Discovery and biological evaluation of a small-molecule inhibitor of CRM1 that suppresses the growth of triple-negative breast cancer cells.

Dysregulation of the nuclear export machinery mediated by chromosomal maintenance 1 (CRM1, also known as exportin-1), is closely associated with various human disorders, such as breast cancer. Previously, we identified sulforaphene and its synthetic analogues as covalent inhibitors of CRM1. Herein, we describe the discovery and biological evaluation of another sulforaphene synthetic analogue, LFS-31, as a potential CRM1 inhibitor. In addition, we investigated the reversible binding mechanism of LFS-31 with CRM1 through molecular simulations coupled with bio-layer interferometry (BLI) and found relatively high binding affinity (KD = 43.1 ± 35.3 nM) between the LFS-31 and CRM1 groups. We found that LFS-31 exhibited a stronger growth suppression of triple-negative breast cancer (TNBC) cells than non-TNBC cells, and had minimal effect on normal breast cells. Pharmacological treatment of TNBC cells with LFS-31 at nanomolar concentrations led to the nuclear retention of IkBα resulting in strong suppression of NF-κB transcriptional activity and attenuated cell growth and proliferation, which collectively contributed to the antitumor responses. To the best of our knowledge, this is the first study to demonstrate the use of a sulforaphene analogue as a potent CRM1 inhibitor that targets the NF-κB signaling pathway for the targeted therapy of TNBC.

Construction of a high-density genetic linkage map and QTL mapping of growth and cold tolerance traits in Takifugu fasciatus.

Takifugu fasciatus is an aquaculture species with high economic value. In recent years, problems such as environmental pollution and inbreeding have caused a serious decline in T. fasciatus germplasm resources. In this study, a high-density genetic linkage map was constructed by whole-genome resequencing. The map consists of 4891 bin markers distributed across 22 linkage groups (LGs), with a total genetic coverage of 2381.353 cM and a mean density of 0.535 cM. Quantitative trait locus (QTL) localization analysis showed that a total of 19 QTLs associated with growth traits of T. fasciatus in the genome-wide significance threshold range, distributed on 11 LGs. In addition, 11 QTLs associated with cold tolerance traits were identified, each scattered on a different LG. Furthermore, we used QTL localization analysis to screen out three candidate genes (IGF1, IGF2, ADGRB) related to growth in T. fasciatus. Meanwhile, we screened three candidate genes (HSP90, HSP70, and HMGB1) related to T. fasciatus cold tolerance. Our study can provide a theoretical basis for the selection and breeding of cold-tolerant or fast-growing T. fasciatus.

XPO1 is a new target of homoharringtonine (HHT): Making NPMc+ AML cells much more sensitive to HHT treatment.

Acute myeloid leukemia (AML) is a heterogeneous disease and about one third of AML patients carry nucleophosmin (NPM1) mutation. Because 95% mutations give NPM1 an additional nuclear export signaling (NES) and dislocate NPM1 in cytoplasm (NPMc+), relocating NPM1 in nucleus provide an innovative strategy for treating this type of AML. The nuclear export of NPM1 depends on the nuclear protein export receptor XPO1, which recognizes the NES sequence on NPM1. Homoharringtonine (HHT) is a first-line chemotherapy drug of AML, yet the exact mechanism of its anti-AML activity is elusive. In this study, we found that HHT can directly target XPO1 to its NES-binding cleft, bind to Cys528 of XPO1, and inhibits its nuclear transport function. In addition, HHT can block NPMc+ proteins nuclear export and thus make NPMc+ AML cells much more sensitive to HHT treatment. Furthermore, the sensitivity of NPMc+ AML cells to HHT is a universal phenomenon irrespective of the different genetic lesions of AML. Taken together, our findings suggest that XPO1 is a new target of HHT and provide a novel strategy for NPMc+ AML treatment.

Discovery of selective and potent USP22 inhibitors via structure-based virtual screening and bioassays exerting anti-tumor activity.

