In vitro study of efficacy of pramlintide in osteosarcoma

Abstract

Osteosarcoma (OS) is the most common primary bone cancer in young adults and children. After the development of chemotherapy in 1970, the survival rate improved from 50 to 70%; however, it has reached its plateau at 70% for almost 30 years. Using comprehensive analysis of proteomic studies, our in-house proteomic database, and underlying pathogenic genetic studies of OS, cardiac glycosides and pramlintide were identified as potential targeted therapeutic agents in OS. Cytotoxic assay and flow cytometry were performed to test in vitro efficacy against OS cells. OS cell lines including Saos-2 responded to digoxin at the concentrations of less than 1 µM. MNNG-HOS and MG-63 responded to digoxin at the concentrations of less than 0.1 µM. 52% of primary OS (11/21) responded at the concentrations of less than 1 µM. 25% (5/21) responded at the concentrations of less than 0.1 µM. For the pramlintide, mRNA expression levels of the CALCR and RAMP3, which are pramlintide receptors, were measured by real-time PCR (qRT-PCR) in four known p53 status cancer cell lines including NCI-H1299, U-2 OS, Saos-2, and MMNG/HOS and 27 primary osteosarcoma lines. They were co-cultured with pramlintide acetate at various concentrations ranging from 0.001-100 µg/ml for 24, 48, and 72 hours. qRT-PCR showed that NCI-H1299, U-2 OS, and a primary osteosarcoma cell line expressed CALCR and RAMP3 while Saos-2, MNNG/HOS, and other 26 primary osteosarcoma cell cultures did not express or expressed at very low levels. Whole exome sequencing (WES) revealed a p53 somatic missense mutation in one primary osteosarcoma cell culture expressing CALCR and RAMP3. At the concentration of 0.001-10 µg/ml, pramlintide acetate could not induce cancer cell apoptosis or cell death including osteosarcoma cells regardless of p53 status or CALCR and RAMP3 expressions. At the concentration of 100 µg/ml, more cells were found to die in the pramlintide acetate treatment group compared to the non-treatment group. However, this was resulted from acidity of the acetate group, not pramlintide per se. In conclusion, this is the first study that demonstrates an in vitro antiosteosarcoma effect of digoxin. These findings should prompt further in vivo and clinical studies to verify its effectiveness.