Oroxylin A reverses the drug resistance of chronic myelogenous leukemia cells to imatinib through CXCL12/CXCR7 axis in bone marrow microenvironment.

Imatinib (IM), a tyrosine-kinase inhibitor, is used in treatment of multiple cancers, most notably Philadelphia chromosome-positive (Ph+ ) chronic myelogenous leukemia (CML). However, the majority of patients continue to present with minimal residual disease occurred in the bone marrow (BM) microenvironment. One of the key factors that contribute to leukemia cell drug resistance is chemokine CXCL12. In the current study, co-culturing CML cell K562 and KU812 with BM stromal cell M2-10B4 attenuated IM-induced apoptosis. CXCL12/CXCR7 pathway was activated in co-culture models, which was further proved to be related to drug resistance by silencing CXCR7. ERK phosphorylation and downstream apoptosis related proteins' activation were also observed in co-culture group after the activation of CXCR7. Moreover, oroxylin A, a bioactive flavonoid isolated from the root of Scutellaria baicalensis Georgi, was found to be effective in reversing BM stroma induced CML resistance to IM. After cells were treated with weakly toxic concentration of oroxylin A, cell apoptosis induced by IM in co-culture model was enhanced. And the activated CXCL12/CXCR7 pathway, the expression of p-ERK and downstream apoptosis related proteins were suppressed. The in vivo study also showed that oroxylin A increased apoptosis of CML cells with low systemic toxicity, and the mechanism was consistent with the in vitro study. In conclusion, oroxylin A improved sensitivity of CML cells to IM treatment in BM microenvironment through regulating CXCL12/CXCR7 pathway. © 2016 Wiley Periodicals, Inc.

Pharmacological strategies to lower crosstalk between nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondria.

Reactive oxygen species (ROS) are the metabolites of oxygen that plays a significant role in cell signaling and homeostasis. Under normal conditions, ROS formation is stabilized by various antioxidant defense systems (ROS scavengers). Several studies in both in-vitro and in-vivo models, together with clinical data indicated that increased production ROS and oxidative stress plays a crucial role in the development and progression of endothelial dysfunction. The interactions between the main cellular sources of ROS, such as mitochondria and NADPH oxidases, however, remain unclear. The purpose of this review is to outline various sources of ROS and describe the crosstalk between NADPH oxidase and mitochondria. Further, we will discuss different antioxidants that lower ROS production and ROS-induced pathological conditions such as aging, atherosclerosis, diabetes, hypertension, and degenerative neurological disorders. In this review, we have mainly focused on antioxidants that inhibit NADPH oxidase and mitochondrial sources of ROS. Moreover, the modification of antioxidants (targeted therapy) may be a significant approach for management of oxidative stress induced pathophysiological complications.

Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ARE pathway.

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, the incidence of cardiotoxicity compromises its therapeutic index. Oxidative stress and apoptosis are believed to be involved in DOX-induced cardiotoxicity. Chitosan oligosaccharides (COS), the enzymatic hydrolysates of chitosan, have been reported to possess diverse biological activities including antioxidant and anti-apoptotic properties. The objective of the present study was to investigate the potential role of COS against DOX-induced cardiotoxicity, and the effects of COS on apoptosis and oxidative stress in rats and H9C2 cells. Furthermore, we also shed light on the involved pathways during the whole process. For this purpose, first, we demonstrated that COS exhibited a significant protective effect on cardiac tissue by not only inducing a decrease in body and heart growth but also ameliorated oxidative damage and ECG alterations in DOX-treated rats. Second, we found that COS reversed the decrease of cell viability induced by DOX, reduced the intracellular reactive oxygen species (ROS), increased the mitochondrial membrane potential (MMP) and Bcl-2/Bax ratio. COS treatment also results in reduced caspase-3 and caspase-9 expressions, and an increase in the phosphorylation of MAPKs (mitogen-activated protein kinases) in DOX-exposed H9C2 cells. Additionally, cellular homeostasis was re-established via stabilization of MAPK mediated nuclear factor erythroid 2-related factor 2/antioxidant-response element (Nrf2/ARE) signaling and transcription of downstream cytoprotective genes. In summary, these findings suggest that COS could be a potential candidate for the prevention and treatment of DOX-induced cardiotoxicity.

