Stepwise candidate drug screening for myopia control by using zebrafish, mouse, and Golden Syrian Hamster myopia models.

BACKGROUND: We developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia. METHODS: A morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-ß2 (TGF-ß2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice. FINDINGS: In the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes. INTERPRETATION: Stepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials. FUNDING: This research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare.

Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.

Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer.