Identification and quantification of five impurities in cloperastine hydrochloride.

Cloperastine hydrochloride, a piperidine derivative, is a drug substance with a central antitussive effect and widely used in cough treatment; and its impurities have not been reported. Herein we isolated and identified five impurities (named as impurity A, B, C, D and E) in cloperastine hydrochloride bulk drug and developed a quantitative HPLC method. First, impurity A, B, C were enriched by ODS column chromatography and isolated by semi-preparative HPLC, at the same time, impurity D was purified by ODS column chromatography. Then, impurity E was enriched by strong acid degradation and purified by semi-preparative HPLC. At last, their structures were characterized by a variety of spectral data (MS, 1H NMR, 13C NMR, HSQC, HMBC and 1H-1H COSY). Impurity A was confirmed as 1-[2-(diphenylmethoxy)ethyl]piperidine, which having one less chloro-substituent compared with cloperastine. Impurity B was confirmed as 1-[2-[(2-chlorophenyl)(phenyl)methoxy]ethyl]piperidine, which was the isomer of cloperastine with 2-chloro-substituent. Impurity C was confirmed as 1-[2-[(3-chlorophenyl)(phenyl)methoxy]ethyl]piperidine, which was the isomer of cloperastine with 3-chloro-substituent. Impurity D was confirmed as (4-chlorophenyl)(phenyl)methanone, which was the raw material for the synthesis of cloperastine. Impurity E was confirmed as (4-chlorophenyl)(phenyl)methanol, which was an intermediate in the synthesis of cloperastine, and it was also a hydrolysate of cloperastine. Finally, the developed method was validated in terms of specificity, linearity, sensitivity, precision and accuracy.

HMGB1-Induced p62 Overexpression Promotes Snail-Mediated Epithelial-Mesenchymal Transition in Glioblastoma Cells via the Degradation of GSK-3ß.

Rationale: Glioblastoma (GBM) is the most common and aggressive brain tumor, characterized by its propensity to invade the surrounding brain parenchyma. The effect of extracellular high-mobility group box 1 (HMGB1) protein on glioblastoma (GBM) progression is still controversial. p62 is overexpressed in glioma cells, and has been associated with the malignant features and poor prognosis of GBM patients. Hence, this study aimed to clarify the role of p62 in HMGB1-induced epithelial-mesenchymal transition (EMT) of GBM both in vitro and in vivo. Methods: Immunoblotting, immunofluorescence and qRT-PCR were performed to evaluate EMT progression in both human GBM cell line and primary GBM cells. Transwell and wound healing assays were used to assess the invasion and migration of GBM cells. shRNA technique was used to investigate the role of p62 in HMGB1-induced EMT both in vitro and in vivo orthotopic tumor model. Co-immunoprecipitation assay was used to reveal the interaction between p62 and GSK-3ß (glycogen synthase kinase 3 beta). Immunohistochemistry was performed to detect the expression levels of proteins in human GBM tissues. Results: In this study, GBM cells treated with recombinant human HMGB1 (rhHMGB1) underwent spontaneous EMT through GSK-3ß/Snail signaling pathway. In addition, our study revealed that rhHMGB1-induced EMT of GBM cells was accompanied by p62 overexpression, which was mediated by the activation of TLR4-p38-Nrf2 signaling pathway. Moreover, the results demonstrated that p62 knockdown impaired rhHMGB1-induced EMT both in vitro and in vivo. Subsequent mechanistic investigations showed that p62 served as a shuttling factor for the interaction of GSK-3ß with proteasome, and ultimately activated GSK-3ß/Snail signaling pathway by augmenting the degradation of GSK-3ß. Furthermore, immunohistochemistry analysis revealed a significant inverse correlation between p62 and GSK-3ß, and a combination of the both might serve as a more powerful predictor of poor survival in GBM patients. Conclusions: This study suggests that p62 is an effector for HMGB1-induced EMT, and may represent a novel therapeutic target in GBM.

HERC3-Mediated SMAD7 Ubiquitination Degradation Promotes Autophagy-Induced EMT and Chemoresistance in Glioblastoma.

