kn-93 and Bone-Neoplasms

Wnt5b, a member of Wnt family, plays multiple roles in tumor progression and metastasis. However, whether Wnt5b contributes to the sensitization of dorsal root ganglia (DRG) neurons and pathogenesis of bone cancer pain still remains unclear. Here, we found that the protein expression of Wnt5b and its atypical tyrosine protein kinase receptor Ryk was upregulated in ipsilateral DRGs in tumor-bearing mice. Application of Wnt5b evoked an increased discharge frequency in isolated DRG neurons and pain hypersensitivity in naïve mice which were almost completely prevented by anti-Ryk antibody. Moreover, intrathecal injection of anti-Ryk antibody to tumor-bearing mice significantly inhibited bone cancer-induced mechanic allodynia and thermal hyperalgesia. Subsequently, we also demonstrated that application of Wnt5b to cultured DRG neurons could enhance membrane P2X3 receptors and α,β-meATP-induced currents. Intrathecal injection of calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 or P2X3 receptors antagonist A317491 almost completely abolished Wnt5b-induced mechanical allodynia and thermal hyperalgesia in mice. Meanwhile, pretreatment with anti-Ryk antibody or CaMKII inhibitor KN93 can attenuate bone-cancer induced the upregulation of P2X3 membrane protein as well as pain hypersensitivity. These findings suggested that Wnt5b/Ryk promoted the trafficking of P2X3 receptors to the membrane via the activation of CaMKII in primary sensory neurons, resulting in peripheral sensitization and bone cancer-induced pain. Our results may offer a potential therapeutic strategy for bone cancer pain.

Topics: Animals; Benzylamines; Bone Neoplasms; Cancer Pain; Cell Line, Tumor; Male; Mice; Mice, Inbred C3H; Pain Measurement; Phenols; Polycyclic Compounds; Protein Kinase Inhibitors; Protein Transport; Purinergic P2X Receptor Antagonists; Receptor Protein-Tyrosine Kinases; Receptors, Purinergic P2X3; Sulfonamides; Wnt Proteins

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease.

Topics: Animals; Autocrine Communication; Benzylamines; Bone Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line, Tumor; Cell Proliferation; Endothelial Growth Factors; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Neoplasm Invasiveness; Osteosarcoma; Peptides, Cyclic; Protein Kinase Inhibitors; RNA Interference; RNA, Small Interfering; Signal Transduction; Sulfonamides; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation; Transplantation, Heterologous; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

The N-methyl-d-aspartate receptor (NMDAR) containing subunit 2B (NR2B) is critical for the regulation of nociception in bone cancer pain, although the precise molecular mechanisms remain unclear. KIF17, a kinesin motor, plays a key role in the dendritic transport of NR2B. The up-regulation of NR2B and KIF17 transcription results from an increase in phosphorylated cAMP-response element-binding protein (CREB), which is activated by calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we hypothesized that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer-related pain behaviors. The expression of spinal t-CaMKII, p-CaMKII, NR2B and KIF17 after inoculation was also evaluated. These results showed that inoculation of osteosarcoma cells induced progressive bone cancer pain and resulted in a significant up-regulation of p-CaMKII, NR2B and KIF17 expression after inoculation. Intrathecal administration of KN93, a CaMKII inhibitor, down-regulated these three proteins and attenuated bone cancer pain in a dose- and time-dependent manner. These findings indicated that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain, and inhibition of CaMKII may be a useful alternative or adjunct therapy for relieving cancer pain.

Topics: Animals; Benzylamines; Bone Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line, Tumor; Injections, Spinal; Kinesins; Mice; Mice, Inbred C3H; Pain; Protein Kinase Inhibitors; Protein Transport; Receptors, N-Methyl-D-Aspartate; Sulfonamides

Osteosarcoma is the most frequent type of primary bone cancer in children and adolescents. These malignant osteoid forming tumors are characterized by their uncontrolled hyperproliferation. Here, we investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the growth of human osteosarcoma. We show that alpha-CaMKII is expressed in human osteosarcoma cell lines and in primary osteosarcoma tissue derived from patients. The pharmacologic inhibition of CaMKII in MG-63 and 143B human osteosarcoma cells by KN-93 resulted in an 80 and 70% decrease in proliferation, respectively, and induced cell cycle arrest in the G(0)/G(1) phase. The in vivo administration of KN-93 to mice xenografted with human osteosarcoma cells significantly decreased intratibial and subcutaneous tumor growth. Mechanistically, KN-93 and alpha-CaMKII siRNA increased p21((CIP/KIP)) gene expression, protein levels, and decreased the phosphorylation of retinoblastoma protein and E2F transactivation. Furthermore, the inhibition of CaMKII decreased membrane-bound Tiam1 and GTP-bound Rac1, which are known to be involved in p21 expression and tumor growth in a variety of solid malignant neoplasms. Our results suggest that CaMKII plays a critical role in the growth of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat conventional high-grade osteosarcoma in children.

Topics: Animals; Benzylamines; Bone Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Line, Tumor; Humans; Male; Mice; Osteosarcoma; Protein Kinase Inhibitors; Signal Transduction; Sulfonamides; Transplantation, Heterologous