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Convallatoxin suppresses osteosarcoma cell proliferation, migration, invasion, and enhances osteogenic differentiation by downregulating parathyroid hormone receptor 1 (PTHR1) expression and inactivating Wnt/β-catenin pathway.

ABSTRACT: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Convallatoxin, a natural cardiac glycoside, exhibits potent anti-tumor activities. Literature has confirmed that PTHR1 is highly expressed in OS tissues and cells and downregulation of PTHR1 could decrease the invasion and growth of OS cells and increase tumor differentiation. In addition, PTHR1 could activate Wnt signaling pathway to promote the malignant functions of OS. In the present study, MG63 and U2OS cells were treated with 0, 12.5, 25, and 50 nM convallatoxin in order to elucidate the precise function of convallatox on the malignant behaviors of OS cells. Moreover, MG63 and U2OS cells treated with convallatoxin were transfected with Ov-PTHR1 or sh-DKK1, aiming to explore whether convallatoxin impeded the malignant progression of OS by modulating PTHR1 and Wnt/β-catenin pathway. CCK-8, wound healing and transwell assays were employed to assess the proliferation, migration, and invasion of OS cells. Differentiation markers (collagen 1, osteopontin, RANKL, Runx2, osteocalcin) were measured to evaluate OS cell differentiation. Results illuminated that convallatoxin suppressed proliferation, migration, and invasion as well as promoted osteogenic differentiation of OS cells. Besides, convallatoxin inhibited PTHR1 expression and inactivated Wnt/β-catenin pathway and PTHR1 overexpression activated Wnt/β-catenin pathway. Furthermore, PTHR1 overexpression or DKK1 knockdown reversed the suppressing effects of convallatoxin on OS cell proliferation, migration, and invasion, as well as the enhancing effect of convallatoxin on OS cell osteogenic differentiation. Collectively, convallatoxin may repress the malignant progression of OS by blocking PTHR1 and Wnt/β-catenin pathway.

SUBMITTER: Liu X

PROVIDER: S-EPMC9275893 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Disorders related to parathyroid hormone (PTH) resistance and PTH signaling pathway impairment are historically classified under the term of pseudohypoparathyroidism (PHP). The disease was first described and named by Fuller Albright and colleagues in 1942. Albright hereditary osteodystrophy (AHO) is described as an associated clinical entity with PHP, characterized by brachydactyly, subcutaneous ossifications, round face, short stature and a stocky build. The classification of PHP is further divided into PHP-Ia, pseudo-PHP (pPHP), PHP-Ib, PHP-Ic and PHP-II according to the presence or absence of AHO, together with an in vivo response to exogenous PTH and the measurement of Gs? protein activity in peripheral erythrocyte membranes in vitro. However, PHP classification fails to differentiate all patients with different clinical and molecular findings for PHP subtypes and classification become more complicated with more recent molecular characterization and new forms having been identified. So far, new classifications have been established by the EuroPHP network to cover all disorders of the PTH receptor and its signaling pathway. Inactivating PTH/PTH-related protein signaling disorder (iPPSD) is the new name proposed for a group of these disorders and which can be further divided into subtypes - iPPSD1 to iPPSD6. These are termed, starting from PTH receptor inactivation mutation (Eiken and Blomstrand dysplasia) as iPPSD1, inactivating Gs? mutations (PHP-Ia, PHP-Ic and pPHP) as iPPSD2, loss of methylation of GNAS DMRs (PHP-Ib) as iPPSD3, PRKAR1A mutations (acrodysostosis type 1) as iPPSD4, PDE4D mutations (acrodysostosis type 2) as iPPSD5 and PDE3A mutations (autosomal dominant hypertension with brachydactyly) as iPPSD6. iPPSDx is reserved for unknown molecular defects and iPPSDn+1 for new molecular defects which are yet to be described. With these new classifications, the aim is to clarify the borders of each different subtype of disease and make the classification according to molecular pathology. The iPPSD group is designed to be expandable and new classifications will readily fit into it as necessary.

Project description:Parathyroid hormone-related protein (PTHrP) expression in breast cancer is enriched in bone metastases compared to primary tumors. Human MCF7 breast cancer cells "home" to the bones of immune deficient mice following intracardiac inoculation, but do not grow well and stain negatively for Ki67, thus serving as a model of breast cancer dormancy in vivo. We have previously shown that PTHrP overexpression in MCF7 cells overcomes this dormant phenotype, causing them to grow as osteolytic deposits, and that PTHrP-overexpressing MCF7 cells showed significantly lower expression of genes associated with dormancy compared to vector controls. Since early work showed a lack of cyclic AMP (cAMP) response to parathyroid hormone (PTH) in MCF7 cells, and cAMP is activated by PTH/PTHrP receptor (PTHR1) signaling, we hypothesized that the effects of PTHrP on dormancy in MCF7 cells occur through non-canonical (i.e., PTHR1/cAMP-independent) signaling. The data presented here demonstrate the lack of cAMP response in MCF7 cells to full length PTHrP(1-141) and PTH(1-34) in a wide range of doses, while maintaining a response to three known activators of adenylyl cyclase: calcitonin, prostaglandin E2 (PGE2), and forskolin. PTHR1 mRNA was detectable in MCF7 cells and was found in eight other human breast and murine mammary carcinoma cell lines. Although PTHrP overexpression in MCF7 cells changed expression levels of many genes, RNAseq analysis revealed that PTHR1 was unaltered, and only 2/32 previous PTHR1/cAMP responsive genes were significantly upregulated. Instead, PTHrP overexpression in MCF7 cells resulted in significant enrichment of the calcium signaling pathway. We conclude that PTHR1 in MCF7 breast cancer cells is not functionally linked to activation of the cAMP pathway. Gene expression responses to PTHrP overexpression must, therefore, result from autocrine or intracrine actions of PTHrP independent of PTHR1, through signals emanating from other domains within the PTHrP molecule.

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Convallatoxin suppresses osteosarcoma cell proliferation, migration, invasion, and enhances osteogenic differentiation by downregulating parathyroid hormone receptor 1 (PTHR1) expression and inactivating Wnt/β-catenin pathway.

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