Pharmacokinetics, biodistribution, efficacy and safety of N-octyl-O-sulfate chitosan micelles loaded with paclitaxel.

Paclitaxel (Taxol), PTX) is a promising anti-cancer drug and has been successfully used to treat a wide variety of cancers. Unfortunately, serious clinical side effects are associated with it, which are caused by PTX itself and non-aqueous vehicle containing Cremophor EL. Development of new formulation of PTX with better efficacy and fewer side effects is extremely urgent. In the present study, a N-octyl-O-sulfate chitosan (NOSC) micelle was developed and used as the delivery system for PTX. The pharmacokinetics, biodistribution, efficacy and safety of PTX-loaded NOSC micelles (PTX-M) were evaluated. The results showed that NOSC micelles had high drug loading capacity (69.9%) and entrapment efficiency (97.26%). The plasma AUC of PTX-M was 3.6-fold lower than that of Taxol; but the V(d) and CL of PTX-M were increased by 5.7 and 3.5-fold, respectively. Biodistribution study indicated that most of the PTX were distributed in liver, kidney, spleen, and lung and the longest retention effect was observed in the lung. Drug safety assessment studies including acute toxicity, hemolysis test, intravenous stimulation and injection anaphylaxis revealed that the PTX-M was safe for intravenous injection. Furthermore, the comparable antitumor efficacy of PTX-M and Taxol was observed at the same dose of 10 mg/kg in in vivo antitumor mice models inoculated with sarcoma180, enrich solid carcinoma (EC), hepatoma solidity (Heps), Lewis lung cancer cells and A-549 human lung cancer cells. These results clearly showed that PTX-M had the similar antitumor efficacy as Taxol, but significantly reduced the toxicity and improved the bioavailability of PTX.

Biological evaluation of N-octyl-O-sulfate chitosan as a new nano-carrier of intravenous drugs.

An amphiphilic chitosan derivate, N-octyl-O-sulfate chitosan (NOSC) was prepared by octylation of amino group at C-2 position and sulfonylation at C-6 position. Micelle formed by NOSC has great capability in solubilization of water-insoluble drug paclitaxel. Enormous attention was attracted by the potential application of NOSC as a new drug delivery system. Tritium labeled NOSC ((3)H NOSC) was injected by tail vein at dose of 13.44 mg/kg in mice; kidney retained the maximum amount of NOSC all the time even after 24h following the injection. Pharmacokinetic parameters (the area under the plasma concentration-time curve, maximum plasma concentration, apparent plasma half-life of distribution phase and elimination phase, mean residence time, apparent volume of distribution, total body clearance) were obtained by fluorometric method in rats. The results showed a linear pharmacokinetics proceeding of FITC-NOSC in vivo. 75.4+/-11.6% (3)H NOSC of dose was excreted in urine over a 7-day period, urinary excretion was the predominant way of excretion of NOSC compared with bilary or fecal pathway. A series of safety studies consisted of acute toxicity study, intravenous stimulation study, injection anaphylaxis study, hemolysis study and cell viability assay were performed to warrant the biocompatibility of the NOSC as intravenous materials. The LD(50) value of NOSC administrated by i.v. and i.p. were calculated as 102.59 and 130.53 mg/kg, respectively. No intravenous stimulation, injection anaphylaxis, hemolysis and cytotoxicity were observed in the safety studies. The tissue distribution, pharmacokinetics, excretion and safety study were persuasive for the potential application of NOSC as a new drug carrier.

Antioxidant Properties of Novel Dimers Derived from Natural ß-Elemene through Inhibiting H2O2-Induced Apoptosis.

A series of novel ß-elemene dimer derivatives were synthesized and evaluated for their antioxidant activities. The results indicated that most of the target compounds showed more potent cytoprotective effects than positive control vitamin E. In particular, dimer D5 exhibited the strongest antioxidant activity, which was significantly superior to the active compound D1 obtained in our previous study. Besides, D5 did not produce obvious cytotoxicity in normal human umbilical vein endothelial cells (HUVECs) and increased the viability of HUVECs injured by H2O2 in a concentration-dependent manner. Further studies suggested that the cytoprotective action of D5 might be mediated, at least in part, by increasing the intracellular superoxide dismutase activity and nitric oxide secretion as well as decreasing the intracellular malonyldialdehyde content and lactate dehydrogenase release. Furthermore, D5 observably inhibited ROS generation and prevented H2O2-induced apoptosis in HUVECs possibly via inhibiting the activation of the MAPK signaling pathway.