Ubiquitin-specific protease 22 (USP22) plays a prominent role in tumor development, invasion, metastasis and immune reprogramming, which has been proposed as a potential therapeutic target for cancer. Herein, we employed a structure-based discovery and biological evaluation and discovered that Rottlerin (IC50 = 2.53 µM) and Morusin (IC50 = 8.29 µM) and as selective and potent USP22 inhibitors. Treatment of HCT116 cells and A375 cells with each of the two compounds resulted in increased monoubiquitination of histones H2A and H2B, as well as reduced protein expression levels of Sirt1 and PD-L1, all of which are known as USP22 substrates. Additionally, our study demonstrated that the administration of Rottlerin or Morusin resulted in an increase H2Bub levels, while simultaneously reducing the expression of Sirt1 and PD-L1 in a manner dependent on USP22. Furthermore, Rottlerin and Morusin were found to enhance the degradation of PD-L1 and Sirt1, as well as increase the polyubiquitination of endogenous PD-L1 and Sirt1 in HCT116 cells. Moreover, in an in vivo syngeneic tumor model, Rottlerin and Morusin exhibited potent antitumor activity, which was accompanied by an enhanced infiltration of T cells into the tumor tissues. Using in-depth molecular dynamics (MD) and binding free energy calculation, conserved residue Leu475 and non-conserved residue Arg419 were proven to be crucial for the binding affinity and inhibitory function of USP22 inhibitors. In summary, our study established a highly efficient approach for USP22-specific inhibitor discovery, which lead to identification of two selective and potent USP22 inhibitors as potential drugs in anticancer therapy.

An oleanolic acid derivative, K73-03, inhibits pancreatic cancer cells proliferation in vitro and in vivo via blocking EGFR/Akt pathway.

Oleanolic acid (OA) and its derivatives show potent anticancer function. Pancreatic cancer (PC) is the fourth core motive of cancer-related deaths worldwide. Epidermal growth factor receptor (EGFR) has been implicated in PC and has been validated as a therapeutic target. Our study demonstrated that K73-03, an OA derivative, was identified as a potent inhibitor of EGFR by using reverse pharmacophore screening and molecular dynamics simulation assays. Moreover, Western blot analysis showed that K73-03 markedly suppressed the levels of phosphorylated-EGFR (p-EGFR) and phosphorylated-Akt (p-Akt). The inhibitory effect of K73-03 on PC cells was assessed in vitro and in vivo. Mechanistically, K73-03 effectively inhibited the cell proliferation of PC cells, and induced apoptosis and autophagy of ASPC-1 cells in a dose-dependent manner. Additionally, pretreatment with chloroquine, an autophagy inhibitor, significantly inhibited K73-03-induced autophagy and enhanced K73-03-induced apoptotic cell death. K73-03 also strongly repressed ASPC-1 cells xenograft growth in vivo. Thus, all these findings provided new clues about OA analog K73-03 as an effective anticancer agent targeted EGFR against ASPC-1 cells, it is worth further evaluation in the future.

Protection of pancreatic ß-cell by phosphocreatine through mitochondrial improvement via the regulation of dual AKT/IRS-1/GSK-3ß and STAT3/Cyp-D signaling pathways.

