lncRNA SNHG9 enhances liver cancer stem cell self-renewal and tumorigenicity by negatively regulating PTEN expression via recruiting EZH2.

Liver cancer stem cell (CSC) self-renewal and tumorigenesis are important causes of hepatocellular carcinoma (HCC) recurrence. We purposed to investigate the function of long noncoding RNA small nucleolar RNA host gene 9 (SNHG9) in liver CSC self-renewal and tumorigenesis in this study. Flow cytometry was carried out to separate CD133+ Populations and CD133- Populations from HCC cell lines. A combination of CD133+ cells and Matrigel matrix was subcutaneously injected to create the NOD-SCID mouse xenograft tumor model. Colony formation test and spheroids formation assay were carried out to clarify the impact of SNHG9 on the self-renewal of liver CSCs. RNA immunoprecipitation, RNA-pull down, and chromatin immunoprecipitation were performed on CD133+ cells to elucidate the mechanism of SNHG9 regulating PTEN expression. We found that SNHG9 was highly expressed in HCC clinical samples, HCC cells, and CD133+ cells. In vitro, interference with SNHG9 prevented the formation of colonies and spheroids in liver CSC cells and primary HCC cells. In vivo, interference with SNHG9 reduced the tumor volume and weight. SNHG9 could bind to EZH2, and SNHG9 interference suppressed EZH2 recruitment and H3K27me3 levels in the PTEN promoter region. In addition, SNHG9 inhibition promoted PTEN expression while having little impact on EZH2 levels. Interference with SNHG9 inhibited liver CSC self-renewal and tumorigenesis by up-regulating PTEN levels. In conclusion, by binding to EZH2, SNHG9 down-regulated PTEN levels, promoting liver CSC self-renewal and tumor formation, and exacerbating HCC progression.

Berbamine Exerts an Anti-oncogenic Effect on Pancreatic Cancer by Regulating Wnt and DNA Damage-related Pathways.

OBJECTIVE: This study aimed to determine the effects of berbamine on pancreatic cancer as well as the underlying mechanisms. METHODS: The pancreatic cancer cells were treated with different concentrations of berbamine and then subjected to cell viability assay, colony formation assay, cell cycle analysis, and apoptosis detection. Western blotting and immunofluorescence analyses were performed to investigate the mechanisms underlying the biological effects of berbamine on the pancreatic cancer cells. Furthermore, the in vivo anti-pancreatic cancer effect of berbamine was examined using a mouse xenograft model. RESULTS: Berbamine significantly inhibited the proliferation and colony-forming ability of BxPC3 and PANC-1 pancreatic cancer cells while inducing a cell cycle arrest and apoptosis. Moreover, berbamine decreased the expression of ß- catenin and phosphorylation of GSK3ß but increased the expression of γ-H2AX and 53BP1. Meanwhile, in vivo studies revealed that berbamine attenuated the growth of xenograft tumors derived from PANC-1 cells. Notably, berbamine treatment led to an increase in the expression of Cleaved Caspase 3 and γ-H2AX, as well as a decrease in the expression of Ki-67 and ß-catenin in the tumor xenografts. CONCLUSION: Berbamine exerts an anti-pancreatic cancer effect, possibly by regulating Wnt and DNA damage-related pathways, suggestive of its therapeutic potential for pancreatic cancer.

Transrectal intracolon cooling prevents paraplegia and mortality in a rat model of aortic occlusion-induced spinal cord ischemia.

