PURPOSE: Our goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma. METHODS: The methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels. RESULTS: WP760 induced G(2)/M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27(Kip1) protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated. CONCLUSIONS: Our results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.
PURPOSE: Our goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma. METHODS: The methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels. RESULTS: WP760 induced G(2)/M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27(Kip1) protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated. CONCLUSIONS: Our results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.
Authors: Luiz C Godoy; Chase T M Anderson; Rajdeep Chowdhury; Laura J Trudel; Gerald N Wogan Journal: Proc Natl Acad Sci U S A Date: 2012-11-26 Impact factor: 11.205