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Enhancing the phagocytosis of immune cells with medicines provides benefits to the physiological balance by removing foreign pathogens and apoptotic cells. The fungal immunomodulatory protein (FIP) possessing various immunopotentiation functions may be a good candidate for such drugs. However, the effect and mechanism of FIP on the phagocytic activity is limitedly investigated. Therefore, the present study determined effects of Cordyceps militaris immunomodulatory protein (CMIMP), a novel FIP reported to induce cytokines secretion, on the phagocytosis using three different types of models, including microsphere, Escherichia Coli and Candida albicans. CMIMP not only significantly improved the phagocytic ability (p &lt, 0.05), but also enhanced the bactericidal activity (p &lt, 0.05). Meanwhile, the cell size, especially the cytoplasm size, was markedly increased by CMIMP (p &lt, 0.01), accompanied by an increase in the F-actin expression (p &lt, 0.001). Further experiments displayed that CMIMP-induced phagocytosis, cell size and F-actin expression were alleviated by the specific inhibitor of TLR4 (p &lt, 0.05). Similar results were observed in the treatment with the inhibitor of the NF-κB pathway (p &lt, 0.05). In conclusion, it could be speculated that CMIMP promoted the phagocytic ability of macrophages through increasing F-actin expression and cell size in a TLR4-NF-κB pathway dependent way.

Aim Drug resistance is an intractable issue urgently needed to be overcome for improving efficiency of antiepileptic drugs in treating refractory epilepsy. microRNAs (miRNAs) have been proved as key regulators and therapeutic targets in epilepsy. Accordingly, the aim of the present study was to identify a novel differentially expressed miRNA which could improve the efficiency of antiepileptic drugs during the treatment of refractory epilepsy. Methods and Results Serum samples were collected from children with refractory epilepsy. An in vivo refractory epilepsy model was developed in SD rats by electrical amygdala kindling. We identified that miR‐139‐5p was decreased and multidrug resistance‐associated protein 1 (MRP1) was remarkably upregulated in the serum samples from children with refractory epilepsy and the brain tissues from rat models of refractory epilepsy. After phenobarbitone injection in rat models of refractory epilepsy, the after discharging threshold in kindled amygdala was detected to screen out drug‐resistant rats. Dual‐luciferase reporter gene assay demonstrated that MRP1 was a target of miR‐139‐5p. In order to evaluate the effect of miR‐139‐5p/MRP1 axis on drug resistance of refractory epilepsy, we transfected plasmids into the hippocampus of drug‐resistant rats to alter the expression of miR‐139‐5p and MRP1. TUNEL staining and Nissl staining showed that miR‐139‐5p overexpression or MRP1 downregulation could reduce the apoptosis and promote survival of neurons, accompanied by alleviated neuronal damage. Conclusion Collectively, these results suggest an important role of miR‐139‐5p/MRP1 axis in reducing the resistance of refractory epilepsy to antiepileptic drugs.

Phagocytosis suppression induced by nanoparticles (NPs) exposure is increasingly reported in marine species. However, the mechanisms underlying this impact remain poorly understood. In order to improve our present understanding of the immunotoxicity of NPs, acute (96 h) TiO 2 NP exposure and rescue trials via exogenous supply of Ca 2+ were performed in the blood clam, Tegillarca granosa . The results show that the phagocytosis rate, cell viability, and intracellular Ca 2+ concentration of haemocytes were significantly suppressed, whereas the intracellular ROS concentration of haemocytes significantly increased upon nTiO 2 exposure. Exposure to nTiO 2 also led to the significant downregulation of Caspase-3, Caspase-6, apoptosis regulator Bcl-2, Bcl-2-associated X, calmodulin kinase II, and calmodulin kinase kinase II. Furthermore, the toxic impacts of nTiO 2 were partially mitigated by the addition of exogenous Ca 2+ , as indicated by the recovery tendency in almost all the measured parameters. The present study indicates that Ca 2+ signaling could be one of the key pathways through which nTiO 2 attacks phagocytosis.

Myocardial infarction (MI) is a serious cardiovascular disease associated with myocardial ischemia/reperfusion (I/R) injury. Dexmedetomidine (Dex), an α2-adrenoceptor agonist, has been reported to protect against I/R injury. We examined the cardioprotective effects of Dex on cardiomyocytes under hypoxia/reoxygenation (H/R) conditions and explored the underlying mechanisms.A H/R model was established to mimic the MI injury. The CCK-8 assay was performed to measure cell viability. Cellular apoptosis was measured using the Annexin V fluorescein isothiocyanate (FITC)-propidium iodide (PI) staining. The levels of interleukin (IL)-1α and tumor necrosis factor (TNF)-α, and the activity of lactate dehydrogenase (LDH) were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit. Reactive oxygen species (ROS) were measured using the 2'-7' dichlorofluorescein diacetate (DCFH-DA) staining assay. In addition, the levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), catalase (CAT), and caspase-3 were measured using a commercial kit. siRNA was used to silence Bcl-2, catalase, or STAT3. Western blotting was used to measure the change in the levels of proteins.Dex improved the cell viability and inhibited the inflammatory response in H9c2 cells exposed to H/R treatment. In addition, Dex inhibited apoptosis and alleviated the endoplasmic reticulum (ER) stress and oxidative stress in H9c2 cells under the H/R treatment. Mechanism investigation showed that Dex inhibited the intrinsic pathway of apoptosis. Moreover, Dex enhanced the activation of the JAK2/STAT3 signaling pathway in H/R-treated H9c2 cells.Altogether, our findings suggested Dex as a promising therapeutic agent for myocardial I/R.

Background: The proximal renal tubule plays a critical role in diabetic kidney disease (DKD) progression. Early glomerular disease in DKD triggers a cascade of injuries resulting in renal tubulointerstitial disease. These pathophysiological responses are collectively described as diabetic tubulopathy (DT). Thus, therapeutic strategies targeting DT hold significant promise for early DKD treatment. Shenkang injection (SKI) has been widely used to treat renal tubulointerstitial fibrosis in patients with chronic kidney disease in China. However, it is still unknown whether SKI can alleviate DT. We designed a series of experiments to investigate the beneficial effects of SKI in DT and the mechanisms that are responsible for its effect on epithelial-to-mesenchymal transition (EMT) and endoplasmic reticulum (ER) stress-induced apoptosis in DT.Methods: The modified DKD rat models were induced by uni-nephrectomy, streptozotocin intraperitoneal injection, and a high-fat diet. Following the induction of renal injury, these animals received either SKI, rosiglitazone (ROS), or vehicle, for 42 days. For in vitro research, we exposed NRK-52E cells to high glucose (HG) and 4-phenylbutyric acid (4-PBA) with or without SKI or ROS. Changes in parameters related to renal tubular injury and EMT were analyzed in vivo. Changes in the proportion of apoptotic renal tubular cells and ER stress, and the signaling pathways involved in these changes, were analyzed both in vivo and in vitro.Results: SKI and ROS improved the general condition, the renal morphological appearance and the key biochemical parameters, and attenuated renal injury and EMT in the rat model of DKD. In addition, SKI and ROS alleviated apoptosis, inhibited ER stress, and suppressed PERK-eIF2α-ATF4-CHOP signaling pathway activation both in vivo and in vitro. Notably, our data showed that the regulatory in vitro effects of SKI on PERK-eIF2α-ATF4-CHOP signaling were similar to those of 4-PBA, a specific inhibitor of ER stress.Conclusion: This study confirmed that SKI can alleviate DT in a similar manner as ROS, and SKI achieves this effect by inhibiting EMT and ER stress-induced apoptosis. Our findings thereby provide novel information relating to the clinical value of SKI in the treatment of DT.

Background: Nuclear factor-kB is highly activated in cardiovascular disorders. However, few articles have targeted at the role of nuclear factor-kB inhibitor in heart failure. Aims: To evaluate the effects of nuclear factor-kB inhibitor pyrrolidine dithiocarbamate on cardiocyte apoptosis and cardiac function in a rat heart failure model. Study Design: Animal experiment. Methods: A stable and reproducible rat heart failure model (n=64) was prepared by injecting homologous microthrombotic particles into the left ventricle of Sprague–Dawley rats while obstructing the ascending aorta to produce coronary microembolization. Rats with heart failure were randomized into untreated (HFu) and pyrrolidine dithiocarbamate-treated (HFp) groups; the latter received an intraperitoneal injection of pyrrolidine dithiocarbamate (100 mg/kg/day) 1 h prior to surgery as well as on postoperative days 1-7. The sham group comprised 32 Sprague–Dawley rats. Eight rats from each group were sacrificed on days 1, 3, 7, and 14 postoperatively. Masson’s trichrome staining was used to determine the micro-fibrotic area to indicate the severity of myocardial loss. Terminal transferase uridine triphosphate nick end labeling staining was used to detect apoptotic cardiomyocytes. Echocardiography and hemodynamics were performed to evaluate left ventricular function. Results: Rats with heart failure exhibited pathological changes evidenced by patchy myocardial fibrosis, remarkably elevated severity of myocardial loss, and persistently reduced left ventricular function. At the end of the study, compared with the HFu group, myocardial infarct size was reduced by 28% (p=0.001), cardiocyte apoptosis was suppressed (7.17%±1.47% vs 2.83%±0.75%, p

Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide and a major global public health problem. With the rapid development of the economy, the incidence of CRC has increased linearly. Accumulating evidence indicates that changes in the gut microenvironment, such as undesirable changes in the microbiota composition, provide favorable conditions for intestinal inflammation and shaping the tumor growth environment, whereas administration of certain probiotics can reverse this situation to a certain extent. This review summarizes the roles of probiotics in the regulation of CRC, such as enhancing the immune barrier, regulating the intestinal immune state, inhibiting pathogenic enzyme activity, regulating CRC cell proliferation and apoptosis, regulating redox homeostasis, and reprograming intestinal microbial composition. Abundant studies have provided a theoretical foundation for the roles of probiotics in CRC prevention and treatment, but their mechanisms of action remain to be investigated, and further clinical trials are warranted for the application of probiotics in the target population.