Diabetes mellitus (DM) is a metabolic syndrome, caused by insufficient insulin secretion or insulin resistance (IR). DM enhances oxidative stress and induces mitochondrial function in different kinds of cell types, including pancreatic ß-cells. Our previous study has showed phosphocreatine (PCr) can advance the mitochondrial function through enhancing the oxidative phosphorylation and electron transport ability in mitochondria damaged by methylglyoxal (MG). Our aim was to explore the potential role of PCr as a molecule to protect mitochondria from diabetes-induced pancreatic ß-cell injury with insulin secretion deficiency or IR through dual AKT/IRS-1/GSK-3ß and STAT3/Cyclophilin D (Cyp-D) signaling pathways. MG-induced INS-1 cell viability, apoptosis, mitochondrial division and fusion, the morphology, and function of mitochondria were suppressed. Flow cytometry was used to detect the production of intracellular reactive oxygen species (ROS) and the changes of intracellular calcium, and the respiratory function was measured by oxygraph-2k. The expressions of AKT, IRS-1, GSK-3ß, STAT3, and Cyp-D were detected using Western blot. The result showed that the oxidative stress-related kinases were significantly restored to the normal level after the pretreatment with PCr. Moreover, PCr pretreatment significantly inhibited cell apoptosis, decreased intracellular calcium, and ROS production, and inhibited mitochondrial division and fusion, and increased ATP synthesis damaged by MG in INS-1 cells. In addition, pretreatment with PCr suppressed Cytochrome C, p-STAT3, and Cyp-D expressions, while increased p-AKT, p-IRS-1, p-GSK-3ß, caspase-3, and caspase-9 expressions. In conclusion, PCr has protective effect on INS-1 cells in vitro and in vivo, relying on AKT mediated STAT3/ Cyp-D pathway to inhibit oxidative stress and restore mitochondrial function, signifying that PCr might become an emerging candidate for the cure of diabetic pancreatic cancer ß-cell damage.

Synthesis and biological evaluation of selenogefitinib for reducing bleomycin-induced pulmonary fibrosis.

Selenium has demonstrated effectiveness in the reduction of oxidative stress and inflammation in vitro and in vivo, both of which are key indicators of the pathogenesis of pulmonary fibrosis. Gefitinib, an FDA-approved EGFR inhibitor, effectively reverses the deterioration of bleomycin-induced pulmonary fibrosis. Based on this, we proposed introducing a selenium atom into the structure of gefitinib, resulting in the generation of selenogefitinib. Compared to gefitinib, selenogefitinib was significantly less hepatotoxic and cytotoxic in cells. The results of the H&E staining of lung tissue validated that Selenogefitinib effectively protected the structure of the alveolar tissue and mitigated the infiltration of inflammatory cells in bleomycin-induced pulmonary fibrosis models. The reduction in the deposition of collagen fibers in lung tissue determined by Masson staining and hydroxyproline (HYP) content also corroborated the efficacy of selenogefitinib in the treatment of pulmonary fibrosis. Furthermore, Selenogefitinib decreased the levels of pro-inflammatory markers IL-4, IL-6, and TNF-α more significantly than gefitinib, which indicated that it exhibited a higher anti-inflammatory activity. In addition, the presence of selenium manifested a greater reduction in oxidative stress based on the decrease in the levels of MDA in mice blood. These results suggested that Selenogefitinib may be a potential candidate for the treatment of IPF.

Silencing of Long Non-coding RNA TTN-AS1 Inhibits Hepatocellular Carcinoma Progression by the MicroRNA-134/ITGB1 Axis.

BACKGROUND: Hepatocellular carcinoma (HCC) causes considerable mortality worldwide. Long non-coding RNA (lncRNA) TTN-AS1 has been recently identified as an oncogene in several cancers, but its role in HCC and the molecules remain largely unknown. AIMS: The study aims to probe the function of lncRNA TTN-AS1 in HCC progression and the molecules involved. METHODS: Differentially expressed lncRNAs between HCC and the adjacent normal tissues were analyzed using a microarray. TTN-AS1 expression in HCC and normal tissues and cells was determined. Targeting relationships between TTN-AS1 and miR-134 and between miR-134 and ITGB1 were validated. Artificial up-regulation or down-regulation of TTN-AS1, miR-134 and ITGB1 was introduced in HCC cells to probe their effects on the biological behaviors of HCC cells. Xenograft tumors were induced in nude mice for in vivo experiments. RESULTS: TTN-AS1 and ITGB1 were highly expressed, while miR-134 was poorly expressed in HCC tissues. TTN-AS1 enforced ITGB1 expression through sequestering miR-134. Silencing of TTN-AS1 or over-expression of miR-134 inhibited proliferation, invasion, migration, and resistance to death of Huh7 cells. Following miR-134 silencing, further down-regulation of ITGB1 suppressed the malignant behaviors of HUH7 cells. The similar results were reproduced in vivo. CONCLUSION: The current study provided evidence that TTN-AS1 might promote HCC progression through sponging miR-134 and the following ITGB1 up-regulation. TTN-AS1 may serve as a potential target for HCC treatment.