OBJECTIVE: Spinal cord ischemia-reperfusion injury (SC-IRI) occurs in many medical conditions such as aneurysm surgical repair but no treatment of SC-IRI is available in clinical practice. The objective of the present study was to develop a novel medical device for the treatment of SC-IRI. METHODS: A rat model of SC-IRI was used. A novel transrectal intracolon (TRIC) temperature management device was developed to maintain an intracolon wall temperature at either 37°C (TRIC37°C) or 12°C (TRIC12°C). The upper body temperature was maintained as close as possible to 37°C in both groups. A 2F Fogarty balloon catheter was inserted via the left common carotid artery to block the distal aortic blood flow to the spinal cord. The proximal blood pressure was controlled by the withdrawal and infusion of blood via the jugular vein catheter, such that the distal tail artery blood pressure was maintained at ∼10 mmHg for 13 and 20 minutes, respectively. Next, the balloon was deflated, and TRIC temperature management was continued for an additional 30 minutes to maintain the colon wall temperature at either 37°C or 12°C during the reperfusion period. RESULTS: All the rats subjected to 13 minutes of spinal cord ischemia in the TRIC37°C group had developed paraplegia during the postischemic phase. In striking contrast, TRIC at 12°C completely prevented the paraplegia, dramatically improved the arterial blood gas parameters, and avoided the histopathologic injuries to the spinal cord in rats subjected to 13 minutes of spinal cord ischemia. Furthermore, TRIC12°C allowed for the extension of the ischemia duration from 13 minutes to 20 minutes, with significantly reduced functional deficits. CONCLUSIONS: Directly cooling the intestine focally with the TRIC device offered an exceptional survival rate and functional improvement after aortic occlusion-induced spinal cord ischemia.

Interruption of endolysosomal trafficking leads to stroke brain injury.

BACKGROUND AND PURPOSE: Dysfunction of the endolysosomal system can cause cell death. A key molecule for controlling the endolysosomal trafficking activities is the N-ethylmaleimide-sensitive factor (NSF) ATPase. This study investigates the cascades of NSF ATPase inactivation events, endolysosomal damage, cathepsin release, and neuronal death after focal brain ischemia. METHODS: A total of 62 rats were used in this study. They were subjected to sham surgery or 2 h of focal brain ischemia followed by 1, 4, and 24 h of reperfusion. Confocal microscopy and Western blot analysis were utilized to analyze the levels, redistribution, and co-localization of key proteins of the Golgi apparatus, late endosomes, endolysosomes, and lysosomes. Light and electron microscopy were used to examine the histopathology, protein aggregation, and endolysosomal ultrastructures. RESULTS: Two hours of focal brain ischemia in rats led to acute neuronal death at the striatal core in 4 h and a slower type of neuronal death in the neocortical area during 1-24 h reperfusion periods. Confocal microscopy showed that NSF immunoreactivity was irreversibly and selectively depleted from most, if not all, post-ischemic penumbral neurons. Western blot analysis further demonstrated that NSF depletion from brain sections was due to its deposition into dense inactive aggregates that could not be recognized by the NSF antibody. Commitantly, the Golgi apparatus was completely fragmented and cathepsin B (CTSB)-containing endolysosomal structures, as well as p62/SQSTM1- and EEA1-immunopositive structures were massively accumulated in the post-ischemic penumbral neurons. Ultimately, CTSB was released into the cytoplasm and extracellular space, causing stroke brain injury. CONCLUSION: Stroke Inactivates NSF, resulting in disruption of the reforming of functional endolysosomal compartments, blockade of the endocytic and autophagic pathways, a large scale of CTSB release into the cytoplasm and extracellular space, and stroke brain injury in the rat model.

A novel ferroptosis phenotype-related clinical-molecular prognostic signature for hepatocellular carcinoma.

Ferroptosis is a newly identified cell death mechanism and potential biomarker for hepatocellular carcinoma (HCC) therapy; however, its clinical relevance and underlying mechanism remain unclear. In this study, transcriptome and methylome data from 374 HCC cases were investigated for 41 ferroptosis-related genes to identify ferroptosis activity-associated subtypes. These subtypes were further investigated for associations with clinical and pathological variables, gene mutation landscapes, deregulated pathways and tumour microenvironmental immunity. A gene expression signature and predictive model were developed and validated using an additional 232 HCC cases from another independent cohort. Two distinct ferroptosis phenotypes (Ferroptosis-H and Ferroptosis-L) were identified according to ferroptosis gene expression and methylation in the patients with HCC. Patients with the Ferroptosis-H had worse overall and disease-specific survival, and the molecular subtypes were significantly associated with different clinical characteristics, mRNA expression patterns, tumour mutation profiles and microenvironmental immune status. Furthermore, a 15-gene ferroptosis-related prognostic model (FPM) for HCC was developed and validated which demonstrated accurate risk stratification ability. A nomogram included the FPM risk score, ECOG PS and hepatitis B status was developed for eventual clinical translation. Our results suggest that HCC subtypes defined by ferroptosis gene expression and methylation may be used to stratify patients for clinical decision-making.