Previous studies have suggested that the anticancer agent, arsenic trioxide (ATO), could attenuate T cell mediated immunity by not only inhibiting the proliferative response of T cells but by also increasing the frequency of regulatory T cells (Tregs). Furthermore, ATO represents a reasonable salvage treatment in some patients with refractory severe aplastic anemia (SAA). The current study aimed to evaluate the function of ATO on the Tregs percentage and cytokines changes in the peripheral blood mononuclear cells (PBMCs) of SAA patients. PBMCs were collected from 20 newly diagnosed SAA patients in Henan Cancer Hospital and treated with different concentrations of ATO (0, 1, 2.5, and 5 μmol/L). Then we investigated the efficacy of ATO on Tregs ratio and the levels of interferon (IFN)-γ, interleukin (IL)-4, IL-17 and transforming growth factor (TGF)-β1 in the peripheral blood of SAA patients in vitro. The results showed that ATO significantly increased the proportion of Tregs (P .05). ATO could mediate the immune regulation, which might contribute to improve hematopoietic recovery in SAA patients.

Background Pro‐NTs (precursor of neurotrophins) and their receptor p75 are potential targets for preventing microvascular dysfunction induced by myocardial ischemia–reperfusion injury ( IRI ). p75ECD (ectodomain of neurotrophin receptor p75) may physiologically produce neurocytoprotective effects by scavenging pro‐ NT s. We therefore hypothesized that p75 ECD may have a cardioprotective effect on IRI through microvascular mechanisms. Methods and Results Myocardial IRI was induced in Sprague‐Dawley rats by occluding the left main coronary arteries for 45 minutes before a subsequent relaxation. Compared with the ischemia–reperfusion group, an intravenous injection of p75 ECD (3 mg/kg) 5 minutes before reperfusion reduced the myocardial infarct area at 24 hours after reperfusion (by triphenyltetrazolium chloride, 44.9±3.9% versus 34.6±5.7%, P NT s expression at 24 hours and 28 days after reperfusion (by Western blotting). A simulative IRI model using rat microvascular pericytes was established in vitro by hypoxia–reoxygenation (2/6 hours) combined with pro‐ NT s treatment (3 nmol/L) at R. p75 ECD (3 μg/mL) given at R improved pericyte survival (by methyl thiazolyl tetrazolium assay) and attenuated apoptosis (by terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labeling). In the reperfused hearts and hypoxia–reoxygenation +pro‐ NT s‐injured pericytes, p75 ECD inhibited the expression of p‐JNK (phospho of c‐Jun N‐terminal kinase)/caspase‐3 (by Western blotting). SP 600125, an inhibitor of JNK , did not enhance the p75 ECD ‐induced infarct‐sparing effects and pericyte protection. Conclusions p75 ECD may attenuate myocardial IRI via pro‐ NT s reduction‐induced inhibition of p‐ JNK /caspase‐3 pathway of microvascular pericytes in rats.

Osteosarcoma is the most common aggressive sarcoma of the bone in children and adolescents. It is characterized by a high level of genetic instability and recurrent DNA deletions and amplifications. microRNAs (miRNAs) play a key role in cancer initiation, progression and metastasis; however, the potential role of miRNAs in osteosarcoma remains largely unknown. In the present study, miR-433 was shown to be overexpressed in osteosarcoma tissues compared with normal human osteoblasts. Transfection of miR-433 mimics into osteosarcoma cell lines significantly decreased apoptosis by targeting programmed cell death 4, a tumor suppressor that is involved in apoptosis. In contrast, inhibition of miR-433 enhanced apoptosis. Furthermore, in vivo miR-433 overexpression inhibited the apoptosis of tumor cells and increased tumor growth. The results of the present study suggested that miR-433 is a potential molecular target for osteosarcoma therapy.

// Ran Zhang 1, * , Xiaoxiao Wang 1, * , Mei Hong 1, * , Ting Luo 1 , Miaomiao Zhao 1 , Haorui Shen 1 , Jun Fang 1 , Xiaojie Li 1 , Sibin Zang 1 , Ping Chen 1 , Dimin Nie 1 , Peng Zheng 1 , Qiuling Wu 1 and Linghui Xia 1 1 Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China * These authors contributed equally to this work Correspondence to: Linghui Xia, email: Linghuixia@mail.hust.edu.cn Qiuling Wu, email: wuqiuling927@hotmail.com Keywords: acute graft-versus-host disease, endothelial microparticles, microRNA-155, T lymphocytes, differentiation Received: October 24, 2016 Accepted: February 12, 2017 Published: February 21, 2017 ABSTRACT Endothelial microparticles (EMPs) upregulation has been observed in acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the role of EMPs remains unclear. We found that EMPs derived from TNF-α-stimulated human umbilical vein endothelial cells (EA.hy926) concentrated more microRNA-155 (miR-155) compared with maternal cells. The miR-155 levels in MPs from peripheral blood of aGVHD patients and mice were remarkably elevated and significantly higher than the levels in plasma. Moreover, the rising peak of miR-155 in MPs occurred significantly prior to the peak in T lymphocytes. Additionally, we observed fluorescently-labeled miR-155 in EMPs actively transported into recipient T lymphocytes. Inhibition of miR-155 in EMPs by antagomir-155 did not influence the proliferation and apoptosis of T lymphocytes, but induced defective differentiation toward Th1, Th9 and Th17 cells and skewed differentiation toward Th2 and Treg cells. Furthermore, intravenous injection of miR-155-deficient-EMPs into aGVHD mice significantly attenuated the exacerbation of aGVHD manifestations and abnormal T lymphocytes differentiation induced by high concentration EMPs. Taken together, these data provide a mechanistic framework in which miR-155 delivered by EMPs is involved in aGVHD pathogenesis by activating specific T lymphocytes functions. The results may provide new therapeutic approaches for aGVHD while preserving graft-versus-leukemia (GVL) effect.

Inflammatory cytokines are released by immune cells and are able to induce vascular smooth muscle cells (VSMCs) to undergo apoptosis, causing atherosclerotic plaque rupture. Changes in the expression levels of connexins (Cxs) have been demonstrated in VSMCs to be involved in the pathogenesis of atherosclerotic progression. The present study examined the effect of tumor necrosis factor‑α (TNF‑α) on Cx43 expression levels and apoptosis in human VSMCs. Overexpression of Cx43 plasmids notably stimulated VSMC proliferation. TNF‑α directly inhibited Cx43 expression levels in a dose‑ and time‑dependent manner in VSMCs, however this was blocked by c‑Jun N‑terminal kinase inhibitor. TNF‑α also increased caspase‑3 activity and apoptosis of VSMCs through the inhibition of Cx43. These data suggested that TNF‑α induced the apoptosis of VMSCs and prompted the destabilization of atherosclerotic plaques by downregulating Cx43.

As a widely used deoxyribonuclease, DNase I is involved in many physiological processes including tumor cell proliferation, metastasis and apoptosis. Furthermore, the level of this enzyme in serum can act as a functional biomarker for the therapeutic monitoring of systemic lupus erythematosus and other diseases. We report here a low cost and sensitive DNase I detecting system based on the single-stranded fluorogenic substrate and nanographene oxide (NGO) and use it for DNase-targeted natural product screening. The system with a detection limit of 0.005 U was then used to evaluate the effect of external factors on DNase I. The results show that Hg2+, As2+, Pb2+, Cd2+ and Cu2+ can inhibit DNase I activity in a concentration-dependent manner with IC50 values of 0.37 mM (Hg2+), 2.7 mM (As2+), 5 mM (Pb2+), 5.3 mM (Cd2+) and 7.8 mM (Cu2+), respectively. Meanwhile, 10 natural compounds isolated from Cyclocarya paliurus leaves were screened as DNase I inhibitors, while 5 compounds were identified as activators. Finally, the system was used to discriminate DNase activity of serum samples with and without HBV. The results showed that HBV infection significantly decreased the level of DNase I in serum samples. In summary, these data indicate that this method with the advantages of rapidity, low cost and high sensitivity is hopeful for DNase assay in biological samples as well as compound screening in vitro.

