Purinergic Signaling in Bone As a Potential Mechanism in Prostate Cancer Proliferation and Cancer-Induced Bone Pain
Date
2017-05
Authors
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Publisher
University of Delaware
Abstract
Prostate cancer (PCa) is the third leading cause of cancer-related deaths in men in the United States. PCa preferentially metastasizes to bone and these metastases occur in approximately 2 out of 3 patients whose cancer has spread to other parts of the body. PCa that metastasizes to bone forms osteosclerotic tumors. Osteosclerotic tumors result from in increased bone formation, but reduces bone strength resulting in increased mechanical strain in the bone microenvironment. The increased strain in bone resulting from tumor formation correlates with increased pain sensation. Prostate Cancer-Induced Bone Pain (PCIBP) is one of the most detrimental symptoms to patients with bone-localized cancer, greatly reducing patient’s quality of life. Current treatment for PCIBP is ineffective. In addition to PCIBP, PCa proliferates quickly once in the bone microenvironment, making it difficult to slow the disease progression and clinical outlook for the patient. Therefore, I seek to discover therapeutic targets that can elucidate why PCa proliferates quickly in bone and how this process and PCIBP could be linked. Discovering therapeutic targets could potentially increase the quality of life in patients with stage IV PCa.
In Aim 1, I proposed that nerve-growth factor (NGF), a molecule implicated in PCa proliferation, enhances the potential for PCIBP by indirectly inducing PCa cell proliferation through upregulating purinergic receptors and sensitizing metastatic PCa cells to the mitogenic effects of ATP. To address the role of NGF in PCa proliferation and purinergic signaling, I applied NGF to C4-2B4 and performed cell counts as well as MTS cell proliferation assays. To determine the mitogenic properties of ATP on PCa cells, I added apyrase to the media to determine the effect of hydrolyzing ATP on C4-2B4 proliferation. NGF treatment demonstrated an increase in cell proliferation in
comparison to control media at all times tested. Addition of NGF with apyrase in an MTS assay showed a reduction in metabolism and thus proliferation over 48 hours. The proliferation studies indicate that NGF and ATP may play important roles together in the proliferation of metastatic PCa cells in bone.
In Aim 2, I proposed that mechanical load on PCa cells results in the release of ATP to activate local nociceptors through P2X3 receptors ultimately resulting in PCIBP. I mechanically loaded human bone-derived metastatic PCa cells, C4-2B4s, via hypotonic swelling. Resultant C4-2B4 load-response conditioned media (CM) +/- apyrase was applied to primary mouse dorsal root ganglia (DRG) and changes in intracellular calcium were measured. Apyrase is an enzyme that hydrolyzes ATP to AMP and should reduce the amount of ATP in the CM. To determine the role of P2X3 receptors in response to CM, TNP-ATP, a P2X3 specific antagonist was utilized. The data indicated that ATP in the CM of mechanically loaded C4-2B4 cells increased intracellular calcium in DRG which was reduced with apyrase. TNP-ATP did not significantly block response to CM suggesting another purinergic receptor may be responsible for this response.
These data overall indicate that proliferation of PCa and PCIBP could be linked through the mechanical release of molecules in bone from PCa and osteogenic cells. These data indicate purinergic receptors and NGF receptors as potential therapeutic targets for treating PCIBP.
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Keywords
Biological Sciences, Purinergic Signaling in Bone,Prostate Cancer Proliferation, Cancer-Induced Bone Pain