Gas6 Exerts Neuroprotective Effects via Restoring the Blood-Brain Barrier in Mice with Sepsis-Associated Encephalopathy.

OBJECTIVE: Sepsis-associated encephalopathy (SAE), characterized by cognitive and emotional impairments, is not well investigated in sepsis survivors. Growth arrest-specific gene 6 (Gas6) has been extensively used to treat cerebral diseases. This study aimed to evaluate the neuroprotective effects of Gas6 in post-septic mice and to determine the underlying mechanisms of action. METHODS: Mice underwent cecal ligation and puncture (CLP) for sepsis induction. Mice were then immediately injected with 6 µg of Gas6 via the tail vein, and the effect was evaluated after 24 hours. The neurological severity score (NSS) was used to assess neurological deficits in post-septic mice. In addition, brain edema was evaluated by measuring the brain water content and blood-brain barrier (BBB) permeability using Evans blue (EB) dye extravasation. Western blotting and immunofluorescence assays were performed to determine the expression of tight junction (TJ)-associated proteins such as occludin and zonula occludens-1 (ZO-1). RESULTS: Post-septic mice exhibited increased NSS, brain edema, and BBB permeability. However, acute Gas6 treatment attenuated the severe effects of sepsis on neurologic function in mice. Therefore, Gas6 attenuates brain edema and restores BBB permeability. These findings suggest that Gas6 could alleviate neurological deficits, brain edema, BBB damage, and reverse the decreased expression of occludin and ZO-1 in the brain tissue to protect against SAE. CONCLUSION: Gas6 protects against SAE by restoring the impaired BBB permeability.

Reduced plasma coagulation factor XIII in patients with acute leukemia in remission during consolidation chemotherapy cycles.

Acute leukemia (AL) is a malignancy from hematologic stem cells (HSC). Consolidation with intensive chemotherapy is required after induced remission and repeatedly causes treatment-related bleeding that is usually attributed to chemotherapy-induced thrombocytopenia (CIT). However, our previous study demonstrated that severe deficiency of plasma coagulation factor XIII (pFXIII) also participated in the bleeding of CIT in AL. However, the relationship between pFXIII deficiency and consolidation chemotherapy was unknown. Here, we observed the concentration of pFXIII in patients with AL before and after consolidation chemotherapy and reevaluated the correlation to bleeding in myelosuppression. Thus, we found that the concentration of pFXIII before chemotherapy in all patients was markedly lower than in the control data and was further decreased by chemotherapy, related to bleeding in myelosuppression. These findings indicated that chemotherapy-induced pFXIII deficiency should be of concern and explored in depth.

ROS-dependent catalytic mechanism of melatonin metabolism and its application in the measurement of reactive oxygen.

Melatonin (Mel) is an endogenous active molecule whose metabolism progress significantly influences its bioactivity. However, the detailed metabolic pathway of Mel in the pathological state has not yet been fully illustrated. In this study, 16 metabolites of Mel in cancer cells and human liver microsomes were identified, of which seven novel metabolites were newly discovered. Among them, 2-hydroxymelatonin (2-O-Mel), as the major metabolite in cancer cells, was revealed for the first time, which was different from the metabolite found in the human liver. Furthermore, CYP1A1/1A2- and reactive oxygen species (ROS)-mediated 2-hydroxylation reactions of Mel were verified to be the two metabolic pathways in the liver and cancer cells, respectively. ROS-dependent formation of 2-O-Mel was the major pathway in cancer cells. Furthermore, the underlying catalytic mechanism of Mel to 2-O-Mel in the presence of ROS was fully elucidated using computational chemistry analysis. Therefore, the generation of 2-O-Mel from Mel could serve as another index for the endogenous reactive oxygen level. Finally, based on the ROS-dependent production of 2-O-Mel, Mel was successfully used for detecting the oxygen-carrying capacity of hemoglobin in human blood. Our investigation further enriched the metabolic pathway of Mel, especially for the ROS-dependent formation of 2-O-Mel that serves as a diagnostic and therapeutic target for the rational use of Mel in clinics.

Intravenous Patient-Controlled Analgesia Versus Oral Opioid to Maintain Analgesia for Severe Cancer Pain: A Randomized Phase II Trial.