Long noncoding RNAs (lncRNAs) play important roles in regulating the apoptosis of gastric cancer (GC) cells. This study aims to investigate the underlying mechanism of lncRNA CASC9 in regulating the apoptosis of GC cells. The expressions of lncRNA and protein in GC tissues and cell lines were detected by qRT-PCR and western blot. GC cell apoptosis was detected by flow cytometry analysis. RNA pull-down and RNA immunoprecipitation (RIP) assays were conducted to verify the interaction between CASC9 and BMI1. LncRNA CASC9 was upregulated in GC tissue and GC cells, and high CASC9 expression was positively correlated with TNM stage and lymph node metastasis. Silencing CASC9 promoted the apoptosis of GC cells. LncRNA CASC9 could interact with BMI1 and positively regulate BMI1 expression. Silencing CASC9 promoted the ubiquitination of BMI1. In addition, lncRNA CASC9 regulated the apoptosis of GC cells through BMI1. Furthermore, interfering CASC9 inhibited the tumor growth of GC. LncRNA CASC9 could interact with BMI1 to regulate the degradation of BMI1, thus to affect the apoptosis of GC cells and suppressed tumor growth.

BACKGROUND Inflammation is one of the most significant mechanisms of hepatic ischemia-reperfusion injury (IRI). Sufentanil has a protective effect against liver injury by reducing inflammatory response. In this study, we used a cellular hepatic ischemic/reoxygenated (IR) model to determine whether sufentanil preconditioning protects against hepatic IRI. MATERIAL AND METHODS The human normal liver cells line L-O2 was studied. The levels of glutamic oxaloacetic transaminase (AST), lactate dehydrogenase (LDH), malonaldehyde (MDA), and superoxide dismutase (SOD) were measured using corresponding assay kits. The protein levels of total and phosphorylated ERK1/2, JNK, and p38, and the expression of p65 and COX2 genes, were measured by Western blotting. The levels of inflammatory factors were examined by ELISA. The Cell Counting Kit-8 (CCK-8) was used to determine if the viability of L-O2 cells was affected by sufentanil. The effects of sufentanil on IR-induced cell apoptosis were examined by flow cytometry. RESULTS IR-induced caused L-O2 cells to become rounded and to have a lower adhesive rate than normal cells. The levels of AST, LDH, and MDA were higher but the level of SOD was lower in the IR group than in the control group. The phosphorylated protein levels of ERK1/2, JNK, and p38, along with the expression of p65 and COX2, were upregulated in the IR group compared to the normal group. In addition, a variety of inflammatory factors were secreted in L-O2 cells after IR. The viability of L-O2 cells decreased and cell apoptosis increased significantly after IR treatment. All indexes of cell injury were reversed by sufentanil in a concentration-dependent manner. CONCLUSIONS Sufentanil stimulation triggers downregulation of inflammatory factors such as HIF-1alpha, TNF-alpha, IL-1s, and IL-6, possibly through suppressing the p38/ERK/JNK/NF-kappaB-p65/COX2 pathways, and thereby reduces the damage to IR hepatic cells.

Phagocytosis suppression induced by nanoparticles (NPs) exposure is increasingly reported in marine species. However, the mechanisms underlying this impact remain poorly understood. In order to improve our present understanding of the immunotoxicity of NPs, acute (96 h) TiO

Cx43 has been documented to be involved in ischemic preconditioning (IPC). However, the participation of Cx43-formed hemichannels in IPC and the potential underlying mechanisms remain unclear. The present study focused on cardiomyocytes' volume regulation during IPC to investigate the role of hemichannels in the IPC-induced cardioprotection. In the study, mice cardiomyocytes were respectively treated with a hemichannel blocker, octanol or 18a-Glycyrrhizic acid (18a-GA), and a Cx43-silenced lentivirus. They were subsequently cultured in hypotonic solution to simulate ischemic reperfusion (SIR) and systemic ischemic preconditioning (SIP). Cell morphology and volumetric (area) change were detected by inverted microscopy at 30 min following the addition of hypotonic solution. Cardiomyocyte mortality was assessed by trypan blue stain assay. The analyses revealed that regardless of the treatments, hypotonic solution aggravated cell edema: Compared with the initial condition (the moment before the solution addition, 0 min), the volumetric area increased significantly 30 min later (for hypotonic+DMSO, 5,050±1,511 vs. 3,464±723 µm(2); for hypotonic+scramble lentiviral vector, 5,517±1,128 vs. 2,331±536 µm(2); P0.05 for all). Notably, results indicated that the SIP group had lower mortality rates compared with its SIR counterpart; the hypotonic+octanol, hypotonic+18a-GA, and hypotonic+Cx43-silenced group showed markedly-declined mortality when compared with their respective control groups (respectively, 35.70±1.02, 30.76±2.20 vs. 53.58±2.14%; 30.89±2.37 vs. 54.12±2.55%; P

Adropin is a secreted polypeptide that has been demonstrated to serve an important role in protecting the vascular endothelium. Pharmacological activation of pro-survival kinases, such as PI3K-Akt and ERK1/2, are involved in the reperfusion injury salvage kinase (RISK) pathway. In the present study, the effects of adropin in cardiomyocyte injury induced by simulated ischemia/reperfusion (SI/R) were assessed. Additionally, the current study also assessed the mechanisms that govern SI/R in a H9c2 cardiomyoblast cell model. Cell viability was measured using an MTT assay. Cell injury was assessed using creatine kinase MB measurements. Apoptosis was assessed using flow cytometry and caspase-3 activity. The inflammatory response was measured using tumor necrosis factor α and interleukin-10 expression. Oxidative stress was assessed using malondialdehyde and superoxide dismutase. The expression levels of Akt, ERK1/2, glycogen synthase kinase 3β (GSK3β), Bcl-2 and Bax were determined using western blot analysis. The results of the current study revealed that moderate-dose adropin increased cell viability, reduced early apoptosis and caspase-3 activity, promoted Bcl-2 expression, inhibited Bax and increased the Bcl-2/Bax ratio. Adropin significantly increased the phosphorylation of Akt, ERK1/2 and GSK3β, whereas inhibitors of PI3K and ERK1/2, respectively, LY294002 and PD98059, abolished the cardioprotective role of adropin. Furthermore, no significant difference was observed in phosphorylated-STAT3/total-STAT3 expression between the adropin and SI/R groups and Janus kinase 2 inhibitor AG490 did not significantly inhibit the protective role of adropin. These results indicate that adropin exerts a protective effect against SI/R injury through the RISK pathway instead of the survivor activating factor enhancement pathway.

The effect and mechanism of Galectin-3 gene expression on proliferation, invasion, and apoptosis of oral squamous cell carcinoma (OSCC) were investigated.Reverse transcription-polymerase chain reaction (RT- PCR) and Western blotting were used to detect the mRNA and protein of Galectin-3 gene in OSCC. OSCC Tca8113 was divided into control, negative control, and Galectin-3 transfection groups. Western blotting was used to detect the expression of Galectin-3, matrix metalloproteinase (MMP)-2, MMP-9, Cleaved Caspase-3, β-catenin, and Cyclin D1 protein after transfection for 48 h in each group. Cell proliferation was detected by CCK8. Cell invasion ability was detected by using a Transwell chamber. Cell apoptosis was detected by flow cytometry.The mRNA and protein expression levels of Galectin-3 gene in OSCC were significantly higher than those in adjacent tissues (P0.01). Galectin-3 protein expression in Tca8113 cells significantly decreased after RNA interference. Cell survival rate and invasion as well as MMP-2, MMP-9, β-catenin, and Cyclin D1 protein expression were significantly lower than the blank group. Apoptosis rate and Cleaved Caspase-3 protein expression were significantly higher than the control group (P0.01).Inhibition of Galectin-3 gene expression in OSCC can significantly reduce the proliferation and invasion of cancer cells and induce apoptosis. The mechanism is related to downregulation of the Wnt/β-catenin signaling pathway.目的 探讨抑制口腔鳞状细胞癌（OSCC）中半乳糖凝集素-3（Galectin-3）基因的表达对癌细胞增殖、侵袭、凋亡的影响及机制。方法 逆转录聚合酶链反应（RT-PCR）和蛋白质印迹（Western blotting）分别检测OSCC中Galectin-3 mRNA和蛋白表达；人OSCC Tca8113分为空白组、阴性对照组和Galectin-3转染组，转染48 h后，Western blotting检测各组细胞中Galectin-3、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）、β-连环蛋白（β-catenin）、细胞周期素D1（Cyclin D1）蛋白表达；CCK8检测细胞增殖；Transwell小室检测细胞侵袭能力；流式细胞术检测细胞凋亡。结果 OSCC中Galectin-3 mRNA及蛋白表达均显著高于癌旁组织（P0.01）；RNA干扰Galectin-3表达后Tca8113细胞中Galectin-3蛋白表达明显降低；Galectin-3转染组细胞存活率、细胞侵袭能力及MMP-2、MMP-9、β-catenin、Cyclin D1蛋白表达均显著低于空白组，细胞凋亡率及Cleaved Caspase-3蛋白表达均显著高于空白组（P0.01）。结论 抑制OSCC中Galectin-3基因表达可显著降低癌细胞的增殖及侵袭能力，诱导细胞凋亡，其机制与下调Wnt/β-catenin信号通路有关。.

