Purinergic signaling: a potential therapeutic target for ischemic stroke.

Pathogenesis of ischemic stroke is mainly characterized by thrombosis and neuroinflammation. Purinergic signaling pathway constitutes adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO). ATP is hydrolyzed to ADP and then to AMP by extracellular nucleotidase CD39; AMP is subsequently converted to adenosine by CD73. All these nucleotides and nucleosides act on purinergic receptors protecting against thrombosis and inhibit inflammation. In addition, many physical methods have been found to play a neuroprotective role through purinergic signaling. This review mainly introduces the role and potential mechanism of purinergic signalings in the treatment of ischemic stroke, so as to provide reference for seeking new treatment methods for stroke.

Advances in Medical Treatment of Primary Aldosteronism.

Primary aldosteronism (PA) is the most common form of secondary hypertension, with its main manifestations including hypertension and hypokalemia. Early identification of PA is extremely important as PA patients can easily develop cardiovascular complications such as atrial fibrillation, stroke, and myocardial infarction. The past decade has witnessed the rapid advances in the genetics of PA, which has shed new light on PA treatment. While surgery is the first choice for unilateral diseases, bilateral lesions can be treated with mineralocorticoid receptor antagonists (MRAs). The next-generation non-steroidal MRAs are under investigations. New medications including calcium channel blockers, macrophage antibiotics, and aldosterone synthase inhibitors have provided a new perspective for the medical treatment of PA.

Ornithinimicrobium laminariae sp. nov., isolated from the kelp Laminaria japonica.

A Gram-stain-positive, aerobic, non-sporulating, yellow-pigmented and rod or cocci-shaped bacterium, designated Arc0846-15T, was isolated from the kelp Laminaria japonica. Strain Arc0846-15T was found to grow at 16-35 °C (optimum, 28 °C), at pH 6.0-9.5 (optimum, 7.0) and in the presence of 0-6 % (w/v) NaCl (optimum, 2 %). Cells were positive for catalase and negative for oxidase activity. Phylogenetic analyses, based on 16S rRNA gene sequence comparisons, revealed that the nearest phylogenetic neighbour strains of strain Arc0846-15T were Ornithinimicrobium murale 01 Gi-040T (96.2 %), Ornithinimicrobium kibberense K22-20T (96.1 %) and Ornithinimicrobium humiphilum HKI 0124T (95.2 %). Based on phylogenomic analysis, the average nucleotide identity values between strain Arc0846-15T and the neighbour strains were 69.8, 69.7 and 69.8 %, respectively; the digital DNA-DNA hybridization values between strain Arc0846-15T and its three closest neighbour strains were 18.8, 19.1 and 19.3 %, respectively. The predominant menaquinone was MK-8 (H4). The dominant cellular fatty acids were C17 : 1 ω8c, iso-C15 : 0, iso-C16 : 0 and C17 : 0. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, glycolipid, one unidentified aminolipid and four unidentified lipids. The DNA G+C content of strain Arc0846-15T was 61.6 mol% based on the whole genome sequence. Based on the phylogenetic and phenotypic characteristics, strain Arc0846-15T is considered to represent a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium laminariae sp. nov. is proposed, with Arc0846-15T (=KCTC 49655T=MCCC 1K06093T) as the type strain.

Shewanella nanhaiensis sp. nov., a marine bacterium isolated from sediment of South China Sea, and emended descriptions of Shewanella woodyi, Shewanella hanedai and Shewanella canadensis.

