Factors influencing pathological changes in the liver tissue in hepatitis B virus carriers with low-level viremia.

OBJECTIVES: To investigate the optimal timing for initiating antiviral therapy in hepatitis B virus (HBV) carriers with low-level viremia (LLV). METHODS: We retrospectively enrolled 126 HBV carriers with LLV who underwent liver biopsy. Patients' clinical data, routine blood test results, portal vein diameter, splenic vein diameter and thickness, and measurements (LSM) within 1 week before liver biopsy were obtained. Single-factor and multifactor statistical methods were used to analyze factors that affected inflammation and fibrosis in pathological liver tissues. The receiver operating characteristic curve was used to analyze liver stiffness and HBV DNA levels to determine liver tissue inflammation and fibrosis. R -Studio software was used to draw nomograms, calibration plots, and model decision curves. RESULTS: Infection duration and HBV DNA levels affected liver tissue inflammation. Albumin(ALB), aspartate aminotransferase (AST), HBV DNA, liver stiffness, age, and splenic thickness affected liver fibrosis. The best cutoff value of the LSM for diagnosing liver inflammation and fibrosis was 7.45 (specificity, 92%). The best cutoff value of HBV DNA for diagnosing liver inflammation and fibrosis was 39.5 (specificity, 96%). HBV DNA,and splenic thickness affected the treatment decision in naive chronic hepatitis Bpatients with LLV CONCLUSIONS: HBV carriers with LLV have high incidences of liver tissue inflammation and fibrosis. The infection duration and HBV DNA levels affected liver inflammation whereas the ALB, AST AST levels, HBV DNA, LSM, age, and splenic thickness affected liver fibrosis. Eligible expansion of antiviral treatment indications is necessary, however, a universal treatment approach may be inefficient. HBV DNA can be a reference for initiating antiviral therapy.

Current Status of Metabolic Compliance and Risk of Cardiovascular Disease in Patients with Type 2 Diabetes in the Zhuang Population in China.

Introduction: This study aimed to investigate the blood glucose, blood pressure, and blood lipid status in Zhuang patients with T2DM and to analyze the correlation between compliance with metabolic monitoring and cardiovascular risk factors. Methods: A total of 1975 Zhuang patients with T2DM were evaluated in four Class III Grade A hospitals in three prefecture-level cities in the Guangxi Zhuang Autonomous Region between January and August 2022. Laboratory indicators, lifestyle, and demographic characteristics were collected. Results: The compliance rates for blood glucose, blood pressure, and blood lipids were 26.08%, 45.77%, and 30.58%, respectively, and only 5.06% of the patients reached the standard in all three indices. The compliance rates for blood glucose, blood pressure, and blood lipids in the CVD group were 32.92%, 21.74%, and 9.94%, respectively. In the CVD group, the usage rates of hypoglycemic, antihypertensive, and lipid-lowering drugs were 77.54%, 3.17%, and 4.11%, respectively. Binary logistic regression analysis showed that older age (OR = 1.033, 95% CI [1.016, 1.050]), female (OR = 0.402, 95% CI [0.260, 0.621]), smoke (OR = 1.994, 95% CI [1.361, 2.922]), blood pressure noncompliance + use of antihypertensive drugs (OR = 0.348, 95% CI [0.230, 0.527]), and blood lipid noncompliance + use of lipid-lowering drugs (OR = 0.244, 95% CI [0.142, 0.417]) were risk factors for CVDs, and moderate-intensity exercise (OR = 0.439, 95% CI [0.300,0.640]) was protective against CVD. Conclusions: Older age, female, smoke, blood lipid levels, and blood pressure noncompliance were risk factors for CVD while moderate-intensity exercise was observed to be protective.

Quantitative disease risk scores from EHR with applications to clinical risk stratification and genetic studies.

