the case for the pre-treatment of the cells with N-acetyl-L-cysteine (NAC), an ROS scavenger, or 2-APB. Indeed, the knockdown of C/EBP homologous protein (CHOP), a transcription factor that functions under ER stress conditions, markedly reduced DHA-mediated apoptosis, indicating that CHOP plays an essential role in the anti-cancer activity of DHA. These results suggest that GPR120 mediates DHA-induced apoptosis by regulating IP3R, ROS, and ER stress levels in cisplatin-resistant cancer cells, and that GPR120 is an effective chemotherapeutic target for cisplatin resistance.

Apoptotic and Anti-Inflammatory Effects of Eupatorium japonicum Thunb. in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovitis, hyperplasia, and the destruction of bone and cartilage. A variety of immunosuppressive biological agents have been developed because the pathogenesis of RA is related predominantly to the inflammatory response. However, rheumatoid arthritis fibroblast-like synovial cells (RAFLS), which are known to play an important role in RA progression, exhibit resistance to immunosuppressants through cancer-like properties. In this study, we identified a novel therapeutic compound for RA, which reduced inflammation and the abnormal proliferation of RAFLS in natural product library made from Korean native plants. Eupatorium japonicum Thunb. (EJT) extract, a component of the natural product library, most effectively reduced viability through the induction of ROS-mediated apoptosis in a dose-dependent manner. In addition, the increased ROS induced the expression of ATF4 and CHOP, key players in ER stress-mediated apoptosis. Interestingly, EJT extract treatment dose-dependently reduced the expression of IL-1ß and the transcription of MMP-9, which were induced by TNF-α treatment, through the inhibition of NF-κB and p38 activation. Collectively, we found that EJT extract exerted apoptotic effects through increases in ROS production and CHOP expression and exerted anti-inflammatory effects through the suppression of NF-κB activation, IL-1ß expression, and MMP-9 transcription.

Angelica gigas Nakai Has Synergetic Effects on Doxorubicin-Induced Apoptosis.

Natural products are valuable sources for drug discovery because they have a wide variety of useful chemical components and biological properties. A quick reevaluation of the potential therapeutic properties of established natural products was made possible by the recent development of the methodology and improvement in the accuracy of an automated high-throughput screening system. In this study, we screened natural product libraries to detect compounds with anticancer effects using HeLa cells. Of the 420 plant extracts screened, the extract of Angelica gigas Nakai (AGN) was the most effective in reducing cell viability of HeLa cells. Markers of apoptosis, such as exposure of phosphatidylserine and cleavage of caspase-7 and PARP, were increased by treatment with the AGN extract. Treatment of the AGN extract increased expression of PKR as well as ATF4 and CHOP, the unfolded protein response genes. In addition, cotreatment of doxorubicin and the AGN extract significantly increased doxorubicin-induced apoptosis in HeLa cells. Decursin and decursinol angelate, which were known to have anticancer effects, were the main components of the AGN extract. These results suggest that the extract of AGN containing, decursin and decursinol angelate, increases doxorubicin susceptibility.

Salubrinal-Mediated Upregulation of eIF2α Phosphorylation Increases Doxorubicin Sensitivity in MCF-7/ADR Cells.

Eukaryotic translation initiation factor 2 alpha (eIF2α), which is a component of the eukaryotic translation initiation complex, functions in cell death and survival under various stress conditions. In this study, we investigated the roles of eIF2α phosphorylation in cell death using the breast cancer cell lines MCF-7 and MCF-7/ADR. MCF-7/ADR cells are MCF-7-driven cells that have acquired resistance to doxorubicin (ADR). Treatment of doxorubicin reduced the viability and induced apoptosis in both cell lines, although susceptibility to the drug was very different. Treatment with doxorubicin induced phosphorylation of eIF2α in MCF-7 cells but not in MCF-7/ADR cells. Basal expression levels of Growth Arrest and DNA Damage 34 (GADD34), a regulator of eIF2α, were higher in MCF-7/ADR cells compared to MCF-7 cells. Indeed, treatment with salubrinal, an inhibitor of GADD34, resulted in the upregulation of eIF2α phosphorylation and enhanced doxorubicin-mediated apoptosis in MCF-7/ADR cells. However, MCF-7 cells did not show such synergic effects. These results suggest that dephosphorylation of eIF2α by GADD34 plays an important role in doxorubicin resistance in MCF-7/ADR cells.