Background Glioma is the most common primary central nervous system tumor derived from glial cells. Kininogen-1 (KNG1) can exert antiangiogenic properties and inhibit proliferation of endothelial cells. The effect of KNG1 on the glioma is rarely reported, so our purpose in to explore its mechanism in glioma cells. Methods The differentially expressed genes (DEGs) were identified based on The Cancer Genome Atlas (TCGA) database. The KNG1-vector was transfected into the two glioma cells. The viability, apoptosis and cell cycle of glioma cells and microvessel density (MVD) were detected by cell counting kit-8 assay, flow cytometry and immunohistochemistry, respectively. The expression were measured by quantitative real-time PCR and Western blot, respectively. A tumor mouse model was established to determine apoptosis rate of brain tissue by terminal deoxynucleotidyl transfer-mediated dUTP nick end labeling (TUNEL) analysis. Results KNG1 was identified as the core gene and lowly expressed in the glioma cells. Overexpression of KNG1 inhibited cell viability and angiogenesis of glioma cells. Overexpression of KNG1 promoted the apoptosis and G1 phase cell cycle arrest of glioma cells. Moreover, the expressions of VEGF, cyclinD1, ki67, caspase-3/9 and XIAP were regulated by overexpression of KNG1. In addition, overexpression of KNG1 inhibited the activity of PI3K/Akt. Furthermore, overexpression of KNG1 decreased the tumor growth and promoted the apoptosis of decreased by overexpression of KNG1 in vivo. . Conclusions Overexpression of KNG1 suppresses glioma progression by inhibiting the proliferation and promoting apoptosis of glioma cells, providing a therapeutic strategy for the malignant glioma.

Background Memory CD8+ T cell responses play an essential role in protection against persistent infection. However, HIV-1 evades vaccine-induced memory CD8+ T cell response by mechanisms that are not fully understood. Methods We analyzed the temporal dynamics of CD8+ T cell recall activity and function during EcoHIV infection in a potent PD1-based vaccine immunized immunocompetent mice. Findings Upon intraperitoneal EcoHIV infection, high levels of HIV-1 GAG-specific CD8+ T lymphocytes recall response reduced EcoHIV-infected cells significantly. However, this protective effect diminished quickly after seven days, followed by a rapid reduction of GAG-specific CD8+ T cell number and activity, and viral persistence. Mechanistically, EcoHIV activated dendritic cells (DCs) and myeloid cells. Myeloid cells were infected and rapidly expanded, exhibiting elevated PD-L1/-L2 expression and T cell suppressive function before day 7, and were resistant to CD8+ T cell-mediated apoptosis. Depletion of myeloid-derived suppressor cells (MDSCs) reduced EcoHIV infection and boosted T cell responses. Interpretation This study provides an overview of the temporal interplay of persistent virus, DCs, MDSCs and antigen-specific CD8+ T cells during acute infection. We identify MDSCs as critical gatekeepers that restrain antiviral T cell memory responses, and highlight MDSCs as an important target for developing effective vaccines against chronic human infections. Funding Hong Kong Research Grant Council (T11–709/18-N, HKU5/CRF/13G), General Research Fund (17122915 and 17114114), Hong Kong Health and Medical Research Fund (11100752, 14130582, 16150662), Grant RGC-ANR A-HKU709/14, the San-Ming Project of Medicine (SZSM201512029), University Development Fund of the University of Hong Kong and Li Ka Shing Faculty of Medicine Matching Fund to HKU AIDS Institute.

Accumulating evidence suggests that endothelial microparticles (EMPs), a marker of endothelial damage, are elevated in acute graft-versus-host disease (aGVHD), and that endothelial damage is implicated in the pathogenesis of aGVHD, but the mechanisms remain elusive. In this study, we detected the plasma EMP levels and endothelial damage in patients and mice with aGVHD in vivo and then examined the effects of EMPs derived from injured endothelial cells (ECs) on endothelial damage and the role of hedgehog-interacting protein (HHIP) carried by EMPs in these effects in vitro. Our results showed that EMPs were persistently increased in the early posttransplantation phase in patients and mice with aGVHD. Meanwhile, endothelial damage was continuous in aGVHD mice, but was temporary in non-aGVHD mice after transplantation. In vitro, EMPs induced endothelial damage, including increased EC apoptosis, enhanced reactive oxygen species, decreased nitric oxide production and impaired angiogenic activity. Enhanced expression of HHIP, an antagonist for the Sonic hedgehog (SHH) signaling pathway, was observed in patients and mice with aGVHD and EMPs from injured ECs. The endothelial damage induced by EMPs was reversed when the HHIP incorporated into EMPs was silenced with an HHIP small interfering RNA or inhibited with the SHH pathway agonist, Smoothened agonist. This work supports a feasible vicious cycle in which EMPs generated during endothelial injury, in turn, aggravate endothelial damage by carrying HHIP into target ECs, contributing to the continuously deteriorating endothelial damage in the development of aGVHD. EMPs harboring HHIP would represent a potential therapeutic target for aGVHD.

The correlation between the expression levels of p65 and TNF-α in patients with acute myelocytic leukemia (AML) and AML cell lines were investigated. The bone marrow samples of 30 AML patients and 10 non-leukemia controls were studied. The mRNA expression levels of p65 and TNF-α were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Pearson's Correlation test was used to demonstrate the correlation between TNF-α and p65 expression levels in AML specimens. Receiver operating characteristic (ROC) curves were plotted to determine whether TNF-α and p65 expression levels could be used to differentiate AML samples from non-leukemia samples. MG132 and anti-TNF-α antibody were used to inhibit the expression of p65 and TNF-α in the AML cell line, HL-60. The expression of p65 and TNF-α were detected by RT-qPCR and western blot analysis. The mRNA expression levels of p65 and TNF-α were significantly increased in AML patients compared with non-leukemia control bone marrow samples by RT-qPCR, and the two molecules expression pattern's exhibited sufficient predictive power to distinguish AML patients from non-leukemia control samples. Pearson's correlation analysis demonstrated that TNF-α expression was strongly correlated with p65 expression in AML bone marrow samples. In HL-60 cells, inhibition of TNF-α reduced the expression of p65; in addition, inhibition of p65 reduced the expression of TNF-α as assessed by RT-qPCR and western blot analysis. p65 and TNF-α were highly expressed in AML patients, and these 2 molecules were strongly correlated. The present study indicates that p65 and TNF-α have potential as molecular markers to distinguish AML patients from non-leukemia control samples, and that these 2 molecules may be useful prognostic factor for patients with AML.

Objective To explore the role of miR-181b in alterations of apoptosis and autophagy in the kainic acid (KA)-induced epileptic juvenile rats via modulating TLR4 and P38/JNK signaling pathway. Methods Dual-luciferase reporter assay was performed to testify the targeting relationship between miR-181b and TLR4. After intracerebroventricular injection (i.c.v.) of KA, rats were injected with miR-181b agomir and TLR4 inhibitor (TAK-242). The TLR-4 activator lipopolysaccharide (LPS) was also administered into rats immediately after injection with miR-181b agomir. Quantitative real-time-polymerase chain reaction (qRT-PCR) was used for detections of miR-181b and TLR4 expressions, hematoxylin-eosin (HE) and Nissl staining for observation of the hippocampus morphological changes, and TUNEL staining for apoptosis analysis. Moreover, western blot was determined to detect TLR4 and P38/JNK pathway proteins, as well as autophagy- and apoptosis-related proteins. Results TLR4 was identified as a direct target of miR-181b using Dual-luciferase reporter assay. KA rats injected with miR-181b agomir or TAK-242 had improved learning and memory abilities, reduced seizure severity of Racine's scale, and lessened neuron injury. Additionally, miR-181b agomir or TAK-242 could significantly inhibit P38/JNK signaling, decrease LC3II/I, Beclin-1, ATG5, ATG7, ATG12, Bax, and cleaved caspases-3, but increase p62 and Bcl-2 expression. No significances were found between KA group and KA + miR-181b + LPS group. Conclusion MiR-181b could inhibit P38/JNK signaling pathway via targeting TLR4, thereby exerting protective roles in attenuating autophagy and apoptosis of KA-induced epileptic juvenile rats.

