CA-5f is a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion.

Treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. CA-5f exhibited strong cytotoxicity against A549 NSCLC cells, but low cytotoxicity to normal HUVECs, by increasing mitochondrial-derived reactive oxygen species (ROS) production.

CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data.

A549 cells or HUVECs were seeded into 100-mm culture dishes. When cells reached 80% confluency, they were exposed to DMSO or CA-5f for 6 h or 24 h. Then the cells were fixed, dehydrated, embedded, sectioned, and stained as previously described. Finally, the ultrathin sections of these samples were observed under a JEM-1230 transmission electron microscopy.

Four-week-old BALB/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Lt and kept in specific pathogen-free conditions at the Experimental Animal Center in the Institute of Medicine in Zhengzhou University. Nude mice underwent subcutaneous injection of A549 cells (1 × 10^6) at the right scapula. 8 days after A549 cell implantation, mice exhibited palpable tumors, then they were randomly divided into 2 groups (each group n = 5). CA-5f was injected via caudal vein every 2 days for up to 30 days. DMSO was used as a control. From the initial injection, the tumor size was measured every 2 days. Tumor volumes were calculated as tumor length × (square of width)/2. At the end of the experiment, all mice were sacrificed and the tumors were resected immediately and photographed. The weight of each tumor was measured.