Mechanistic characterization of disulfide bond reduction of an ERAD substrate mediated by cooperation between ERdj5 and BiP.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase family member that accelerates ERAD by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates. Biochemical analyses using purified proteins revealed that J-chain oligomers were reduced to monomers by ERdj5 in a stepwise manner via trimeric and dimeric intermediates, and BiP synergistically enhanced this action in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using high-speed atomic force microscopy, demonstrated the stochastic release of small J-chain oligomers through repeated actions of ERdj5 on peripheral and flexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected at the molecular level.

Development and validation of a rapid LC-MS/MS method for the determination of JCC76, a novel antitumor agent for breast cancer, in rat plasma and its application to a pharmacokinetics study.

JCC76 is a novel nimesulide analog that selectively inhibits the human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer cell proliferation and tumor progression. To support further pharmacological and toxicological studies of JCC76, a novel and rapid method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated for the quantification of the compound in rat plasma. A C18 column was used for chromatographic separation, and the mobile phase was aqueous ammonium formate (pH 3.7; 5 mm)-methanol (1:9, v/v) with an isocratic elution. With a simple liquid-liquid extraction procedure using the mixture of methyl tert-butyl ether-hexane (1:2, v/v), the mean extraction efficiency of JCC76 in rat plasma was determined as 89.5-97.3% and no obvious matrix effect was observed. This method demonstrated a linear calibration range from 0.3 to 100 ng/mL for JCC76 in rat plasma and a small volume of sample consumption. The intra- and inter-assay accuracy and precision were within ±10%. The pharmacokinetics of JCC76 was also profiled using this validated method in rats. In conclusion, this rapid and sensitive method has been proven to effectively quantify JCC76 for pharmacokinetics study.

From COX-2 inhibitor nimesulide to potent anti-cancer agent: synthesis, in vitro, in vivo and pharmacokinetic evaluation.

Cyclooxygenase-2 (COX-2) inhibitor nimesulide inhibits the proliferation of various types of cancer cells mainly via COX-2 independent mechanisms, which makes it a good lead compound for anti-cancer drug development. In the presented study, a series of new nimesulide analogs were synthesized based on the structure-function analysis generated previously. Some of them displayed very potent anti-cancer activity with IC(50)s around 100 nM-200 nM to inhibit SKBR-3 breast cancer cell growth. CSUOH0901 (NSC751382) from the compound library also inhibits the growth of the 60 cancer cell lines used at National Cancer Institute Developmental therapeutics Program (NCIDTP) with IC(50)s around 100 nM-500 nM. Intraperitoneal injection with a dosage of 5  mg/kg/d of CSUOH0901 to nude mice suppresses HT29 colorectal xenograft growth. Pharmacokinetic studies demonstrate the good bioavailability of the compound.

Nuclear accumulation of an uncapped RNA produced by Drosha cleavage of a transcript encoding miR-10b and HOXD4.

Patterning of the animal embryo's antero-posterior (AP) axis is dependent on spatially and temporally regulated Hox gene expression. The murine Hoxd4 gene has been proposed to harbour two promoters, an upstream promoter P2, and a downstream promoter P1, that lie 5.2 and 1.1 kilobase pairs (kb) upstream of the coding region respectively. The evolutionarily conserved microRNA-10b (miR-10b) gene lies in the Hoxd4 genomic locus in the intron separating the non-coding exons 4 and 5 of the P2 transcript and directly adjacent to the proposed P1 promoter. Hoxd4 transcription is regulated by a 3' neural enhancer that harbours a retinoic acid response element (RARE). Here, we show that the expression profiles of Hoxd4 and miR-10b transcripts during neural differentiation of mouse embryonal carcinoma (EC) P19 cells are co-ordinately regulated, suggesting that both Hoxd4 and miR-10b expression is governed by the neural enhancer. Our observation that P1 transcripts are uncapped, together with the mapping of their 5' ends, strongly suggests that they are generated by Drosha cleavage of P2 transcripts rather than by transcriptional initiation. This is supported by the colocalization of P1 and P2 transcripts to the same posterior expression domain in the mouse embryo. These uncapped P1 transcripts do not appear to possess an Internal Ribosomal Entry Site (IRES), but accumulate within multiple punctate bodies within the nucleus suggesting that they play a functional role. Finally, similar uncapped Drosha-cleaved P1-like transcripts originating from the paralogous Hoxb4/miR-10a locus were also identified. We propose that these transcripts may belong to a novel class of regulatory RNAs.

In vitro and in vivo effects of a cyclooxygenase-2 inhibitor nimesulide analog JCC76 in aromatase inhibitors-insensitive breast cancer cells.

Third generation aromatase inhibitors (AIs) are more effective than tamoxifen in the treatment of estrogen receptor (ER) positive breast cancer. However, long-term use of AIs commonly results in resistance. We examined whether compound JCC76{Cyclohexanecarboxylic acid [3-(2,5-dimethyl-benzyloxy)-4-(methanesulfonyl-methyl-amino)-phenyl]-amide}, an analog of Cyclooxygenase-2 (COX-2) inhibitor nimesulide, can inhibit the growth of AI-insensitive breast cancer cells and the mechanisms by which the compound affects cell proliferation. LTEDaro (long term estrogen deprived MCF-7aro cell) cells, which are a model for AI resistance, were used in this study. JCC76 effectively inhibited LTEDaro cell proliferation with an IC(50) of 2.75 ± 0.31 µM. Further investigations reveal that the compound significantly induced apoptosis in LTEDaro cells by decreasing pAKT, BCL-2 and pBad protein levels, which were all up regulated in the cells after long term estrogen deprivation. LTEDaro tumor size and weight were decreased in ovariectomized nude mice treated with the compound, and cell apoptosis in the tumor tissue was increased compared to the control. The animal weight remained almost unchanged which indicated the low toxicity of the compound. These results suggest that JCC76 overcame AI resistance by inducing cell apoptosis as illustrated in the in vitro and in vivo models. Collectively, results from this study provide data to support that nimesulide analog JCC76 may be a new drug candidate to treat AI-resistant breast cancers. It could be also used as a lead to design and synthesize more potent derivatives.

COX-2 inhibitor nimesulide analogs are aromatase suppressors in breast cancer cells.

Cyclooxygenase-2 (COX-2) inhibitor nimesulide derivatives compounds A and B decreased aromatase activity in breast cancer cells via a novel mechanism different to aromatase inhibitors (AIs), and were defined as "aromatase suppressors". Breast carcinoma cells (MCF-7aro and T47Daro) transfected with aromatase full gene were used to explore the mechanisms of the two compounds. They dose and time-dependently suppressed aromatase activity in MCF-7aro and T47Daro cells in the nanomole range. However, they neither directly inhibited aromatase, nor improved aromatase degradation even at much higher concentrations. They could also suppress androgen stimulated cell growth, but did not affect estrogen enhanced cell proliferation. These results suggest that compounds A and B selectively interfere with aromatase in breast cancer cells, but not estrogen receptor (ER) downstream to disrupt androgen mediated cell growth. Interestingly, compound B effectively inhibited LTED (long-term estrogen deprived MCF-7aro cell) cell growth, which is a model for AIs resistance, with an IC(50) of 4.68 ± 0.54 µM. The results indicate that compound B could potentially overcome AI resistance in breast cancer cell and could be used as a lead to design more potent derivatives.