A Gram-stain-negative, motile, facultative anaerobic and rod-shaped bacterium, designated strain NR704-98T, was isolated from marine sediment of the northern South China Sea. Cells were positive for oxidase and catalase activity. Growth was observed at 4-30 °C (optimum 20-25 °C), at pH 6-9 (pH 7) and with 0.5-7 % NaCl (2 %). The 16S rRNA gene-based phylogenetic analysis revealed that the nearest phylogenetic neighbours of strain NR704-98T were Shewanella woodyi MS32T (97.9 %), Shewanella hanedai 281T (97.1 %), Shewanella sediminis HAW-EB3T (96.8 %) and Shewanella canadensis HAW-EB2T (96.7 %). Based on the results of phylogenomic analysis, the average nucleotide identity and the digital DNA-DNA hybridization values between strain NR704-98T and the previously mentioned type strains of species of the genus Shewanella were in the range of 74.9-93.1 % and 20.6-51.4 %, respectively. The respiratory quinones were Q-7 and Q-8. The predominant fatty acids (>10 %) of strain NR704-98T were C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C15 : 0. Phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminophospholipids and five unidentified lipids were detected in strain NR704-98T. Based on the phylogenetic and phenotypic characteristics, strain NR704-98T is considered to represent a novel species of the genus Shewanella, for which the name Shewanella nanhaiensis sp. nov. is proposed. The type strain is NR704-98T (=KCTC 82799T=MCCC 1K06091T).

Experimental and Model Predictive Investigation into the Relationship of pH Alternation with Addition of Lime in Acid Tin Tailing Treatments.

Lime is one of the commonly used amendments for acidic soils. The reasonable application of lime can effectively improve the current status of acid tailings and reduce harm to the environment. In this study, we analyzed the pH alternation of acid tin tailing as a function of lime dose based on three methods-single titration method, K-bicarbonate titration method, and buffer curve method-to predict the accurate lime requirement (LR) in acid tin tailing treatments. Of these prediction methods, the buffer curve method was best suited for the prediction of lime dose, and the prediction values agreed with the experimental data by factors of 1.0‒1.4. Thus, we determined that the buffer curve method was more suitable for predicting the lime requirement of acid tailings. This study of acid tailings lime requirement provides scientific research for the subsequent modification of tailings.

Salinimicrobium nanhaiense sp. nov. and Salinimicrobium oceani sp. nov., two novel species of the family Flavobacteriaceae isolated from the South China Sea.

Strains J15B81-2T and J15B91T were isolated from a sediment sample collected from the South China Sea. Cells of both strains were observed to be rod-shaped, non-gliding, Gram-stain-negative, yellow-pigmented, facultatively anaerobic, catalase-positive, oxidase-negative and showing optimum growth at 30 °C. Strains J15B81-2T and J15B91T could tolerate up to 9 and 10  % (w/v) NaCl concentration and grow at pH 6.5-9.5 and 6.0-9.0, respectively. The strains shared 97.4 % 16S rRNA gene sequence similarity to each other but were identified as two distinct species based on 81.1-85.8 % ANIb and 31.5 % dDDH values calculated using whole genome sequences. Strains J15B81-2T and J15B91T shared highest 16S rRNA gene sequence similarity to Salinimicrobium xinjiangense CGMCC 1.12522T (98.4 %) and Salinimicrobium sediminis CGMCC 1.12641T (98.0 %), respectively. Among species with validly published names, S. sediminis CGMCC 1.12641T shared close genetic relatedness with strains J15B81-2T [85.1-85.3% average nucleotide identity based on blastBlast+ (ANIb) and 30.6 % digital DNA-DNA hybridization (dDDH)] and J15B91T (76.6-79.1 % ANIb and 21.5 % dDDH). The major fatty acid of strains J15B81-2T and J15B91T were identified as iso-C15 : 0 and iso-C16 : 0, respectively, and the major polar lipids of the two strains consisted of phosphatidylethanolamine, one unidentified phospholipid, one unidentified aminolipid and one unidentified lipid. The strains contained MK-6 as their predominant menaquinone. The genomic G+C contents of strains J15B81-2T and J15B91T were determined to be 41.7 and 41.8 mol %, respectively. Both strains are considered to represent two novel species of the genus Salinimicrobium and the names Salinimicrobium nanhaiense sp. nov. and Salinimicrobium oceani sp. nov. are proposed for strains J15B81-2T (=KCTC 72867T=MCCC 1H00410T) and J15B91T (=KCTC 72869T=MCCC 1H00411T), respectively.

Formosa maritima sp. nov., isolated from coastal sediment.

