A novel hybrid drug between two potent anti-tubulin agents as a potential prolonged anticancer approach

We report the design, synthesis and biological characterisation of a novel hybrid drug by conjugation of two tubulin inhibitors, a hemiasterlin derivative A (H-Mpa-Tle-Aha-OH), obtained by condensation of three non-natural amino acids, and cis-3,4',5-trimethoxy-3'aminostilbene (B). As we have previously demonstrated synergy between A and B, we used a monocarbonyl derivative of triethylene glycol as linker (L) to synthesise compounds A-L and A-L-B; via HPLC we analysed the release of its potential hydrolysis products A, A-L, B and B-L in physiological fluids: the hybrid A-L-B undergo hydrolysis in rat whole blood of the ester bond between A and L (half-life = 118.2 +/- 9.5 min) but not the carbamate bond between B and L; the hydrolysis product B-L was further hydrolyzed, but with a slower rate (half-life = 288 +/- 12 min). The compound A-L was the faster hydrolyzed conjugate (half-life = 25.4 +/- 1.1 min). The inhibitory activity of the compounds against SKOV3 ovarian cancer cell growth was analysed. The IC50 values were 7.48 +/- 1.27 nM for A, 40.3 +/- 6.28 nM for B, 738 +/- 38.5 nM for A-L and 37.9 +/- 2.11 nM for A-L-B. The anticancer effect of A-L-B was evidenced to be obtained via microtubule dynamics suppression. Finally, we stated the expression of the active efflux transporters P-gp (ABCB1) and MRP1 (ABCC1) in the human normal colon epithelial NCM460 cell line by reverse-transcription PCR. Via permeation studies across NCM460 monolayers we demonstrate the poor aptitude of A to interact with active efflux transporters (AET): indeed, the ratio between its permeability coefficients for the basolateral (B). apical (A) and B. A transport was 1.5 +/- 0.1, near to the ratio of taltobulin (1.12 +/- 0.06), an hemiasterlin derivative able to elude AETs, and significantly different form the ratio of celiprolol (3.4 +/- 0.2), an AET substrate. (C) 2016 Elsevier B.V. All rights reserved

Effects of lipopolysaccharide on the expression of plasma membrane monoamine transporter (PMAT) at the blood-brain barrier and its implications to the transport of neurotoxins

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter that is highly expressed in the central nervous system. This study aimed to investigate the effect of lipopolysaccharide on PMAT expression at the blood-brain barrier and the interaction between PMAT and neurotoxins. As a result, PMAT mRNA was identified in brain microvessels (BMVs), brain microvascular endothelial cells (BMECs), astrocytes, and pericytes isolated from C57BL/6 mice and/or Wistar rats using RT-qPCR. The immunofluorescence staining confirmed the expression of PMAT protein in BMVs and striatum of C57BL/6 mice. Western blotting demonstrated its localization at the luminal and abluminal sides of BMECs. In C57BL/6 mice, PMAT protein was significantly increased in BMVs 24 h after an intraperitoneal injection of 3 mg/kg lipopolysaccharide. Lipopolysaccharide treatment also significantly increased PMAT expression in cerebral cortex and the striatum in a time-dependent manner, as well as the brain-to-plasma ratio of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-benzyl-TIQ). In isolated cells, lipopolysaccharide treatment significantly increased PMAT mRNA in brain astrocytes and the BMECs co-cultured with astrocytes. In addition to 1-methyl-4-phenylpyridinium, the kinetic study indicated that both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-benzyl-TIQ are substrates of human PMAT. These findings suggest that inflammation can change PMAT expression at the blood-brain barrier, which may affect PMAT-mediated transport of neurotoxins

Reevesioside A, a Cardenolide Glycoside, Induces Anticancer Activity against Human Hormone-Refractory Prostate Cancers through Suppression of c-myc Expression and Induction of G1 Arrest of the Cell Cycle