BACKGROUND: Optimal analgesic maintenance for severe cancer pain is unknown. This study evaluated the efficacy and safety of intravenous patient-controlled analgesia (IPCA) with continuous infusion plus rescue dose or bolus-only dose versus conventional oral extended-release morphine as a background dose with normal-release morphine as a rescue dose to maintain analgesia in patients with severe cancer pain after successful opioid titration. METHODS: Patients with persistent severe cancer pain (≥7 at rest on the 11-point numeric rating scale [NRS]) were randomly assigned to 1 of 3 treatment arms: (A1) IPCA hydromorphone with bolus-only dose where dosage was 10% to 20% of the total equianalgesic over the previous 24 hours (TEOP24H) administered as needed, (A2) IPCA hydromorphone with continuous infusion where dose per hour was the TEOP24H divided by 24 and bolus dosage for breakthrough pain was 10% to 20% of the TEOP24H, and (B) oral extended-release morphine based on TEOP24H/2 × 75% (because of incomplete cross-tolerance) every 12 hours plus normal-release morphine based on TEOP24H × 10% to 20% for breakthrough pain. After randomization, patients underwent IPCA hydromorphone titration for 24 hours to achieve pain control before beginning their assigned treatment. The primary endpoint was NRS over days 1 to 3. RESULTS: A total of 95 patients from 9 oncology study sites underwent randomization: 30 into arm A1, 32 into arm A2, and 33 into arm B. Arm B produced a significantly higher NRS over days 1 to 3 compared with arm A1 or A2 (P<.001). Daily NRS from day 1 to day 6 and patient satisfaction scores on day 3 and day 6 were worse in arm B. Median equivalent-morphine consumption increase was significantly lower in A1 (P=.024) among the 3 arms. No severe adverse event occurred in any arm. CONCLUSIONS: Compared with oral morphine maintenance, IPCA hydromorphone for analgesia maintenance improves control of severe cancer pain after successful titration. Furthermore, IPCA hydromorphone without continuous infusion may consume less opioid.

Cytotoxic diterpenoid dimer containing an intricately caged core from Euphorbia fischeriana.

Bislangduoids A and B, a novel class of dimeric diterpenoids based on ent-abietanes tethered by C-17-C-15' bridge, were identified as trace components from a traditional Chinese medicine Euphorbia fischeriana (Langdu). Bislangduoid A features a highly oxidized scaffold incorporating a cage-like pentacyclic core. Their structures were elucidated by extensive spectroscopic techniques, electronic circular dichroism, and NMR calculations. The biosynthetic pathway for the dimeric skeleton and the unique caged moiety via Michael and acetal-formation reactions was proposed. Bislangduoid A showed pronounced cytotoxicity against HepG2 cells through the mitochondria-dependent apoptosis pathway.

Combining diffusion kurtosis imaging and clinical data for predicting the extramural venous invasion of rectal adenocarcinoma.

OBJECTIVE: The aim of this study is to investigate the diagnostic ability of diffusion kurtosis imaging (DKI) -derived parameters combining with clinical data as risk factors for EMVI's involvement status in rectal adenocarcinoma. MATERIALS AND METHODS: Preoperative MR examination including DKI and conventional diffusion-weighted imaging (DWI) was performed on 154 rectal adenocarcinoma patients enrolled in this respective study. Kmean, Dmean, and apparent diffusion coefficient (ADC) values were calculated. Clinical information, serum tumor markers, MR and pathological assessment of EMVI were recorded. The Shapiro-Wilk test, two-sample t-test, Mann-Whitney U test, Spearman's rank-order correlation, univariate and multivariate logistic regression analyses were used for statistical analysis. Receiver operating characteristic (ROC) curve analyses were performed to identify risk factors in EMVI involvement. RESULTS: Of the 154 patients, pEMVI-positive rectal tumors had significantly higher Kmean values, lower ADCmean values compared to pEMVI-negative rectal tumors. Kmean values positively correlated with mrEMVI scores, whereas ADCmean values showed a negative correlation with mrEMVI scores. However, there was no significant correlation between the Dmean values and the mrEMVI scores. Univariate analysis demonstrated increased Kmean values, decreased ADCmean values, nodal involvement, an advanced tumor stage, and a G2 tumor grade were significantly related to the pEMVI of rectal adenocarcinoma. The multivariate analysis demonstrated that the Kmean values, lymph node involvement and an advanced tumor stage (T3) were independent risk factors for EMVI. CONCLUSION: The potential for diffusion kurtosis imaging as a biomarker for evaluating the EMVI of rectal cancer is feasible, especially given DKI's capability of detecting tumor heterogeneity noninvasively.