Hypericum ascyron L. (Great St. Johnswort), which belongs to the Hypericaceae family, has been used for the treatment of hematemesis, metrorrhagia, rheumatism, swelling, stomach ache, abscesses, dysentery and irregular menstruation for >2,000 years in China. The aim of the present study was to clarify the anticancer activity compounds from H. ascyron L. and the underlying molecular mechanism. Anticancer activity of H. ascyron L. extract was evaluated using an MTT assay. To confirm the anticancer mechanism of activity compounds, Hoechst 33258, Annexin V-fluorescein isothiocyanate/propidium iodide, 2',7'-dichlorodihydrofluorescein diacetate, rhodamine 123 staining and caspase-3 activity analysis were performed. The results demonstrated that the anti-proliferative action of the mixture of kaempferol 3-O-β-(2″-acetyl) galactopyranoside (K) and quercetin (Q) (molar ratio, 1:1) was significantly increased compared with either of these two compounds separately, and the active fraction of the H. ascyron L. extract |(HALE). HALE, indicating that the anti-proliferative function of H. ascyron L. may be a synergic effect of K and Q. Furthermore, the inhibitory effect of KQ on the growth of HeLa cells was mediated by the induction of apoptosis. To the best of our knowledge, the present study is the first to identify that KQ exhibits significant anti-proliferation activity on HeLa cells via the apoptotic pathway, and is also the first to evaluate the anticancer potential of H. ascyron L. The results of the present study may provide a rational base for the use of H. ascyron L. in the clinic, and shed light on the development of novel anticancer drugs.

Cancer treatment alters microRNA (miRNA) expression, revealing potential therapeutic targets (oncotarget). Here we treated pancreatic cancer (ASPC-1) cells with either recombinant human endostatin (rh-endostatin) or gemcitabine. Then high-throughput sequencing assay was performed to screen for altered miRNAs. Both treatments decreased levels of MiR-19a. We found that miR-19a stimulated cell proliferation, migration, invasion in vitro and tumor growth in vivo. High levels of miR-19a correlated with poor prognosis in patients. Ras homolog family member B (RHOB) was identified as a direct target of miR-19a. Furthermore, RHOB was down-regulated in human pancreatic cancer samples. Restoration of RHOB induced apoptosis, inhibited proliferation and migration of ASPC-1 cells. SP-1 was identified as an upstream transcription factor of miR-19a gene, promoting miR-19a transcription. Rh-endostatin decreased miR-19a expression by down-regulating SP-1. These findings suggest that miR-19a is a potential therapeutic target in pancreatic cancer.

Chondrocytes play a critical role in the repair process of osteoarthritis, which is also known as degenerative arthritis. Integrins, as the key family of cell surface receptors, are responsible for the regulation of chondrocyte proliferation, differentiation, survival and apoptosis through the recruitment and activation of downstream adaptor proteins. Moreover, G-protein-coupled receptor kinase interacting protein-1 (GIT1) exerts its effects on cell proliferation and migration through interaction with various cytokines. It has been previously suggested that GIT1 acts as a vital protein downstream of the integrin-mediated pathway. In the present study, we investigated the effects of integrin-β1 on cell proliferation and apoptosis, as well as the underlying mechanisms in chondrocytes in vitro. Following transfection with a vector expressing integrin-β1, our results revealed that the overexpression of integrin-β1 enhanced GIT1 expression, whereas the knockdown of integrin-β1 by siRNA suppressed GIT1 expression. However, no significant effect was observed on integrin-β1 expression following the enforced overexpression of GIT1, which suggests that GIT1 is localized downstream of integrin-β1. In other words, integrin-β1 regulates the expression of GIT1. Furthermore, this study demonstrated that integrin-β1 and GIT1 increased the expression levels of aggrecan and type II collagen, thus promoting chondrocyte proliferation; however, they inhibited chondrocyte apoptosis. Taken together, our data demonstrate that integrin-β1 plays a vital role in chondrocyte proliferation, differentiation and apoptosis. GIT1 exerts effects similar to those of integrin-β1 and is a downstream target of integrin-β1.

Autophagy is one of the major causes of drug resistance. For example, the angiogenesis inhibitor bevacizumab shows only transient and short-term therapeutic effects, whereas long-term therapeutic benefits are rarely observed, probably due to hypoxia-induced autophagy. Nitric oxide (NO) is an important molecule with multiple functions, and it has recently been reported to function as a regulator of autophagy. Therefore, a reasonable therapeutic strategy for overcoming drug resistance by NO would involve it being directly delivered to the tumor. Here, we investigated the inhibitory effect of NO on autophagy by using a macromolecular NO donor S-nitrosated human serum albumin (SNO-HSA) with a high degree of NO loading and tumor targeting potential. In colon 26 (C26) cells, SNO-HSA significantly suppressed hypoxia-induced autophagy by inhibiting the phosphorylation of JNK1 and the expression of its downstream molecule Beclin1. The effect of SNO-HSA was also confirmed in vivo by combining it with Bev. In C26-bearing mice, significant suppression of tumor growth as well as lung metastasis was achieved in the combination group compared to the SNO-HSA or bevacizumab alone group. Similar to the in vitro experiments, the immunostaining of tumor tissues clearly showed that SNO-HSA inhibited the autophagy of tumor cells induced by bevacizumab treatment. In addition to other known antitumor effects of SNO-HSA, that is, the induction of apoptosis and the inhibition of multidrug efflux pumps, these data may open alternate strategies for cancer chemotherapy by taking advantage of the ability of SNO-HSA to suppress autophagy-mediated drug resistance and enhance the efficacy of chemotherapy.

Tanshinone IIA, one of the active ingredients in the Chinese medicine Danshen, is cardioprotective when applied prior to sustained myocardial ischemia. The present study aimed to investigate whether pharmacological postconditioning with tanshinone IIA attenuates myocardial ischemia-reperfusion injury when applied prior to prolonged reperfusion following a sustained ischemia. A total of 88 Sprague-Dawley rats received 30 min myocardial ischemia followed by 5 or 120 min reperfusion. Compared with the ischemia-reperfusion model group, the group that received an intravenous injection of 10 mg/kg tanshinone IIA prior to reperfusion had a reduced myocardial infarct size, higher levels of phospho-Akt and phospho-endothelial nitric oxide synthase and less reduction in the optical density of the mitochondria at 540 nm, indicating that the mitochondrial permeability transition (MPT) was attenuated. The cardioprotective effect conferred by tanshinone IIA was abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). These results demonstrate that tanshinone IIA postconditioning protects the myocardium from ischemia-reperfusion injury through the PI3K/Akt pathway, and the MPT may be also involved in this process.

Cisplatin (CDDP) is a commonly used drug for gallbladder cancer (GBC) chemotherapy. However, resistance to CDDP treatment results in relapse. Therefore, there is a need for the development of more effective treatment strategies to overcome chemoresistance. Dual-specificity phosphatase 1 (DUSP1) was reported to be involved in the resistance of a number of chemotherapeutic agents and was revealed to be highly expressed in CDDP-resistant GBC cells and CDDP-treated tumor types compared with normal cells or tissues in the present study. DUSP1 was revealed to inhibit the cytotoxicity of CDDP in two GBC cell lines, SGC996 and GBC-SD. P38 mitogen-activated protein kinases may be involved in the mechanism of chemoresistance. Furthermore, the number of DNA double-strand breaks in SGC996 OE cells was reduced compared with SGC996 vector cells indicating DUSP1 may attenuate the chemotherapeutic efficiency. Due to its potency against CDDP treatment, DUSP1 may be a promising target to overcome chemoresistance in GBC therapy.

The formation of fibrocartilage, cartilaginous and bony calluses is vital for bone healing following a fracture. Fibroblasts, chondrocytes and osteoblasts are critical functional cells that are involved in these three processes, respectively. Platelet‑derived growth factor (PDGF), a growth factor that is released from platelet particles and appears during the early stages at the site of fractures, is essential in bone healing via regulation of cell proliferation and differentiation. However, the effects of PDGF on the chondrocytes remain unclear. In the present study, PDGF promoted phosphorylation of Src and upregulated the expression level of G‑protein‑coupled receptor kinase interacting protein‑1 (GIT1) according to the results of the cell culture of chondrocytes in vitro and western blotting. However, the effect of PDGF on the upregulation of GIT1 expression was mostly inhibited by the Src inhibitor, PP2. After knocking down GIT1 expression using siRNA, the phosphorylation of Src continued to be induced by PDGF, although the expression of GIT1 was inhibited. Furthermore, the results indicated that PDGF promoted chondrocyte proliferation and migration, however, the effect on cell apoptosis induction was suppressed after adding the Src inhibitor, PP2. Additionally, when knocking down GIT1 using siRNA, the expression level of GIT1 decreased, which is similar to the effect of the Src inhibitor, PP2. The current study demonstrates that PDGF may initially activate the phosphorylation of Src, and subsequently induce GIT1 expression to promote chondrocyte proliferation and migration, but suppress cell apoptosis.