In the past decade, there has been a profound increase in the number of studies revealing that cardenolide glycosides display inhibitory activity on the growth of human cancer cells. The use of potential cardenolide glycosides may be a worthwhile approach in anticancer research. Reevesioside A, a cardenolide glycoside isolated from the root of Reevesia formosana, displayed potent anti-proliferative activity against human hormone-refractory prostate cancers. A good correlation (r(2) = 0.98) between the expression of Na+/K+-ATPase alpha(3) subunit and anti-proliferative activity suggested the critical role of the alpha(3) subunit. Reevesioside A induced G1 arrest of the cell cycle and subsequent apoptosis in a thymidine block-mediated synchronization model. The data were supported by the down-regulation of several related cell cycle regulators, including cyclin D1, cyclin E and CDC25A. Reevesioside A also caused a profound decrease of RB phosphorylation, leading to an increased association between RB and E2F1 and the subsequent suppression of E2F1 activity. The protein and mRNA levels of c-myc, which can activate expression of many downstream cell cycle regulators, were dramatically inhibited by reevesioside A. Transient transfection of c-myc inhibited the down-regulation of both cyclin D1 and cyclin E protein expression to reevesioside A action, suggesting that c-myc functioned as an upstream regulator. Flow cytometric analysis of JC-1 staining demonstrated that reevesioside A also induced the significant loss of mitochondrial membrane potential. In summary, the data suggest that reevesioside A inhibits c-myc expression and down-regulates the expression of CDC25A, cyclin D1 and cyclin E, leading to a profound decrease of RB phosphorylation. G1 arrest is, therefore, induced through E2F1 suppression. Consequently, reevesioside A causes mitochondrial damage and an ultimate apoptosis in human hormone-refractory prostate cancer cells

Zerumbone, a ginger sesquiterpene, induces apoptosis and autophagy in human hormone-refractory prostate cancers through tubulin binding and crosstalk between endoplasmic reticulum stress and mitochondrial insult

Zerumbone, a natural monocyclic sesquiterpene, is the main component of the tropical plant Zingiber zerumbet Smith. Zerumbone induced antiproliferative and apoptotic effects against PC-3 and DU-145, two human hormone-refractory prostate cancer (HRPC) cell lines. Zerumbone inhibited microtubule assembly and induced an increase of MPM-2 expression (specific recognition of mitotic proteins). It also caused an increase of phosphorylation of Bcl-2 and Bcl-xL, two key events in tubulin-binding effect, indicating tubulin-binding capability and mitotic arrest to zerumbone action. Furthermore, zerumbone induced several cellular effects distinct from tubulin-binding properties. First, zerumbone significantly increased, while paclitaxel (as a tubulin-binding control) decreased, Mcl-1 protein expression. Second, paclitaxel but not zerumbone induced Cdk1 activity. Third, zerumbone other than paclitaxel induced Cdc25C downregulation. The data suggest that, in addition to targeting tubulin/microtubule, zerumbone may act on other targets for signaling transduction. Zerumbone induced mitochondrial damage and endoplasmic reticulum (ER) stress as evidenced by the loss of mitochondrial membrane potential and upregulation of GRP-78 and CHOP/GADD153 expression. Zerumbone induced an increase of intracellular Ca2+ levels, a crosstalk marker between ER stress and mitochondrial insult, associated with the formation of active calpain I fragment. It induced apoptosis through a caspase-dependent way and caused autophagy as evidenced by dramatic LC3-II formation. In summary, the data suggest that zerumbone is a multiple targeting compound that inhibits tubulin assembly and induces a crosstalk between ER stress and mitochondrial insult, leading to apoptosis and autophagy in HRPCs

Can autism be screened using Chinese CHAT in Hong Kong?

published_or_final_versionMedical SciencesMasterMaster of Medical Science

Repurposing of nitroxoline as a potential anticancer agent against human prostate cancer - a crucial role on AMPK/mTOR signaling pathway and the interplay with Chk2 activation

Nitroxoline is an antibiotic by chelating Zn2+ and Fe2+ from biofilm matrix. In this study, nitroxoline induced G1 arrest of cell cycle and subsequent apoptosis in prostate cancer cells through ion chelating-independent pathway. It decreased protein levels of cyclin D1, Cdc25A and phosphorylated Rb, but activated AMP-activated protein kinase (AMPK), a cellular energy sensor and signal transducer, leading to inhibition of downstream mTOR-p70S6K signaling. Knockdown of AMPKa significantly rescued nitroxoline-induced inhibition of cyclin D1-Rb-Cdc25A axis indicating AMPK-dependent mechanism. However, cytoprotective autophagy was simultaneously evoked by nitroxoline. Comet assay and Western blot analysis demonstrated DNA damaging effect and activation of Chk2 other than Chk1 to nitroxoline action. Instead of serving as a DNA repair transducer, nitroxoline-mediated Chk2 activation was identified to function as a pro-apoptotic inducer. In conclusion, the data suggest that nitroxoline induces anticancer activity through AMPK-dependent inhibition of mTOR-p70S6K signaling pathway and cyclin D1-Rb-Cdc25A axis, leading to G1 arrest of cell cycle and apoptosis. AMPK-dependent activation of Chk2, at least partly, contributes to apoptosis. The data suggest the potential role of nitroxoline for therapeutic development against prostate cancers