Identification of Escherichia coli β-glucuronidase inhibitors from Polygonum cuspidatum Siebold & Zucc

Abstract Gut bacterial β-glucuronidase (GUS) can reactivate xenobiotics that exert enterohepatic circulation- triggered gastrointestinal tract toxicity. GUS inhibitors can alleviate drug-induced enteropathy and improve treatment outcomes. We evaluated the inhibitory effect of Polygonum cuspidatum Siebold & Zucc. and its major constituents against Escherichia coli GUS (EcGUS), and characterized the inhibitory mechanism of each of the components. Trans-resveratrol 4'-O-β-D-glucopyranoside (HZ-1) and (-)-epicatechin gallate (HZ-2) isolated from P. cuspidatum were identified as the key components and potent inhibitors. These two components displayed strong to moderate inhibitory effects on EcGUS, with Ki values of 9.95 and 1.95 μM, respectively. Results from molecular docking indicated that HZ-1 and HZ-2 could interact with the key residues Asp163, Ser360, Ile 363, Glu413, Glu504, and Lys 568 of EcGUS via hydrogen bonding. Our findings demonstrate the inhibitory effect of P. cuspidatum and its two components on EcGUS, which supported the further evaluation and development of P. cuspidatum and its two active components as novel candidates for alleviating drug-induced damage in the mammalian gut.

In vitro and in vivo evaluation of self-assembled chitosan nanoparticles selectively overcoming hepatocellular carcinoma via asialoglycoprotein receptor.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related mortality worldwide. Nowadays, liver-targeting drug delivery system has been proven as a promising strategy for overcoming HCC. Asialoglycoprotein receptor (ASGPR) is an ideal receptor for liver targeting, which is mainly expressed on hepatocytes. In this study, we developed several novel liver-targeting chitosan nanoparticles to selectively overcome HCC via ASGPR. Chitosan nanoparticles (Gly-CS-VE, Gal-Gly-CS-VE, Gly-CS-DCA, and Gal-Gly-CS-DCA) were prepared by grafting hydrophilic group (glycidol, Gly), hydrophobic group (deoxycholic acid, DCA or vitamin E succinate, VE), and ASGPR recognizing group (galactose, Gal). Subsequently, their characterizations were measured by 1H NMR, FT-IR, TEM, and DLS. Doxorubicin (DOX) was loaded in nanoparticles and released out in a pH-dependent manner. Most importantly, the galactosylated Gal-Gly-CS-VE and Gal-Gly-CS-DCA nanoparticles exhibited significantly stronger in vitro cell internalization, cytotoxicity, anti-migration capabilities and in vivo anticancer efficacies than the corresponding Gly-CS-VE and Gly-CS-DCA nanoparticles, as well as free DOX. Finally, the four chitosan nanoparticles exhibited good biocompatibility without causing any obvious histological damage to the major organs. Overall, the galactosylated chitosan nanoparticles were proven to be promising pharmaceutical formulations for selectively overcoming HCC, with great potential for clinical applications.

UV-light-driven photooxidation of harmaline catalyzed by riboflavin: Product characterization and mechanisms.

ß-Carboline alkaloid harmaline (HA) is a candidate drug molecule that has been proven to have broad and significant biological activity. Herein, the effects of HA on the riboflavin (RF)-sensitized photooxidation under aerobic conditions were studied for the first time. The photooxidation reaction of HA catalyzed by RF is triggered by UV light at 365 nm and shows a time-dependent stepwise reaction process. Seven transformed products, including five undescribed compounds, oxoharmalines A-E (1-4 and 7), and two known compounds, N-(2-(6-Methoxy-2-oxoindolin-3-yl)ethyl)acetamide (5) and harmine (6), were isolated and identified from the reaction system, following as the gradual oxidation mechanisms. The rare polymerization and dehydrogenation processes in radical-mediated photocatalytic reactions were involved in the process. The transformed products 2-7 exhibited significant neuroprotective activity in a model of H2O2-introduced injury in SH-SY5Y cells, which suggested that the products of the interaction between HA and vitamins may be beneficial to health.