A Gram-stain-negative, strictly aerobic, gliding-motile, rod-shaped and orange-pigmented bacterium, designated 1494T, was isolated from marine sediment collected off the coast of Weihai, PR China. Strain 1494T was found to grow at 4-37 °C (optimum, 30 °C), at pH 6.0-9.0 (pH 7.0) and in the presence of 0-8 % (w/v) NaCl (2 %). Cells were positive for oxidase and catalase activity. The results of 16S rRNA gene based phylogenetic analysis revealed that strain 1494T belonged to the genus Formosa and exhibited the highest sequence similarity to Formosa spongicola KCTC 22662 T (98.4 %). Menaquinone-6 (MK-6) was detected as the major respiratory quinone. The dominant cellular fatty acids were iso-C15 : 1 G and iso-C15 : 0. The DNA G+C content of strain 1494T was 31.1 mol%. The major polar lipids included phosphatidylethanolamine, one unidentified phospholipid and one unidentified lipid. Based on its phylogenetic and phenotypic characteristics, strain 1494T is considered to represent a novel species from the genus Formosa, for which the name Formosa maritima sp. nov. is proposed. The type strain is 1494T (=KCTC 72531T=MCCC 1H00385T).

Transparency and AIE tunable supramolecular polymer hydrogel acts as TEA-HCl vapor controlled smart optical material.

Stimuli-responsive optical materials attract lots of attention due to their broad applications. Herein, a novel smart stimuli-responsive supramolecular polymer was successfully constructed using a simple tripodal quaternary ammonium-based gelator (TH). The TH self-assembles into a supramolecular polymer hydrogel (TH-G) and shows aggregation-induced emission (AIE) properties. Interestingly, the transparency and fluorescence of the TH-G xerogel film (TH-GF) could be reversibly regulated by use of triethylamine (TEA) and hydrochloric acid (HCl) vapor. When alternately fumed with TEA and HCl vapor, the optical transmittance of the TH-GF was changed from 8.9% to 92.7%. Meanwhile, the fluorescence of the TH-G shows an "ON/OFF" switch. The reversible switching of the transparency and the fluorescence of the TH-GF is attributed to the assembly and disassembly of the supramolecular polymer TH-G. Based on these stimuli-response properties, the TH-GF could act as an optical material and shows potential applications as smart windows or fluorescent display material controlled by TEA and HCl vapor.

Epidemiological, clinical characteristics and drug resistance situation of culture-confirmed children TBM in southwest of China: a 6-year retrospective study.

BACKGROUND: Sichuan is a province located in southwestern China, which have a higher incidence of tuberculosis (TB). This study aimed to analyze the epidemiological and clinical characteristics, as well as drug resistance in culture-confirmed children with Tuberculosis meningitis (TBM) in Southwest of China. METHODS: We performed a retrospective study on children (< 14 years old) with cerebrospinal fluid (CSF) culture-confirmed TBM between January 2013 and December 2018 at Public Health Clinical Center of Chengdu (PHCCC). Mycobacterium tuberculosis (MTB) drug sensitivity testing (DST) was performed using the MicroDST™ method. The age, gender, family history of tuberculosis, status of Bacillus Calmette-Guérin (BCG) vaccination, residential areas information, clinical, laboratory, and radiological features were recorded. Data were analyzed using SPSS Statistics Client 25.0, and the change in drug resistance rate was examined using the Cruskal-Wallis test. RESULTS: Among 319 patients clinically diagnosed with TBM, 42 (13.2%) were Mycobacterial culture positive. Their median age was nine years, and the distribution was equal among female and male patients. Among 42 patients who were enrolled in the study, 1/42 (2.38%) passed away. Children with TBM were concentrated in the minority areas of western Sichuan, where 34/42 (81.0%) patients with TBM belonged to ethnic minorities, and only 2/42 (4.76%) received BCG vaccination in the past. Chest X-rays changes were observed in all patients. Fever and headache were the most common presenting symptom. Thirty-five (83.3%) patients suffered from neck stiffness, and 30/42 (71.4%) had high CSF pressure. DST results showed that the resistance rate was high; resistance to any anti-tuberculosis drug (ATD) was observed in 13 (31.0%) patient isolates, while multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) were found in 2 (4.8%) and 1 (2.4%) patients, respectively. CONCLUSIONS: TBM among children in Southwest China was mainly concentrated in the minority areas of western Sichuan and more than 95% of patients did not receive BCG vaccination at birth. The most common symptoms were fever, headache, and neck stiffness and all patients had positive chest X-ray findings. In addition, high rates of drug resistance were found.