Labeling clinical data from electronic health records (EHR) in health systems requires extensive knowledge of human expert, and painstaking review by clinicians. Furthermore, existing phenotyping algorithms are not uniformly applied across large datasets and can suffer from inconsistencies in case definitions across different algorithms. We describe here quantitative disease risk scores based on almost unsupervised methods that require minimal input from clinicians, can be applied to large datasets, and alleviate some of the main weaknesses of existing phenotyping algorithms. We show applications to phenotypic data on approximately 100,000 individuals in eMERGE, and focus on several complex diseases, including Chronic Kidney Disease, Coronary Artery Disease, Type 2 Diabetes, Heart Failure, and a few others. We demonstrate that relative to existing approaches, the proposed methods have higher prediction accuracy, can better identify phenotypic features relevant to the disease under consideration, can perform better at clinical risk stratification, and can identify undiagnosed cases based on phenotypic features available in the EHR. Using genetic data from the eMERGE-seq panel that includes sequencing data for 109 genes on 21,363 individuals from multiple ethnicities, we also show how the new quantitative disease risk scores help improve the power of genetic association studies relative to the standard use of disease phenotypes. The results demonstrate the effectiveness of quantitative disease risk scores derived from rich phenotypic EHR databases to provide a more meaningful characterization of clinical risk for diseases of interest beyond the prevalent binary (case-control) classification.

Improved YOLO-V3 with DenseNet for Multi-Scale Remote Sensing Target Detection.

Remote sensing targets have different dimensions, and they have the characteristics of dense distribution and a complex background. This makes remote sensing target detection difficult. With the aim at detecting remote sensing targets at different scales, a new You Only Look Once (YOLO)-V3-based model was proposed. YOLO-V3 is a new version of YOLO. Aiming at the defect of poor performance of YOLO-V3 in detecting remote sensing targets, we adopted DenseNet (Densely Connected Network) to enhance feature extraction capability. Moreover, the detection scales were increased to four based on the original YOLO-V3. The experiment on RSOD (Remote Sensing Object Detection) dataset and UCS-AOD (Dataset of Object Detection in Aerial Images) dataset showed that our approach performed better than Faster-RCNN, SSD (Single Shot Multibox Detector), YOLO-V3, and YOLO-V3 tiny in terms of accuracy. Compared with original YOLO-V3, the mAP (mean Average Precision) of our approach increased from 77.10% to 88.73% in the RSOD dataset. In particular, the mAP of detecting targets like aircrafts, which are mainly made up of small targets increased by 12.12%. In addition, the detection speed was not significantly reduced. Generally speaking, our approach achieved higher accuracy and gave considerations to real-time performance simultaneously for remote sensing target detection.

Co-localization between Sequence Constraint and Epigenomic Information Improves Interpretation of Whole-Genome Sequencing Data.

The identification of functional regions in the noncoding human genome is difficult but critical in order to gain understanding of the role noncoding variation plays in gene regulation in human health and disease. We describe here a co-localization approach that aims to identify constrained sequences that co-localize with tissue- or cell-type-specific regulatory regions, and we show that the resulting score is particularly well suited for the identification of rare regulatory variants. For 127 tissues and cell types in the ENCODE/Roadmap Epigenomics Project, we provide catalogs of putative tissue- or cell-type-specific regulatory regions under sequence constraint. We use the newly developed co-localization score for brain tissues to score de novo mutations in whole genomes from 1,902 individuals affected with autism spectrum disorder (ASD) and their unaffected siblings in the Simons Simplex Collection. We show that noncoding de novo mutations near genes co-expressed in midfetal brain with high confidence ASD risk genes, and near FMRP gene targets are more likely to be in co-localized regions if they occur in ASD probands versus in their unaffected siblings. We also observed a similar enrichment for mutations near lincRNAs, previously shown to co-express with ASD risk genes. Additionally, we provide strong evidence that prioritized de novo mutations in autism probands point to a small set of well-known ASD genes, the disruption of which produces relevant mouse phenotypes such as abnormal social investigation and abnormal discrimination/associative learning, unlike the de novo mutations in unaffected siblings. The genome-wide co-localization results are available online.