Comparing Patient-Controlled Analgesia Versus Non-PCA Hydromorphone Titration for Severe Cancer Pain: A Randomized Phase III Trial.

BACKGROUND: Opioid titration is necessary to achieve rapid, safe pain relief. Medication can be administered via patient-controlled analgesia (PCA) or by a healthcare provider (non-PCA). We evaluated the efficacy of intravenous PCA versus non-PCA hydromorphone titration for severe cancer pain (≥7 at rest on the 11-point numeric rating scale [NRS]). PATIENTS AND METHODS: Patients with severe cancer pain were randomized 1:1 to PCA or non-PCA titration, stratified by opioid-tolerant or opioid-naïve status. The PCA pump was set to no continuous dose, with a hydromorphone bolus dose 10% to 20% of the total previous 24-hour equianalgesic (for opioid-tolerant patients) or 0.5 mg (for opioid-naïve patients). For the non-PCA group, the initial hydromorphone bolus dose was identical to that in the PCA group, with the subsequent dose increased by 50% to 100% (for NRS unchanged or increased) or repeated at the current dose (for NRS 4-6). Hydromorphone delivery was initiated every 15 minutes (for NRS ≥4) or as needed (for NRS ≤3). The primary endpoint was time to successful titration (TST; time from first hydromorphone dose to first occurrence of NRS ≤3 in 2 consecutive 15-minute intervals). RESULTS: Among 214 patients (PCA, n=106; non-PCA, n=108), median TSTs (95% CI) were 0.50 hours (0.25-0.50) and 0.79 hours (0.50-1.42) for the PCA and non-PCA groups, respectively (hazard ratio [HR], 1.64; 95% CI, 1.23-2.17; P=.001). TSTs in opioid-tolerant patients were 0.50 hours (0.25-0.75) and 1.00 hours (0.50-2.00) for the PCA and non-PCA groups, respectively (HR, 1.92; 95% CI, 1.32-2.78; P=.003); in opioid-naive patients, TST was not significantly different for the PCA versus non-PCA groups (HR, 1.35; 95% CI, 0.88-2.04; P=.162). Pain score (median NRS; interquartile range) over 24 hours was significantly lower in the PCA group (2.80; 2.15-3.22) than in the non-PCA group (3.00; 2.47-3.53; P=.020). PCA administration produces significantly higher patient satisfaction with pain control than non-PCA administration (P<.001). CONCLUSIONS: Intravenous hydromorphone titration for severe cancer pain was achieved more effectively with PCA than with non-PCA administration.

lncRNA NEAT1 aggravates sepsis-induced lung injury by regulating the miR-27a/PTEN axis.

Sepsis is an acute inflammatory reaction and a cause of acute respiratory distress syndrome (ARDS). In the present study, we explored the roles and underlying mechanism of the lncRNA Nuclear enriched abundant transcript 1 (NEAT1) in ARDS. The expression levels of genes, proteins and pro-inflammatory cytokines in patients with ARDS, LPS-stimulated cells and septic mouse models were quantified using qPCR, western blotting and ELISA assays, respectively. The molecular targeting relationship was validated by conducting a dual-luciferase reporter assay. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. The cell cycle phase was determined by flow cytometry assay. The expression levels of NEAT1 and pro-inflammatory cytokines were higher in patients with ARDS and septic models than in controls. Knockdown of NEAT1 significantly increased cell proliferation and cycle progression and prolonged mouse survival in vitro and in vivo. Mechanistically, miR-27a was identified as a downstream target of NEAT1 and directly inhibited PTEN expression. Further rescue experiments revealed that inhibition of miR-27a impeded the promoting effects of NEAT1 silence on cell proliferation and cycle progression, whereas inhibition of PTEN markedly weakened the inhibitory effects of NEAT1 overexpression on cell proliferation and cycle progression. Altogether, our study revealed that NEAT1 plays a promoting role in the progression of ARDS via the NEAT1/miR-27a/PTEN regulatory network, providing new insight into the pathologic mechanism behind ARDS.

Prognostic values of anti-Ro52 antibodies in anti-MDA5-positive clinically amyopathic dermatomyositis associated with interstitial lung disease.

OBJECTIVE: Anti-Ro52 antibody often co-occurs with anti-Jo1 antibody in antisynthetase syndrome and their co-occurrence correlates with a more aggressive clinical phenotype and poorer prognosis. The strong association of anti-Ro52 antibody with anti-melanoma differentiation-associated protein-5 (anti-MDA5) antibody has been indicated in juvenile myositis. The aim of this study was to assess the clinical significance of anti-Ro52 antibody in a cohort of adult patients with anti-MDA5-positive clinically amyopathic dermatomyositis with interstitial lung disease (CADM-ILD). METHODS: We assessed a cohort of 83 consecutive patients with anti-MDA5-positive CADM-ILD. Anti-MDA5 antibodies and anti-Ro52 antibodies were detected in immunoblotting and semi-quantitatively analysed by densitometry. Clinical features and the 24 month survival were compared between anti-MDA5-positive patients with and without anti-Ro52 antibodies. RESULTS: Anti-Ro52 antibodies were found in 74.7% of anti-MDA5-positive CADM-ILD patients and were associated with an increased frequency of rapidly progressive interstitial lung disease (RP-ILD; 54.8% vs 23.8%; P = 0.014) and cutaneous ulcerations (27.4% vs 4.8%; P = 0.033). The cumulative 24 month survival rate tended to be lower in patients with anti-Ro52 antibodies than patients without (59.9% vs 85.7%; P = 0.051). The combination of anti-Ro52 antibody status and anti-MDA5 antibody levels further stratified patients' survival rates, showing that the survival rate of patients who were dual positive for anti-MDA5 antibody and anti-Ro52 antibody was significantly lower than patients with mild positive anti-MDA5 antibody alone (59.9% vs 100%; P = 0.019). CONCLUSION: Anti-Ro52 antibody is highly prevalent in anti-MDA5-positive CADM-ILD patients and their coexistence correlates with a subgroup of patients with more aggressive phenotypes. The combination of anti-MDA5 antibody levels and anti-Ro52 antibody status could help to predict patients' prognosis and guide risk-based therapy.

Low Circulating Monocytes Is in Parallel With Lymphopenia Which Predicts Poor Outcome in Anti-melanoma Differentiation-Associated Gene 5 Antibody-Positive Dermatomyositis-Associated Interstitial Lung Disease.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis (DM)-associated interstitial lung disease (ILD) may progress rapidly and lead to high mortality within 6 or 12 months. Except for reported prognostic factors, simple but powerful prognostic biomarkers are still in need in practice. In this study, we focused on circulating monocyte and lymphocyte counts and their variation tendency in the early stage of ILD. A total of 351 patients from two inception anti-MDA5 antibody-positive cohorts were included in this study, with various treatment choices. Lymphocyte count remained lower in the first month after admission in the non-survivor patients. Although baseline monocyte count showed no significant differences, average monocyte count in the following 4 weeks was also lower in the non-survivor group. Based on the C-index and analysis by the "survminer" R package in the discovery cohort, we chose 0.24 × 109/L as the cutoff value for Mono W0-2, 0.61 × 109/L as the cutoff value for lymph W0-2, and 0.78 × 109/L as the cutoff value for peripheral blood mononuclear cell (PBMC) W0-2, to predict the 6-month all-cause mortality. The Kaplan-Meier survival curves and adjusted hazard ratio with age, gender, and the number of immunosuppressants used all validated that patients with lower average monocyte count, lower average lymphocyte count, or lower average PBMC count in the first 2 weeks after admission had higher 6-month death risk, no matter in the validation cohort or in the pooled data. Furthermore, flow cytometry figured out that non-classical monocytes in patients with anti-MDA5 antibody-positive DM were significantly lower than healthy controls and patients with DM without anti-MDA5 antibodies. In conclusion, this study elucidated the predictive value of monocyte and lymphocyte counts in the early stage and may help rheumatologists to understand the possible pathogenesis of this challenging disease.

Spontaneous Intramuscular Hemorrhage in Anti-MDA5 Positive Dermatomyositis: A Case Series and Literature Review.

