CDER167, a dual inhibitor of URAT1 and GLUT9, is a novel and potent uricosuric candidate for the treatment of hyperuricemia.

Urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) are important targets for the development of uric acid-lowering drugs. We previously showed that the flexible linkers of URAT1 inhibitors could enhance their potency. In this study we designed and synthesized CDER167, a novel RDEA3710 analogue, by introducing a linker (methylene) between the naphthalene and pyridine rings to increase flexibility, and characterized its pharmacological and pharmacokinetics properties in vitro and in vivo. We showed that CDER167 exerted dual-target inhibitory effects on both URAT1 and GLUT9: CDER167 concentration-dependently inhibited the uptake of [14C]-uric acid in URAT1-expressing HEK293 cells with an IC50 value of 2.08 ± 0.31 µM, which was similar to that of RDEA3170 (its IC50 value was 1.47 ± 0.23 µM). Using site-directed mutagenesis, we demonstrated that CDER167 might interact with URAT1 at S35 and F365. In GLUT9-expressing HEK293T cells, CDER167 concentration-dependently inhibited GLUT9 with an IC50 value of 91.55 ± 15.28 µM, whereas RDEA3170 at 100 µM had no effect on GLUT9. In potassium oxonate-induced hyperuricemic mice, oral administration of CDER167 (10 mg·kg-1 · d-1) for 7 days was more effective in lowering uric acid in blood and significantly promoted uric acid excretion in urine as compared with RDEA3170 (20 mg·kg-1 · d-1) administered. The animal experiment proved the safety of CDER167. In addition, CDER167 displayed better bioavailability than RDEA3170, better metabolic stability and no hERG toxicity at 100 µM. These results suggest that CDER167 deserves further investigation as a candidate antihyperuricemic drug targeting URAT1 and GLUT9.

ML365 inhibits TWIK2 channel to block ATP-induced NLRP3 inflammasome.

Dysregulation of NLRP3 inflammasome results in uncontrolled inflammation, which participates in various chronic diseases. TWIK2 potassium channel mediates potassium efflux that has been reported to be an essential upstream mechanism for ATP-induced NLRP3 inflammasome activation. Thus, TWIK2 potassium channel could be a potential drug target for NLRP3-related inflammatory diseases. In the present study we investigated the effects of known K2P channel modulators on TWIK2 channel expressed in a heterologous system. In order to increase plasma membrane expression and thus TWIK2 currents, a mutant channel with three mutations (TWIK2I289A/L290A/Y308A) in the C-terminus was expressed in COS-7 cells. TWIK2 currents were assessed using whole-cell voltage-clamp recording. Among 6 known K2P channel modulators tested (DCPIB, quinine, fluoxetine, ML365, ML335, and TKDC), ML365 was the most potent TWIK2 channel blocker with an IC50 value of 4.07 ± 1.5 µM. Furthermore, ML365 selectively inhibited TWIK2 without affecting TWIK1 or THIK1 channels. We showed that ML365 (1, 5 µM) concentration-dependently inhibited ATP-induced NLRP3 inflammasome activation in LPS-primed murine BMDMs, whereas it did not affect nigericin-induced NLRP3, or non-canonical, AIM2 and NLRC4 inflammasomes activation. Knockdown of TWIK2 significantly impaired the inhibitory effect of ML365 on ATP-induced NLRP3 inflammasome activation. Moreover, we demonstrated that pre-administration of ML365 (1, 10, 25 mg/kg, ip) dose-dependently ameliorated LPS-induced endotoxic shock in mice. In a preliminary pharmacokinetic study conducted in rats, ML365 showed good absolute oral bioavailability with F value of 22.49%. In conclusion, ML365 provides a structural reference for future design of selective TWIK2 channel inhibitors in treating related inflammatory diseases.

Versatile Nanodrugs Containing Glutathione and Heme Oxygenase 1 Inhibitors Enable Suppression of Antioxidant Defense System in a Two-Pronged Manner for Enhanced Photodynamic Therapy.

