Augmentation of autophagy with rapamycin protects against tumor metastasis.
C57BL/6 mice were injected with B16-F10 melanoma cells (5×105) and were humanely sacrificed 14 days after inoculation of tumor cells. Mice were intraperitoneally administered with the TLR4/TLR9 agonist complex (dosage and frequency as in the legend of Fig. 1) with or without rapamycin (10 mg/kg, once a day) after tumor cell inoculation. (A) Rapamycin protects against metastasis. Metastatic nodules were counted and data are presented as the mean ± S.E. (n = 10). (B) Rapamycin activates autophagy and regulates STAT1/3 signaling. The expression of STAT1/3 singling and autophagy-related molecules in the lung tissue was detected by Western blot. The lungs were excised and the cytoplasmic and nuclear fractions were extracted as described in the Methods. The expression of p-STAT1, STAT1, p-STAT3, STAT3, and histone H3 in nucleic extracts and IRGM1, LC3B, cleaved caspase-3, P62, PCNA, and β-actin in the cytoplasm were detected with Western blotting. Left panel is representative western blots and right panels are summary results. Data are presented as the mean ± S.E. of 5 mice per group. (C) Rapamycin treatment decreases p62 accumulation in the lung tissue. Representative immunofluorescence microphotographs were presented to show p62 accumulation in melanoma cells. Lung sections were stained for p62 (green) and DAPI (blue). Scale bar: 30 µm.