PURPOSE: Glioblastoma, a common malignant intracranial tumor, has the most dismal prognosis. Autophagy was reported to act as a survival-promoting mechanism in gliomas by inducing epithelial-to-mesenchymal transition (EMT). Here, we determined the critical molecules involved in autophagy-induced EMT and elucidated the possible mechanism of chemoradiotherapy resistance and tumor recurrence. EXPERIMENTAL DESIGN: We used isobaric tags for relative and absolute quantitation to identify the critical proteins and pathway mediating EMT via autophagy inducer treatment, and tested the expression of these proteins using tissue microarray of gliomas and clinical glioblastoma samples as well as tissues and cells separated from the core lesion and tumor-peripheral region. Analysis of the Cancer Genome Atlas database and 110 glioblastoma cases revealed the prognostic value of these molecules. The functional role of these critical molecules was further confirmed by in vitro experiments and intracranial xenograft in nude mice. RESULTS: Autophagy inducers significantly upregulated the expression of HERC3, which promotes ubiquitination-mediated degradation of SMAD7 in an autolysosome-dependent manner. The corresponding increase in p-SMAD2/3 level and TGFß pathway activation finally induced EMT in cell lines and primary glioblastoma cells. Moreover, HERC3 overexpression was observed in pseudo-palisade cells surrounding tumor necrosis and in tumor-adjacent tissue; high HERC3 and low SMAD7 levels predicted poor clinical outcome in glioblastoma; xenograft of nude mice and in vitro experiments confirmed these findings. CONCLUSIONS: Together, our findings reveal the indispensable role of HERC3 in regulating canonical SMAD2/3-dependent TGFß pathway involvement in autophagy-induced EMT, providing insights toward a better understanding of the mechanism of resistance to temozolomide and peripheral recurrence of glioblastoma.

Endoscopic ultrasound elastography in the diagnosis of pancreatic masses: A meta-analysis.

BACKGROUND AND AIMS: Endoscopic ultrasound (EUS) elastography is a novel non-invasive technique that can be used for distinguishing benign from malignant pancreatic masses. However, the studies have reported widely varied sensitivities and specificities. A meta-analysis was performed to assess the performance of EUS elastography for the differentiation of benign and malignant pancreatic masses. METHODS: All the eligible studies were searched by PubMed, Medline, Embase, and the Cochrane Library. Sensitivity, specificity, positive likelihood ratio (LR), negative LR, and area under the curve (AUC) were calculated to examine the accuracy. RESULTS: A total of nineteen studies which included 1687 patients were analyzed. The pooled sensitivity and specificity for the diagnosis of malignant pancreatic masses were 0.98 (95% confidence interval [CI] 0.96-0.99) and 0.63 (95% CI 0.58-0.69) for qualitative EUS elastography, 0.95 (95% CI 0.93-0.97) and 0.61 (95% CI 0.56-0.66) for quantitative EUS elastography, respectively. The positive and negative LR were 2.60 (95% CI 1.84-3.66) and 0.05 (95% CI 0.02-0.10) for qualitative EUS elastography, 2.64 (95% CI 1.82-3.82) and 0.10 (95% CI 0.06-0.16) for quantitative EUS elastography, respectively. The summary diagnostic odds ratio (DOR) and the AUC were 60.59 (95% CI 28.12-130.56) and 0.91 (Q* = 0.842) for qualitative EUS elastography, 30.09 (95% CI 15.40-58.76) and 0.93 (Q* = 0.860) for quantitative EUS elastography. CONCLUSIONS: Our meta-analysis shows that both qualitative and quantitative EUS elastography have high accuracy in the detection of malignant pancreatic masses, which could be used as a valuable complementary method to EUS-FNA for the differentiation of pancreatic masses in the future.

Characterization of a novel rice metallothionein gene promoter: its tissue specificity and heavy metal responsiveness.

The rice (Oryza sativa L.) metallothionein gene OsMT-I-4b has previously been identified as a type I MT gene. To elucidate the regulatory mechanism involved in its tissue specificity and abiotic induction, we isolated a 1 730 bp fragment of the OsMT-I-4b promoter region. Histochemical ß-glucuronidase (GUS) staining indicated a precise spacial and temporal expression pattern in transgenic Arabidopsis. Higher GUS activity was detected in the roots and the buds of flower stigmas, and relatively lower GUS staining in the shoots was restricted to the trichomes and hydathodes of leaves. No activity was observed in the stems and seeds. Additionally, in the root of transgenic plants, the promoter activity was highly upregulated by various environmental signals, such as abscisic acid, drought, dark, and heavy metals including Cu²(+) , Zn²(+) , Pb²(+) and Al³(+) . Slight induction was observed in transgenic seedlings under salinity stress, or when treated with Co²(+) and Cd²(+) . Promoter analysis of 5'-deletions revealed that the region -583/-1 was sufficient to drive strong GUS expression in the roots but not in the shoots. Furthermore, deletion analysis indicated important promoter regions containing different metal-responsive cis-elements that were responsible for responding to different heavy metals. Collectively, these findings provided important insight into the transcriptional regulation mechanisms of the OsMT-I-4b promoter, and the results also gave us some implications for the potential application of this promoter in plant genetic engineering.