The antioxidant defense system in malignant cells, which involves antioxidant enzymes and antioxidant molecules, is an innate barrier to photodynamic therapy (PDT). Because of the complexity of the endogenous antioxidant mechanisms of these cells, simply inhibiting individual antioxidant pathways has a limited effect on improving the lethality of ROS. To enhance the efficacy of PDT for tumor treatment, a versatile nanoparticle (NP)-based drug is developed, which the authors call PZB NP, containing the glutathione inhibitor l-buthionine sulfoximine (BSO) and the heme oxygenase 1 (HO-1) inhibitor protoporphyrin zinc(II) (ZnPP) to suppress the innate antioxidant defense system of cancer cells in a two-pronged manner. BSO reduces intracellular glutathione levels to minimize ROS elimination and protein protection during PDT, and ZnPP inhibits the ROS-stimulated upregulation of the antioxidant HO-1, thus preventing ROS removal by cells after PDT. Thus, BSO and ZnPP synergistically suppress the antioxidant defense systems of cancer cells both during and after protoporphyrin-IX-mediated PDT in a two-pronged manner, resulting in tumor cell death through excess oxidative pressure. The results demonstrate that the construction of nanodrugs having dual antioxidation defense suppression properties is a promising route for the development of highly efficient ROS-based therapies.

In Situ Detection of Plasma Exosomal MicroRNA-1246 for Breast Cancer Diagnostics by a Au Nanoflare Probe.

Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, and metastasis monitoring are of great importance to favorable prognosis. Although conventional diagnostic methods, such as breast X-ray mammography and image positioning biopsy, are accurate, they could cause radioactive or invasive damage to patients. Liquid biopsy as a noninvasive method is convenient for repeated sampling in clinical cancer prognostic, metastatic evaluation, and relapse monitoring. MicroRNAs encased in exosomes circulating in biofluids are promising candidate cancer biomarkers because of their cancer-specific expression profiles. Here, we report an in situ detection of microRNA-1246 (miR-1246) in human plasma exosomes as breast cancer biomarker by a nucleic acid functionalized Au nanoflare probe. Needing neither time-consuming and costly isolation of exosomes from the plasma sample nor transfection means, the Au nanoflare probe can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting miR-1246. Only 40 µL of plasma is needed to incubate 4 h with the probe, giving signal sensitive enough to distinguish samples of breast cancer to normal control. Using plasma miR-1246 level detected by our assay as a marker, we differentiated 46 breast cancer patients from 28 healthy controls with 100% sensitivity and 92.9% specificity at the best cutoff. This simple, accurate, sensitive, and cost-effective liquid biopsy by the Au nanoflare probe is potent to be developed as a noninvasive breast cancer diagnostic assay for clinical adaption.

miR-133a Promotes TRAIL Resistance in Glioblastoma via Suppressing Death Receptor 5 and Activating NF-κB Signaling.

Recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), as a novel cancer therapeutic, is being tested in phase II and III clinical trials; however, TRAIL resistance remains a big obstacle preventing its clinical application. Considering that TRAIL-induced apoptosis through death receptors DR4 and DR5, their activation may be an alternative pathway to suppress TRAIL resistance. In this study, a negative correlation between DR5 expression and TRAIL resistance was observed, and miR-133a was predicted to be the most promising candidate to suppress DR5 expression. Further investigation demonstrated that miR-133a knockdown dramatically suppressed TRAIL resistance in glioblastoma in vitro and in vivo. An NF-κB family member, phosphorylated IκBα (P-IκBα), was shown to be stimulated by miR-133a, leading to the activation of this signaling. Finally, miR-133a was found to be inversely correlated with DR5 expression in human clinical specimens. In conclusion, our data demonstrate that miR-133a promotes TRAIL resistance in glioblastoma by suppressing DR5 expression and activating NF-κB signaling.

Usp5 functions as an oncogene for stimulating tumorigenesis in hepatocellular carcinoma.

As deubiquitinases, several ubiquitin specific protease members have been reported to mediate tumorigenesis. Although ubiquitin specific protease 5 (Usp5) was previously demonstrated to suppress p53 transcriptional activity and DNA repair, its role in carcinogenesis remains elusive. In this study, we sought to define a novel role of Usp5 in tumorigenesis. It was found that Usp5 was significantly upregulated in hepatocellular carcinoma (HCC) cells and most clinical specimens. Further functional investigation also showed that Usp5 knockdown suppressed cell proliferation, migration, drug resistance and induced apoptosis; on the other hand, Usp5 overexpression promoted colony formation, migration, drug resistance and tumorigenesis. Additionally, the inactivated p14ARF-p53 signaling was observed in Usp5 overexpressed HCC cells, while this signaling was activated by Usp5 knockdown. Therefore, our data demonstrated that Usp5 contributed to hepatocarcinogenesis by acting as an oncogene, which provides new insights into the pathogenesis of HCC and explores a promising molecular target for HCC diagnosis and therapy.

Ubiquitin specific peptidase 5 mediates Histidine-rich protein Hpn induced cell apoptosis in hepatocellular carcinoma through P14-P53 signaling.

