Yuanzheng Yang 1, Zhanglong Peng 1, Elsa R Flores 2, Eugenie S Kleinerman 1,3,*
Posted: Sep 2, 2022
Abstract
Simple Summary
Outcomes for patients with osteosarcoma have remained stagnant for more than 3 decades. Novel therapeutic agents are desperately needed. Osteosarcoma cells often express abnormal form of the p53, p63, and p73 genes, which promote glucose uptake, cell glycolysis, and rapid proliferation. Pramlintide, an FDA-approved drug for type 2 diabetes, interfered with tumor glycolysis leading to decreased cell growth and increased cell death. When we injected Pramlintide into osteosarcoma tumor nodules of the mice, the tumors were significantly smaller after 21 days while the control tumors continued to grow. Tumor hypoxia was also decreased. This is the first report showing the potential efficacy of Pramlintide against osteosarcoma, indicating that Pramlintide may be a novel therapeutic approach for patients with relapsed osteosarcoma.
Abstract
Despite aggressive combination chemotherapy and surgery, outcomes for patients with osteosarcoma have remained stagnant for more than 25 years, and numerous clinical trials have identified no new therapies. p53 deletion or mutation is found in more than 80% of osteosarcoma tumors. In p53-deficient cancers with structurally altered p63 and p73, interfering with tumor cell metabolism using Pramlintide (an FDA-approved drug for type 2 diabetes) results in tumor regression. Pramlintide response is mediated through upregulation of islet amyloid polypeptide (IAPP). Here, we showed that osteosarcoma cells have altered p63, p73, and p53, and decreased IAPP expression but have the two main IAPP receptors, CalcR and RAMP3, which inhibit glycolysis and induce apoptosis. We showed that in osteosarcoma cells with high- or mid-range glycolytic activity, Pramlintide decreased cell glycolysis, resulting in decreased proliferation and increased apoptosis in vitro. In contrast, Pramlintide had no effect in osteosarcoma cells with low glycolytic activity. Using a subcutaneous osteosarcoma mouse model, we showed that intratumoral injection of Pramlintide-induced tumor regression. Tumor sections showed increased apoptosis and a decrease in Ki-67 and HIF-1α. These data suggest that in osteosarcoma cells with altered p53, p63, and p73 and a high glycolytic function, Pramlintide therapy can modulate metabolic programming and inhibit tumor growth.
NeoBiotechnologies’ products were used in this study:
Based on the corresponding hematoxylin and eosin staining, 10 fresh frozen tumor sections (5 µm) were selected from control and pramlintide treatment groups to do Ki67, CD31, and HIF1-α immunofluorescence staining. The sections were air dried at room temperature for 40 min, fixed in cool acetone for 10 min, and permeabilized by 0.25% TritonX-100 for 5 min. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide for 10 min. After three PBS washes, the sections were blocked by 10% normal goat serum for 1 h at room temperature in a humid chamber box, then incubated with Ki67 (1:200), CD31 (1:100), HIF1-α (1:100), CD86 (1:200), and CD163 (1:200) primary antibodies (Ki67, Invitrogen, MA5-14520; CD31, Abcam, ab28364; HIF1-α, Invitrogen, PA1-16601, CD86, NeoBiotechnologies, 942-RBM4-P0; CD163, Abcam, ab182422). Samples were stored at 4 °C overnight with a parafilm coverslip in a humid chamber box to prevent evaporation. Then, the sections were incubated with a secondary antibody (1:500, Alexa Fluor 488 goat anti-rabbit, Invitrogen, A11034) at room temperature for 1 h and counterstained with DAPI in mounting medium. Two to four tumor areas (hot spots) from each section were imaged at 200× by Leica DMi8 microscopy. Quantification of Ki-67 was performed by Image J software to count the number of Ki67-positive cells and total cells same field. This measurement was expressed as the percentage of Ki67-positive cells. Quantification of CD31 and HIF1-α were performed by Image J software to measure the fluorescence intensity of CD31 and HIF1-α staining and expressed as relative fluorescence units (RFUs).
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Recombinant CD86 (Dendritic Cells Maturation Marker) Antibody [C86/2160R]
$249.00 – $539.00Catalog Number:942-RBM4Gene:CD86Application:ELISA, Flow Cytometry, Immunofluorescence, Immunohistochemistry, Western BlotReactivity:HumanHost:RabbitIsotype:IgG
Keywords: osteosarcoma, pramlintide, IAPP, p53, p63, p73, glycolytic metabolism
Publication History:
Cancers (Basel). 2022 Sep 2;14(17):4310. doi: 10.3390/cancers14174310
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