A predictive nomogram for lymph node metastasis of incidental gallbladder cancer: a SEER population-based study.

BACKGROUND: Existing imaging techniques have a low ability to detect lymph node metastasis (LNM) of gallbladder cancer (GBC). Gallbladder removal by laparoscopic cholecystectomy can provide pathological information regarding the tumor itself for incidental gallbladder cancer (IGBC). The purpose of this study was to identify the risk factors associated with LNM of IGBC and to establish a nomogram to improve the ability to predict the risk of LNM for IGBC. METHODS: A total of 796 patients diagnosed with stage T1/2 GBC between 2004 and 2015 who underwent surgery and lymph node evaluation were enrolled in this study. We randomly divided the dataset into a training set (70%) and a validation set (30%). A logistic regression model was used to construct the nomogram in the training set and then was verified in the validation set. Nomogram performance was quantified with respect to discrimination and calibration. RESULTS: The rates of LNM in T1a, T1b and T2 patients were 7, 11.1 and 44.3%, respectively. Tumor diameter, T stage, and tumor differentiation were independent factors affecting LNM. The C-index and AUC of the training set were 0.718 (95% CI, 0.676-0.760) and 0.702 (95% CI, 0.659-0.702), respectively, demonstrating good prediction performance. The calibration curves showed perfect agreement between the nomogram predictions and actual observations. Decision curve analysis showed that the LNM nomogram was clinically useful when the risk was decided at a possibility threshold of 2-63%. The C-index and AUC of the validation set were 0.73 (95% CI: 0.665-0.795) and 0.692 (95% CI: 0.625-0.759), respectively. CONCLUSION: The nomogram established in this study has good prediction ability. For patients with IGBC requiring re-resection, the model can effectively predict the risk of LNM and make up for the inaccuracy of imaging.

Identification of APEX2 as an oncogene in liver cancer.

BACKGROUND: DNA damage is one of the critical contributors to the occurrence and development of some cancers. APEX1 and APEX2 are the most important molecules in the DNA damage, and APEX1 has been identified as a diagnostic and prognostic biomarker in liver hepatocellular carcinoma (LIHC). However, the expression of APEX2 and its functional mechanisms in LIHC are still unclear. AIM: To examine the expression of APEX2 and the potential mechanism network in LIHC. METHODS: We conducted a pan-cancer analysis of the expression of APEX1 and APEX2 using the interactive TIMER tool. GEO datasets, including GSE14520, GSE22058, and GSE64041, were used to compare the APEX2 expression level in tumor tissues and adjacent non-tumor tissues. Then, we calculated the 5-year survival rate according to the web-based Kaplan-Meier analysis. We included the TCGA liver cancer database in GSEA analysis based on the high and low APEX2 expression, showing the potential mechanisms of APEX2 in LIHC. After that, we conducted Pearson correlation analysis using GEPIA2. Next, we performed quantitative polymerase chain reaction (qPCR) assay to examine the APEX2 levels in normal liver cell line LO2 and several liver cancer cell lines, including HepG2, Huh7, SMMC7721, and HCCLM3. APEX2 in HCCLM3 cells was knocked down using small interfering RNA. The role of APEX2 in cell viability was confirmed using CCK-8. Dual-luciferase reporter assay was performed to examine the promoter activity of CCNB1 and MYC. RESULTS: APEX1 and APEX2 are both highly expressed in the tumor tissues of BLCA, BRCA, CHOL, COAD, ESCA, HNSC, LIHC, LUAD, LUSC, READ, and STAD. APEX2 overexpression in LIHC was validated using GSE14520, GSE22058, and GSE64041 datasets. The survival analysis showed that LIHC patients with high expression of APEX2 had a lower overall survival rate, even in the AJCC T1 patients. High level of APEX2 could indicate a lower overall survival rate in patients with or without viral hepatitis. The GSEA analysis identified that kinetochore and spindle microtubules are the two main cellular components of APEX2 in GO Ontology. APEX2 was also positively associated with molecular function regulation of chromosome segregation and DNA replication. The results of KEGG analysis indicated that APEX2 expression was positively correlated with cell cycle pathway and pro-oncogenic MYC signaling. Pearson correlation analysis showed that APEX2 had a significant positive correlation with CCNB1 and MYC. APEX2 level was higher in liver cancer cell lines than in normal liver LO2 cells. Small interfering RNA could knock down the APEX2 expression in HCCLM3 cells. Knockdown of APEX2 resulted in a decrease in the viability of HCCLM3 cells as well as the expression and promoter activity of CCNB1 and MYC. CONCLUSION: APEX2 is overexpressed in LIHC, and the higher APEX2 level is associated with a worse prognosis in overall survival. APEX2 is closely involved in the biological processes of chromosome segregation and DNA replication. APEX2 expression is positively correlated with the pro-oncogenic pathways. Knockdown of APEX2 could inhibit the cell viability and CCNB1 and MYC pathways, suggesting that APEX2 is an oncogene in LIHC, which could be a potential pharmaceutic target in the anti-tumor therapy.