Hempseed oil induces reactive oxygen species- and C/EBP homologous protein-mediated apoptosis in MH7A human rheumatoid arthritis fibroblast-like synovial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal efficacy of hempseed (Cannabis sativa L.), which is rich in polyunsaturated fatty acids, in atopic dermatitis, inflammation, and rheumatoid arthritis (RA) has been suggested for centuries. Hempseed has been used as a treatment for these diseases in Korean and Chinese folk medicine. The aim of the study is to investigate the effects of hempseed oil (HO) on MH7A human RA fibroblast-like synovial cells. MATERIALS AND METHODS: MH7A cells were used to study the anti-rheumatoid effects of hempseed (Cannabis sativa L., cv. Cheungsam/Cannabaceae) oil by investigating cell viability, apoptosis, lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress-induced apoptosis. RESULTS: HO treatment reduced the survival rate of MH7A cells and promoted apoptotic cell death in a time- and dose-dependent manner. Both lipid accumulation and the level of intracellular reactive oxygen species (ROS) increased in HO-treated MH7A cells. Co-treatment with the antioxidant Tiron effectively abrogated the cytotoxic effects of HO; the ROS level was reduced, cell viability was recovered, and apoptotic cell death was significantly diminished. Moreover, HO-treated cells exhibited increased expression of the major ER stress markers, glucose-regulated protein 78 and C/EBP homologous protein (CHOP). The siRNA-mediated knockdown of CHOP prevented HO-induced apoptosis. CONCLUSIONS: Our results suggest that HO treatment induced lipid accumulation, ROS production, CHOP expression, and apoptosis in MH7A cells, and that CHOP functions as an anti-rheumatoid factor downstream of HO in MH7A cells.

28-Day Oral Toxicity of Cadmium Selenide in Sprague-Dawley Rats.

This study was performed to evaluate the toxicity of cadmium selenide for a period of 28 days in Sprague-Dawley rats. Each of 10 healthy male and females rats per group received daily oral administration for 28-day period at dosage levels 30, 300 and 1,000 mg/kg of body weight. Mortality and clinical signs were checked, and body weight, water intake and food consumption were also recorded weekly. There were no dose-related changes in food consumption or urine volume. All animals survived to the end of study with no clinical signs or differences in body weight gain observed when compared with the control group. At the end of study, all animals including control group, were subjected to necropsy. Blood samples were collected for hematology tests including coagulation time and biochemistry analysis. Blood coagulation time and relative organ weight were unaffected by all received doses. White Blood Cell (WBC) counts significantly increased in the 300 mg/ kg administered male animal group when compared to the control. Monocyte (MO) value were also increased significantly in both 300 and 1,000 mg/kg male animal group. However, Mean Corpuscular Volume (MCV) were significantly decreased compared with the control in the 1,000 mg/kg dose groups for male and female animals. Mean Corpuscular Hemoglobin (MCH) decreased significantly for female in the 300 and 1,000 mg/kg group compared to the control. Blood biochemical values of Inorganic phosphorus (IP) were significantly increased in both the 300 and 1,000 mg/kg dose groups in male animals when compared to the control. Creatinine (CRE) levels indicated significant increase in kidney function for the female, 30 mg/kg dose group when compared with control. There was a significant decrease in thymus absolute organ weight in the female, 1,000 mg/kg dose group when compared with control. Histopathological findings revealed no evidence of injury related to cadmium selenide except for one case of focal hepatic inflammation in the high dose (1,000 mg/kg) group. One case of lung inflammation was also seen in the control group. Basis on these result, the No Observable Adverse Effect Level (NOAEL) of cadmium selenide was determined to be more than 1,000 mg/kg/day for male and female rats under conditions in this study.