Adhesion of renal carcinoma cells to endothelial cells depends on PKCmu

Abstract

Background: The formation of metastases includes the separation of tumor cells from the primary tumor, cell migration into subendothelial tissue and cell proliferation in secondary organ. In this process, cell adhesion of tumor cells to the endothelium is an essential requirement for formation of metastases. Protein kinase C (PKC) regulates adhesion and proliferation. To identify a relation between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell adhesion and proliferation, and possible influences of integrins were analyzed in RCC cells.

Methods: The experiments were performed in the RCC cell lines CCF-RC1 and CCF-RC2 after pre-incubation (16 h) with the PKC inhibitors GF109203X (inhibits PKCalpha, betaI, betaII, gamma, delta and epsilon), GO6976 (inhibits PKCalpha, betaI and mu), RO31-8220 (inhibits PKCalpha, betaI, betaII, gamma and epsilon) and rottlerin (inhibits PKCdelta). Cell adhesion was assessed through adherence of RCC cells to an endothelial monolayer. Cell proliferation was analyzed by a BrdU incorporation assay. The expression of beta1 integrins was analyzed by flow cytometry.

Results: In CCF-RC1 cells, cell adhesion was significantly reduced by GO6976 to 55% and by RO31-8220 to 45% of control. In CCF-RC2 cells, only GO6976 induced a significant reduction of cell adhesion to 50% of control levels. Proliferation of both cell lines was reduced by rottlerin to 39% and 45% of control, respectively. The beta1 integrin expression on the cell surface of CCF-RC1 and CCR-RC2 cells was decreased by RO31-8220 to 8% and 7% of control, respectively. beta2 and beta3 integrins were undetectable in both cell lines.

Conclusions: The combination of the PKC inhibitors leads to the assumption that PKCmu influences cell adhesion in CCF-RC1 and CCF-RC2 cells, whereas in CCF-RC1 cells PKCepsilon also seems to be involved in this process. The expression of beta1 integrins appears to be regulated in particular by PKCepsilon. Cell proliferation was inhibited by rottlerin, so that PKCdelta might be involved in cell proliferation in these cells.

Figure 1

Cell vitality after PKC inhibition. CCF-RC1 (A) and CCF-RC2 (B) cells were treated with PKC isoform specific inhibitors GF109203X, GÖ6976, RO31-8220 and rottlerin in different concentrations, cell vitality was determined by MTT assay. Only treatment of CCF-RC2 cells with GÖ6976 resulted in a slight reduction of cell vitality. The columns and bars represent mean value and standard error in % of control (untreated cells).

Figure 2

Cell proliferation after PKC inhibition. CCF-RC1 (A) and CCF-RC2 (B) cells were treated with the PKC isoform specific inhibitors GF109203X, GÖ6976 and RO31-8220 in concentrations between 1 μM and 5 μM, and with Rottlerin in concentrations between 1 μM and 10 μM. The proliferation value is determined as a percentage of the proliferation of control (untreated cells). The columns and bars represent mean value and standard error in % of control.

Figure 3

Cell adhesion to endothelial cells after PKC inhibition. Adhesion of CCF-RC1 (A) and CCF-RC2 (B) cells to a monolayer of human umbilical endothelial cells (HUVEC) was measured after treatment with the PKC isoform specific inhibitors GF109203X, GÖ6976 and RO31-8220 in concentrations between 1 μM and 5 μM, and with Rottlerin in concentrations between 1 μM and 10 μM. The adhesion value is determined as a percentage of the adhesion of control (untreated cells). The columns and bars represent mean value and standard error in % of control.

Figure 4

Expression of β1, β2 and β3 integrins. Integrin expression was analyzed in untreated CCF-RC1 and CCF-RC2 cells by flow cytometry. The columns represent mean values of 10 000 counted cells. For control, an isotype specific IgG control was used. Only β1 integrins were expressed in both cell lines.

Figure 5

Expression of β1 integrins after PKC inhibition. CCF-RC1 (A) and CCF-RC2 (B) cells were treated with the PKC isoform specific inhibitors GF109203X, GÖ6976 and RO31-8220 in concentrations between 1 μM and 5 μM, and with Rottlerin in concentrations between 1 μM and 10 μM, integrin expression was determined by flow cytometry. The columns represent mean value of 10 000 counted cells in % of control (untreated cells).

Figure 6

Expression of PKCδ, ε and μ in CCF-RC2 cells. Expression of PKCδ (78 kDa), ε (83 kDa) and μ (115 kDa) in CCF-RC2 cell was determined by Western blot (30 μg protein extract per lane). β-actin was used as loading control. All three PKC isoforms are expressed in CCF-RC2 cells.