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. Author manuscript; available in PMC: 2015 Nov 19.
Published in final edited form as: Sci Transl Med. 2014 Nov 19;6(263):263ra161. doi: 10.1126/scitranslmed.3010382

Fig. 3. Bisphosphonates inhibit KRAS prenylation and induce ER stress and autophagy.

Fig. 3

(A) U2OS cells expressing Flag-KRAS G12D were treated with bisphosphonates (BPH-1222, 10 μM; BPH-714, 10 μM; zoledronate, 20 μM) or chloroquine (30 μM) for 48 h. Cellular distribution of proteins (HRAS, KRAS, and RAP1A) that require prenylation was examined by immunoblotting. p, pellet contains correctly prenylated proteins which bind avidly to membrane; s, supernatant contains unmodified proteins in the cytoplasm. *, HRAS signal left on the membrane. **, autophagy activation determined by the presence of LC3-II (bottom band, PE-conjugated form). (B) Same cells were treated with FTI-277 (15 μM), GGTI-298 (15 μM) or BPH-1222 (5, 10, 15 μM) for 48 h. Cell lysates were separated with 15-cm SDS-PAGE and blotted with indicated antibodies. *, KRAS mobility shift was observed. (C) Mouse lung cancer cells (M3L2) were treated with BPH-1222 for 48 h and analyzed for KRAS, AKT, caspase-3 activation, and LC3 conversion. KRAS-GTP was pulled down from whole cell lysate with RAF-1 RBD beads and immunoblotted with total KRAS antibody. (D) Human pancreatic cancer cells harboring KRAS mutations (Panc-1 and MiaPaCa2) were treated with BPH-1222 (10 μM) for 48 h and analyzed the same way as in (C). (E) BPH-1222 treatment (10 μM) for 1, 2, or 3 days induced ER stress (CHOP, BiP), autophagy (PE-conjugated LC3II) and apoptosis (caspase-3) in mouse lung cancer cells (6#) in a time-dependent manner.