Preparation of Biodegradable Polymeric Nanocapsules for Treatment of Malignant Tumor Using Coaxial Capillary Microfluidic Device.

Objective: Nanocapsules play a role in the targeted delivery of chemotherapy drugs. However, the traditional technology for preparation of nanocapsules is relatively complex with poor controllability, leading to large differences batch to batch. This study aimed to evaluate the quality of drugs-loaded nanocapsules (Drugs-NCs) fabricated by coaxial capillary microfluidic device, and inhibitory effect on malignant tumors. Materials and Methods: In this study, oxaliplatin, irinotecan, and 5-fluorouracil were selected as chemotherapy drugs, and Drugs-NCs were prepared by coaxial glass capillary microfluidic device. Next, transmission electron microscope was utilized to characterize surface morphology and particle size distribution of Drugs-NCs. Then, high performance liquid chromatography was used to determine the drug loading and encapsulation efficiency. Dialysis method was performed to measure the drug release of Drugs-NCs in vitro. To study the effects of Drugs + NCs on tumor growth in vivo, BALB/c (nu/nu) nude mice were used in vivo experiments. Results: The Drugs-NCs were spherical and uniform in size (103.4 nm). Besides, the encapsulation efficiencies of oxaliplatin, irinotecan, and 5-fluorouracil were 97.0%, 95.7%, and 15.6%, respectively. Moreover, drugs encapsulated in the nanocapsules released less and was pH-dependent, with more rapid release being observed at pH 5.5 group compared with pH 7.4 group. MTT assay and in vivo experiments indicated the inhibitory effect of Drugs-NCs on malignant tumors. Conclusion: The prepared nanocapsules had potential tumor targeting. Furthermore, coaxial capillary microfluidic device could be used as a promising microfluidic technology to fabricate multiple Drug-NCs.

LncRNA SAMMSON negatively regulates miR-9-3p in hepatocellular carcinoma cells and has prognostic values.

In the present study, we investigated the role of lncRNA SAMMSON in hepatocellular carcinoma (HCC). We found that SAMMSON was up-regulated in HCC tissues, and patients with high levels of SAMMSON in HCC tissues had significantly lower overall rate within 5 years after admission. miR-9-3p was down-regulated in HCC tissues and inversely correlated with SAMMSON. SAMMSON expression was not significantly affected by HBV and HCV infections in HCC patients. In HCC cells, SAMMSON overexpression resulted in down-regulated miR-9-3p expression, while miR-9-3p overexpression caused no significant changes in expression levels of SAMMSON. SAMMSON overexpression led to increased, while miR-9-3p overexpression resulted in decreased migration and invasion rates of HCC cells. Therefore, SAMMSON negatively regulated miR-9-3p in HCC cells to promote cancer cell migration and invasion.