Involvement of xanthine oxidase and paraoxonase 1 in the process of oxidative stress in nonalcoholic fatty liver disease.

Xanthine oxidase (XOD) and paraoxonase 1 (PON1) are important enzymes in redox reactions in vivo, and are predominantly synthesized by the liver. The aim of the present study was to investigate the redox state in nonalcoholic fatty liver disease, and determine the association between the activities of XOD and PON1 and the severity of NAFLD. Sprague­Dawley rats were randomly divided into control, model and α­lipoic acid (high and low dose) groups. The rats in the NAFLD model were induced by feeding a high fat diet for 12 weeks and the in vitro cell model of hepatocyte steatosis was induced by treating L­02 cells with oleic acid for 24 h. The body weight, liver function, lipid and oxidative stress indices, and histological features of the liver were examined in the rats. Compared with the control group, the rats in the NAFLD model group showed impaired liver function, lipid disorders and damage from oxidative stress. The serum activity of XOD increased significantly from the 4th week and was markedly higher, compared with that in the control group, reaching a peak in the 12th week. The activity of PON1 was negatively correlated with that of XOD. Compared with the control cells, the activity of XOD and levels of free­fatty acids were significantly higher, and the activity of PON1 was significantly lower in the NAFLD L­02 cell model. All the above indicators were significantly improved by treatment with the antioxidant, α­lipoic acid. The activities of XOD and PON1 may be promising as markers in a noninvasive approach for detecting the severity of NAFLD clinically. α­lipoic acid had protective effects on the NAFLD rats, and the potential mechanism may be associated with the inhibition of oxidative stress and lipid peroxidation.

Clematichinenoside inhibits VCAM-1 and ICAM-1 expression in TNF-α-treated endothelial cells via NADPH oxidase-dependent IκB kinase/NF-κB pathway.

Proinflammatory cytokine TNF-α-induced adhesion of leukocytes to endothelial cells plays a critical role in the early stage of atherosclerosis. Oxidative stress and redox-sensitive transcription factors are implicated in the process. Thus, compounds that mediate intracellular redox status and regulate transcription factors are of great therapeutic interest. Clematichinenoside (AR), a triterpene saponin isolated from the root of Clematis chinensis Osbeck, was previously demonstrated to have anti-inflammatory and antioxidative properties. However, little is known about the exact mechanism underlying these actions. Thus we performed a detailed study on its effect on leukocytes-endothelial cells adhesion with TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) and cell-free systems. First, we found that AR reduced TNF-α-induced VCAM-1 and ICAM-1 expression and their promoter activity, inhibited translocation of p65 and phosphorylation of IκBα, suppressed IκB kinase-ß (IKK-ß) activity, lowered O2(∙-) and H2O2 levels, tackled p47(phox) translocation, and decreased NOX4 NADPH oxidase expression. Second, we showed that AR exhibited no direct free radical scavenging ability in cell-free systems at concentrations that were used in intact cells. Besides, AR had no direct effect on the activity of IKK-ß that was extracted from TNF-α-stimulated HUVECs. We also found that p47 translocation, NOX4 expression, and reactive oxygen species (ROS) levels were up-regulated before IκB phosphorylation in TNF-α-induced HUVECs. Moreover, TNF-α-enhanced IKK-ß activity was also inhibited by (polyethylene glycol) PEG-catalase, N-acetylcysteine (NAC), and vitamin E. In conclusion, these results suggest that AR reduces VCAM-1 and ICAM-1 expression through NADPH oxidase-dependent IKK/NF-κB pathways in TNF-α-induced HUVECs, which finally suppress monocyte-HUVECs adhesion. This compound is potentially beneficial for early-stage atherosclerosis.

HQS-3, a newly synthesized flavonoid, possesses potent anti-tumor effect in vivo and in vitro.

