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Autophagy is a homeostatic process that recycles damaged organelles and long-lived proteins by delivering them in double-membrane vesicles to lysosomes for degradation. Autophagy has a prominent role in survival, proliferation, and resistance of tumors in metabolic and chemotherapeutic stress conditions. Clinical trials with chloroquine-a known autophagy inhibitor-were unable to achieve complete autophagy inhibition in vivo, warranting the search for more potent autophagy inhibitors. In a process of exploring the mechanism of action of previously identified cytotoxic s-triazine analogs, we discovered that both IITZ-01 and IITZ-02 act as potent autophagy inhibitors. Treatment with these compounds resulted in the vacuolated appearance of cells due to their specific accumulation in lysosomes. In addition, these basic compounds also deacidify lysosomes as evidenced by the decrease in lysotracker red staining and inhibit maturation of lysosomal enzymes leading to lysosomal dysfunction. IITZ-01 and IITZ-02 enhance autophagosome accumulation but inhibit autophagosomal degradation by impairing lysosomal function, finally resulting in the inhibition of autophagy. Interestingly, compound IITZ-01 exhibited more than 10-fold potent autophagy inhibition along with 12- to 20-fold better cytotoxic action than CQ. IITZ-01 and IITZ-02 also abolished mitochondrial membrane potential and triggered apoptosis through the mitochondria-mediated pathway. Furthermore, IITZ-01 and IITZ-02 displayed potent antitumor action in vivo through autophagy inhibition and apoptosis induction in MDA-MB-231 breast cancer xenograft model with IITZ-01 exhibiting superior anticancer efficacy. Overall, these data demonstrate that IITZ-01 is potent autophagy inhibitor with single-agent anticancer activity and awaits further preclinical development as potential anticancer therapeutic.

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Osteoporosis is a major debilitating cause of fractures and decreases the quality of life in elderly patients. Bone homeostasis is maintained by bone forming osteoblasts and bone resorpting osteoclasts. Substantial evidences have shown that targeting osteoclasts using natural products is a promising strategy for the treatment of osteoporosis. In the current study, we investigated the osteoprotective effect of Abietic acid (AA) in in vitro and in vivo models of osteolysis. In vitro experiments demonstrated that, AA suppressed receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and F-actin ring formation in a concentration dependent manner. Mechanistically, AA abrogated RANKL-induced phosphorylation of IKKα/β (ser 176/180), IkBα (ser 32), and inhibited the nuclear translocation of NF-κB. We also found that, AA attenuated the RANKL-induced phosphorylation of MAPKs and decreased the expression of osteoclast specific genes such as TRAP, DC-STAMP, c-Fos, and NFATc1. Consistent with in vitro results, in vivo Lipoploysaccharide (LPS)-induced osteolysis model showed that AA inhibited the LPS-induced serum surge in cytokines TNF-α and IL-6. μ-CT analysis showed that AA prevented the LPS-induced osteolysis. Furthermore, histopathology and TRAP staining results suggested that AA decreased the number of osteoclasts in LPS-injected mice. Taken together, we demonstrated that the osteoprotective action of AA is coupled with the inhibition of NF-κB and MAPK signaling and subsequent inhibition of NFATc1 and c-Fos activities. Hence, AA may be considered as a promising drug candidate for the treatment of osteoporosis.