in mice: SKOV3 cells resuspended injected subcutaneously in to the flanks of 4-wk-old BALB/C athymic mice. When mice exhibited palp able tumors, they were randomly assigned to treatment groups (6 to 8 mice per group) to avoid treatment bias. Elaiophylin (Azalomycin-B) was given i.p. (1 or 2mg/kg) every2 d for 21 d, or DMSO was used as a control. After the initial treatment, the tumor size was determined every day.

Autophagic flux was inhibited by elaiophylin treatment. Increased numbers of autophagosomes can be associated either with increased autophagosome synthesis or decreased autophagosome turnover. To distinguish between these 2 possibilities, there were measured the autophagic flux. First, was analyzed autophagy in the presence of autophagy inhibitors, such as 3-methyladenine (3-MA) and chloroquine (CQ) in GFP-LC3B-SKOV3 cells. Coincubation of elaiophylin (0.5 μM) with 3-MA (10 mM), which blocked the upstream steps of autophagy, reduced the accumulation of (GFP)-tagged microtubule-associated protein 1 light chain 3 β (LC3B) (GFP-LC3B). puncta. In contrast, coincubation of elaiophylin (0.5 μM) with CQ (25 μM), which blocked the downstream steps of autophagy, did not induce a significant increase in GFP-LC3B puncta compared with elaiophylin treatment alone, indicating that elaiophylin was not an autophagy inducer. Additionally, elaiophylin-treated cells did not exhibit an increase in LC3B-II levels in CQ-treated cells compared with control cells.