miR-421 up-regulation by the oleanolic acid derivative K73-03 regulates epigenetically SPINK1 transcription in pancreatic cancer cells leading to metabolic changes and enhanced apoptosis.

SPINK1 overexpression promotes cancer cell aggressiveness and confers chemo-resistance to multiple drugs in pancreatic cancer. Oleanolic acid (OA) derivatives possess active effects against different cancers. Here we report the effect of K73-03, a new novel OA derivative, against pancreatic cancer through mitochondrial dysfunction via miR-421/SPINK1 regulation. We examined the binding ability of miR-421 with SPINK1-3'UTR Luciferase reporter assays. Moreover, miR-421/SPINK1 expressions in pancreatic cancer, with or without K73-03 treatment, were evaluated. Cells viability, migration, autophagy, mitochondrial function and apoptosis were examined with or without K73-03 treatment. We established that the K73-03 effect on the miR-421 that plays a crucial role in the regulation of SPINK1 in pancreatic cancer. Our findings indicated that K73-03 inhibited the mitochondrial function that led to inducing autophagy and apoptosis through epigenetic SPINK1 down-regulation via miR-421 up-regulation in pancreatic cancer. Furthermore, the inhibition of miR-421 expression in pancreatic cancer cells abolished the efficacy of K73-03 against SPINK1 oncogenic properties. We found an interesting finding that the interaction between miR-421 and SPINK1 is related to mitochondrial function through the effect of K73-03. Further, SPINK1 appear to be the molecular targets of K73-03 especially more than gemcitabine.

Synthesis and antitumor activity of α,ß-unsaturated carbonyl moiety- containing oleanolic acid derivatives targeting PI3K/AKT/mTOR signaling pathway.

Oleanolic acid (OA) and its semi-synthetic derivatives have been reported to have a wide range of biological activities. The introduction of electrophilic Michael acceptor group can increase the reactivity of OA to cellular targets and thus improve the anti-tumor activity. In this work, a series of novel α,ß-unsaturated carbonyl derivatives of OA were designed and synthesized. Their in vitro cytotoxic activity against MCF-7, HepG2 and HeLa cells were tested. Most derivatives exhibited improved cell growth inhibitory activity, especially for 3d with an IC50 of 0.77 µM in MCF-7 cells. Moreover, 3d inhibited the migration of MCF-7 and HeLa cells at the concentration of 4 µM. Flow cytometric analysis revealed that 3d induced cell apoptosis and S phase arrest in a concentration-dependent manner. Western blotting experiment demonstrated that 3d inhibited the phosphorylation of AKT and mTOR. These results suggest that this series of OA derivatives bearing exocyclic methylene ketone pharmacophore are promising anticancer agents as potential PI3K/AKT/mTOR pathway inhibitors.

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

Uncoupled endothelial nitric oxide synthase (eNOS) produces O2- instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O2- production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O2- burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O2- releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis.

Target Enzyme-Activated Two-Photon Fluorescent Probes: A Case Study of CYP3A4 Using a Two-Dimensional Design Strategy.