HQS-3 is a newly baicalein derivative with a benzene substitution. We investigated the anticancer effect of HQS-3 in vivo and in vitro. HQS-3 significantly decreased tumor growth in mice inoculated with Heps and HepG2 cells; and had little influence on the state and weight of animals. After treatment with 20 mg/kg HQS-3, the inhibitory rate of tumor weight in mice inoculated with Heps and HepG2 cells were 63.62% and 68.03%, respectively. Meanwhile, HQS-3 inhibited the viability of various kinds of tumor cells with IC50 values in the range of 22.98-54.32 µM after 48 h treatment measured by MTT-assay. HQS-3 remarkably inhibited viability of hepatoma cells in a concentration- and time-dependent manner and induced apoptosis in HepG2 cells by DAPI staining and Annexin V/PI double staining. The apoptosis-induction effect of HQS-3 was attributed to its ability to modulate the activity of caspase-9, caspase-3 and PARP. Moreover, the expression of bax protein was increased while the bcl-2 protein was decreased, leading to an increase in Bax/Bcl-2 ratio. The accumulation of ROS induced by HQS-3 in HepG2 cells was also observed. The further results suggested that HQS-3 induced mitochondrial-mediated apoptosis by increasing ROS level and inhibiting the expression of anti-oxidative protein SOD2. HQS-3 exerted anti-tumor activity both in vitro and in vivo via inducing tumor cells apoptosis, and these results suggested that it deserves further investigation as a novel chemotherapy for human tumors.

PPARγ agonist pioglitazone reverses memory impairment and biochemical changes in a mouse model of type 2 diabetes mellitus.

AIMS: Pioglitazone, known as a peroxisome proliferator-activated receptor γ (PPARγ) agonist, is used to treat type 2 diabetes mellitus (T2DM). T2DM has been associated with reduced performance on numerous domains of cognitive function. Here, we investigated the effects of pioglitazone on memory impairment in a mouse model with defects in insulin sensitivity and secretion, namely high-fat diet (HFD) streptozotocin (STZ)-induced diabetic mice. METHODS: ICR mice were fed with HFD for 4 weeks and then injected with a single low dose of STZ followed by continued HFD feeding for an additional 4 weeks. Pioglitazone (18 mg/kg, 9 mg/kg body weight) was orally administered for 6 weeks once daily. Y-maze test and Morris water maze test (MWM) were employed for testing learning and memory. Serum glucose, serum insulin, serum triglyceride, brain ß-amyloid peptide (Aß), brain ß-site amyloid precursor protein cleaving enzyme (BACE1), brain nuclear factor κB (NF-κB), and brain receptor for advanced glycation end products (RAGE) were also tested. RESULTS: The STZ/HFD diabetic mice, characterized by hyperglycemia, hyperlipemia and hypoinsulinemia, performed poorly on Y-maze and MWM hence reflecting impairment of learning and memory behavior with increases of Aß40/Aß42, BACE1, NF-κB, and RAGE in brain. Treatment of PPARγ agonist, pioglitazone (18 or 9 mg/kg body weight), significantly reversed diabetes-induced impairment of learning and memory behavior, which is involved in decreases of Aß40/Aß42 via inhibition of NF-κB, BACE1 and RAGE in brain as well as attenuation of hyperglycemia, hyperlipemia, and hypoinsulinemia. CONCLUSIONS: It is concluded that PPARγ agonist pioglitazone may be considered as potential pharmacological agents for the management of cognitive dysfunction in T2DM.

Effect of baicalein on experimental prostatic hyperplasia in rats and mice.

We determined the effect of baicalein on prostatic hyperplasia in experimental animal models. Prostatic hyperplasia was induced by testosterone propionate in mice and castrated rats and by transplantation of homologous strain fetal mice urogenital sinus in mice. With the histopathological examination, the efficacy of baicalein on prostate hyperplasia in experimental animals was evaluated by the activity of serum acid phosphatase (ACP) and the following norm of the prostate gland: the volume, wet weight, wet weight index, dry weight index, DNA contents and prostatic epithelial height and cavity diameter. Results showed that baicalein at doses of 260 and 130 mg/kg administrated intragastrically (i.g.) significantly inhibited prostatic hyperplasia in castrated rats induced by testosterone propionate compared with the negative control group (p<0.01). Baicalein at doses of 520 and 260 mg/kg (i.g.) also significantly inhibited prostatic hyperplasia in mice induced by transplantation of homologous strain fetal mouse urogenital sinus and by testosterone propionate (p<0.01). These results suggested that baicalein has an inhibitory effect on prostatic hyperplasia in experimental animals.