Fig 1: In Cellula Activity of Rocaglates(A) Cytotoxicity of rocaglates toward NIH 3T3 (gray circle) and eIF4A1em1JP (red triangle) cells. Cells were exposed to 40 nM compound for 4 days, and viability was measured using the sulforhodamine B (SRB) assay; n = 3 ± SEM.(B) Rocaglates show different sequence preferences for inhibiting cap-dependent translation in cellula. HEK293T cells were transfected with the indicated mRNA reporters, compounds added 1 h later, and luciferase activity measured; n = 3 ± SD.(C) Dose response of the indicated mRNAs to hippuristanol and pateamine in HEK293T cells; n = 3 ± SD.(D) Comparison of the frequencies of quadruplet motifs in 5′ leaders of eIF4A1-bound mRNAs upon CR-1–13-B and silvestrol treatments relative to DMSO. The quadruplet motifs are color-coded: blue is for W4, orange is for R4, and cyan is for NAGT and AGTN. W = A, T; R = A, G. Data are compiled in Table S2.See also Figure S4 and Table S1.
Fig 2: Different Rocaglates Exhibit Distinct Biological Activities(A) Polypurine clamping is a correlative, but not universal, predictor of cap-dependent inhibition. The ΔmP obtained with eIF4A1:poly r(AG)8 RNA was measured for each compound (10 μM) and is plotted against the fold inhibition for cap-dependent translation (2 μM) of FF-HCV-Ren mRNA in Krebs-2. Note the duplicate values for RHT (open circles) are due to two preparations of different enantiomeric purity, and the duplicate values for CR-1–31-B (dotted circles) are due to two different compound batches (see Table S1). Pearson r = −0.62; p < 0.0001.(B) Rocaglates preferentially stimulate eIF4A binding onto purine-rich RNAs. Different RNA probes were incubated in the presence of 500 nM eIF4A1 and compound for 30 min before measurement. The ΔmP in the presence of compound relative to vehicle control is presented; n = 3 ± SEM.(C) mRNA sensitivity toward CR-1–31-B is correlated to 5′ leader purine content. The inhibition of cap-dependent (FLuc) and -independent (RLuc) translation was measured in response to CR-1–31-B; n = 3 ± SEM.(D) Dose response of the indicated rocaglates in Krebs-2 extracts programmed with the indicated mRNAs; n = 3 ± SEM.See also Figures S1, S2, and S3 and Table S1.
Fig 3: Human hepatocellular carcinoma cell lines express eIF4A1, eIF4A2, and DDX3 proteins. (A) The levels of the three proteins were assessed in HLE, HLF, PLF/PRF/5, SNU182, and SNU449 cell lines by western blot analysis. GAPDH was used as a loading control, and protein band intensities of eIF4A1, eIF4A2, and DDX3 were normalized to GAPDH levels (B).
Fig 4: Levels of eIF4A1 protein negatively correlate with the survival of patients with hepatocellular carcinoma (HCC) (n = 47). Kaplan-Meyer curves in HCC patients for eIF4A1 (A), eIF4A2 (B), and DDX3 (C) proteins.
Fig 5: Screening for the target of eIF4A1 in PDAC. a c-MYC expression was significantly downregulated (log2FC = − 1.05, FDR = 0.00192) in the rocaglate CR-31-B-treated group compared with the control group, ranking in the top 0.6% of all the regulated genes. b The expression of eIF4A1 and c-MYC in tumor tissues from the same PDAC patients were analyzed by IHC staining and the results showed that c-MYC is positively correlated with eIF4A1 (n = 44, r = 0.5041, P = 0.0005, Spearman correlation). c The DEPs were enriched in multiple metastasis-associated functions. d GSEA was performed with ribosomal sequencing results. Gene enrichment plots showed that the enrichment of EMT-related gene sets was reduced substantially after eIF4A-intervention
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