Design, synthesis and biological evaluation of novel osimertinib derivatives as reversible EGFR kinase inhibitors.

A series of osimertinib derivatives without acrylamide groups were synthesized and their inhibitory rates against L858R/T790M/C797S mutated EGFR kinase and antiproliferation activities against non-small cell lung cancer cell lines (A549, H1975) were evaluated. The preferred compounds were selected and their in vitro inhibitory activities against various EGFR kinases (wild-type, L858R/T790M, L858R/T790M/C797S) and c-Met kinase were tested. Compound 9h showed remarkable inhibitory activity against the wild type (IC50 = 29 nM), L858R/T790M mutant type (IC50 = 10 nM) and L858R/T790M/C797S mutant type (IC50 = 242 nM) as reversible EGFR kinase inhibitor, which was selected to further perform the AO/EB staining assays, cell cycle distribution assays and wound-healing assays on A549 and/or H1975 cell lines. The results showed dose-dependent activities of the induction of cell apoptosis, G1/G0-phase arrestation and inhibition of migration. Compound 22a showed remarkable inhibitory activity against the L858R/T790M/C797S mutant EGFR kinase (IC50 = 137 nM), which was nearly three times compared to osimertinib (IC50 = 410 nM). It's worth noting that 22a exhibited excellent kinase selectivity against the L858R/T790M/C797S mutant EGFR kinase rather than the wild-type, which reached 5.4 times and far more than the 0.012 times of osimertinib. Additionally, molecular docking analyses were performed to explain the action modes between the compounds and the corresponding EGFR kinases. In conclusion, compounds 9h and 22a have been demonstrated as promising candidates and worth further study.

Design, synthesis and biological evaluation of novel N-(3-amino-4-methoxyphenyl)acrylamide derivatives as selective EGFRL858R/T790M kinase inhibitors.

On the basis of N-(3-amino-4-methoxyphenyl)acrylamide scaffold, a series of novel compounds containing 3-substitutional-1-methyl-1H-indole, 2-substitutional pyrrole or thiophene moieties were synthesized and their in vitro antiproliferation activities against A549 and H1975 cell lines were evaluated. The results indicated that most of the compounds showed moderate to excellent antitumor activities. Especially, compounds 9a (A549 IC50 = 1.96 µM, H1975 IC50 = 0.095 µM), 17i (A549 IC50 = 4.17 µM, H1975 IC50 = 0.052 µM), 17j (A549 IC50 = 1.67 µM, H1975 IC50 = 0.061 µM) exhibited comparable antitumor activities and selectivity ratios compared to the positive control osimertinib (A549 IC50 = 2.91 µM, H1975 IC50 = 0.064 µM). In vitro inhibitory activities against EGFR kinases containing different mutations were also tested. Compound 17i showed remarkable inhibitory activity (with IC50 value of 1.7 nM) to EGFRL858R/T790M kinase and selectivity (22-folds compared to EGFRWT kinase). Furthermore, acridine orange/ethidium bromide (AO/EB) staining assay, cell apoptosis assay, cell cycle distribution assay and wound-healing assay of the compounds 9a and 17i were performed on H1975 cell line. The results showed dose-dependent activities of the induction of apoptosis, G0/G1-phase arrestation and inhibition of migration, which were similar to the positive control osimertinib. Additionally, molecular docking analysis was performed to seek the possible binding mode between the selected compounds (9a, 17i-17j) and EGFRL858R/T790M kinase. The results demonstrated that compound 17i is a promising candidate and worth further study.

Starvation-induced autophagy promotes the invasion and migration of human bladder cancer cells via TGF-ß1/Smad3-mediated epithelial-mesenchymal transition activation.

The biological characteristics of bladder cancer include enhanced invasion and migration, which are the main causes of death in patients. Starvation is a typical feature of the bladder cancer microenvironment and can induce autophagy. Autophagy has an important relationship with the invasion and migration of tumors. However, the role of autophagy in the invasion and migration of bladder cancer cells remains unclear. Hence, the aim of the current study was to clarify this role and underlying mechanism. In this study, we found that starvation enhanced the epithelial-mesenchymal transition (EMT)-mediated invasion and migration of T24 and 5637 cells while inducing autophagy. The inhibition of autophagy with chloroquine (CQ) or 3-methyladenine (3MA) decreased EMT-mediated invasion and migration. In addition, the expression of transforming growth factor 1 (TGF-ß1) and phosphorylated Smad3 (p-Smad3) increased after starvation. The inhibition of autophagy with CQ or 3MA also decreased the expression of TGF-ß1 and p-Smad3. The inhibitor of TGF-ß receptor sb431542 also inhibited the invasion, migration, and EMT of T24 and 5637 cells during starvation. Furthermore, recombinant TGF-ß1 induced autophagy and inhibition of the TGF-ß/Smad signaling pathway with sb431542 suppressed autophagy. In summary, our results suggested that autophagy promotes the invasion and migration of bladder cancer cells by inducing EMT through the TGF-ß1/Smad3 signaling pathway. Moreover, autophagy and TGF-ß1 can form a positive feedback loop to synergistically promote invasion and migration. Thus, our findings may provide a theoretical basis for the prevention of invasion and migration in bladder cancer.