Hpn is a small histidine-rich cytoplasmic protein from Helicobacter pylori and has been recognized as a high-risk factor for several cancers including gastric cancer, colorectal cancer, and MALT lymphoma. However, the relationship between Hpn and cancers remains elusive. In this study, we discovered that Hpn protein effectively suppressed cell growth and induced apoptosis in hepatocellular carcinoma (HCC). A two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics was performed to find the molecular targets of Hpn in HCC cells. It was identified that twelve proteins were differentially expressed, with USP5 being one of the most significantly downregulated protein. The P14ARF -P53 signaling was activated by USP5 knockdown in HCC cells. Furthermore, USP5 overexpression significantly rescued the suppressive effect of Hpn on the viability of HCC cells. In conclusion, our study suggests that Hpn plays apoptosis-inducing roles through suppressing USP5 expression and activating the P14ARF -P53 signaling. Therefore, Hpn may be a potential candidate for developing novel anti-HCC drugs.

H19 mediates methotrexate resistance in colorectal cancer through activating Wnt/ß-catenin pathway.

Colorectal cancer (CRC) is a common malignancy, most of which remain unresponsive to chemotherapy. As one of the earliest cytotoxic drugs, methotrexate (MTX) serves as an anti-metabolite and anti-folate chemotherapy for various cancers. Unfortunately, MTX resistance prevents its clinical application in cancer therapy. Thereby, overcoming the drug resistance is an alternative strategy to maximize the therapeutic efficacy of MTX in clinics. Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years. More and more emerging evidences have demonstrated that they play important regulatory roles in various biological activities and disease progression including drug resistance. In the present study, a MTX-resistant colorectal cell line HT-29 (HT-29-R) was developed, which displayed the active proliferation and shortened cell cycle. LncRNA H19 was found to be significantly upregulated in this resistant cell line. Further investigation showed that H19 knockdown sensitized the MTX resistance in HT-29-R cells while its overexpression improved the MTX resistance in the parental cells, suggesting that H19 mediate MTX resistance. The Wnt/ß-catenin signaling was activated in HT-29-R cells, and H19 knockdown suppressed this signaling in the parental cells. In conclusion, H19 mediated MTX resistance via activating Wnt/ß-catenin signaling, which help to develop H19 as a promising therapeutic target for MTX resistant CRC.

Synthesis and potent inhibitory activities of carboxybenzyl-substituted 8-(3-(R)-aminopiperidin-1-yl)-7-(2-chloro/cyanobenzyl)-3-methyl-3,7-dihydro-purine-2,6-diones as dipeptidyl peptidase IV (DPP-IV) inhibitors.

Fourteen 3-methyl-3,7-dihydro-purine-2,6-dione derivatives 1-14 bearing carboxybenzyl and 2-chloro/cyanobenzyl groups at the N-1 and N-7 positions, respectively, were synthesized as dipeptidyl peptidase IV (DPP-IV) inhibitors. These compounds were characterized on the basis of NMR ((1)H and (13)C) and ESI MS data. In vitro bioassay indicates that most of these compounds showed moderate to good inhibitory activities against DPP-IV. Among them, compound 13 (IC50=36 nM) exhibited comparable activity with a positive control, Sitagliptin (IC50=16 nM). In addition, the structure-activity relationship of these compounds is also briefly discussed.

Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells.

Plumbagin (PLB) has shown anti-cancer activity but the mechanism is unclear. This study has found that PLB has a potent pro-apoptotic and pro-autophagic effect on A549 and H23 cells. PLB arrests cells in G2/M phase, and increases the intracellular level of reactive oxygen species in both cell lines. PLB dose-dependently induces autophagy through inhibition of PI3K/Akt/mTOR pathway as indicated by reduced phosphorylation of Akt and mTOR. Inhibition or induction of autophagy enhances PLB-induced apoptosis. There is crosstalk between PLB-induced apoptosis and autophagy. These findings indicate that PLB initiates both apoptosis and autophagy in NSCLC cells through coordinated pathways.

[Clinicopathological diagnosis of hypersensitivity pneumonitis].

