U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving Phosphoinositide-specific Phospholipases C

Abstract

The definition of the number and nature of the signal transduction pathways involved in the pathogenesis and the identification of the molecules promoting metastasis spread might improve the knowledge of the natural history of osteosarcoma, also allowing refine the prognosis and opening the way to novel therapeutic strategies. Phosphatydil inositol (4,5) bisphosphate (PIP2), belonging to the Phosphoinositide (PI) signal transduction pathway, was related to the regulation of ezrin, an ezrin-radixin-moesin protein involved in metastatic osteosarcoma spread. The levels of PIP2 are regulated by means of the PI-specific Phospholipase C (PLC) enzymes. Recent literature data suggested that in osteosarcoma the panel of expression of PLC isoforms varies in a complex and unclear manner and is related to ezrin, probably networking with Ras GTPases, such as RhoA and Rac1. We analyzed the expression and the subcellular localization of PLC enzymes in cultured human osteosarcoma MG-63 cells, commonly used as an experimental model for human osteoblasts, using U-73122 PLC inhibitor, U-73343 inactive analogue, and by silencing ezrin. The treatment with U-73122 significantly reduces the number of MG-63 viable cells and contemporarily modifies the expression and the subcellular localization of selected PLC isoforms. U-73122 reduces the cell growth in cultured MG-63 ostesarcoma cell line involving PI-specific Phospholipases C.

Keywords: DMSO; MG-63; Oncology; Osteosarcoma; Phosphoinositide; Phospholipase C; U-73122; U-73343.

Fig. 1

MG-63 cultures. a Phase contrast microscopy of MG-63 cells. Cell morphology cultured 24 h: (upper line) untreated control cells (CTRL) and cells treated with U-73122 1 μΜ (centre line) and 10 μΜ (lower line) after 1, 3, 6 h from seeding b, c MG-63 cells after 1, 3, 6 h from seeding: untreated (NT), DMSO, U-73122 1 μΜ, U-73122 10 μΜ and U-73343 10 μΜ treated cells. c Comparison histogram of cell growth with error bars

Fig. 2

Immunofluorescence images of MG-63 cells (controls and cells treated with 10 micrm U-73122). For each PLC isoform, on the left immunomarking with the corresponding antibody Red Texas (red) or FITC (green) conjugated; DAPI counterstain for nuclei in the centre; on the right merge (inverted fluorescence microscope, 40×)