Sesamin exerts anti-tumor activity in esophageal squamous cell carcinoma via inhibition of TRIM44 and NF-κB signaling.

Tripartite motif-containing 44 (TRIM44) is known to play an oncogenic role in multiple human cancers, including esophageal cancer. Sesamin possesses potent anti-inflammatory and anti-cancer properties for various cancers. This study is designed to unravel the biological functions of sesamin and TRIM44 in esophageal cancer. TRIM44 expression in esophageal squamous cell cancer (ESCC) cell lines and tissues was determined by RT-qPCR assay and Western blot. The effects of sesamin and TRIM44 on ESCC cell growth in vivo and in vitro were assessed by the mouse model and CCK-8 assay, respectively. We found that TRIM44 was significantly upregulated in ESCC cell lines and tissues when compared to their counterparts. Sesamin treatment or depletion of TRIM44 markedly reduced ESCC cell proliferation. The nuclear factor kappa B (NF-κB) and toll-like receptor 4 (TLR4) signaling pathway may be involved in sesamin-mediated TRIM44 suppression. Finally, we showed that oral administration of sesamin dramatically inhibited tumor growth or ESCC in nude mice. Our results suggest that sesamin exerts anti-tumor activity in ESCC via inhibition of NF-κB signaling pathway, demonstrating its potential for the treatment of esophageal cancer.

Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

Electrophysiological characterization of drug response in hSC-derived cardiomyocytes using voltage-sensitive optical platforms.

INTRODUCTION: Voltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays. METHODS: Three commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites. RESULTS: Baseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition. DISCUSSION: In conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses.

Effect of ischaemic brain injury on sexual function in adult mice.

OBJECTIVE: Priapism refers to a condition with persistent abnormal erection of the penis, which is usually caused by disease or injury in the brain or spinal cord, or obstruction to the outflow of blood through the dorsal vein at the root of the penis, without sexual desires. The effect of cerebral ischaemia on sexual function is unknown. The aim of this study is to explore whether priapism occurs in adult mice. Furthermore, we examined the relationship between priapism and the region of infarct in the brain. DESIGN: Adult male CD-1 mice who underwent permanent middle cerebral artery occlusion (pMCAO) were closely examined from 2 hours to 14 days postoperation. RESULTS: We found that priapism occurs in ∼80% of the mice with pMCAO, which could persist up to 14 days. Further study has demonstrated that the occurrence of priapism is related to the infarct region: priapism is found only in mice with ischaemic injury extending to the hypothalamus and the hippocampus regions. CONCLUSION: Our result suggested priapism may be used as a deep brain injury marker for evaluating brain injury in mice after pMCAO.

Melatonin Pretreatment Improves the Survival and Function of Transplanted Mesenchymal Stem Cells after Focal Cerebral Ischemia.

Mesenchymal stem cell (MSC) transplantation has been shown to be beneficial in treating cerebral ischemia. However, such benefit is limited by the low survival of transplanted MSCs in an ischemic microenvironment. Previous studies showed that melatonin pretreatment can increase MSC survival in the ischemic kidney. However, whether it will improve MSC survival in cerebral ischemia is unknown. Our study examined the effect of melatonin pretreatment on MSCs under ischemia-related conditions in vitro and after transplantation into ischemic rat brain. Results showed that melatonin pretreatment greatly increased survival of MSCs in vitro and reduced their apoptosis after transplantation into ischemic brain. Melatonin-treated MSCs (MT-MSCs) further reduced brain infarction and improved neurobehavioral outcomes. Angiogenesis, neurogenesis, and the expression of vascular endothelial growth factor (VEGF) were greatly increased in the MT-MSC-treated rats. Melatonin treatment increased the level of p-ERK1/2 in MSCs, which can be blocked by the melatonin receptor antagonist luzindole. ERK phosphorylation inhibitor U0126 completely reversed the protective effects of melatonin, suggesting that melatonin improves MSC survival and function through activating the ERK1/2 signaling pathway. Thus, stem cells pretreated by melatonin may represent a feasible approach for improving the beneficial effects of stem cell therapy for cerebral ischemia.

Purification and immunogenicity study of human papillomavirus 58 virus-like particles expressed in Pichia pastoris.