[Clinical features of catch-up growth after kidney transplantation in children].

OBJECTIVE: To study the clinical features of catch-up growth of body height after kidney transplantation in children and related influencing factors. METHODS: A retrospective analysis was performed from the chart review data of 15 children who underwent kidney transplantation in Guangzhou Women and Children's Medical Center from July 2017 to November 2019. According to whether the increase in height standard deviation score (ΔHtSDS) in the first year after kidney transplantation reached ≥0.5, the children were divided into a catch-up group with 8 children and a non-catch-up group with 7 children. According to whether final HtSDS was ≥-2, the children were divided into a standard group with 6 children and a non-standard group with 9 children. The features of catch-up growth of body height and related influencing factors were compared between groups. RESULTS: The data showed that median ΔHtSDS was 0.8 in the first year after transplantation, which suggested catch-up growth of body height. There was a significant difference in HtSDS between the non-catch-up and catch-up groups (P<0.05). Baseline HtSDS before transplantation was positively correlated with HtSDS at the end of follow-up (r=0.622, P<0.05) and was negatively correlated with ∆HtSDS in the first year after transplantation (r=-0.705, P<0.05). Age of transplantation and mean dose of glucocorticoid (GC) per kg body weight were risk factors for catch-up growth after kidney transplantation (OR=1.23 and 1.74 respectively; P<0.05), while baseline HtSDS and use of antihypertensive drugs were independent protective factors for catch-up growth (OR=0.08 and 0.18 respectively; P<0.05); baseline HtSDS and ΔHtSDS in the first year after kidney transplantation were influencing factors for final HtSDS (ß=0.984 and 1.271 respectively; P<0.05). CONCLUSIONS: Kidney transplantation should be performed for children as early as possible, growth retardation before transplantation should be improved as far as possible, and multiple treatment methods (including the use of GC and antihypertensive drugs) should be optimized after surgery, in order to help these children achieve an ideal body height.

Cloud-Point Extraction Combined with Thermal Degradation for Nanoplastic Analysis Using Pyrolysis Gas Chromatography-Mass Spectrometry.

The contamination of micro- and nanoplastics in marine systems and freshwater is a global issue. Determination of micro- and nanoplastics in the aqueous environment is of high priority to fully assess the risk that plastic particles will pose. Although microplastics have been detected in a variety of aquatic ecosystems, the analysis of nanoplastics remains an unsolved challenge. Herein, for the first time, a Triton X-45 (TX-45)-based cloud-point extraction (CPE) was proposed to preconcentrate trace nanoplastics in environmental waters. Under the optimum extraction conditions, an enrichment factor of 500 was obtained for two types of nanoplastics with different compositions, polystyrene (PS) and poly(methyl methacrylate) (PMMA), without disturbing their original morphology and sizes. Additionally, following thermal treatment at 190 °C for 3 h, the CPE-obtained extract could be submitted to pyrolysis gas chromatography-mass spectrometry (Py-GC/MS) analysis for mass quantification of nanoplastics. Taking 66.2 nm PS nanoplastics and 86.2 nm PMMA nanoplastics as examples, the proposed method showed excellent reproducibility, and high sensitivity with respective detection limits of 11.5 and 2.5 fM. Feasibility of the proposed approach was verified by application of the optimized procedure to four real water samples. Recoveries of 84.6-96.6% at a spiked level of 88.6 fM for PS nanoplastics and 76.5-96.6% at a spiked level of 50.4 fM for PMMA nanoplastics were obtained. Consequently, this work provides an efficient approach for nanoplastic analysis in environmental waters.

