Discovery of [1,2,3]triazolo[4,5-d]pyrimidine derivatives as highly potent,selective, and cellularly active USP28 inhibitors

Ubiquitin specific peptidase 28 (USP28) is closely associated to the occurrence and development of various malignancies, and thus has been validated as a promising therapeutic target for cancer therapy. To date, only few USP28 inhibitors with moderate inhibitory activity have been reported, highly potent and selective USP28 inhibitors with new chemotypes remain to be discovered for pathologically investigating the roles of deubiquitinase. In this current study, we reported the synthesis and biological evaluation of new [1,2,3]triazolo[4,5-d]pyrimidine derivatives as potent USP28 inhibitors. Especially, compound 19 potently inhibited USP28 (IC₅₀ = 1.10 ± 0.02 μmol/L, K_d = 40 nmol/L), showing selectivity over USP7 and LSD1 (IC₅₀ > 100 μmol/L). Compound 19 was cellularly engaged to USP28 in gastric cancer cells. Compound 19 reversibly bound to USP28 and directly affected its protein levels, thus inhibiting the proliferation, cell cycle at S phase, and epithelial-mesenchymal transition (EMT) progression in gastric cancer cell lines. Docking studies were performed to rationalize the potency of compound 19. Collectively, compound 19 could serve as a new tool compound for the development of new USP28 inhibitors for exploring the roles of deubiquitinase in cancers.     

Targeting USP1‐dependent KDM4A protein stability as a potential prostate cancer therapy

The histone demethylase lysine‐specific demethylase 4A (KDM4A) is reported to be overexpressed and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 or H3K36 methylation marks. However, the biological role and mechanism of KDM4A in prostate cancer (PC) remain unclear. Herein, we reported KDM4A expression was upregulation in phosphatase and tensin homolog knockout mouse prostate tissue. Depletion of KDM4A in PC cells inhibited their proliferation and survival in vivo and vitro. Further studies reveal that USP1 is a deubiquitinase that regulates KDM4A K48‐linked deubiquitin and stability. Interestingly, we found c‐Myc was a key downstream effector of the USP1‐KDM4A/androgen receptor axis in driving PC cell proliferation. Notably, upregulation of KDM4A expression with high USP1 expression was observed in most prostate tumors and inhibition of USP1 promotes PC cells response to therapeutic agent enzalutamide. Our studies propose USP1 could be an anticancer therapeutic target in PC.     

Cutaneous nodular fasciitis with genetic analysis: a case series

Nodular fasciitis is a benign self‐limited myofibroblastic neoplasm, which usually involves the upper extremities and trunk of young patients. These tumors have been shown to harbor a translocation involving the MYH9 and USP6 genes, leading to overexpression of the latter. We report seven cases of nodular fasciitis with cutaneous presentations. All cases involved the dermis, with six involving the superficial subcutis, and one auricular tumor extending into cartilage. All cases showed USP6 rearrangement by fluorescence in situ hybridization; in two of three cases, the characteristic MYH9‐USP6 fusion was shown by RT‐PCR. All patients underwent conservative resection. Nodular fasciitis is an uncommon mesenchymal neoplasm that can occasionally present in superficial locations and is sometimes mistaken for a malignant process. Molecular testing can be useful to distinguish this entity from other cutaneous spindle cell tumors.     

ShRNA-mediated silencing of the ubiquitin-specific protease 22 gene restrained cell progression and affected the Akt pathway in nasopharyngeal carcinoma

Ubiquitin-specific protease 22 (USP22) is closely related with poor prognosis of cancer patients. However, the role of USP22 expression in nasopharyngeal carcinoma (NPC) has not been determined. The main aim of this study was to determine the role of USP22 in the pathologic processes of NPC. Immunohistochemistry (IHC), western blot (WB), and real-time polymerase chain reaction (RT-PCR) were used to measure the expression of USP22 in cell lines and tissues of NPC in comparison with expression in non-cancerous cells and tissues. USP22-specific short hairpin RNA (shRNA) was used to knock down USP22 expression in the NPC cell line CNE-1 and CNE-2. Furthermore, the impact of USP22 in cellular proliferation, growth, and cell cycle were detected respectively. WB was used to determine the role of USP22 in the AKT/GSK-3/Cyclin signaling pathway. The expression levels of USP22 were remarkably higher in NPC cell lines and tissues. With cell counting and the MTS assay, cellular growth and proliferation progression of USP22 knockdown cell line was shown to be effectively restrained. The USP22 silencing both in CNE-1 and CNE-2 cells caused them to accumulate in the G0/G1 phase of the cell cycle. USP22 knockdown was also found to modulate the AKT/GSK-3/Cyclin pathway, resulting in downregulation of p-AKT, p-GSK-3β, and cyclinD1. This study suggests that USP22 plays a critical regulatory role in the pathologic processes of NPC, and that it may be a potential biological treatment target in the future.     