The rapid development of fluorescent probes for monitoring target enzymes is still a great challenge owing to the lack of efficient ways to optimize a specific fluorophore. Herein, a practical two-dimensional strategy was designed for the development of an isoform-specific probe for CYP3A4, a key cytochrome P450 isoform responsible for the oxidation of most clinical drugs. In first dimension of the design strategy, a potential two-photon fluorescent substrate (NN) for CYP3A4 was effectively selected using ensemble-based virtual screening. In the second dimension, various substituent groups were introduced into NN to optimize the isoform-selectivity and reactivity. Finally, with ideal selectivity and sensitivity, NEN was successfully applied to the real-time detection of CYP3A4 in living cells and zebrafish. These findings suggested that our strategy is practical for developing an isoform-specific probe for a target enzyme.

Anticancer effect of SZC015 on pancreatic cancer via mitochondria-dependent apoptosis and the constitutive suppression of activated nuclear factor κB and STAT3 in vitro and in vivo.

Pancreatic cancer is the fourth leading cause of cancer-related death worldwide. Advances in therapeutic strategies such as chemotherapy have improved the clinical outcomes for pancreatic cancer patients. However, developing new therapeutic compounds against pancreatic cancer is still urgent due to the poor prognosis. Here, we show that SZC015, an oleanolic acid derivative, exhibits potent inhibitory effect on both pancreatic cancer cells in vitro and the corresponding xenograft tumors in vivo. Mechanistically, the activation of intrinsic apoptosis and G1 phase arrest resulting from mitochondria damage caused by SZC015 contribute significantly to the anticancer effects of SZC015. SZC015 also has remarkably inhibitory effects on the transcription factors that are extensively activated in pancreatic cancer tissues. As a constitutively activated transcription factor in pancreatic cancer, the nuclear factor κB is highly suppressed after SZC015 treatment in vitro or administration in vivo. Based on the bioinformatics analysis of microarray data, we validate that JAK2/STAT3 signaling is indeed activated in the human pancreatic cancer tissues and SZC015 also shows inhibitory effect on this signaling both in vitro and in vivo. These data suggest the potent effects of SZC015 on pancreatic cancer and also provided novel insights into the mechanisms of SZC015 as a new potent candidate for treating pancreatic cancer.

Phosphocreatine attenuates endoplasmic reticulum stress-mediated hepatocellular apoptosis ameliorates insulin resistance in diabetes model.

Diabetes mellitus (DM) associated liver damage is a major health burden. Hepatocellular-damage in DM characterized with elevated endoplasmic reticulum stress (ER) and may enhanced insulin-resistance. Phosphocreatine (PCr) a rapidly high-energy-reserve molecule of phosphates naturally occurs in liver, brain and skeletal muscle. This study aimed to investigate the protective effect of PCr on the liver-injury-associated with DM and to report the mechanism involved. Wistar rat's diabetes model was induced using streptozotocin (STZ), and the animals were treated with 20 mg/kg, or 50 mg/kg PCr injection. Blood glucose level, and body wt were recorded. Liver tissues homogenate were analyzed for liver damage markers alanine transaminase (ALT), aspartate transaminase (AST). Liver tissues proteins further evaluated for apoptosis, endoplasmic reticulum stress (ER), and insulin resistance biomarkers using western blotting. Our results revealed that PCr reduced blood glucose level, improved body wt, ameliorates liver function enzymes. Furthermore, PCr upregulates anti-apoptotic Bcl2 proteins expression, and down-regulates significantly pro-apoptotic casp3 and Bax proteins expression in vivo and invitro. Moreover, ER stress CHOP, GRP78 and ATF4 biomarkers level were significantly attenuated in PCr treated animals comparing to STZ diabetes associated liver-damage model with significant improving in insulin-resistance Akt and IRS-1. Our results revealed that treating with PCr in diabetes-associated liver injury models decreased blood glucose level and possess protective effect in-vitro and in-vivo, which could be suggested as potential therapeutic strategy for diabetes associated liver injury patients.

EGCG protects against homocysteine-induced human umbilical vein endothelial cells apoptosis by modulating mitochondrial-dependent apoptotic signaling and PI3K/Akt/eNOS signaling pathways.

