AIM The mammalian target of rapamycia (mTOR) signaling pathway has been shown to play important role in regulating cell growth, proliferation and survival.mTOR signal pathway is over-activated in many cancer cells which results in cancer invasion and metastasis.A number of evidences have demonstrated that mTOR signal transduction pathway is an active target for antitumor drug discovery.In this study, we established a cell-based assay and screened natural compounds inhibiting mTOR signal pathway.METHODS The phosphorylation of 4EBP1 by active mTOR signal regulates the nuclear location of eIF4E, which could be readout of the state of mTOR signaling pathway.The inhibitory effect of compounds was monitored in MEF cells after treatment for 5 h at 10 μmol·L-1 concentration, with the rapamycin and PI-103 as the positive controls.Nuclear-to-cytoplasmic elF4E intensity ratios were collected using high content screening after immunofluorescence.RESULTS Total 1235 compounds from a natural compound library were screened using the assay and 10 of them turned out to be able to promote the elF4E nuclear translocation.To verify the inhibitory effect of these positive compounds in cancer cells, we have determined the nuclei-to-cytoplasm elF4E intensity ratios in six cancer cell lines, SMMC7721, Hep G2, MCFT, MD-MAB-231, A549 and SW480 at 20 μmol·L-1 , l0 μmol·L-1 and 5 μmol·L-1 for 5 h.The results demonstrated that two of the positive compounds showed markedly inhibitory effect on the roTOR signaling in a dose dependent manner.CONCLUSION A cell-based assay with the eIF4E nuclear translocation as the indicator of roTOR signaling activation was established and with this assay natural compounds with anti-tumor potential were screened and discovered.