An eco-friendly HPLC-UV method for the determination of risedronate in its bulk and tablet dosage form with application to content uniformity,dissolution and stability testing

Risedronate is a nitrogen-containing bisphosphonate for the treatment and prevention of postmenopausal osteoporosis. The current work aims to develop a novel green HPLC-UV method for the rapid analysis of risedronate sodium in bulk and tablet formulation. The analyzed samples were separated on Waters Atlantis dC18 (150 mm × 3.9 mm; 5 μm) column using a green mobile phase consisting of potassium phosphate buffer pH 2.9 and potassium edetate buffer pH 9.5 in a ratio of 1:2, the final pH was adjusted to 6.8 with phosphoric acid, the mobile phase was pumped at a rate of 1.0 mL/min, with column temperature set at 30 °C, eluted samples were detected at 263 nm and the chromatographic run time was 3.0 min. The method was found to be linear over the concentration range of 14–140 μg/mL with a correlation coefficient (r²) of 0.9994. Accuracy and precision were evaluated from three QC samples (LQC, MQC and HQC) together with the five calibrators where the percentage accuracy was found to be 101.84%. Processed quality control samples of risedronate sodium were tested for stability at different conditions, short term, long term and freeze- thaw stability. The current method was further extended to study the content uniformity of Actonel® tablets following United States Pharmacopoeia (USP) guidelines. The proposed method was fully validated as per ICH guidelines.     

CyclinB1 deubiquitination by USP14 regulates cell cycle progression in breast cancer

Breast cancer is the most common malignant tumor among women in China, which seriously threatens women's physical and mental health. Tumorigenesis is closely related to the dysregulation of cell cycle. The cell cycle progression includes interphase and mitotic phase (M phase). Cyclin B1 is a key protein in regulating M phase, which is essential for the whole cell cycle progression. CyclinB1 can be degraded through ubiquitination mediated by the anaphase promoting complex/cyclosome (APC/C). However, the mechanism of how CyclinB1 is deubiquitinated in breast cancer still remains unclear. In this study, we discovered that CyclinB1 interacted with ubiquitin-specific peptidase 14 (USP14). Based on the deubiquitinating function of USP14, we detected the effect of USP14 on the ubiquitination of CyclinB1. Inhibiting the activity of USP14 or USP14 knockdown significantly increased the ubiquitination of CyclinB1. In accordance with this, knocking down USP14 arrested cell cycle at G2/M phase. Knocking down USP14 with siRNAs significantly inhibited the proliferation and migration of breast cancer cells. In conclusion, our study demonstrated that USP14 regulated the cell cycle of breast cancer cells by regulating the ubiquitination of CyclinB1, which will provide a solid theoretical basis for the development of anti-cancer drugs targeting USP14.     

超声诊断在结肠癌术前分期中的临床应用

[ Objective]The aim of this study was to assess the value of ultrasonic probe (USP) under colonoscope in the preoperative staging of colorectal carcinoma. [ Methods]75 patients with colorectal carcinomas proven pathologically were given USP (Olympus UM-2R, 12MHz; UM -3R, 20 MHz) under colonoscope before operation. The results were compared with histopathologic findings of resected specimens. [ Results] Colorectal carcinoma appeared as a hypoechoic mass under USP. USP had an overall accuracy rate of 82.7% in the diagnosis of T staging of colorectal carcinoma. In determining lymph node metastasis , the sensitivity and specificity were 53.2% and 61.5%, the positive predictive value and the negative predictive value were 0.87 and 0. 22, respectively. [ Conclusions ] USP is valuable for the staging of colorectal carcinoma and has a high accuracy rate in determining the depth of tumor invasion. The preoperative information obtained by this tool may influence the choice of therapy.     