Influence of miR-520e-mediated MAPK signalling pathway on HBV replication and regulation of hepatocellular carcinoma cells via targeting EphA2.

We determined the role of miR-520e in the replication of hepatitis B virus (HBV) and the growth of hepatocellular carcinoma (HCC) cells. MiR-520e and EPH receptor A2 (EphA2) in HBV-positive HCC tissues and cells were detected, and we studied the impact of miR-520e and the EphA2 receptor in cellular and murine HBV replication models. We find that MiR-520e was upregulated and EphA2 was downregulated in HBV-positive HCC tissues and cells. MiR-520e was decreased in Huh7-X and HepG2-X cells in which HBx was stably expressed, but was dose-dependently elevated after interfering with HBx. Additionally, miR-520e mimic and si-EphA2 groups were reduced in association with increases in HBV DNA content, HBsAg and HBeAg levels, cell proliferation and were enhanced in the expressions of EphA2, p-p38MAPK/p38MAPK, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2)/ERK1/2 and cell apoptosis. Furthermore, si-EphA2 reversed the promotion effect of miR-520e inhibitor on HBV replication and tumour cell growth. Upregulating miR-520e in rAAV8-1.3HBV-infected mouse resulted in reduced EphA2 in liver tissues and HBV DNA content in serum. We find that MiR-520e was decreased in HBV-positive HCC, while overexpression of miR-520e blocked p38MAPK and ERK1/2 signalling pathways by an inhibitory effect on EphA2 and ultimately reduced HBV replication and inhibited tumour cell growth. These data indicate a role for miR-520e in the regulation of HBV replication.

Mechanisms of Probiotic VSL#3 in a Rat Model of Visceral Hypersensitivity Involves the Mast Cell-PAR2-TRPV1 Pathway.

BACKGROUND: Mast cells (MCs), PAR2 and TRPV1, play a key role in the regulation of visceral pain. Several studies have found that probiotics regulate visceral sensitivity. AIMS: The purpose of the current study was to explore the role of MC-PAR2-TRPV1 in VH and the mechanism of VSL#3 in a rat model of VH. METHODS: A total of 64 rats were randomly divided into eight groups: Control VH, VH + ketotifen, VH + FSLLRY-NH2, VH + SB366791, VH + VSL#3, VH + VSL#3 + capsaicin, and VH + VSL#3 + SLIGRL-NH2. The rat model of VH was induced by acetic acid enema and the partial limb restraint method. VH was assessed by the abdominal withdrawal reflex score. MCs in colonic tissue were detected by the toluidine blue staining assay. The expression of PAR2 and TRPV1 in DRGs (L6-S1) was measured by immunohistochemistry and Western blotting. RESULTS: The established VH was abolished by treatment with ketotifen, a mast cell stabilizer FSLLRY-NH2, a PAR2 antagonist SB366791 a TRPV1 antagonist, and probiotic VSL#3 in rats. The administration of ketotifen or probiotic VSL#3 caused a decrease in mast cell number in the colon and decreased PAR2 and TRPV1 expression in DRGs. Intrathecal injection of FSLLRY-NH2 or SB366791 caused decreased expression of PAR2 and/or TRPV1 in DRGs in VH rats. SLIGRL-NH2, a PAR2 agonist, and capsaicin, a TRPV1 agonist, blocked the effects of probiotic VSL#3. CONCLUSIONS: The probiotic VSL#3 decreases VH in rat model of VH. The mechanism may be related with the mast cell-PAR2-TRPV1 signaling pathway.

[Renal physiology of urea transporters].

Urea transporters (UTs) are transmembrane urea-selective channel proteins that include two UT subfamilies, UT-A and UT-B. UT-A subfamily includes six members, UT-A1 to UT-A6, which are mainly expressed in kidney. UT-B subfamily has only one member that has a wide distribution in the body. UTs have been confirmed to play important roles in urinary concentration via the phenotypic analysis of 6 UT selective knockout mouse models. Experimental results suggest that UTs might be diuretic targets and that UT inhibitors might be developed as novel diuretics. This article reviews the physiological function and drug discovery of UT.

miR-218 suppresses epithelial-to-mesenchymal transition by targeting Robo1 and Ecop in lung adenocarcinoma cells.