Berbamine Enhances the Efficacy of Gefitinib by Suppressing STAT3 Signaling in Pancreatic Cancer Cells.

BACKGROUND: Small molecular inhibitors such as gefitinib (Gefi), which target EGF receptor (EGFR), are considered to be a viable pathway for the selective inhibition of pancreatic cancer (PC) development. However, the large difference in Gefi response between PC patient individuals and PC cell lines severely limits the clinical efficacy of Gefi. Berbamine (BBM) is a well-known natural-derived antitumor agent. However, no study yet exists on whether BBM can enhance the sensitivity of PC cells to Gefi or its underlying mechanisms. METHODS: MTS assay and clonogenic assay were used to determine whether BBM could enhance the anti-PC activity of Gefi by. Flow cytometric analysis was performed to study the cell cycle progression and rate of apoptosis after combined treatment with BBM and Gefi. Surface plasmon resonance (SPR) and Western blot experiments were carried out to detect the STAT3 binding affinity and the STAT3 inhibitory effect of BBM. Molecular docking and Molecular dynamic simulation were used to predicting the dominant interaction between BBM and STAT3. RESULTS: This study found that BBM synergizes with Gefi to inhibit cell growth and induce cell cycle arrest and PC cell apoptosis. Mechanistically, our results showed that BBM and Gefi have synergistic inhibitory effects on STAT3 phosphorylation, but have little effect on other EGFR downstream pathways, suggesting that BBM may exert sensitization through the inhibition of STAT3. Besides, BBM has a high affinity for STAT3 and a good inhibitory effect on STAT3 activation, further indicating that BBM was a potent direct STAT3 inhibitor. Molecular modeling between STAT3 and BBM suggested that BBM formed several key hydrophilic interactions with STAT3. CONCLUSION: Our findings suggest that the combination of BBM and Gefi could be further developed as a potential PC therapy.

CircRNA_100290 promotes colorectal cancer progression through miR-516b-induced downregulation of FZD4 expression and Wnt/ß-catenin signaling.

Accumulating evidence indicates that circular RNAs (circRNA) exert crucial functions in the development and advance of cancers. CircRNA_100290 has been reported to promote proliferation in oral cancer. However, whether it participates in colorectal cancer (CRC) remains unclear. Here, our report showed that circRNA_100290 level was significantly increased in CRC tissues and cell lines. Besides, circRNA_100290 expression was positively correlated with tumor metastasis while inversely correlated with prognosis. Silencing circRNA_100290 markedly reduced cell proliferation rate, inhibited migration and invasion abilities, but promoted apoptosis in vitro. Mechanistically, our data revealed circRNA_100290 was a competing endogenous RNA (ceRNA) of FZD4 by sponging miR-516b, leading to activation of Wnt/ß-catenin pathway. Rescue assay indicated that FZD4-induced activation of ß-catenin pathway is indispensable for the function of circRNA_100290 in CRC. In summary, our study for the first time revealed a novel regulatory loop of circRNA_100290/miR-516b/FZD4/Wnt/ß-catenin implicated in CRC progression.

Nest-building activity as a reproducible and long-term stroke deficit test in a mouse model of stroke.

INTRODUCTION: Neuroprotective therapeutics achieved from animal studies have not been able to translate into clinical stroke therapies. A major reason may be that the functional tests and outcomes between animal stroke studies and clinical trials are significantly different. Ultimately, functional recovery is most important for stroke patients, but it remains challenging to identify animal functional tests that reflect human stroke deficits. This study aimed to explore whether the nest-building activity can be used as a functional test of mouse stroke deficit. METHODS: Forty-one C57B6 male mice were randomly assigned into a sham-operated control group and 20-, 40- and 60-min middle cerebral artery occlusion (MCAO) groups. Mice were perfusion-fixed at 21 days following sham surgery or MCAO. Infarct volumes were assessed under the light microscopy. The nest-building activity was characterized and quantitatively evaluated. RESULTS: The results show that only a small portion of striatum was damaged after 20-min MCAO. The brain damage areas were expanded from striatum to the neocortex and hippocampus proportionally after 40-min and 60-min MCAO, respectively. Consistently, relative to that of the sham-operated mice, the nest-building activity was insignificantly altered after 20-min MCAO, but dramatically and significantly reduced proportionally following 40-min and 60-min MCAO, respectively. The nest-building deficit was a long-lasting event and could be seen for as long as 14-21 days of recovery, the longest endpoint of this study. CONCLUSIONS: The results suggest that the nest-building activity may be a novel, objective, easy to use, highly sensitive, and long-lasting test that may reflect the multifaceted sensorimotor and cognitive deficits after stroke in humans. Our findings may provide a novel multifaceted test for bridging the gap between animal stroke studies and clinical trials.