miR-4319通过靶向调控USP2抑制乳腺癌细胞侵袭

目的探讨miR-4319与泛素特异性蛋白酶2(USP2)表达的相关性以及miR-4319靶向USP2通过核转录因子κB(NF-κB)信号通路对乳腺癌细胞侵袭的影响。方法实时荧光定量PCR(qRT-PCR)检测miR-4319在正常乳腺癌上皮细胞(MCF10A)、低侵袭性乳腺癌细胞(MCF7)和高侵袭性乳腺癌细胞(MDA-MB-231)中的表达量。将MCF10A、MCF7和MDA-MB-231细胞分为6组进行转染:(1)MDA-MB-231/NC组,瞬时转染插入一段乱码序列(scramble 1),即作为miR-4319过表达对照质粒;(2)MDA-MB-231/miR-4319组,瞬时转染插入目的片段miR-4319质粒过表达miR-4319,即miR-4319过表达;(3)MDA-MB-231/miR-4319+Con组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达对照质粒,即miR-4319过表达以及USP2过表达对照;(4)MDA-MB-231/miR-4319+USP2组,瞬时转染同时转入miR-4319过表达质粒和USP2过表达质粒,即miR-4319过表达和USP2过表达;(5)MDA-MB-231/miR-4319inhibitor组,瞬时转染miR-4319的反义序列抑制miR-4319的表达,即抑制miR-4319的表达;(6)MDA-MB-231/miR-4319inhibitor NC组,瞬时转染插入一段乱码序列(scramble 2),即作为抑制miR-4319组的对照组。qRT-PCR检测miR-4319在MDA-MB-231细胞中的转染效率。荧光素酶实验检测miR-4319与USP2 mRNA是否存在结合位点。qRT-PCR检测miR-4319在乳腺癌细胞中过表达后的USP2 mRNA表达水平。蛋白质印迹法检测过表达miR-4319或抑制miR-4319表达后的USP2蛋白表达水平。Transwell侵袭实验检测过表达miR-4319或USP2后MDA-MB-231细胞侵袭能力。双荧光素酶实验检测miR-4319对NF-κB信号通路活性的影响以及过表达USP2后miR-4319对NF-κB信号通路活性的影响。结果 qRT-PCR结果显示,miR-4319相对表达量在细胞MDA-MB-231(t=14.860,P<0.001)和MCF7(t=12.770,P<0.001)中分别为0.330±0.075和0.570±0.082,均低于细胞MCF10A(1.012±0.051);miR-4319相对表达量MDA-MB-231/miR-4319组(3.980±0.083)高于MDA-MB-231/NC组(1.009±0.058),差异有统计学意义,t=102.90,P<0.001。荧光素酶实验结果显示,pGL3-USP2 3’-UTR-WT报告载体与miR-4319质粒共转染后的荧光素酶活性下降,差异有统计学意义,t=15.740,P<0.001。qRT-PCR结果显示,USP2 mRNA相对表达量MDA-MB-231/NC组(1.013±0.058)和MDA-MB-231/miR-4319组(0.988±0.062)基本没有变化,差异无统计学意义,t=1.201,P=0.296。而蛋白质印迹法结果显示,USP2蛋白相对表达量miR-4319组(0.371±0.083)低于miR-4319/NC组(1.003±0.064),miR-4319/inhibitor组(1.982±0.093)高于inhibitor/NC组(1.104±0.072),USP2蛋白相对表达量的组间比较差异有统计学意义,F=212.4,P<0.001。Transwell侵袭实验结果显示,穿过Matrigel细胞数MDA-MB-231/miR-4319组(58.337±5.972)低于MDA-MB-231/NC组(192.371±5.476),差异有统计学意义,t=29.720,P<0.001;穿过Matrigel细胞数MDA-MB-231/miR-4319+USP2组(167.197±8.292)高于MDA-MB-231/miR-4319+Con组(63.283±10.397),差异有统计学意义,t=14.150,P=0.001。双荧光素酶结果显示,miR-4319/NF-κB-luc组荧光素酶活性(0.324±0.06)低于NC/pRL-TK组(1.024±0.080)、NC/NF-κB-luc组(2.023±0.110)和miR-4319/pRL-TK组(1.109±0.050),组间比较差异有统计学意义,F=237.1,P<0.001;miR-4319+USP2/NF-κB-luc组荧光素酶活性(2.032±0.12)高于miR-4319+USP2/pRL-TK组(1.094±0.100)、miR-4319+Con/pRL-TK组(1.063±0.080)和miR-4319+Con/NF-κB-luc组(0.334±0.050),组间比较差异有统计学意义,F=164.2,P<0.001。结论 miR-4319在乳腺癌细胞中低表达,miR-4319靶向结合USP2,过表达USP2逆转了miR-4319对乳腺癌细胞侵袭能力的抑制作用和NF-κB转录活性的抑制作用。miR-4319通过靶向结合USP2抑制NF-κB信号通路,从而抑制乳腺癌细胞的侵袭。     

Calendering as a direct shaping tool for the continuous production of fixed-dose combination products via co-extrusion

In this study calendering is used as a downstream technique to shape monolithic co-extruded fixed-dose combination products in a continuous way. Co-extrudates with a metoprolol tartrate-loaded sustained-release core and a hydrochlorothiazide-loaded immediate-release coat were produced and immediately shaped into a monolithic drug delivery system via calendering, using chilled rolls with tablet-shaped cavities. In vitro metoprolol tartrate release from the ethylcellulose core of the calendered tablets was prolonged in comparison with the sustained release of a multiparticulate dosage form, prepared manually by cutting co-extrudates into mini-matrices. Analysis of the dosage forms using X-ray micro-computed tomography only detected small differences between the pore structure of the core of the calendered tablet and the mini-matrices. Diffusion path length was shown to be the main mechanism behind the release kinetics. Terahertz pulsed imaging visualized that adhesion between the core and coat of the calendered tablet was not complete and a gradient in coat thickness (varying from 200 to 600 μm) was observed. Modulated differential scanning calorimetry and X-ray diffraction indicated that the solid-state properties of both drugs were not affected by the calendering procedure.