AIM: Although, miR-218 has been implicated in epithelial-to-mesenchymal transition process, the detailed mechanisms of miR-218 involvement in epithelial-to-mesenchymal transition in human lung adenocarcinoma cell are still unclear. MATERIALS & METHODS: miR-218 function assays and its target gene analysis were performed. RESULTS: miR-218 suppresses human lung adenocarcinoma cell migration and invasion and inhibits its target gene, Ecop and Robo1 expression, which subsequently suppresses NF-κB activity and its downstream targets. CONCLUSION: miR-218 inhibits human lung adenocarcinoma cell migration and invasion via the suppression of Ecop and Robo1 expression, thus suggesting that miR-218 could serve as a potential therapeutic target.

MicroRNA-455 suppresses non-small cell lung cancer through targeting ZEB1.

MicroRNA-455 (miRNA-455), which is downregulated in human cancer, potently mediates the multiple steps of carcinogenesis. However, the role of miR-455 in non-small cell lung cancer (NSCLC) carcinogenesis remains unclear. In present study, we determined the mature miRNA-455 expression in NSCLC tissues and cells by real-time PCR. Follow-up studies examined the effects of a miR-455 mimic (gain of function) on cell proliferation, migration, and invasion. Our data indicate that miR-455 was significantly down-regulated in NSCLC cell lines and tissues. In functional assays, overexpression of miR-455 suppressed the proliferation, migration, and invasion of NSCLC cell lines. Data from reporter assays showed that miR-455 directly binds to 3'UTR of ZEB1 and suppresses the endogenous level of ZEB1 protein expression. Furthermore, overexpression of ZEB1 reverses miR-455-suppressed malignant phenotype of NSCLC cells. Moreover, we found that upregulation of ZEB1 expression is inversely associated with miR-455 expression in NSCLC tissues. Taken together, miR-455 as an anti-oncogene in non-small cell lung cancer through up-regulation of ZEB1 and serve as a potential therapeutic target in NSCLC.

The effect of ferrous-chelating hairtail peptides on iron deficiency and intestinal flora in rats.

BACKGROUND: Chelating agents, such as small peptides, can decrease free iron content and increase iron bioavailability. They may have promising therapeutic potential and may prevent the pro-oxidant effects of low molecular weight iron. Hairtail is a species of fish that is rich in easily digestible proteins. We extended this strategy for iron delivery by using an enzymatic hydrolysate of hairtail as the chelating agent and found that the ferrous-chelating hairtail peptides have anti-anaemic activity in Sprague-Dawley rats with anaemia. RESULTS: The anti-anaemic activity of ferrous-chelating hairtail peptides prepared by enzymatic hydrolysis of the hairtail and ferrous chelation was studied in rat models of iron deficiency anaemia. After the end of the 35 d experiment, we noted significant differences in haemoglobin, mean corpuscular volume, haemoglobin distribution width, and ferritin concentrations between those animals supplemented with ferrous-chelating hairtail peptides and FeSO4 and healthy animals. There were no negative side effects on the animals' growth or behaviour. There was no obvious inflammation in the intestinal mucosa lamina propria and no unbalance of intestinal flora. CONCLUSION: The novel ferrous-chelating hairtail peptides may be a suitable fortificant for improving iron-deficiency status. Our findings demonstrated that this multi-tracer technique has many applications in nutritional research. © 2015 Society of Chemical Industry.

Betulinic Acid and its Derivatives as Potential Antitumor Agents.

