Predicting Agents That Can Overcome 5-FU Resistance in Colorectal Cancers via Pharmacogenomic Analysis.

5-Fluorouracil (5-FU) is one of several chemotherapeutic agents in clinical use as a standard of care to treat colorectal cancers (CRCs). As an antimetabolite, 5-FU inhibits thymidylate synthase to disrupt the synthesis and repair of DNA and RNA. However, only a small proportion of patients benefit from 5-FU treatment due to the development of drug resistance. This study applied pharmacogenomic analysis using two public resources, the Genomics of Drug Sensitivity in Cancer (GDSC) and the Connectivity Map, to predict agents overcoming 5-FU resistance in CRC cells based on their genetic background or gene expression profile. Based on the genetic status of adenomatous polyposis coli (APC), the most frequent mutated gene found in CRC, we found that combining a MEK inhibitor with 5-FU exhibited synergism effects on CRC cells with APC truncations. While considering the gene expression in 5-FU resistant cells, we demonstrated that targeting ROCK is a potential avenue to restore 5-FU response to resistant cells with wild-type APC background. Our results reveal MEK signaling plays a pivotal role in loss-of-function, APC-mediated 5-FU resistance, and ROCK activation serves as a signature in APC-independent 5-FU resistance. Through the use of these available database resources, we highlight possible approaches to predict potential drugs for combinatorial therapy for patients developing resistance to 5-FU treatment.

Transgenic expression of salmon delta-5 and delta-6 desaturase in zebrafish muscle inhibits the growth of Vibrio alginolyticus and affects fish immunomodulatory activity.

Marine fish are an important nutritional source for highly polyunsaturated fatty acids (PUFAs). PUFA biosynthesis requires the following key enzymes: delta-4 (Δ-4) desaturase, delta-5 (Δ-5) desaturase, delta-6 (Δ-6) desaturase, delta-5 (Δ-5) elongase, and delta-6 (Δ-6) elongase. The effect of overexpressing delta-5 desaturase and/or delta-6 desaturase in zebrafish muscle has not previously been reported. Herein, we investigated the effects of these proteins on antibacterial and immunomodulatory activity in transgenic zebrafish infected with Vibrio alginolyticus. Overexpression of delta-5 and delta-6 desaturase enhanced antibacterial activity at 4 and 12 h after injection of bacteria into muscle, as compared to controls. Furthermore, expression of immune-related genes (IL-1ß, IL-22, and TNF-α) was observed to be altered in transgenic fish after 4 h of bacterial infection, resulting in a significant decrease in the inflammatory response, as compared to control fish. These results demonstrate that muscle-specific expression of transgenic desaturases in zebrafish not only enhance PUFA production, but also enhance antibacterial and anti-inflammatory activity. Overall, these results identify delta-5 and delta-6 desaturase as novel candidate genes for use in aquaculture, to enhance both disease resistance and fish oil production.

Expression characterization and promoter activity analysis of the tilapia (Oreochromis niloticus) myosin light chain 3 promoter in skeletal muscle of fish.

A tilapia (Oreochromis niloticus) myosin light chain 3 (Mlc3) promoter region (~4.3 kb) was isolated and characterized. Sequence analysis of the clone revealed high similarity with a tilapia gene encoding the Mlc3 promoter region, exon 1, and intron 1. The clone contained several putative binding sequences for transcription factors, including MEF-2, MYOG, MyoD, PKNOX1, and AREB6. Deletion of a region of the tilapia Mlc3 promoter (801 to -3,881 bp) enhanced promoter activity, as determined by direct injection of a luciferase reporter construct into skeletal muscle of Archocentrus nigrofasciatus. These findings suggest that the region between -801 and -3,881 bp may contain negative regulatory elements. Stable germline transgenic strains of the ornamental fish species A. nigrofasciatus var. carrying the Taiwan coral red fluorescent protein (TcRFP) driven by the Mlc3 promoter were established. F1 adult transgenic A. nigrofasciatus var. exhibited brilliant pink fluorescence in skeletal muscles in the daylight. Therefore, our current study demonstrates the feasibility of using the tilapia Mlc3 promoter to drive fluorescence in new fish species, such as Perciformes.

Characterization of tilapia (Oreochromis niloticus) viperin expression, and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle.

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.

Five different piscidins from Nile tilapia, Oreochromis niloticus: analysis of their expressions and biological functions.

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.

Transgenic expression of tilapia hepcidin 1-5 and shrimp chelonianin in zebrafish and their resistance to bacterial pathogens.

