Phosphoproteins Involved in the Inhibition of Apoptosis and in Cell Survival in the Leiomyoma

Abstract

Uterine leiomyomas are benign smooth muscle cell tumors originating from the myometrium. In this study we focus on leiomyoma and normal myometrium phosphoproteome, to identify differentially phosphorylated proteins involved in tumorigenic signaling pathways, and in anti-apoptotic processes and cell survival. We obtained paired tissue samples of seven leiomyomas and adjacent myometria and analyzed the phosphoproteome by two-dimensional gel electrophoresis (2-DE) combined with immobilized metal affinity chromatography (IMAC) and Pro-Q Diamond phosphoprotein gel stain. We used mass spectrometry for protein identification and Western blotting for 2-DE data validation. Quantities of 33 proteins enriched by the IMAC approach were significantly different in the leiomyoma if compared to the myometrium. Bioinformatic analysis revealed ten tumorigenic signaling pathways and four phosphoproteins involved in both the inhibition of apoptosis and cell survival. Our study highlights the involvement of the phosphoproteome in leiomyoma growth. Further studies are needed to understand the role of phosphorylation in leiomyoma. Our data shed light on mechanisms that still need to be ascertained, but could open the path to a new class of drugs that not only can block the growth, but could also lead to a significant reduction in tumor size.

Keywords: 2-DE; leiomyoma; mass spectrometry; myometrium; phosphoproteomics.

Figure 1

(A.) Images of gels stained with Pro-Q Diamond to detect phosphoproteins. (B) Images of gels stained with SYPRO Ruby for total proteins.

Figure 2

Two-dimensional electrophoresis map of normal myometrium and leiomyoma phosphoproteome enriched by IMAC columns. Immobilized pH gradient 4–7 strips were used for the first dimension and 12% polyacrylamide gel was used for the second dimension. The numbered circles indicate the differentially phosphorylated spots.

Figure 3

Western blot analysis was utilized to confirm the alteration of phosphorylation of proteins HSPB1, VINC and HSPA5 in paired myometrium (M) and leiomyoma (L). The intensity of immunostained bands was normalized against the total protein intensities measured from the same blot stained with Red Ponceau. Number 1-5 indicate the patients. The bar graph shows the relative expression (band density) of HSPB1, VINC and HSPA5 in the myometrium and the leiomyoma. Results are shown as a histogram (P < 0.05) and each bar represents mean ± standard deviation.