Homocysteine (Hcy) induced vascular endothelial injury leads to the progression of endothelial dysfunction in atherosclerosis. Epigallocatechin gallate (EGCG), a natural dietary antioxidant, has been applied to protect against atherosclerosis. However, the underlying protective mechanism of EGCG has not been clarified. The present study investigated the mechanism of EGCG protected against Hcy-induced human umbilical vein endothelial cells (HUVECs) apoptosis. Methyl thiazolyl tetrazolium assay (MTT), transmission electron microscope, fluorescent staining, flow cytometry, western blot were used in this study. The study has demonstrated that EGCG suppressed Hcy-induced endothelial cell morphological changes and reactive oxygen species (ROS) generation. Moreover, EGCG dose-dependently prevented Hcy-induced HUVECs cytotoxicity and apoptotic biochemical changes such as reducing mitochondrial membrane potential (MMP), decreasing Bcl-2/Bax protein ratio and activating caspase-9 and 3. In addition, EGCG enhanced the protein ratio of p-Akt/Akt, endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) formation in injured cells. In conclusion, the present study shows that EGCG prevents Hcy-induced HUVECs apoptosis via modulating mitochondrial apoptotic and PI3K/Akt/eNOS signaling pathways. Furthermore, the results indicate that EGCG is likely to represent a potential therapeutic strategy for atherosclerosis associated with Hyperhomocysteinemia (HHcy).

Induction of autophagy by an oleanolic acid derivative, SZC017, promotes ROS-dependent apoptosis through Akt and JAK2/STAT3 signaling pathway in human lung cancer cells.

The signal transducers and activators of transcription 3 (STAT3) signaling pathway is a common feature in many solid tumors including non-small cell lung cancer, whereas current therapies usually fail to treat this disease in majority of cases. In the present study, we aimed to investigate the cytotoxic effect and the underlying mechanisms of SZC017, an oleanolic acid derivative, on human lung cancer cells. Cell viability was significantly decreased in SZC017-treated lung cancer cells. Mechanistically, SZC017 reduced A549 cell viability by activating both apoptosis and autophagy pathways. SZC017 was able to inhibit the phosphorylation of Akt, JAK2, and STAT3 in A549 cells, resulting in the inactivation of Akt and JAK2/STAT3 signaling pathways. In addition, SZC017 could induce ROS generation and Ca2+ release. Pretreatment with N-Acetyl L-Cysteine, a ROS scavenger, could fully reverse SZC017-induced ROS and increase the expression of Akt, p-STAT3, and procaspase-3, while decrease the ratio of LC3-II/I and the expression of Beclin-1. In summary, our study provides pharmacological evidence that SZC017 exhibits potential use in the treatment of lung cancer.

Erratum to: Phosphocreatine protects against LPS-induced human umbilical vein endothelial cell apoptosis by regulating mitochondrial oxidative phosphorylation.

The original version of this article unfortunately contained a mistake. The arrow marks in Fig. 5 were incorrect. It is now corrected with this erratum. The correct version of Fig. 5 is given below. The authors apologise for this error and the inconvenience it has caused to the readers.

Phosphocreatine protects against LPS-induced human umbilical vein endothelial cell apoptosis by regulating mitochondrial oxidative phosphorylation.

Phosphocreatine (PCr) is an exogenous energy substance, which provides phosphate groups for adenosine triphosphate (ATP) cycle and promotes energy metabolism in cells. However, it is still unclear whether PCr has influenced on mitochondrial energy metabolism as well as oxidative phosphorylation (OXPHO) in previous studies. Therefore, the aim of the present study was to investigate the regulation of PCr on lipopolsaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs) and mitochondrial OXPHO pathway. PCr protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c (Cyt C), Ca(2+), reactive oxygen species and subsequent activation of caspases, and increasing Bcl2 expression, while suppressing Bax expression. More importantly, PCr significantly improved mitochondrial swelling and membrane potential, enhanced the activities of ATP synthase and mitochondrial creatine kinase (CKmt) in creatine shuttle, influenced on respiratory chain enzymes, respiratory control ratio, phosphorus/oxygen ratio and ATP production of OXPHO. Above PCr-mediated mitochondrial events were effectively more favorable to reduced form of flavin adenine dinucleotide (FADH2) pathway than reduced form of nicotinamide-adenine dinucleotid pathway in the mitochondrial respiratory chain. Our results revealed that PCr protects against LPS-induced HUVECs apoptosis, which probably related to stabilization of intracellular energy metabolism, especially for FADH2 pathway in mitochondrial respiratory chain, ATP synthase and CKmt. Our findings suggest that PCr may play a certain role in the treatment of atherosclerosis via protecting endothelial cell function.