OBJECTIVE: To enhance the understandings of clinical, radiological and pathological features of hypersensitivity pneumonitis (HP). METHODS: Six HP cases with pathological data, clinical and radiological data were retrospectively analyzed during the period from February 2009 to September 2011 at Beijing Hospital of Ministry of Health. There were 2 males and 4 females with a mean age of 51.5 years (range: 38-61). Clinically, the patients presented with chronic cough, shortness of breath and dyspnea (n = 2). The disease course was 1-8 months. Five cases had fed pigeons and other contact histories. Specimens obtained by transbronchial lung biopsy (n = 3) and open lung biopsy (n = 3) were paraffin embedded and stained by hematoxylin and eosin, special stains and immunohistochemistry. RESULTS: Four cases had subacute HP and 2 cases chronic HP. Three cases of subacute HP underwent transbronchial lung biopsy. One case of subacute HP and 2 cases of chronic HP were diagnosed by open lung biopsy. High-resolution computed tomography of lungs showed diffuse ground glass and patch shadow along the bronchial and centrilobular distributions. There was a predominance of upper half zone. Typical visible mosaic syndrome was present. There was poorly formed granuloma without cheesy necrosis. With an insidious medical history and complicated radiological features, chronic HP cases were characterized by pulmonary interstitial fibrosis. There were usual interstitial pneumonitis (UIP)-like fibrosis and fibrosis with an airway-centered distribution type. The lesions were distributed around bronchioles. Continuous bridge fibrosis might be present. There were bronchiolar metaplasia of peribronchiolar alveoli, poorly formed granuloma and multinucleated giant cells in interstitium. Schaumann body was identified in 1 case. CONCLUSIONS: Because of its diverse clinical, radiological and pathological features, HP may be easily confused with other interstitial lung diseases. Aggregate analyses yield a definite diagnosis.

Praeruptorin A inhibits lipopolysaccharide-induced inflammatory response in murine macrophages through inhibition of NF-κB pathway activation.

Praeruptorin A (PA) is a pyranocoumarin compound isolated from the dried root of Peucedanum praeruptorum Dunn (Umbelliferae). However, the antiinflammatory effect of PA has not been reported. The present study investigated the antiinflammatory effect of PA in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. PA significantly inhibited the LPS-induced production of nitric oxide (NO), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). The mRNA and protein expressions of inducible nitric oxide synthase (iNOS), IL-1ß and TNF-α were also suppressed by this compound. Further study showed that PA decreased the cytoplasmic loss of inhibitor κB-α (IκB-α) protein and inhibited the translocation of NF-κB from cytoplasm to nucleus. Taken together, the results suggest that PA may exert antiinflammatory effects in vitro in LPS-stimulated RAW 264.7 macrophages through inhibition of NF-κB signal pathway activation.

Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells.

Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5'-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.

[Clinicopathologic characteristics of metastatic carcinomas to spleen].

OBJECTIVE: To study the clinicopathologic characteristics of metastatic carcinomas to the spleen. METHODS: Sixteen cases of metastatic carcinoma to the spleen were retrieved from archival clinical, surgical pathology and autopsy records. The demographic data (including sex and age of patients), clinical symptoms, primary sites, tumor histologic types, gross appearance of spleen and growth patterns within the spleen were analyzed. RESULTS: Among the 16 patients studied, 12 were males and 4 were females. The male predilection was obvious. The age ranged from 48 to 90 years, the median age 66.5 years. Major clinical symptoms included discomfort in the left upper quadrant, pain, emaciation and loss of appetite. Splenomegaly was noted in some patients and computerized tomography could show space-occupying lesions in the spleen. In general, lung was the most common primary site for splenic metastasis and accounted for 43.8% of all cases (7/16). In male patients, primary lung tumor was found in 50.0% cases (6/12). On the other hand, primary ovarian tumor was commonly seen in females (2/4). Histologically, undifferentiated carcinoma of lung was frequently encountered (25.0%, 4/16), including 3 cases of small cell undifferentiated carcinoma and 1 case of large cell undifferentiated carcinoma. Other histologic tumor types included bronchioloalveolar carcinoma (2 cases), colonic adenocarcinoma (2 cases), ovarian serous papillary adenocarcinoma (2 cases), and prostatic adenocarcinoma (2 cases). The commonest histologic tumor type found in male patients was pulmonary undifferentiated carcinoma. The growth patterns of metastatic carcinoma in spleen included nodular, diffuse and multinodular. Most cases presented as a single splenic nodule. Sometimes, tumors with high metastatic potential (5/16) showed diffuse and multinodular growth patterns. Examples of these tumors included small cell undifferentiated carcinoma (3 cases), pulmonary adenocarcinoma (1 case) and prostatic adenocarcinoma (1 case). CONCLUSIONS: Metastatic carcinoma to the spleen is rare. Understanding of the clinicopathologic characteristics is helpful in guiding clinical management and pathologic diagnosis.

[Functional changes of dendritic cells after infection by recombinant retrovirus carrying human telomerase reverse transcriptase gene fragment].