Hemiasterlin derivative (R)(S)(S)-BF65 and Akt inhibitor MK-2206 synergistically inhibit SKOV3 ovarian cancer cell growth

We reported previously that a hemiasterlin derivative BF65 is a potent anticancer agent that can inhibit microtubule assembly. Here we show that a more potent stereospecific diastereomer (R)(S)(S)-BF65 can synergize with an allosteric Akt inhibitor MK-2206 to suppress the growth of SKOV3 ovarian cancer cells with constitutively active Akt. (R)(S)(S)-BF65 induced mitotic arrest and MK-2206 caused G0/G1 arrest, while the combination of both induced simultaneous G0/G1 and G2/M cell cycle arrest. (R)(S)(S)-BF65 induced phosphorylation and inactivation of Bcl-2, and downregulated Mcl-1, consequently may lead to apoptosis. (R)(S)(S)-BF65 inhibited mitogen-activated protein kinases (MAPKs), which may stimulate cell proliferation upon activation. (R)(S)(S)-BF65 also induced DNA damage after long-term treatment. MK-2206 is known to inhibit phosphorylation and activation of Akt and suppress cancer cell growth. The combination of (R)(S)(S)-BF65 and MK-2206 also inhibited the Akt pathway. Interestingly, MK-2206 upregulated Bcl-2 and induced activation of MAPKs in SKOV3 cells; however, when combined with (R)(S)(S)-BF65, these prosurvival effects were reversed. The combination also more significantly decreased Mcl-1 protein, increased PARP cleavage, and induced gamma-H2AX, a DNA damage marker. Remarkably, MK-2206 enhanced the microtubule depolymerization effect of (R)(S)(S)-BF65. The combination of (R)(S)(S)-BF65 and MK-2206 also markedly inhibited cell migration. Thus, MK-2206 synergizes with (R)(S)(S)-BF65 to inhibit SKOV3 cell growth via downregulating the Akt signaling pathway, and enhancing the microtubule disruption effect of (R)(S)(S)-BF65. (R)(S)(S)-BF65 in turn suppresses Bcl-2 and MAPKs induced by MK-2206. (R)(S)(S)-BF65 and MK-2206 compensate each other leading to increased apoptosis and enhanced cytotoxicity, and may also suppress cancer cell invasion. (C) 2016 Elsevier Inc. All rights reserved

The K898E germline variant in the PP1-binding motif of BRCA1 causes defects in DNA Repair

BRCA1 is a phosphoprotein involved in many biological processes, including transcription, ubiquitination, checkpoint control, homologous recombination, and DNA repair. We have demonstrated that protein phosphatase 1 alpha (PP1 alpha) interacts with BRCA1 via a PP1-binding motif (KVTF901)-K-898, and can dephosphorylate multiple serine residues phosphorylated by checkpoint kinases. A K898E germline missense variant in the PP1-binding motif of BRCA1 has been found in an Ashkenazi patient and a non-Ashkenazi Argentinean patient with breast and ovarian cancer, but its clinical significance is still unknown. Here we report that the lysine residue in the PP1-binding motif of BRCA1 is highly conserved across many mammalian species. The K898E mutation interferes with the interaction between BRCA1 and PP1 alpha. Moreover, while the expression of wild-type BRCA1 in Brca1-deficient cells improved cell survival after DNA damage induced by ionizing radiation (IR), expression of BRCA1 K898E proved unable to enhance cell survival. DNA damage repair mechanisms remained defective in these BRCA1 K898E-reconstituted cells, as revealed by the comet assay and IR-induced Rad51 foci formation assay. These results reflect the significance of the interaction between BRCA1 and PP1, and indicate that the K898E variant may render carriers susceptible to DNA damage and malignant transformation