Heme oxygenase-1 (HO-1), an inducible enzyme that catalyzes oxidative degradation of heme to form biliverdin, carbon monoxide and free iron, may protect tumor cells against oxidative stress, thus contributing to rapid tumor growth in vivo. Here, we discuss whether pegylated zinc protoporphyrin (PEG-ZnPP), a potent HO inhibitor, modulates the chemotherapeutic response of tumor cells to treatment that generates reactive oxygen species (ROS). PEG-ZnPP is a water-soluble HO inhibitor that accumulates in tumor tissues after intravenous administration. Cytotoxicity of antitumor agents in vitro was determined by means of MTT and annexin V assays using human colon carcinoma SW480 cells. Mice bearing sarcoma 180 tumors were used as an in vivo model. Pegylated D-amino acid oxidase (PEG-DAO), which behaves as an oxidative chemotherapeutic agent by generating toxic oxidants at tumor tissues, was administered with its substrate D-proline to mice with or without PEG-ZnPP pretreatment. PEG-ZnPP-treated SW480 cells became vulnerable to insults caused by various cytotoxic agents; the 50% lethal doses were reduced by 25%, 39%, 83%, and 61% for hydrogen peroxide, t-butyl hydroperoxide, camptothecin and doxorubicin, respectively. Cells treated with PEG-ZnPP plus cytotoxic oxidants exhibited marked production of intracellular ROS, which paralleled the incidence of apoptosis. PEG-ZnPP pretreatment significantly reduced tumor growth in mice receiving PEG-DAO/D-proline compared to no PEG-ZnPP pretreatment. These findings suggest that HO-1 may become an attractive target for chemotherapeutic intervention. Further study of the effect of PEG-ZnPP plus conventional anticancer drugs that generate ROS, such as cisplatin, camptothecin, doxorubicin, mitomycin C and etoposide, is warranted.

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy due to abnormal proliferation of hematopoietic progenitor cells, with difficulty in treatment and high mortality. Nucleophosmin 1 (NPM1), a restricted nucleolar localization protein, shuttles between nucleus and cytoplasm. NPM1-mutant protein, aberrant cytoplasmic dislocation of nucleophosmin, occurs in about one third of AML. Deguelin exhibits significant inhibitory effects and induces apoptosis in a variety of cancer cell lines in vitro and in vivo. Our previous researches showed deguelin induced differentiation of NPMc+ AML cells potentially by targeting NPM1-mutant protein. Thus, we extend our investigations of deguelin-induced differentiation activity to an AML xenograft mouse model and clinical samples. After sub-lethal whole body radiation, OCI/AML3 cells were injected intravenously in NOD/SCID mice. 7 days later, either deguelin or vehicle (2%DMSO) was injected intraperitoneally every day. Kaplan-Meier estimates showed 1mg/kg/day deguelin extended the longest survival of mice. The percentage of CD11b+ cells was 17.73%±3.03% in control group, 41.33%±13.22% and 76.15%±10.25% after deguelin-treated for 7 days and 14 days (P < 0.05). Thus, we used 14 days in the following experiments. The percent of CD114+ cells in the vehicle- and deguelin-treated group were 13.29%±10.79% and 47.36%±21.66%, respectively (P < 0.05). The percentage of CD14+ cells in the vehicle- and deguelin-treated group were 22.86%±15.54% and 83.49%±8.06%, respectively (P < 0.05). Wright's Giemsa staining of mouse bone marrow cells showed differentiated. The ratios of differentiated cells were 36.92%±4.99% and 77.01%±1.98% in control and deguelin group (P < 0.01). Median OS of vehicle-treated group was 23 days, while that of deguelin-treated group was 55 days (HR, 0.5818, 95% confidence interval [CI], 0.1623-1.001). Survival of deguelin-treated mice was significantly longer compared with mice injected with vehicle (P=0.0002). IHC and IF staining for mutated NPM1 protein showed bone marrow biopsy densities significantly reduced in deguelin-treated mice which was further confirmed by western blot assay (P < 0.05). We analyzed the toxicity of deguelin (1mg/kg/day for 14 days) to healthy NOD/SCID mice. They had steady movement and no rigidity posture treated with deguelin. HE staining indicated no pathological changes in striatum, substantia nigra pars compacta (SNpc), liver, kidney and heart in deguelin-treated mice. Striatum and SNpc, subjected to immunostaining with anti-tyrosine hydroxylase (TH) antibody, showed deguelin did not alter TH immunoreactivity in striatum and SNpc in vivo (P >0.05). Plasmatic levels of liver enzymes (AST and ALT), renal function (Scr and BUN) and cardiac enzymes (CK-MB and cTnT) in deguelin-treated group had not statistical difference comparing with control group (P>0.05). Thus, deguelin did not cause neurotoxicity, hepatotoxicity, nephrotoxicity and cardiotoxicity to mice. We collected 82 cases of patients with acute leukemia. 24 cases out of 68 AML patients carried mutated NPM1 gene (20 cases of type A; 1 case of type B; 2 cases of type D and 1 case of type I). Bone marrow mononuclear cells (BMMCs) of 6 AML patients were analyzed by FACS analysis. We analyzed patient (pt.) 1 to 4 with NPM1 mutation. FACS showed deguelin increased the percentage of CD11b+ and CD14+ cells of pt.1 in a dose-dependent manner. The percent of CD11b+ and CD14+ cells were 55% and 57.9% separately at 16 nM. Deguelin increased CD14 but CD11b expression of BMMCs of pt.2 in the concentration of 8 nM, 12 nM, 16 nM and 20 nM. The highest percentage of CD14+ cells was 85.5%. The expression of CD14 in cells from pt.3 increased after treating with 16 nM or 20 nM and the highest percentage of CD14+ cells was 49.4%. Deguelin induced differentiation of BMMCs from pt.4. The maximum percent of CD11b+ cells was 42.5% at 16 nM. Moreover, FACS analysis showed deguelin did not promote differentiation in BMMCs from pt.5 and 6 without NPM1 mutation. These results demonstrated deguelin induced differentiation of BMMCs cells from AML patients with NPM1 mutation in vitro. Taken together, mutated NPM1 protein may be one of targets of deguelin, which provides molecular basis for inducing differentiation in NPM1-mutant AML cells. Disclosures No relevant conflicts of interest to declare.

Mangiferin is glucosylxanthone extracted from plants of the Anacardiaceae and Gentianaceae families. The aim of this study was to investigate the effects of mangiferin on Nrf2-antioxidant response element (ARE) signaling and the sensitivity to etoposide of human myeloid leukemia cells in vitro. Human HL-60 myeloid leukemia cells and mononuclear human umbilical cord blood cells (MNCs) were examined. Nrf2 protein was detected using immunofluorescence staining and Western blotting. Binding of Nrf2 to ARE was examined with electrophoretic mobility shift assay. The level of NQO1 was assessed with real-time RT-PCR and Western blotting. DCFH-DA was used to evaluate intracellular ROS level. Cell proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. Mangiferin (50 μmol/L) significantly increased Nrf2 protein accumulation in HL-60 cells, particularly in the nucleus. Mangiferin also enhanced the binding of Nrf2 to an ARE, significantly up-regulated NQO1 expression and reduced intracellular ROS in HL60 cells. Mangiferin alone dose-dependently inhibited the proliferation of HL-60 cells. Mangiferin (50 mol/L) did not attenuate etoposide-induced cytotoxicity in HL-60 cells, and combined treatment of mangiferin with low concentration of etoposide (0.8 μg/mL) even increased the cell inhibition rate. Nor did mangiferin change the rate of etoposide-induced apoptosis in HL-60 cells. In MNCs, mangiferin significantly relieved oxidative stress, but attenuated etoposide-induced cytotoxicity. Mangiferin is a novel Nrf2 activator that reduces oxidative stress and protects normal cells without reducing the sensitivity to etoposide of HL-60 leukemia cells in vitro. Mangiferin may be a potential chemotherapy adjuvant.