Recently, tilapia hepcidin (TH)1-5 was characterized, and its antimicrobial functions against several pathogens were reported. The antimicrobial functions of another shrimp antimicrobial peptide (AMP), chelonianin, were also characterized using a recombinant chelonianin protein (rcf) that was expressed by a stably transfected Chinese hamster ovary (CHO) cell line against pathogen infections in fish. The function of the overexpression of both AMPs in zebrafish muscles was not examined in previous studies. Herein, we investigated the antimicrobial functions of TH1-5 and chelonianin against Vibrio vulnificus (204) and Streptococcus agalactiae (SA48) in transgenic TH1-5 zebrafish and transgenic chelonianin zebrafish. The presence of TH1-5 and chelonianin enhanced the inhibitory ability in transgenic AMP zebrafish against the two different bacterial infections. The bacterial number of either V. vulnificus (204) or S. agalactiae (SA48) had decreased at 96 h after injection into transgenic AMP zebrafish muscle compared to non-transgenic zebrafish muscle. Additionally, immune-related gene expressions analyzed by real-time PCR studies showed the modulation of several genes including interleukin (IL)-10, IL-22, IL-26, MyD88, Toll-like receptor (TLR)-1, TLR-3, TLR-4, nuclear factor (NF)-κB, tumor necrosis factor (TNF)-α, and lysozyme, and significant differences were found between transgenic AMP zebrafish and wild-type zebrafish injected with PBS at 1-24 h. These results suggest that several immune-related gene expressions were induced in transgenic TH1-5 and chelonianin zebrafish which effectively inhibited bacterial growth. The survival rate dropped to 86.6% in transgenic chelonianin zebrafish after 28 days of infection compared of the 50% survival rate in transgenic TH1-5 zebrafish after 28 days of infection. Overall, these results indicate that TH1-5 and chelonianin possess the potential to be novel candidate genes for aquaculture applications to treat fish diseases.

Using an improved Tol2 transposon system to produce transgenic zebrafish with epinecidin-1 which enhanced resistance to bacterial infection.

In order to advance the application of antimicrobial peptides in aquaculture, transgenic zebrafish expressing the antimicrobial peptide, epinecidin-1, were developed and are reported on here. First, we cloned the zebrafish mylz2 promoter for this purpose. To characterize the activity of the mylz2 promoter, various fragments of it were analyzed using a firefly luciferase transient expression assay, in which maximum promoter activity was found with a 2.5-kb fragment. In addition, the 2.5-kb fragment also expressed considerable red fluorescent proteins in skeletal muscles of transgenic zebrafish. Second, in order to improve the translation efficiency of the Tol2 transposase, we constructed untranslated regions (UTRs) of zebrafish ba1 globin flanked by a transposase. A transient embryonic excision assay (TEEA) and in vivo fluorescent observations showed high transposition efficiency during embryonic development. After optimization of the promoter and transgene efficiencies, transgenic zebrafish with the Epi-1/DsRed plasmid (pTLR-m2.5 K-K.Epinecidin-1/DsRed vector) were developed, and expressions of Epi-1/DsRed in muscles and blood were demonstrated by immunohistochemical staining techniques. Moreover, we also found that the Epi-1/DsRed gene was efficiently and significantly expressed in vivo against Vibrio vulnificus and Streptococcus agalactiae after injecting the bacteria and determining bacterial counts. A gene expression study using real-time RT-PCR revealed that Epi-1/DsRed itself induced endogenous MyD88 expression in vivo. After Epi-1/DsRed transgenic zebrafish were infected with V. vulnificus 204, interleukin (IL)-10, IL-22, IL-26, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, MyD88, and nuclear factor (NF)-kappaB activating protein-like were upregulated, but IL-1beta and tumor necrosis factor-alpha were downregulated at 12h post-infection; IL-21, complement component c3b, and NF-kappaB activating protein-like were downregulated, but MyD88 was upregulated at 24h post-infection. These results suggest that using epinecidin-1 as a transgene in zebrafish can effectively inhibit bacterial growth for up to 24h after infection.

Stable expression in a Chinese hamster ovary (CHO) cell line of bioactive recombinant chelonianin, which plays an important role in protecting fish against pathogenic infection.

Chelonianin, originally isolated from the shrimp (Penaeus monodon), exhibits antimicrobial effects in vitro and in vivo and is used to treat infectious fish diseases. Herein, we report that the recombinant chelonianin protein fused to a fluorescent protein (rcf protein) was expressed from a stably transfected Chinese hamster ovary (CHO) cells. The in vitro experiments showed that the rcf protein exhibited antimicrobial activity against several bacteria, while the recombinant fluorescent protein alone did not. In addition, pretreatment and post-treatment with the rcf protein were both effective in promoting a significant decrease in fish mortality and decreasing the number of infectious bacteria. We utilized the quantitative reverse-transcriptase polymerase chain reaction technique to survey the levels of gene expressions of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide synthase 1 induced in response to bacterial infection in experiments with tilapia (Oreochromis mossambicus). Our results indicated that the rescue of fish treated with the rcf protein may involve regulation of TNF-alpha expression. Collectively, chelonianin inhibited the production of an inflammatory mediator and reduced mortality in fish during bacterial challenge, suggesting that it has potential as a therapeutic or prophylactic drug for use against bacterial infectious diseases.