OBJECTIVE: Spontaneous intramuscular hemorrhage (SIH) is a rare but life-threatening complication associated with dermatomyositis (DM). This study reported a case series of SIH associated with DM. In addition, the characteristics and prognostic effects for this complication were analyzed based on literature review. METHODS: We reported seven cases of anti-melanoma differentiation-associated gene five positive dermatomyositis (MDA5+DM) complicated by SIH in our single-center cohort, and a comprehensive literature review was performed. Clinical characteristics, treatment, and outcome data of all eligible reported cases were summarized. Potential prognostic effects were identified by comparisons between the deceased and survivors. RESULTS: Among cumulatively reported patients with DM patients and SIH, the overall mortality was 60.9% (14/23) (including our cases). Fourteen out of nineteen (73.7%) hemorrhagic events occurred within 6 months of disease onset. Anti-MDA5 antibody predominated in those myositis-specific antibodies available cases (8/10), although patients with positive anti-NXP2 and anti-Mi2 have also been documented. Iliopsoas (52.2%, 12/23) was the most frequently involved bleeding location. Bleeding in deep muscles was identified to be associated with poorer prognosis. The mortality of patients with DM and deep muscular hematoma (non-palpable) (80%, 12/15) was significantly higher than that of patients with only superficial muscular hematoma (palpable) (25%, 2/8) (p =0.023). CONCLUSION: Spontaneous hematoma in non-palpable deep muscles probably leads to excess mortality in dermatomyositis, particularly for those with anti-MDA5 antibody, which often occurs within 6 months of disease onset. Clinicians should be vigilant to this rare but potentially fatal complication and carefully balance the risks and benefits of prophylactic anti-thrombotic treatment.

Highly potent non-steroidal FXR agonists protostane-type triterpenoids: Structure-activity relationship and mechanism.

Farnesoid X receptor (FXR) is a key regulator in charge of bile acid synthesis, transport, and metabolism. Activation of FXR represses bile acid synthesis and increases its excretion and transport, consequently protecting the liver functions. Thus, FXR is considered as a critical therapeutic target of cholestasis and nonalcoholic steatohepatitis. Herein, we isolated and identified fourteen new protostane-type triterpenoids (1-14) and four known analogues (15-18) from Alisma orientale, and finally constructed a small library of protostane-type triterpenoids (1-70) to investigate their structure-activity relationship with FXR, further leading to obtain compound 15 with potent agonistic activity against FXR (EC50 = 90 nM). Extensive in vitro investigation confirmed high efficacy of compound 15 against FXR in living cell, and revealed its underlying mechanism for FXR activation (amino acid residues Arg331 and Ser332) by molecular docking and site-directed mutagenesis technology.

Facile synthesis of fluorescent carbon dots from Prunus cerasifera fruits for fluorescent ink, Fe3+ ion detection and cell imaging.

Carbon dots (CDs) synthesized from natural products have drawn numerous attentions due to some unique properties. Here, Prunus cerasifera fruits were used as carbon source to synthesize high luminescent CDs by hydrothermal method. The obtained CDs were characterized by TEM, FTIR and XPS methods, founding the CDs were near-spherical and contained abundant nitrogen element. The CDs aqueous solution exhibited bright blue fluorescence under ultraviolet illumination, with the maximum emission at 450 nm. They could be potentially used as invisible fluorescent ink by written on the paper and irradiated by UV light, due to their fluorescent properties. Moreover, the CDs were found being selectively quenched by Fe3+ ion. The quench of CDs was linearly related to the concentration of Fe3+ ion in the range of 0-0.5 mM, meaning they could be developed as fluorescent probe of Fe3+ ion. At last, the CDs were used for cell imaging, founding they were low toxicity to HepG2 cells and exhibited blue and green fluorescence under a fluorescence microscope. In summary, the CDs prepared from Prunus cerasifera fruits exhibited excellent fluorescence properties, and could be potentially applied in the field of fluorescent ink, Fe3+ ion detection and cell imaging.

Adenovirus-Mediated CRM197 Sensitizes Human Glioma Cells to Gemcitabine by the Mitochondrial Pathway.