The Natural Compound Oblongifolin C Exhibits Anticancer Activity by Inhibiting HSPA8 and Cathepsin B In Vitro.

PPAPs (Polycyclic polyprenylated acylphloroglucinols) are a class of compounds with diverse bioactivities, including anticancer effects. Oblongifolin C (OC) is a PPAP isolated from the plant of Garcinia yunnanensis Hu. We previously discovered that OC induces apoptosis, inhibits autophagic flux, and attenuates metastasis in cancer cells. However, the protein targets and the detailed mechanism of action of OC remain unclear. To identify protein targets of OC, a non-labeled protein fishing assay was performed, and it was found that OC may interact with several proteins, including the heat shock 70 kDa protein 8 (HSPA8). Expanding on our previous studies on protein cathepsin B, this current study applied Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) to confirm the potential binding affinity between OC and two protein targets. This study highlights the inhibitory effect of OC on HSPA8 in cancer cells under heat shock stress, by specifically inhibiting the translocation of HSPA8. OC also enhanced the interaction between HSPA8, HSP90, and p53, upregulated the expression of p53 and significantly promoted apoptosis in cisplatin-treated cells. Additionally, a flow cytometry assay detected that OC sped up the apoptosis rate in HSPA8 knockdown A549 cells, while overexpression of HSPA8 delayed the OC-induced apoptosis rate. In summary, our results reveal that OC potentially interacts with HSPA8 and cathepsin B and inhibits HSPA8 nuclear translocation and cathepsin B activities, altogether suggesting the potential of OC to be developed as an anticancer drug.

Screening of FDA-approved drugs identifies sutent as a modulator of UCP1 expression in brown adipose tissue.

BACKGROUND: The pharmacological activation of thermogenesis in brown adipose tissue has long been considered promising strategies to treat obesity. However, identification of safe and effective agents remains a challenge. In this study, we addressed this challenge by developing a cellular system with a fluorescence readout, and applied in a high-throughput manner to screen for FDA-approved drugs that may activate endogenous UCP1 expression in adipocytes. METHODS: We have generated a Ucp1-2A-GFP reporter mouse, in which GFP intensity serves as a surrogate of the endogenous expression level of UCP1 protein; and immortalized brown adipocytes were derived from this mouse model and applied in drug screening. Candidate drugs were further tested in mouse models either fed with normal chow or high fat diet to induce obesity. FINDINGS: By using the cellular screening platform, we identified a group of FDA-approved drugs that can upregulate UCP1 expression in brown adipocyte, including previously known UCP1 activators and new candidate drugs. Further studies focusing on a previously unreported drug-sutent, revealed that sutent treatment could increase the energy expenditure and inhibit lipid synthesis in mouse adipose and liver tissues, resulting in improved metabolism and resistance to obesity. INTERPRETATION: This study offered an easy-to-use cellular screening system for UCP1 activators, and provided a candidate list of FDA-approved drugs that can potentially treat obesity. Further study of these candidates may shed new light on the drug discovery towards obesity. FUND: National Key Research and Development Program and the Strategic Priority Research Program of the Chinese Academy of Sciences, etc. (250 words).

The Association of Weekly pre-Hemodialysis Systolic Blood Pressure and Following Week Mortality.

BACKGROUND/AIMS: Few studies examine the impact of systolic blood pressure (SBP) on mortality in the incident hemodialysis (HD) period, and throughout the first HD year. This large retrospective observational study analyzes the impact of "current" and cumulative low preSBP <110 mmHg (L), and variations in preSBP on short-term (1 week) mortality over the first HD year. METHODS: Weekly mean preSBP for HD weeks 1 to 51 was categorized into L or high preSBP>=110 mmHg (H) for each patient. A generalized linear model (GLM) was used to compute the probability of death in the following week. The model includes age, gender, race and three preSBP-related parameters: (a) percent of prior weeks with L preSBP; (b) percent of prior weeks with switching between L to H; (c) "current" week's preSBP as a binary variable. Separate models were constructed that include demographics and BP-related parameters (a), (b), and (c) separately. RESULTS: In a model combining (a), (b), and (c) above, "current" week L preSBP is associated with increased odds ratio for following week mortality throughout the first HD year. The percent of prior week's L and more switching between L and H are less significantly associated with short-term mortality. In models including (a), (b), and (c) separately, "current" L preSBP is associated with higher mortality. CONCLUSION: This study confirms an association of L preSBP with increased short-term mortality which is maintained over the first HD year. Percent of L preSBP in prior weeks, switching between L and H, and "current" week L are all associated with short-term mortality risk, but "current" week L preSBP is most significant.

Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screening Platform.

Autophagy is an evolutionarily conserved homeostasis process through which aggregated proteins or damaged organelles are enveloped in a double-membrane structure called an autophagosome and then digested in a lysosome-dependent manner. Growing evidence suggests that malfunction of autophagy contributes to the pathogenesis of a variety of diseases, including cancer, viral infection, and neurodegeneration. However, autophagy is a complicated process, and understanding of the relevance of autophagy to disease is limited by lack of specific and potent autophagy modulators. ATG4B, a Cys-protease that cleaves ATG8 family proteins, such as LC3B, is a key protein in autophagosome formation and maturation process. A novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring protease activity of ATG4B was developed, validated, and adapted into a high-throughput screening (HTS) format. HTS was then conducted with a Roche focus library of 57,000 compounds. After hit confirmation and a counterscreen to filter out fluorescence interference compounds, 267 hits were confirmed, constituting a hit rate of 0.49%. Furthermore, among 65 hits with an IC50 < 50 µM, one compound mimics the LC3 peptide substrate (-TFG-). Chemistry modification based on this particular hit gave preliminary structure activity relationship (SAR) resulting in a compound with a 10-fold increase in potency. This compound forms a stable covalent bond with Cys74 of ATG4B in a 1:1 ratio as demonstrated by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, this compound displayed cellular ATG4B inhibition activity. Overall, the novel TR-FRET ATG4B protease assay plus counterscreen assay provides a robust platform to identify ATG4B inhibitors, which would help to elucidate the mechanism of the autophagy pathway and offer opportunities for drug discovery.

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors.

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure-activity relationship (SAR) and protease selectivity are also discussed.

Xanthones from the Leaves of Garcinia cowa Induce Cell Cycle Arrest, Apoptosis, and Autophagy in Cancer Cells.

Two new xanthones, cowaxanthones G (1) and H (2), and 23 known analogues were isolated from an acetone extract of the leaves of Garcinia cowa. The isolated compounds were evaluated for cytotoxicity against three cancer cell lines and immortalized HL7702 normal liver cells, whereby compounds 1, 5, 8, and 15-17 exhibited significant cytotoxicity. Cell cycle analysis using flow cytometry showed that 5 induced cell cycle arrest at the S phase in a dose-dependent manner, 1 and 16 at the G2/M phase, and 17 at the G1 phase, while 16 and 17 induced apoptosis. Moreover, autophagy analysis by GFP-LC3 puncta formation and western blotting suggested that 17 induced autophagy. Taken together, our results suggest that these xanthones possess anticancer activities targeting cell cycle, apoptosis, and autophagy signaling pathways.

Identification and characterization of anticancer compounds targeting apoptosis and autophagy from Chinese native Garcinia species.

Natural compounds from medicinal plants are important resources for drug development. Active compounds targeting apoptosis and autophagy are candidates for anti-cancer drugs. In this study, we collected Garcinia species from China and extracted them into water or ethanol fractions. Then, we performed a functional screen in search of novel apoptosis and autophagy regulators. We first characterized the anti-proliferation activity of the crude extracts on multiple cell lines. HeLa cells expressing GFP-LC3 were used to examine the effects of the crude extracts on autophagy. Their activities were confirmed by Western blots of A549 and HeLa cells. By using bioassay guided fractionation, we found that two caged prenylxanthones from Garcinia bracteata, neobractatin and isobractatin, can significantly induce apoptosis and inhibit autophagy. Our results suggest that different Garcinia species displayed various degrees of toxicity on different cancer cell lines. Furthermore, the use of a high content screening assay to screen natural products was an essential method to identify novel autophagy regulators.

