Synthetic lethality in human bladder cancer cells by curcumin via concurrent Aurora A inhibition and autophagy induction.

Combination therapies to induce mixed-type cell death and synthetic lethality have the potential to overcome drug resistance in cancer. In this study, we demonstrated that the curcumin-enhanced cytotoxicity of cisplatin/carboplatin in combination with gemcitabine was associated with Aurora A suppression-mediated G2/M arrest, and thus apoptosis, as well as MEK/ERK-mediated autophagy in human bladder cancer cells. Animal study data confirmed that curcumin combined with cisplatin/gemcitabine reduced tumorigenesis of xenograft in mice and this phenomenon was associated with elevated expressions of p-ERK and reduced p-Aurora A in tumors. Gene analyses using data repositories further revealed that reduced Aurora A expression alone did not significantly elevate the sensitivity of human bladder carcinoma cells to these anticancer drugs. Unlike other major cancer types, human bladder urothelial carcinoma tissue coexpressed higher AURKA and lower MAP1LC3B than normal tissue, and reduced Aurora A and induction of autophagy have been clinically associated with a better prognosis in patients with early but not advanced stage bladder cancer. Therefore, our results suggest that treatment strategies can utilize the synthetic lethal pair to concurrently suppress oncogenic Aurora A and induce autophagy by coadministrating curcumin with anticancer drugs for early-stage bladder cancer with high expression of Aurora A.

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.

Zebrafish heparin-binding neurotrophic factor enhances neurite outgrowth during its development.

Heparin-binding neurotrophic factor (HBNF) is a secreted heparin-binding protein containing highly basic and cysteine-rich amino acid residues. In this study, we cloned the full-length HBNF cDNA from zebrafish and determined its genomic structure by bioinformatics analysis. Zebrafish HBNF gene is composed of five exons and four introns spanning approximately 82kb. RT-PCR analysis revealed that zebrafish HBNF transcript was highly expressed in adult brain and intestine tissues while less in other tissues. During embryogenesis, zebrafish HBNF transcript was observed to be moderately expressed at earlier stages with a gradual decline. Higher expression level was observed after hatching and maintaining this level into adulthood. The overall amino acid sequence of zebrafish HBNF shows 60% identity to human HBNF, but with approximately 40% identity to other midkine proteins. Like mammalian homolog, zebrafish HBNF could induce significant neurite outgrowth in PC12 cells without NGF stimulation. In addition, zebrafish HBNF was able to enhance extensive neurite outgrowth in zebrafish embryos during embryogenesis. In summary, a feasible in vivo assay for neurite outgrowth was established in zebrafish.

Identification of 5'-upstream region of pufferfish ribosomal protein L29 gene as a strong constitutive promoter to drive GFP expression in zebrafish.

The genomic structure of Tetraodon fluviatilis L29 gene was determined and its promoter activity was analyzed in COS-1 cells and zebrafish embryos. The TfL29 gene comprises four exons and three introns, spanning approximately 1.7kb. The 5(')-upstream 2.2-kb of the first exon contains 10 E-boxes and many putative binding motifs for transcription factors GATA-1, AML-1a, c-Myb, Oct-1, CdxA, and NRF-2. Promoter activity assay showed that the distal 2.2-kb fragment not only had high luciferase activity in COS-1 cells, but also strong and ubiquitous GFP expression in a variety of tissues in zebrafish embryos. On the other hand, there are no TATA or CAAT boxes within a 300-bp region upstream from the transcription initiation site. Although this region has high luciferase activity in COS-1 cells, it is not sufficient to drive GFP expression in zebrafish embryos. In this proximal 300-bp region, there are two E-boxes, two CdxA sites, and one NRF-2 site that is immediately downstream of the transcription start site.

Cloning of zebrafish BAD, a BH3-only proapoptotic protein, whose overexpression leads to apoptosis in COS-1 cells and zebrafish embryos.

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.