OBJECTIVE: To observe the functional changes of dendritic cells (DCs) after infection by recombinant retrovirus carrying human telomerase reverse transcriptase (hTERT) gene fragment. METHODS: Interleukin-12 (IL-12) levels in DC culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The abilities of DCs infected with recombinant retrovirus carrying hTERT gene (hTERT-DCs) and non-infected DCs (N-DCs) to stimulate allogeneic lymphocyte proliferation were evaluated with mixed leukocytes reaction (MLR), and the surface markers of DCs including CD80, CD83, CD86 and HLA-DR were detected by flow cytometry. Cytotoxic T lymphocyte (CTL) assay was performed with CytoTox 96 non-radioactive cytoxicity assay. RESULTS: Compared with N-DCs, hTERT-DCs showed no significant changes in IL-12 secretion and capacity to stimulate allogeneic lymphocytes reaction, but had significantly lower CD83 expression. Specific CTLs induced by hTERT-DCs resulted in higher cytotoxicity against telomerase-positive target cells than that against the negative target cells. CONCLUSION: Infection with the recombinant retrovirus carrying hTERT fragment may jeopardize the maturation of DCs, which, however, still retain their capacity to activate and stimulate lymphocyte proliferation and to prime autologous T lymphocytes to generate specific CTL against hTERT.

[Total alkaloid of Macleaya cordata: in vitro cytotoxic effect on Hep3B cells and in vivo antitumor effect in mice].

OBJECTIVE: To observe the cytotoxic effects in vitro and antitumor effects in vivo of total alkaloid of Macleaya cordata. METHODS: MTT assay was used to assess the proliferation of Hep3B and H22 cells in vitro treated with the alkloid, and the inhibitory effects of the alkaloid on H22 and S180 tumors were observed in mice with subcutaneous inoculation of the tumor cells. RESULTS: The total alkaloid significantly inhibited the proliferation of human Hep3B cells and murine H22 cells in a dose-dependent in vitro. IC(50) of the alkloid in 3 repeated experiments was 3.04, 3.98 and 2.98 mug/ml respectively in Hep3B cells, and was 2.89, 2.21 and 2.34 mug/ml in H22 cells. In the tumor-bearing mice, the alkaloid inhibited the development of H22 tumor and prolonged the survival of S180 tumor-bearing mice. At the daily dose of 1, 2, and 4 mg/kg.b.w (i.p.) and 4 mg/kg.b.w. (i.g.) for 10 days, the inhibition rates of the alkaloid on H22 tumor in 3 repeated experiments were 18.6%, 35.1%, 44.9% and 7.9% respectively, and the rates of survival prolongation of the tumor-bearing mice were 24.8%, 48.9%, and 52.7%, respectively. CONCLUSION: The alkloid possesses antitumor effects both in vitro and in vivo.

[Study of koumine-induced apoptosis of human colon adenocarcinoma LoVo cells in vitro].

OBJECTIVE: To study the apoptosis-inducing effect of koumine on human colon adenocarcinoma LoVo cells in vitro. METHODS: After koumine (50 mmol/L) treatment in vitro, the LoVo cells were examined under light microscope, transmission electron microscope and fluoroscope respectively for apoptosis, and the cell cycle distribution was analyzed using flow cytometry. RESULTS: The percentage of apoptotic cells increased in a time-dependent manner after the cells were treated with koumine, whose action exhibited remarkable cell cycle specificity. The percentage of LoVo cells in G(0)/G(1) phase rose from 31.3% to 42.3% and the percentage of cells in S phase fells from 62.0% to 38.7%. CONCLUSION: Koumine can induce apoptosis of LoVo cells in a time-dependent manner and inhibit the DNA synthesis in LoVo cells, thereby blocking the cell cycle from G1 to S phase.

[Human telomerase reverse transcriptase as a novel target for tumor immunotherapy].

Recently, a new immunotherapy against cancer based on a novel tumor-associated antigen of the human telomerase reverse transcriptase (hTERT) is being investigated. Antigen peptides derived from hTERT are expressed and presented with major histocompatibility complex (MHC) class I molecules in tumor cells, and elicit ex vivo cytotoxic T-lymphocyte (CTL) responses in healthy individuals and cancer patients. Data from both human and murine systems demonstrate that TERT-special CTL kill TERT-positive tumor cells of various histological origins in a MHC-restricted fashion. However, they do not lyze rarely normal cell types such as hematopoietic progenitor cells and activated T-lymphocytes in which telomerase has been detected. Phase I clinical trials targeting hTERT in advanced cancer patients further confirmed that there was little influence on autoimmunity. These results suggested that the possibility of broad spectrum immunotherapy or even immunoprevention therapy based on hTERT could be considered.