Hsa_circ_0103809 promotes cell proliferation and inhibits apoptosis in hepatocellular carcinoma by targeting miR-490-5p/SOX2 signaling pathway.

BACKGROUND: Circular RNAs (circRNAs) represent a class of non-coding RNAs that are emerging as important regulators during tumorigenesis and provide potential targets for cancer intervention. However, the expression profiles and functions of circRNAs in hepatocellular carcinoma (HCC) have not been completely clarified. Herein, the role of hsa_circ_0103809 was investigated in HCC tissues and cell lines. METHODS: High-throughput circRNA sequencing was performed to detect the expression profiles of circRNA in HCC tissues. The CCK-8, wound healing and flow cytometry were performed to measure the cell viability, migration and apoptosis in HCC cells. The expression levels of gene and protein in HCC tissues and cell lines were assayed by RT-qPCR and western blotting, respectively. Immunohistochemical staining was used to assess the protein expression of SOX2 in HCC tissues. RESULTS: We discovered that hsa_circ_0103809 was significantly increased in HCC tissues and cell lines. Knockdown of hsa_circ_0103809 inhibited proliferation and migration and induced apoptosis in HCC cell lines. Investigation to the molecular mechanisms of hsa_circ_0103809 in HCC cells had revealed that hsa_circ_0103809 directly suppressed miR-490-5p, which targeted to the 3'-UTR of SOX2. Hsa_circ_0103809 loss-of-function could increase the expression of miR-490-5p as well as decreased the expression of SOX2. Furthermore, we found that si-0103809 induced growth and migration inhibition and apoptosis could be reversed by transfected with miR-490-5p inhibitors or SOX2 in HCC cells. CONCLUSION: Our findings suggested that hsa_circ_0103809 might facilitate HCC malignant progression, at least partially, by regulating miR-490-5p/SOX2 signaling pathway.

The long non-coding RNA AK023948 enhances tumor progression in hepatocellular carcinoma.

The long non-coding RNAs (lncRNAs) have been demonstrated to play pivotal roles in a broad range of processes including tumor biology. However, the exact contributions of lncRNAs to hepatocellular carcinoma (HCC) remain poorly defined. In current study, we have unraveled a novel function of AK023948 in HCC. We found that AK023948 was substantially upregulated in tumor tissues. Meanwhile, higher AK023948 expression correlated with poor survival. Upregulation of AK023948 expression can promote HepG2 and Hep3B proliferation and invasion in in vitro experiments. Furthermore, AK023948 also decreased tumor growth in vivo. The tumorigenic role of AK023948 was partially ascribed to PI3K/Akt/mTOR signaling and AK023948 knockdown decreased pathway activation and tumor growth. These data collectively suggest an oncogenic role for AK023948 and may provide potential insight into therapeutic intervention.

Long non-coding RNA 657 suppresses hepatocellular carcinoma cell growth by acting as a molecular sponge of miR-106a-5p to regulate PTEN expression.