Laparoscopic vesiculectomy for large seminal vesicle cystadenoma.

Cystadenomas of the seminal vesicles are extremely rare. Here, we report a large seminal vesicle cystadenoma. A 37-year-old man presented a 6-month history of haemospermia, 10 days of Lower Urinary Tract symptoms (LUTSs) and gross haematuria. Transabdominal ultrasonography, computed tomography and magnetic resonance imaging were performed and revealed a large solid-cystic pelvic mass morphometrically measured 7.0 cm × 11.9 cm × 8.6 cm on the right seminal vesicle, which caused hydronephrosis of the right kidney. The prostate-specific antigen of the patient was 27.860 ng/dl. Laparoscopic exploration found the capsule of tumour was complete and the tumour came from the right seminal vesicle, in addition, the mass had a certain space with the bladder and prostate, which could be separated. So a nerve-sparing Laparoscopic Vesiculectomy was performed at last, even though the intraoperative frozen section analysis could not make sure the nature of the tumour either. The postoperative pathology revealed cystadenoma of the seminal vesicle.

Activation of a c-Jun N-terminal kinase-mediated autophagy pathway attenuates the anticancer activity of gemcitabine in human bladder cancer cells.

The role of autophagy in the anticancer activity of gemcitabine (GEM) in bladder cancer is unclear. The aim of this study is to determine whether GEM activates autophagy, the role of autophagy in the anticancer activity of GEM, and the underlying mechanism by which GEM induces autophagy. Human bladder cancer cell lines T24 and BIU87 were treated with GEM in vitro. Cell viability was measured using the Cell Counting Kit-8 assay. Apoptosis was detected by annexin V assay and western blot. Autophagy was measured by western blot and transmission electron microscopy. c-Jun N-terminal kinase (JNK) activation was detected by western blot. Chemical inhibitors were used for intervention of JNK and autophagy. GEM killed bladder cancer cells, which was associated with apoptosis induction. Autophagy was effectively activated by GEM. Suppressing autophagy in GEM-treated cells significantly decreased cell viability, which was associated with increased apoptosis. GEM-induced JNK activation and suppressed B-cell lymphoma 2 expression. The JNK inhibitor SP600125 inhibited GEM-induced autophagy activation and increased GEM's cytotoxicity. GEM kills bladder cancer cells through apoptosis. Meanwhile, JNK-mediated autophagy was activated, which protects the cells against apoptosis. Therefore, inhibition of autophagy could be exploited to enhance the anticancer efficacy of GEM for treating bladder cancer.

Effects of elevated CO2 concentration and nitrogen supply on biomass and active carbon of freshwater marsh after two growing seasons in Sanjiang Plain, Northeast China.

An experiments were carried out with treatments differing in nitrogen supply (0, 5 and 15 g N/m2) and CO2 levels (350 and 700 micromol/mol) using OTC (open top chamber) equipment to investigate the biomass of Calamagrostis angustifolia and soil active carbon contents after two years. The results showed that elevated CO2 concentration increased the biomass of C. angustifolia and the magnitude of response varied with each growth period. Elevated CO2 concentration has increased aboveground biomass by 16.7% and 17.6% during the jointing and heading periods and only 3.5% and 9.4% during dough and maturity periods. The increases in belowground biomass due to CO2 elevation was 26.5%, 34.0% and 28.7% during the heading, dough and maturity periods, respectively. The responses of biomass to enhanced CO2 concentrations are differed in N levels. Both the increase of aboveground biomass and belowground biomass were greater under high level of N supply (15 g N/m2). Elevated CO2 concentration also increased the allocation of biomass and carbon in root. Under elevated CO2 concentration, the average values of active carbon tended to increase. The increases of soil active soil contents followed the sequence of microbial biomass carbon (10.6%) > dissolved organic carbon (7.5%) > labile oxidable carbon (6.6%) > carbohydrate carbon (4.1%). Stepwise regressions indicated there were significant correlations between the soil active carbon contents and plant biomass. Particularly, microbial biomass carbon, labile oxidable carbon and carbohydrate carbon were found to be correlated with belowground biomass, while dissolved organic carbon has correlation with aboveground biomass. Therefore, increased biomass was regarded as the main driving force for the increase in soil active organic carbon under elevated CO2 concentration.