A human leucyl-tRNA synthetase as an anticancer target

Abstract

Several aminoacyl-tRNA synthetases have been reported to be overexpressed for charging essential aminoacyl-tRNAs in many cancer types. In this study, we aimed to explore the potential role of leucyl-tRNA synthetase (LARS) as an anticancer target. MTT assay was performed to screen inhibitors to human LARS (hsLARS) from compounds AN2690 and its derivatives, compounds 1-6, in U2OS and SKOV3 cells. The compound with the strongest inhibitory ability was further investigated for its inhibitory effect in cancer cell lines and in an animal tumor model. Additionally, a LARS-rescue experiment was performed to explore the potential target in U2OS using Western blot and flow cytometry. Luciferase reporter assay was designed to analyze the effect of of hsLARS inhibitor on p21 activation. We identified an hsLARS inhibitor (compound 2) that suppressed the proliferation of U2OS and SKOV3 cells in vitro. A LARS-rescue experiment demonstrated that the proliferation inhibition was induced by targeting intracellular LARS. In addition, the hsLARS inhibition was shown to activate the p21 early transcription and promote cell apoptosis, as well as reduce implanted EMT6 tumor progression in mice. Our results suggest that LARS might serve as a potential anticancer target through the p21 signaling pathway and that the nutritional signaling pathway may provide a valuable anticancer strategy for further investigation.

Keywords: anticancer target; apoptosis; leucyl-tRNA synthetase; mouse model.

Figure 1

Identification of inhibitors against hsLARS and their suppression of tumor cell proliferation. Notes: Selected tbLARS inhibitors were tested for their inhibition against hsLARS and 2 human cancer cell lines in vitro, with the concentration of all the compounds at 100 μM. The inhibition rate of the compounds against tbLARS and hsLARS was calculated through an aminoacylation assay (A). The inhibition rate against U2OS cells and SKOV3 cells was calculated according to MTT assay results (B). Compound 2 inhibited the proliferation of U2OS cells in a dose-dependent manner in an MTT assay. The IC₅₀ of the inhibitor was 66.8 μM (C). The structure of compound AN2690 and the derivative compound 2 (D). *P<0.05 and **P<0.01. Abbreviations: IC₅₀, half maximal inhibitory concentration; LARS, leucyl-tRNA synthetase; hsLARS, Homo sapiens LARS; tbLARS, Trypanosoma brucei LARS.

Figure 2

hsLARS is a cellular target of growth inhibition by LARS inhibitor. Notes: hsLARS expression construct (hsLARS) or control empty vector (vector) were cotransfected with EGFP into U2OS cells. After 24 hours, both groups of transfected cells were mixed in a 1:1 ratio with cells transfected with pDsRed-monomer as an internal control and were treated with hsLARS-specific or control inhibitors. After 24 hours of treatment, the green and red fluorescent cell ratios in each group were compared with their own initial green/red ratio and quantified by fluorescent microscopy, with four random fields acquired for each sample. At least 200 cells per slide were counted. Dose-dependent expression of transfected inhibitor exogenous hsLARS (A). Dose-responsive increase in green/red ratio after hsLARS rescue in cells treated with hsLARS-specific compound 2 (B), but not in cells treated with nonspecific compound 1 (C). *P<0.05 and **P<0.01. Abbreviations: hsLARS, Homo sapiens leucyl-tRNA synthetase; Con, DMSO control; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein.

Figure 3

Inhibition of LARS causes apoptosis in cancer cells. Notes: Cell morphology change was examined with fluorescent chromatin dye DAPI in U2OS cells for 24 hours in DMSO-(negative control), ActD-(apoptosis control), compound 2, and CHX (protein synthesis inhibitor)-treated cells by fluorescent microscopy. Arrows indicate condensed and fragmented nuclei characteristic of apoptotic cells (A). The percentage of apoptotic cells with each treatment is quantified with four random fields (B). Apoptotic cells were also analyzed through flow cytometric analysis with the indicated concentration of compound 2, CHX, and ActD 24 hours after treatment (C). **P<0.01. Abbreviations: ActD, actinomycin D; CHX, cycloheximide; DMSO, dimethyl sulfoxide; LARS, leucyl-tRNA synthetase; PI, propidium iodide.

Figure 4

Inhibition of LARS regulates the transcriptional activity of the p21 promoter. Notes: U2OS cells were transfected with p21 promoter-driven luciferase reporter, with cotransfected pcDNA3-β-Gal as an internal control. The cells were treated with Con, the LARS inhibitor compound 2, or protein synthesis inhibitor CHX for 12 hours (A), 48 hours (B). Abbreviations: CHX, cycloheximide; Con, DMSO control; DMSO, dimethyl sulfoxide; LARS, leucyl-tRNA synthetase.

Figure 5

Compound 2 suppresses transplant-tumor formation in an animal model. Notes: The growth inhibition of mouse mammary carcinoma cell line EMT6 and mouse fibroblast cell line is induced by compound 2 (A). Each point is the mean of three independent experiments consisting of six mice; the error bar represents standard error. Representations of five independent experiments each consisting of six mice are shown with similar results. Compound 2 inhibits tumor growth in mice compared with vehicle-treated mice. The data are expressed as mean ± SD from three independent experiments (*P<0.05; **P<0.01) (B). Compound 2 treatment has little effect on body weight compared with vehicle-treated mice. The data are represented as mean ± SD from three independent experiments (C). Abbreviation: SD, standard deviation.