Previous study has identified the aberrant expression of LINC00657, a long non-coding RNA (lncRNA), in human breast cancer. However, the expression pattern, biological function and underlying mechanism of LINC00657 in human hepatocellular carcinoma (HCC) remain obscure. The expression levels of LINC00657 in HCC tissues and cell lines were determined by quantitative real-time PCR. CCK-8 assay, cell colony formation assay, cell cycle analysis, Transwell assay were performed to determine whether LINC00657 could affect HCC progression. Luciferase reporter assay was used to assess the target of LINC00657. Expressions of the relevant proteins were analyzed by Western blot. Herein, we found that LINC00657 was downregulated in HCC tissue specimens as well as in malignant HCC cell lines. LINC00657 overexpression inhibited the proliferation, migration and invasion of HCC cells, while LINC00657 depletion promoted both cell viability and cell invasion in vitro. We also found that LINC00657 could inhibit tumor growth in vivo. Further experiments demonstrated that down-regulated LINC00657 increased the expression of miR-106a-5p. miR-106a-5p decreased the abundances of PTEN protein, while had no impact on PTEN mRNA. Moreover, we identified that both LINC00657 and PTEN mRNA were targets of miR-106a-5p by using dual-luciferase reporter assay. Our results provide the new evidence supporting the tumor-suppressive role of LINC00657 in HCC, suggesting that LINC00657 might play a role in HCC and can be a novel therapeutic target for treating HCC.

Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs as potential IκB kinase ß inhibitors for the treatment of pancreatic cancer.

Given the important role that inhibitory kappa B (IκB) kinase ß (IKKß) plays in pancreatic cancer (PC) development and progression, inhibitors targeting IKKß are believed to be increasingly popular as novel anti-PC therapies. Two synthetic molecules, named EF24 and EF31, exhibited favorable potential in terms of inhibition of both IKKß activity and PC cell proliferation. Aiming to enhance their cellular efficacy and to analyze their structure-activity relationship, four series of EF24 and EF31 analogs were designed and synthesized. Through kinase activity and vitality screening of cancer cells, D6 displayed excellent inhibition of both IKKß activity and PC cell proliferation. Additionally, multiple biological evaluations showed that D6 was directly bound to IKKß and significantly suppressed the activation of the IKKß/nuclear factor κB pathway induced by tumor necrosis factor-α, as well as effectively inducing cancer cell apoptosis. Moreover, molecular docking and molecular dynamics simulation analysis indicated that the dominant force between D6 and IKKß comprised hydrophobic interactions. In conclusion, D6 may be a promising therapeutic agent for PC treatment and it also provides a structural lead for the design of novel IKKß inhibitors.

Cellular mechanisms of neuronal damage from hyperthermia.

Hyperthermia can cause brain damage and also exacerbate the brain damage produced by stroke and amphetamines. The developing brain is especially sensitive to hyperthermia. The severity of, and mechanisms underlying, hyperthermia-induced neuronal death depend on both temperature and duration of exposure. Severe hyperthermia can produce necrotic neuronal death. For a window of less severe heat stresses, cultured neurons exhibit a delayed death with apoptotic characteristics including cytochrome c release and caspase activation. Little is known about mechanisms of hyperthermia-induced damage upstream of these late apoptotic effects. This chapter considers several possible upstream mechanisms, drawing on both in vivo and in vitro studies of the nervous system and other tissues. Hyperthermia-induced damage in some non-neuronal cells includes endoplasmic reticular stress due to denaturing of nascent polypeptide chains, as well as nuclear and cytoskeletal damage. Evidence is presented that hyperthermia produces mitochondrial damage, including depolarization, in cultured mammalian neurons.

Subcellular stress response after traumatic brain injury.