Steroid receptor coactivator-3 (SRC-3) mediates the transcriptional functions of nuclear receptors and other transcription factors, regulating the cell proliferation, survival and growth. SRC-3 amplification or overexpression, detected in many hormone-dependent or -independent tumors, has been demonstrated its important role in promoting carcinogenesis. It was suggested that development of an effective SRC-3 inhibitor could offer a promising therapeutic strategy. Gambogic acid (GA), the major active ingredient in gamboges, is secreted from Garcinia hanburyi. In recent decades, accumulating evidence has been discovered to elucidate its potent anti-tumor efficacies. In our study, we investigated the effects and molecular mechanisms of GA on non-Hodgkin's lymphoma (NHL) cell lines. GA was demonstrated to induce cell growth inhibition, G1 phase cell cycle arrest and apoptosis in NHL. The IC50 for Daudi or Raji at 24 h was 0.15±0.01 μΜ or 0.79±0.23 μΜ, respectively. In control, the IC50 for Jurkat or normal human lymphocytes was 1.324±0.02 μΜ or 2.54±0.21 μΜ, respectively. After incubation with 0.2 μΜ GA for 12 h, the rate of Daudi cells in G1 phase increased from 33.8±1.83% to 64.4±4.4%, with rate of S phase from 53.0±0.14% to 17.99±3.87%. For Raji cells (0.6 μΜ for 12 h), the rate of cells in G1 phase increased from 44.34±1.55% to 57.07±0.5%, with rate of S phase from 44.75±2.2% to 38.16±0.87%. The G1 phase cell cycle arrest was accompanied by the down-regulation of cyclin D3 and p-Rb and up-regulation of P21 and P27. GA (0.2 μΜ for 24 h) greatly induced apoptosis in Daudi cells (from 6.2±2.8% to 46.8±3.2%), but almost none in Raji cells (from 9.5±0.5% to 9±0.72%). When treated with 0.4 μΜ or 0.6 μΜ GA, the rate of apoptosis in Raji cells increased to 40.5±5.5% or 76.5±4.5%. The apoptosis was further confirmed by evaluating the activation of caspase3 with western blot assay. After GA treatment, cyto C and Apaf-1 increased, and Bcl-2 decreased, all indicating GA induced apoptosis in mitochondrial pathway. The effects of GA on the regulations of five oncoproteins (Bcl-6, c-Myc, NF-κB, SRC-3 and Bcl-2) were measured by western blot analysis. These oncoproteins have been considered to be strongly implicated in B-cell lymphomagenesis. SRC-3, Bcl-2 and c-Myc were down-regulated. NF-κB was down-regulated directly or inhibited via up-regulation of IκB-α. The effects of GA on Bcl-6 were contradictory in Daudi and Raji. Because of the pivotal role of SRC-3 in promoting carcinogenesis, we explored whether the above effects of GA were mainly mediated by the regulation of SRC-3. Western blot analysis showed an obvious down-regulation of SRC-3 while the mRNA levels measured by RT-PCR analysis had no marked decline. SRC-3 protein stability examined by cycloheximide assay showed an accelerated degradation of SRC-3. Since SRC-3 has been reported to be degraded in proteasome pathway, we doubted whether SRC-3 was degraded similarly in response to GA. Cullin3, the component of the ubiquitin E3 ligase for SRC-3, was demonstrated to be up-regulated. The proteasome inhibitor MG132 could weaken GA-induced SRC-3 degradation. SRC-3 was shown to be ubiquitinated with immunoprecipitation method, and the ubiquitinated SRC-3 accumulated in the presence of MG132. All the data suggested that GA induced down-regulation of SRC-3 mainly through ubiquitin-dependent proteasome degradation pathway mediated by cullin3. According to GA-induced apoptosis in five cell lines (Daudi, Raji, Pfeiffer, SU-DHL6 and Jurkat), cells with higher SRC-3 level were observed the higher sensitivity. To figure out the relationship of SRC-3 level and cell sensitivity, stable cell clones expressing lower SRC-3 by lentiviral shRNA transfection was established. We compared the antitumor efficacies of GA in SRC-3 siRNA clones with control clones. After treated with 0.6 μΜ GA for 24 h, the rate of apoptosis in SRC-3 siRNA clones or control clones increased from 14.6±3.3% to 44.3±3.7% or from 16.6±2.24% to 63.6±1.99%, respectively (44.3±3.7% vs. 63.6±1.99%, P Taken together, our results demonstrated the potent antitumor efficacies of GA on NHL and revealed that SRC-3 ubiquitination, mediated by Cullin3, played a critical role in this process, providing the molecular basis for the clinical trial in lymphoma. Disclosures No relevant conflicts of interest to declare.

High expression of the inducible isoform of heme oxygenase (HO-1) is well known in various solid tumors in human and experimental animal models. To investigate the relationship between HO-1 and nasopharyngeal carcinomas, especially its involvement in the response of nasopharyngeal carcinomas to radiotherapy, thirty-two nasopharyngeal carcinomas were semi-quantitatively analyzed by RT-PCR, and the expression of HO-1 was correlated with the consequence after novel radiotherapy, which was evaluated by the reduction of tumor size. Among 32 nasopharyngeal carcinomas, HO-1 expression was found in19 samples (59.4%), in which 9 patients (47.4%) showed no response to radiotherapy. Interestingly, in 13 nasopharyngeal carcinoma patients with negative expression of HO-1, radiotherapy exhibited to be effective (9 patients, 69.2%) or responsive (3 patients, 23.1%). In this study, we first demonstrated the expression of HO-1 in nasopharyngeal carcinomas, and more important, these findings strongly suggest the potential of HO-1as a useful index in identifying patients with well response to radiotherapy, further these data indicate a new therapeutic for nasopharyngeal carcinoma by inhibiting HO-1 activity, which warrants further investigation.

AML with NPM1 mutation (Mt NPM1) showing aberrant cytoplasmic dislocation of NPM1 exhibit relative good response to induction therapy, however, still a proportion of NPMc+ AML can not be cured by conventional treatments. Therefore, the aim of the present study was to determine the therapeutic efficacy of deguelin, a natural rotenoid isolated from several plant species, which has been demonstrated extensive biological activity in various tumors with low lethal effect. In the 54th ASH meeting in 2012, we reported deguelin selectively reduced Mt NPM1, induced differentiation and potentiated apoptosis in acute myeloid leukemia cells carrying NPM1 mutation. In this paper, we will focus on the investigation of the effect and molecular of mechanisms of deguelin treatment in nontoxic concentration with long time duration on AML cells carrying Mt NPM1. Deguelin treatment at the differentiation-induced concentration of 2µM for 5 days significantly induced granulocytic/monocytic differentiation such as a lower nucleocytoplasmic ratio, peripherally located nucleus, and chromatin condensation in 66.7% of OCI-AML3 cells carrying Mt NPM1. However, these phenomena were not presented in the OCIM2 cells harboring Wt NPM1. The NBT reduction assays that used as another marker of differentiation of myeloid leukemia exhibited increased NBT reduction activity, and Fluorescence-activated cell sorting (FACS) analysis demonstrated significantly increased expression levels of CD11b/CD14/CSF-3R in deguelin-treated OCI-AML3 cells (CD11b: from 2.8% to 62%; CD14: from 2.6% to 32.3%, CSF-3R: from 0.7% to 21.6%, P£¼0.01). Furthermore, immunoblot analysis revealed deguelin significantly reduced the levels of Mt NPM1 and HDACs (HDAC1/HDAC3/SIRT1), but not Wt NPM1, accompanied by increasing the expression of proteins and mRNAs of CEBP¦Â and G-CSFR. In addition, immunoprecipitation and immunoblot analysis confirmed deguelin significantly down-regulated Mt NPM1 via the ubiquitin-proteasome pathway. In consistent with above results, sh-Mt NPM1 and sh-SIRT1 treatment induced differentiation of OCI-AML3 (sh-Mt NPM1: CD11b/CD14/CSF-3R respectively: from 15.5% to 62.3%; from 13.9% to 47.1%; from 0.6% to 18.4%; sh-SIRT1: CD11b/CD14/CSF-3R respectively: from 18.2% to 47.1%; from 19.2% to 43.7%; from 0.4% to 13.4%, P£¼0.01), which confirmed by morphological examination and NBT reduction assays, and also upregulated the levels of proteins and mRNAs of CEBP¦Â and G-CSFR. sh-Mt NPM1 treatment reduced the expression of SIRT1, which did not change the levels of HDAC1/3. In other sides, sh-SIRT1 treatment did not alter the levels of Mt NPM1, suggesting that the decline of SIRT1 was partially accountable for granulocytic/monocytic differentiation of AML cells with Mt NPM1. Finally, overexpression of Mt NPM1 was performed to determine whether the depletion of Mt NPM1 directly induces granulocytic/monocytic cell differentiation. Compared to GFP-expressing cells, the proportion of the differentiation antigens (CD11b/CD14) and differentiation gene CSF-3R declined in Mt NPM1-expressing cells after deguelin treatment (Mt NPM1-expressing-treated deguelin-induced CD11b/CD14/CSF-3R-positive cells respectively: from 47.5% to 23.1%; from 29% to 18.8%; from 23.2% to 6.5%, P£¼0.01). Importantly, the upregulation of CEBP¦Â and G-CSFR by deguelin was appreciably hampered in Mt NPM1 expressing cells, compared with GFP-expressing cells. Taken together, our studies suggest deguelin, selective silencing of Mt NPM1, which induces differentiation via the axis of SIRT1/CEBP¦Â/G-CSFR (Fig1) may represent a novel active differentiating agent like ATRA for NPMc+ AML. Footnotes * Asterisk with author names denotes non-ASH members Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

High expression of the inducible isoform of heme oxygenase (HO-1) is now well known in solid tumors in humans and experimental animal models. We reported previously that HO-1 may be involved in tumor growth (Tanaka et al., Br. J. Cancer, 88: 902-909, 2003), in that inhibition of HO activity in tumors by using zinc protoporphyrin (ZnPP) significantly reduced tumor growth in a rat model. We demonstrate here that poly(ethylene glycol)-conjugated ZnPP (PEG-ZnPP), a water-soluble derivative of ZnPP, exhibited potent HO inhibitory activity and had an antitumor effect in vivo. In vitro studies with cultured SW480 cells, which express HO-1, showed that PEG-ZnPP induced oxidative stress, and consequently apoptotic death, of these cells. Pharmacokinetic analysis revealed that PEG-ZnPP-administered i.v. had a circulation time in blood that was 40 times longer than that for nonpegylated ZnPP. More important, PEG-ZnPP preferentially accumulated in solid tumor tissue in a murine model. In vivo treatment with PEG-ZnPP (equivalent to 1.5 or 5 mg of ZnPP/kg, i.v., injected daily for 6 days) remarkably suppressed the growth of Sarcoma 180 tumors implanted in the dorsal skin of ddY mice without any apparent side effects. In addition, this PEG-ZnPP treatment produced tumor-selective suppression of HO activity as well as induction of apoptosis. The major reason for tumor-selective targeting of PEG-ZnPP is attributed to the enhanced permeability and retention effect that is observed commonly in solid tumors for biocompatible macromolecular drugs. These findings suggest that tumor-targeted inhibition of HO activity could be achieved by using PEG-ZnPP, which induces apoptosis in solid tumors, probably through increased oxidative stress.

Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against oxidative stress. Here, we investigated the mechanism of cytoprotection of HO-1 in solid tumour with a focus on the antiapoptotic activity of HO-1. Treatment of rat hepatoma AH136B cells with the HO inhibitor zinc protoporphyrin IX (ZnPP IX) or tin protoporphyrin IX resulted in extensive apoptotic changes of tumour cells both in vivo and in vitro. Caspase-3 activity of the ZnPP IX-treated hepatoma cells increased significantly. Moreover, ZnPP IX-induced apoptosis was completely inhibited by simultaneous incubation with a specific caspase-3 inhibitor and was partially abrogated by bilirubin, a reaction product of HO. In vivo ZnPP IX treatment did not affect nitric oxide (NO) production and tumour blood flow. Western blot analyses showed that HO-1 expression in AH136B cells was strongly upregulated by NO donors, for example, S-nitroso-N-acetyl penicillamine and propylamine NONOate in vitro; conversely, it was remarkably reduced in vivo by pharmacological blockade of NOS. We conclude that HO-1 may function in antiapoptotic defense of the tumour, and thus it may have important protective and beneficial effects for tumour cells against oxidative stress induced by NO, which is produced in excess during solid tumour growth in vivo.

Abstract 2435 Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) gene mutations, leading to aberrant cytoplasmic expression of the nucleolar protein NPM1, accounts for about one-third of adult AML and shows distinctive biological and clinical features. In spite of the relatively good prognosis of NPM1-mutated AML, there are still cases that show poorer outcome, especially those associated with FLT3-ITD mutation and elderly patient population. Therefore new therapeutic strategies need to be explored. Deguelin, a rotenoid isolated from several plant species including Mondulea sericea (Leguminosae), exhibits significant inhibitory effects and induce apoptosis in a variety of cancer cell lines in vitro and in vivo. Researches have been revealed that NPM1 wild type (NPM-wt) and NPM1 mutant protein are potential therapeutic targets for AML cells harboring NPM1 mutations, however, isotype-specific inhibitors remain to be developed. Our previous study found deguelin induced apoptosis on Jurkat cells by disrupting NPM1 expression. Thus, in the present report we investigate the effects and molecular mechanisms of deguelin on AML NPMc+ cells, with focus on the NPM1 protein. Deguelin exerted dose-dependent dural effects on cell line OCI-AML3 carrying NPM1 mutation but not in the OCIM2 cell line (not harboring NPM1 gene mutation). Deguelin strongly induced OCI-AML3 cell apoptosis as shown by annexin V-fluoroisothyocyanate analysis (from 6.8% to 65.9%, 48 h at 32 μM), while apoptosis was minimal in OCIM2 cells (from 2.2% to 11.7%, 48h at 32 μM), which was further confirmed by a western blot assay to evaluate the activation of caspase-3 and the cleavage of PARP. Because of the pivotal role of NPM1 in AML cell survival and proliferation, we explored whether the above effects of deguelin were mediated by interfering with NPM1. Western blot analysis using specific antibodies showed marked downregulation of the leukemic NPM1 mutant protein upon deguelin treatment (0, 4 and 8μM, 48h), while NPM1-wt protein levels reminded unchanged. In addition, western blot and caspase activity assay revealed that the pro-apoptosis effect of deguelin was associated with caspase-6 and −8 activations, which might be caused by NPM mutation protein downregulation. In nontoxic concentration, morphologic features of granulocytic/monocytic differentiation were evident after 8 days treatment with dose of deguelin of 2μM in OCI-AML3 cells whereas no changes were found in OCIM2 cells. A more precise evaluation of cell differentiation by detecting CD11b (from 11.5% to 43.0%, P Disclosures: No relevant conflicts of interest to declare.

Abstract 1820 Background and Objective: Although there have been introductions of novel and more potent therapeutic regimens, multiple myeloma (MM) remains an incurable hematological malignant disorder. Current goals in MM treatment are focused on finding new therapies that target the deregulated signaling cascades which promote MM cell survival and proliferation, such as STAT3 and nuclear factor-κB (NF-κB) (Bharti AC et al, Blood 2004). Cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) is isolated from Alpinia katsumadai (Zingiberaceae), a plant widely used in traditional Chinese medicine. Previous reports have demonstrated that cardamonin possesses diverse pharmacologic actions, such as anti-inflammation (Hatziieremia S et al, Br J Pharmacol 2006), anti-melanogenesis (Cho M et al, Biochem Biophys Res Commun 2009) and anti-platelet properties (Jantan I et al, Phytomedicine 2008). However, very little is known about anti-myeloma activity of cardamonin. Thus, in the present report we investigated the therapeutic potential of cardamonin against MM, specifically studying the capacity of cardamonin to inhibit STAT3 and NF-κB pathways. Methods and Results: Cardamonin directly inhibited the growth of MM cell lines in a dose-dependent (from 0 μM to 100 μM) and time-dependent (from 0 h to 72 h) manner by using a cell counting kit-8 assay kit. Growth inhibition of MM cell lines, including RPMI 8226, U266 and ARH-77 cells, was demonstrated with an IC50 of less than 50 μM cardamonin at 24 h. Cardamonin also induced chemosensitivity to vincristine, doxorubicin, and dexamethasone in MM cells. Flow cytometric analysis showed that cardamonin caused accumulation of MM cells in G2 phase (from 14.6% to 81.9% for RPMI 8226, 24 h at 10 μM). A more precise evaluation of cell cycle by Hochest 33342 and 5-ethynyl-2′-deoxyuridine (EdU) fluorescence staining indicated that the cells in S phase were reduced in cardamonin-treated cells. Indeed, cardamonin strongly induced cell apoptosis as shown by annexin V-fluoroisothyocyanate analysis (from 5.3% to 49.4% for RPMI 8226, 48 h at 25 μM), which was also proved by a combination of acridine orange and ethidium bromide staining assay. Furthermore, cardamonin significantly enhanced the apoptotic effects of bortezomib from 23.1% to 74.3% and of thalidomide from 20.1% to 65.8%. Because of the pivotal role of STAT3 and NF-κB in MM cell survival and proliferation, we explored whether the above effects of cardamonin were mediated by interfering with STAT3 and NF-κB pathways by western blot analysis and immunofluorescence. We found that cardamonin blocked IL-6-inducible STAT3 phosphorylation and sequent STAT3 nuclear translocation. The constitutive phosphorylation of STAT3 found in certain cells was also abrogated by treatment with cardamonin in a dose- and time-dependent manner. In addition, we discovered that NF-κB, constitutively active in all human MM cell lines examined, was downregulated by cardamonin through suppression of phosphorylation of NF-κB p65 as evaluated by western blot. This correlated with reduction of the nuclear retention of p65. Moreover, cardamonin suppressed phosphorylation of IκBα, an inhibitor of NF-κB, and phosphorylation of Akt, which has been shown to phosphorylate p65. Additionally, immunoblotting analysis indicated that the expression of STAT3 and NF-κB-regulated gene products associated with proliferation (cyclin D1, TF and COX2), antiapoptosis (Bcl-2, Bcl-xL, Survivin, XIAP and Bfl-1/A1), invasion (ICAM-1), and angiogenesis (vascular endothelial growth factor) were down-regulated by cardamonin. ELISA assay showed that IL-6 release was also suppressed by cardamonin in certain MM cells. Conclusions: Taken together, our results suggest that cardamonin is a potent in vitro inhibitor of STAT3 and NF-κB pathways, which provides the molecular basis for its anti-myeloma activities, including suppression of proliferation, arrest of cell cycle, and induction of apoptosis. Disclosures: No relevant conflicts of interest to declare.

Background Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines. Methods Gene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively. Results Enforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2. Conclusion This study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF-FVIIa pathway may contribute, at least in part, to chemotherapy resistance in neuroblastoma.