Two human papillomavirus (HPV) prophylactic vaccines are currently available in the market: Gardasil and Cervarix. These two vaccines work against tumor high-risk subtypes HPV 16 and HPV 18. However, they do not include other high-risk subtypes such as HPV 58. Epidemiological research in China shows that HPV 58 is a prevalent high-risk subtype, second only to HPV 16 and HPV 18. Thus, for cervical cancer prevention in China, developing a vaccine against HPV 58 is necessary. In this study, HPV 58 virus-like particles (VLPs) were expressed in the Pichia pastoris, and subsequently purified through pretreatment and a three-step purification process consisting of strong cation exchange chromatography, size-exclusion chromatography, and hydroxyapatite chromatography. The highly purified HPV 58 VLPs were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electron microscopy, dynamic laser scattering, and ultracentrifugation. The purified VLPs were used to immunize mice to test their ability to induce humoral immunity. Enzyme-linked immunosorbent assays were performed on the sera of the immunized mice and significantly high anti-HPV 58 VLP antibody titers were observed. The immunogenicity study demonstrates that the purified HPV 58 VLPs are HPV vaccine candidates.

p38 MAP kinase mediates arsenite-induced apoptosis through FOXO3a activation and induction of Bim transcription.

Sodium arsenite induces apoptosis in PC12 cells by activating the stress-activated p38 MAP kinase and the pro-apoptotic Bcl-2 family protein Bim(EL). However, the relationship between p38 and Bim(EL) in this apoptosis has not been fully defined. Here, we report that sodium arsenite stimulates the protein expression and promoter activity of Bim(EL) in a p38-dependent manner. Sodium arsenite also caused nuclear translocation of FOXO3a, indicative of FOXO3a activation. Addition of a p38 inhibitor prevented FOXO3a nuclear translocation. RNAi knock down of FOXO3a inhibited Bim promoter activity, Bim(EL) protein expression, and arsenite-induced apoptosis. Our data identify p38 activation of FOXO3a and subsequent induction of Bim(EL) expression as a novel apoptotic mechanism. Together with our previous finding that Bim(EL) is phosphorylated and activated by p38, these results demonstrate that p38 induces apoptosis by regulating Bim(EL) at both the transcriptional and post-translational levels.

p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65.

The stress-activated c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) regulate apoptosis induced by several forms of cellular insults. Potential targets for these kinases include members of the Bcl-2 family proteins, which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway. Indeed, the activities of several Bcl-2 family proteins, both pro- and anti-apoptotic, are controlled by JNK phosphorylation. For example, the pro-apoptotic activity of Bim(EL), a member of the Bcl-2 family, is stimulated by JNK phosphorylation at Ser-65. In contrast, there is no reported evidence that p38-induced apoptosis is due to direct phosphorylation of Bcl-2 family proteins. Here we report evidence that sodium arsenite-induced apoptosis in PC12 cells may be due to direct phosphorylation of Bim(EL) at Ser-65 by p38. This conclusion is supported by data showing that ectopic expression of a wild type, but not a non-phosphorylatable S65A mutant of Bim(EL), potentiates sodium arsenite-induced apoptosis and by experiments showing direct phosphorylation of Bim(EL) at Ser-65 by p38 in vitro. Furthermore, sodium arsenite induced Bim(EL) phosphorylation at Ser-65, which was blocked by p38 inhibition. This study provides the first example whereby p38 induces apoptosis by phosphorylating a member of the Bcl-2 family and illustrates that phosphorylation of Bim(EL) on Ser-65 may be a common regulatory point for cell death induced by both JNK and p38 pathways.

Rotenone-induced apoptosis is mediated by p38 and JNK MAP kinases in human dopaminergic SH-SY5Y cells.

Rotenone is a naturally derived pesticide that has recently been shown to evoke the behavioral and pathological symptoms of Parkinson's disease in animal models. Though rotenone is known to be an inhibitor of the mitochondrial complex I electron transport chain, little is known about downstream pathways leading to its toxicity. We used human dopaminergic SH-SY5Y cells to study mechanisms of rotenone-induced neuronal cell death. Our results suggest that rotenone, at nanomolar concentrations, induces apoptosis in SH-SY5Y cells that is caspase-dependent. Furthermore, rotenone treatment induces phosphorylation of c-Jun, the c-Jun N-terminal protein kinase (JNK), and the p38 mitogen activated protein (MAP) kinase, indicative of activation of the p38 and JNK pathways. Importantly, expression of dominant interfering constructs of the JNK or p38 pathways attenuated rotenone-induced apoptosis. These data suggest that rotenone induces apoptosis in the dopaminergic SH-SY5Y cells that requires activation of the JNK and p38 MAP kinases and caspases. These studies provide insights concerning the molecular mechanisms of rotenone-induced apoptosis in neuronal cells.