The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation.

Metabolic stress induces autophagy as an alternative source of energy and metabolites. Insufficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy. Here, we performed a functional screen in search of novel autophagy regulators from natural products. We showed that oblongifolin C (OC), a natural small molecule compound extracted from Garcinia yunnanensis Hu, is a potent autophagic flux inhibitor. Exposure to OC results in an increased number of autophagosomes and impaired degradation of SQSTM1/p62. Costaining of GFP-LC3B with LysoTracker Red or LAMP1 antibody demonstrates that autophagosome-lysosome fusion is blocked by OC treatment. Furthermore, OC inhibits lysosomal proteolytic activity by altering lysosomal acidification and downregulating the expression of lysosomal cathepsins. Importantly, OC can eliminate the tolerance of cancer cells to nutrient starvation. Starvation dramatically increases the susceptibility of cancer cells to OC-induced CASP3-dependent apoptosis in vitro. Subsequent studies in xenograft mouse model showed that OC has anticancer potency as revealed by increased staining of cleaved CASP3, LC3 puncta, and SQSTM1, as well as reduced expression of lysosomal cathepsins. Combined treatment with OC and caloric restriction potentiates anticancer efficacy of OC in vivo. Collectively, these data demonstrated that OC is a novel autophagic flux inhibitor and might be useful in anticancer therapy.

Targeting cadherin-17 inactivates Ras/Raf/MEK/ERK signaling and inhibits cell proliferation in gastric cancer.

Cadherin-17 (CDH17), one member of 7D-cadherin superfamily, was overexpressed in gastric cancer (GC) and was associated with poor survival, tumor recurrence, metastasis, and advanced tumor stage. So far the cellular function and signaling mechanism of CDH17 in GC remains unclear. In this study, we showed that over 66% of GC cell lines (20/30) were CDH17 positive. Tissue microarray (TMA) assay showed that 73.6% Chinese GC tissues (159/216) were CDH17 positive, while 37% respective adjacent normal tissues were CDH17 positive. Knockdown of CDH17 inhibited cell proliferation, migration, adhesion and colony formation, and also induced a cell cycle arrest and apoptosis in AGS human GC cells. On the other side, overexpression of CDH17 facilitated MGC-803 GC tumor growth in nude mice. Antibody array and Western blotting assay demonstrated that knockdown of CDH17 in AGS cells down-regulated integrin ß series proteins, further inactivated the Ras/Raf/MEK/ERK pathway and led to p53 and p21 accumulation, which resulted in proliferation inhibition, cell-cycle arrest and apoptosis induction. Collectively, our data firstly demonstrate the capacity of CDH17 to regulate the activity of Ras/Raf/MEK/ERK pathway for cell proliferation in GC, and suggest that CDH17 can serve as an attractive therapeutic target for future research.

Small molecule R1498 as a well-tolerated and orally active kinase inhibitor for hepatocellular carcinoma and gastric cancer treatment via targeting angiogenesis and mitosis pathways.

Protein kinases play important roles in tumor development and progression. Lots of kinase inhibitors have entered into market and show promising clinical benefits. Here we report the discovery of a novel small molecule, well-tolerated, orally active kinase inhibitor, R1498, majorly targeting both angiogenic and mitotic pathways for the treatment of hepatocellular carcinoma (HCC) and gastric cancer (GC). A series of biochemical and cell-based assays indicated that the target kinase cluster of R1498 included Aurora kinases and VEGFR2 et al. R1498 showed moderate in vitro growth inhibition on a panel of tumor cells with IC50 of micromole range. The in vivo anti-tumor efficacy of R1498 was evaluated on a panel of GC and HCC xenografts in a parallel comparison with another multikinase inhibitor sorafenib. R1498 demonstrated superior efficacy and toxicity profile over sorafenib in all test models with >80% tumor growth inhibition and tumor regression in some xenogratfts. The therapeutic potential of R1498 was also highlighted by its efficacy on three human GC primary tumor derived xenograft models with 10-30% tumor regression rate. R1498 was shown to actively inhibit the Aurora A activity in vivo, and decrease the vascularization in tumors. Furthermore, R1498 presented good in vivo exposure and therapeutic window in the pharmacokinetic and dose range finding studies. Theses evidences indicate that R1498 is a potent, well-tolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic and antiproliferative profile, and provide strong confidence for further development for HCC and GC therapy.