PURPOSE: The cross-reacting material 197 (CRM197) is a mutation of the diphtheria toxin. The protein of CRM197 was used successfully for the therapy of various tumors in the recent studies. In this study, the recombinant adenoviruses containing the CRM197gene(AdCRM197) were used to enhance the cellar toxicity of gemcitabine in human glioma U87, U251, and H4 cells. PROCEDURES: MTT assay and flow cytometric analysis were performed to test the apoptosis of the U87, U251 and H4 cells with the combined treatment of AdCRM197 plus gemcitabine. Western blotting analyses were carried out to detect the cell apoptosis of the mitochondrial pathway. And the xenograft nude mice were used to observe the enhanced antitumor effect of AdCRM197 in vivo. RESULTS: AdCRM197 sensitizes human glioma cells to gemcitabine in vitro by the mitochondrial pathway. Tumor volume was inhibited and survival time was prolonged in the U251 or U87 xenografted nude mice with gemcitabine plus AdCRM197. The enhanced antitumor effect of AdCRM197 was also detected by the immunohistochemical analyses and TUNEL staining. CONCLUSION: The authors found that AdCRM197 sensitized the human glioma to gemcitabine not only in vitro but also in vivo. They provide the first evidence that adenovirus-mediated CRM197 may be a potential chemosensitizing agent for the treatment of cancer. The diphtheria toxin is of great toxicity that even one molecule of diphtheria toxin is enough to kill one cell. However, because of the high toxicity, the diphtheria toxin would kill the packing cells when it is being packaged into the recombinant viruses. Therefore, the diphtheria toxin is hard to be used in the gene therapy for virus vectors. The cross-reacting material 197 (CRM197) is a mutation of the diphtheria toxin. Unlike DTA, CRM197 exhibit a weak toxicity. The week toxicity of CRM197 is a good feature for the virus packaging. In the present study, we used a recombinant adenovirus which carried a CRM197 gene (AdCRM197) to enhance the cellar toxicity of gemcitabine in human glioma cells.

Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases.

Amentoflavone (AMF), an abundant natural biflavonoid found in many medicinal plants, displays various beneficial effects including anti-inflammatory, anti-oxidative and anti-cancer. Despite the extensive studies on pharmacological activities, the toxicity or undesirable effects of AMF are rarely reported. In this study, the inhibitory effects of AMF on human UDP-glucuronosyltransferases (UGTs) were carefully investigated. AMF displayed strong inhibition towards most of human UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4 and 2B17, with the IC50 values ranging from 0.12 µM to 16.81 µM. Inhibition constants (Ki) of AMF against various human UGTs varied from 0.29 µM to 11.51 µM. Further investigation demonstrated that AMF was a noncompetitive inhibitor of UGT1A1 mediated NCHN-O-glucuronidation but functioned as a competitive inhibitor of UGT1A1 mediated 4-MU-O-glucuronidation. In addition, AMF was a competitive inhibitor of UGT1A4 mediated TFP-N-glucuronidation in both UGT1A4 and human liver microsomes, while functioned as a competitive inhibitor of UGT1A9 mediated propofol or 4-MU-O-glucuronidation. These findings demonstrated that AMF was a strong and broad-spectrum natural inhibitor of most human UGTs, which might bring potential risks of herb-drug interactions (HDIs) via UGT inhibition. Additionally, this study provided novel insights into the underlying mechanism of AMF-associated toxicity from the perspective of UGT inhibition.

A Practical and High-Affinity Fluorescent Probe for Uridine Diphosphate Glucuronosyltransferase 1A1: A Good Surrogate for Bilirubin.

This study aimed to develop a practical and high-affinity fluorescent probe for uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a key conjugative enzyme responsible for the elimination and detoxification of many potentially harmful compounds. Several substrates derived from N-butyl-4-phenyl-1,8-naphthalimide were designed and synthesized on the basis of the substrate preference of UGT1A1 and the principle of photoinduced electron transfer (PET). Following the preliminary screening, substrate 2 was found with a high specificity and high affinity toward UGT1A1, while such biotransformation brought remarkable changes in fluorescence emission. Both inhibition kinetic analyses and molecular docking simulations demonstrated that 2 could bind on UGT1A1 at the same ligand-binding site as bilirubin. Furthermore, this newly developed probe was successfully used for sensing UGT1A1 activities and the high-throughput screening of UGT1A1 modulators in complex biological samples. In conclusion, a practical and high-affinity fluorescent probe for UGT1A1 was designed and well-characterized, which could serve as a good surrogate for bilirubin to investigate UGT1A1-ligand interactions.