Betulinic acid (BA) is a lupane-type pentacyclic triterpene, distributed ubiquitously throughout the plant kingdom. BA and its derivatives demonstrate multiple bioactivities, particularly an antitumor effect. This review critically describes the recent research on isolation, synthesis, and derivatization of BA and its natural analogs betulin and 23-hydroxybetulinic acid. The subsequent part of the review focuses on the current knowledge of antitumor properties, combination treatments, and pharmacological mechanisms of these compounds. A 3D-QSAR analysis of 62 BA derivatives against human ovarian cancer A2780 is also included to provide information concerning the structure-cytotoxicity relationships of these compounds.

B4G2 induces mitochondrial apoptosis by the ROS-mediated opening of Ca(2+)-dependent permeability transition pores.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is the most common type of liver cancer. At present, only sorafenib is approved to treat HCC. In this study, we found that a 23-hydroxybetulinic acid derivative, B4G2, exhibited potent antiproliferative activity in HCC cell lines. METHODS: We used four HCC cell lines (HepG2, HepG2/ADM, Hep3B and Bel-7402) to evaluate the anti-tumour activity and explore underlying mechanisms by which B4G2 induces apoptosis. RESULTS: Among these cell lines, HepG2 showed the highest sensitivity to B4G2. HepG2 cells treated with B4G2 showed a depolarized mitochondrial membrane potential, released cytochrome c, activated caspase-9 and caspase-3 and cleaved poly ADP-ribose polymerase (PARP). However, Z-VAD-FMK, a pan-caspase inhibitor, did not attenuate B4G2-induced apoptosis, implying that the induction of mitochondrial apoptosis by B4G2 may be independent of caspases. Moreover, pre-treatment with MgCl2, a blocker of Ca2+-dependent permeability transition (PT) pores, attenuated the depolarization of the mitochondrial potential and decreased the population of apoptotic cells, indicating that B4G2-induced apoptosis was partly dependent on the opening of the Ca2+-dependent PT pores. B4G2 also increased the levels of intracellular calcium and reactive oxygen species (ROS). Furthermore, an ROS scavenger, N-acetyl-cysteine (NAC), markedly decreased the accumulation of intracellular calcium and apoptosis. CONCLUSION: This is the first demonstration that B4G2 inhibits the growth of HCC cells and induces mitochondrial apoptosis in hepatocellular carcinoma cells by the ROS-mediated opening of Ca2+-dependent permeability transition pores.

Cabozantinib reverses multidrug resistance of human hepatoma HepG2/adr cells by modulating the function of P-glycoprotein.

BACKGROUND & AIMS: Cabozantinib, a small-molecule multitargeted tyrosine kinase inhibitor, has entered into a phase III clinical trial for the treatment of hepatocellular carcinoma (HCC). This study assessed the mechanistic effect of cabozantinib on the reversal of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). METHODS: CCK-8 assays and tumour xenografts were used to investigate the reversal of MDR in vitro and in vivo respectively. Substrate retention assays were evaluated by fluorescence microscope and flow cytometry. Western blotting was used to detect protein expression levels. mRNA expression was determined by qPCR. The ATPase activity of P-gp was investigated using Pgp-Glo(™) assay systems. The binding mechanism of cabozantinib to P-gp at the molecular level was evaluated using docking analysis. RESULTS: Cabozantinib enhanced the cytotoxicity of P-gp substrate drugs in HepG2/adr and HEK293-MDR1 cells but had no effect on non-P-gp substrates. In addition, cabozantinib increased the accumulation of P-gp substrates in HepG2/adr cells but had no effect in HepG2 cells. Furthermore, cabozantinib did not alter the expression of P-gp mRNA or protein but did stimulate the activity of P-gp ATPase. The docking study indicated that cabozantinib and verapamil may partially share a binding site on P-gp. The reversal concentrations of cabozantinib did not affect the expression of MET, AKT and ERK1/2. Significantly, cabozantinib increased the inhibitory efficacy of doxorubicin in P-gp-overexpressing HepG2/adr cell xenografts in nude mice. CONCLUSION: Cabozantinib reverses P-gp-mediated MDR by directly inhibiting the efflux function of P-gp, indicating that cabozantinib may help to reverse P-gp-mediated MDR in HCC and other cancer chemotherapy.