5k, a novel ß-O-demethyl-epipodophyllotoxin analogue, inhibits the proliferation of cancer cells in vitro and in vivo via the induction of G2 arrest and apoptosis.

Etoposide (VP-16), a topoisomerase II (Topo II) inhibitor, has been widely used to treat malignancies. Its clinical application, however, has been hindered by the rise of acquired multidrug resistance (MDR). Here, we report that 4ß-{[4-(pyrrolidin-1-ylmethyl)phenyl]amino}-4'-O-Demethyl-4-Epipodophyllotoxin (5k), a novel ß-O-demethyl-epipodophyllotoxin analogue, possesses higher antitumor activity than its parent compound (VP-16) in a panel of various human tumor cell lines. More importantly, it was also effective against MDR cells both in vitro and in vivo. Using a KB/VCR MDR tumor xenograft model that overexpresses P-gp, 5k (2.5 mg/kg) exhibited a 2.4-fold higher growth inhibition rate versus VP-16 (5 mg/kg). In contrast, 5k and VP-16 displayed similar antitumor activities in a KB tumor xenograft model. Molecular and cellular mechanism studies revealed that 5k targeted Topo II by trapping DNA-Topo II cleavage complexes that could directly cause DNA damage. There were two distinct cellular responses to DNA damage elicited by the treatment with 5k: at low concentrations (20-80 nM), mitotic entry was arrested through the suppression of the activity of Cyclin B1/Cdc 2 complexes via the ATM/ATR signaling pathway; at high concentrations (1.25-5.00 µM), 5k-induced apoptotic signaling was mediated by the mitochondrial death pathways. Collectively, these data demonstrate the potential value of 5k as an antitumor drug candidate that should be further developed.

Up-regulation of death receptor 4 and 5 by celastrol enhances the anti-cancer activity of TRAIL/Apo-2L.

Our previous study demonstrated that celastrol combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) exhibited significant synergistic anti-cancer activities, thus we were promoted to investigate the molecular mechanism of this synergy. Here in this study, we show that celastrol up-regulates death receptor 4 (DR4) and 5 (DR5) expression at mRNA, total protein and cell surface levels, and the specific knockdown using DR4- or DR5-targeting siRNA transfection attenuates the PARP cleavage caused by the combination of celastrol and TRAIL/Apo-2L, denoting the critical roles of DR induction in this sensitization. Of note is that although celastrol activates p38 mitogen activated protein kinases (p38 MAPK), SB203580, one specific inhibitor of p38, fails to interrupt celastrol-induced DR4 expression and the enhanced apoptosis caused by celastrol plus TRAIL/Apo-2L. In addition, the protein expression of Mcl-1 and FLIP, two critical antiapoptotic factors, is not decreased upon celastrol treatment under our experimental conditions. Taken together, the present study demonstrates that the enhanced mRNA and protein expression of DR4 and DR5 play prominent roles in the sensitization of celastrol to TRAIL/Apo-2L-induced apoptosis, in a p38 MAPK-independent manner.

Novel 4 beta-anilino-podophyllotoxin derivatives: design synthesis and biological evaluation as potent DNA-topoisomerase II poisons and anti-MDR agents.

