The ability of tumor cells to adhere to and interact with different components of the ECM is a prerequisite for cell migration and cell invasion into the basement membrane.
We investigated the effect of statins on the adhesion of B16BL6 cells to type I and type IV collagen, fibronectin, and laminin. We observed that the number of Selleck MI-503 cells that adhered to type I collagen, type IV collagen, fibronectin, and laminin were significantly decreased in the presence of statins as buy VRT752271 compared to that in the 0.1% DMSO-treated cultures (control) (P < 0.01, Figure 3A-D). Figure 3 Effect of statins on B16BL6 cell adhesion to ECM components. B16BL6 cells, which had been treated with 0.05 μM fluvastatin or 0.1 μM simvastatin for 3 d, were incubated with (A) type I collagen-, (B) type IV collagen-, (C) fibronectin-, or (D) laminin-coated plates for 30 min at 37°C in an atmosphere containing 5% CO2. The results are representative of 5 independent experiments. (E) Image showing the results of RT-PCR analysis of integrins mRNA. B16BL6 cells were treated with 0.05 μM fluvastatin or 0.1 μM simvastatin. After 3 d, equal amounts of RNA were reverse-transcribed to generate cDNA, which was used for PCR analysis of integrins mRNA expression in B16BL6 cells. (E) Image showing western blot of the integrin α2, integrin α4, and integrin α5 proteins. Whole-cell lysates were generated and immunoblotted with antibodies against integrin
α2, integrin α4,
integrin α5, and β-actin (internal standard). Suppression of integrin α2, integrin α4, and integrin α5 mRNA and protein expression by statins To elucidate the effect of statins on cell adhesion CYT387 supplier ifenprodil to ECM components, the mRNA expression of α integrins was assessed by RT-PCR. As shown in Figure 3E, statins suppressed the mRNA expression of integrin α2, integrin α4, and integrin α5 in the B16BL6 cells. There was no substantial change in the level of integrin α1, integrin α3, and integrin α6 mRNA expressions in the statins-treated cells compared with that in the control cells (0.1% DMSO-treated). Further, we investigated whether the protein expression of integrin α2, integrin α4, and integrin α5 was actually inhibited in the B16BL6 cells when statins were administered; we observed that after the administration of statins, the protein expressions of integrin α2, integrin α4, and integrin α5 were significantly reduced (Figure 3F). Inhibitory effects of statins on the Rho signaling pathway To demonstrate whether statins inhibit the functions of Rho by suppressing their prenylation, the protein samples were subjected to a standard western blot assay to detect the presence of small GTPases in both the membrane and cytoplasm lysates of B16BL6 cells incubated with or without statins. The membrane localization of Rho proteins showed a significant decrease in statin-treated cells compared to the control cells (0.1% DMSO-treated).