Traumatic brain injury (TBI) initiates a complex genetic response that may include the expression of organelle specific stress genes. We investigated the effects of brain trauma on the expression of a number of stress genes by in situ hybridization and Western blot analysis including the endoplasmic reticulum (ER) stress gene grp78, ER protein processing enzymes calnexin and protein disulphide isomerase (PDI), the mitochondrial stress gene hsp60, and the cytoplasmic stress gene hsp70. Male Sprague-Dawley rats were subjected either to sham-surgery or moderate (1.8-2.2 atm) parasagittal fluid-percussion (F-P) brain injury followed by 30 min of either normoxic or hypoxic (30-40 mm Hg) gas levels. Expression of grp78 was increased in the ipsilateral cerebral cortex and dentate gyrus beginning 4 h after trauma plus hypoxia. Similarly, mRNA encoding the mitochondrial hsp60 was induced in the ipsilateral outer cortical layers at 4-24 h after TBI plus hypoxia. Calnexin and PDI mRNAs were not significantly altered following TBI with or without secondary hypoxia. In contrast, mRNA of the cytoplasmic hsp70 was strongly induced at 4 h after brain injury in multiple brain regions within the injured hemisphere, and this expression was greatly enhanced by secondary hypoxia. Because subcellular stress gene expression may reflect where unfolded or damaged proteins are abundant, these findings suggest that abnormal proteins are localized mainly in the cytoplasm, and to a lesser degree in the ER lumen and mitochondria after brain trauma. Thus, distinct parts of the cellular machinery respond to traumatic and metabolic stresses in specific ways.

Alterations in mammalian target of rapamycin signaling pathways after traumatic brain injury.

In response to traumatic brain injury (TBI), neurons initiate neuroplastic processes through the activation of intracellular signaling pathways. However, the molecular mechanisms underlying neuroplasticity after TBI are poorly understood. To study this, we utilized the fluid-percussion brain injury (FPI) model to investigate alterations in the mammalian target of rapamycin (mTOR) signaling pathways in response to TBI. Mammalian target of rapamycin stimulates mRNA translation through phosphorylation of eukaryotic initiation factor 4E binding protein-1 (4E-BP1), p70 ribosomal S6 kinase (p70S6K), and ribosomal protein S6 (rpS6). These pathways coordinate cell growth and neuroplasticity via dendritic protein synthesis. Rats received sham surgery or moderate parasagittal FPI on the right side of the parietal cortex, followed by 15 mins, 30 mins, 4 h, 24 h, or 72 h of recovery. Using Western blot analysis, we found that mTOR, p70S6K, rpS6, and 4E-BP1 phosphorylation levels were significantly increased in the ipsilateral parietal cortex and hippocampus from 30 mins to 24 h after TBI, whereas total protein levels were unchanged. Using confocal microscopy to localize these changes, we found that rpS6 phosphorylation was increased in the parietal cortex and all subregions of the hippocampus. In accordance with these results, eIF4E, a key, rate-limiting mRNA translation factor, was also phosphorylated by mitogen-activated protein kinase-interacting kinase 1 (Mnk1) 15 mins after TBI. Together, these results suggest that changes in mRNA translation may be one mechanism that neurons use to respond to trauma and may contribute to the neuroplastic changes observed after TBI.

Changes in trkB-ERK1/2-CREB/Elk-1 pathways in hippocampal mossy fiber organization after traumatic brain injury.

Traumatic brain injury (TBI) leads to mossy fiber reorganization, which is considered to be a causative factor in the development of temporal lobe epilepsy. However, the underlying mechanism is not fully understood. Emerging evidence suggests that TrkB-ERK1/2-CREB/Elk-1 pathways are highly related to synaptic plasticity. This study used the rat fluid-percussion injury model to investigate activation of TrkB-ERK1/2-CREB/Elk-1 signaling pathways after TBI. Rats were subjected to 2.0-atm parasagittal TBI followed by 30 minutes, 4 hours, 24 hours, and 72 hours of recovery. After TBI, striking activation of TrkB-ERK1/2-CREB/Elk-1 signaling pathways in mossy fiber organization were observed with confocal microscopy and Western blot analysis. ERK1/2 was highly phosphorylated predominantly in hippocampal mossy fibers, whereas TrkB was phosphorylated both in the mossy fibers and the dentate gyrus region at 30 minutes and 4 hours of recovery after TBI. CREB was also activated at 30 minutes, peaked at 24 hours of recovery, and returned to the control level at 72 hours of recovery in dentate gyrus granule cells. Elk-1 phosphorylation was seen in CA3 neurons at 4 hours after TBI. The results suggest that the signaling pathways of TrkB-ERK1/2-CREB/Elk-1 are highly activated in mossy fiber organization, which may contribute to mossy fiber reorganization seen after TBI.