A new series of 4 beta-anilino-podophyllotoxin analogs have been designed, synthesized and evaluated their bioactivities as novel DNA-topoisomerase II poisons as well as P-glycoprotein (P-gp)-dependent multidrug resistance (MDR) inhibitors. The new compounds show improved potency and efficacy with respect to the parent molecule etoposide (VP-16), one of the semisynthetic derivatives of podophyllotoxin. The treatment of 5k-n in KB/VCR cells caused G(2)/M phase arrest and finally induced apoptosis. Furthermore, molecular docking is applied to testify that 5k-n could not be the substrates of P-gp, which is consistent with the result of MDR1 and P-glycoprotein express tests. The most potent compound 5n is chosen for in vivo studies, the administration of 5n was effective in treatment of cancer with a lower dose than VP-16 in drug-sensitive xenograft model and drug-resistant xenograft model. Compound 5n is a potential drug candidate for anticancer chemotherapy.

ROS-driven Akt dephosphorylation at Ser-473 is involved in 4-HPR-mediated apoptosis in NB4 cells.

N-(4-hydroxyphenyl) retinamide (4-HPR), as a synthetic retinoid, has been shown to inhibit carcinogenesis in a variety of cancers. Extensive studies have indicated that ROS are involved in 4-HPR-mediated apoptosis. Herein, we provide further evidence that the Akt signaling pathway is involved in 4-HPR-mediated apoptosis. Of note is the fact that the expression of PI3K (p110) does not change obviously, and neither LY294002 nor insulin could influence the apoptosis induced by 4-HPR. These observations implicate the direct interaction between Akt and ROS. Our data also reveal that 4-HPR-mediated ROS evoke Akt conformational change by forming an intramolecular disulfide bond; N-acetylcysteine and glutathione, as thiol antioxidants, significantly abate the ROS generation in 4-HPR-exposed cells. Further experiments indicate that the conformational change in Akt not only disrupts Akt-Hsp90 binding, but also enhances Akt-PP2A interaction. All these results collectively suggest that 4-HPR-induced apoptosis is associated with a ROS-mediated conformational change in Akt, and this change, as a consequence, mediates dephosphorylation of Akt via regulating Akt-Hsp90 or Akt-PP2A complex formation.

Antimultidrug-resistant effect and mechanism of a novel CA-4 analogue MZ3 on leukemia cells.

A major issue in the treatment of leukemia is resistance to chemotherapeutic drugs. The most common mechanism encountered in the laboratory is the increased efflux of hydrophobic cytotoxic drugs that is mediated by a family of energy-dependent transporters. Besides, resistance to apoptosis can also cause failure in the treatment of leukemia. Recently, we have introduced 4-(4-bromophenyl)-2,3-dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-2-oxoidmidazole-1-carboxamide (MZ3) as a novel synthesized combretastatin A-4 analogue which is a potent and specific compound against leukemia cells both in vitro and in vivo. Aim of this study was to evaluate the effect of MZ3 on multidrug-resistant (MDR) cancer cells of leukemia, and explore the antimultidrug-resistant mechanisms. Here, we observed that the MDR leukemia cell models investigated, overexpressing MDR1 (P-gp), were hypersensitive against MZ3. Parental K562, HL60 cells and MDR1-overexpressing K562R, HL60R cells were employed in this study. MZ3 hypersensitivity was confirmed to be based on great apoptosis induction and cell cycle arrest at unaltered intracellular drug accumulation. Cell proliferation assay demonstrated that, compared with HL60 and K562 cells, HL60R and K562R cells exhibited 1.3-fold and 2.4-fold resistance to MZ3, showing 26.9-fold and 92.2-fold resistance to daunorubicin (DNR) respectively. Moreover, real-time RT-PCR result showed that MZ3 impacted the transcription of MDR1 gene and western blotting results indicated that MZ3 can activate apoptosis on MDR cells by downregulating the anti-apoptotic protein XIAP levels and inducing the decrease in the phosphorylation state of ERK. Summarizing, our data demonstrate that MZ3 can inhibit the MDR function of leukemia cells, and it exerts the effect through altering the transcription of MDR1 genes and downregulating the anti-apopotic protein levels. MZ3 may be a potential candidate for further research and development in anti-MDR territory.