Increased Risk of Stroke in Patients with Isolated Third, Fourth, or Sixth Cranial Nerve Palsies: A Nationwide Cohort Study.

BACKGROUND AND PURPOSE: The aim of this nationwide cohort study was to evaluate whether the occurrence of isolated 3rd, 4th or 6th cranial nerve (CN) palsies is associated with a higher risk of ischemic stroke. METHODS: This study utilized data from Taiwan Longitudinal Health Insurance Database during 1995-2012. Subjects aged 20 years or older who had isolated CN 3/4/6 palsies diagnosed by a neurologist or ophthalmologist between January 2000 and December 2011 were included. A set of propensity score matched, randomly sampled patients who had never been diagnosed with CN 3/4/6 palsies were extracted to constitute the control group (cases and controls = 1:4). All subjects were followed until death, loss due to follow-up or completion of the study. Cox proportional hazard regression model stratified by matched pairs was used to estimate the hazards ratio (HR) of ischemic stroke. RESULTS: A total of 657 patients with isolated CN 3/4/6 palsies (61.1% male, mean age 54.8 years) were identified. Compared with control group, the patients with isolated CN 3/4/6 palsies exhibited an increased risk of ischemic stroke (CN3: adjusted HR 3.69 (95% CI 2.20-6.19); CN4: 2.71 (95% CI 1.11-6.64); CN6: 2.15 (95% CI 1.31-3.52)). The association between CN 3/4/6 palsies and ischemic stroke was detected in both separate subgroup and sensitivity analyses. CONCLUSIONS: The patients with CN 3/4/6 palsies exhibited an increased risk of developing ischemic stroke. Therefore, isolated ocular motor nerves palsies appear to represent an unrecognized risk factor for ischemic stroke, and these require further confirmation and exploration.

SZC017, a novel oleanolic acid derivative, induces apoptosis and autophagy in human breast cancer cells.

Oleanolic acid (OA) and its derivatives such as 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), CDDO-Me, and CDDO-Im show potent anticancer function. In this study, we elucidated the anticancer effect of SZC017, a novel OA derivative and identified the mechanisms by which SZC017 induces MCF-7 cell death. We found that SZC017 effectively decreased the cell viability of these breast cancer cells, but was less toxic to MCF10A mammary epithelial cells. Mechanisms underlying the inhibition of cell viability are apoptosis, autophagy induction, and G0/G1 phase arrest. SZC017 treatment suppressed the levels of Akt, phosphorylated-Akt (p-Akt), p-IκBα, total p65, and total p-p65, in addition to p-p65 in both the cytoplasm and nucleus. Furthermore, the inhibition of p65 nuclear translocation was confirmed by immunofluorescence staining. Cell viability was increased after pretreatment with chloroquine, an inhibitor of autophagy, whereas the level of procaspase-3 was significantly decreased. A concentration-dependent increase in the intracellular reactive oxygen species (ROS) level was observed in both MCF-7 and MDA-MB-231 cells. Additionally, pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, increased cell viability and the expression of Akt and procaspase-3, but decreased the ratio of LC3-II/I. These data show that SZC017 is an effectively selective anticancer agent against breast cancer cells, highlighting the potential use of this derivative as a breast cancer therapeutic agent.