Bisebromoamide, an extract from Lyngbya species, induces apoptosis through ERK and mTOR inhibitions in renal cancer cells

Abstract

Advanced renal cell carcinoma (RCC) remains an incurable disease, and newer anticancer drugs are needed. Bisebromoamide, a novel cytotoxic peptide, was isolated from the marine cyanobacterium Lyngbya species at our laboratory in 2009. This compound specifically inhibited the phosphorylation of ERK in platelet-derived growth factor-activated normal rat kidney cells. The aim of this study was to evaluate the effect and elucidate the potential mechanism of Bisebromoamide actions on human RCC cells. Two renal cancer cell lines, 769-P and 786-O, were used. The effects of Bisebromoamide were analyzed employing assays for water-soluble Tetrazolium-1 salts. Apoptosis was determined by flow cytometric TUNEL analysis. Cell-cycle distributions were analyzed by flow cytometry using BrdU/propidium iodide (PI) staining. Kinases of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of Rapamycin (mTOR) pathway and Raf/MEK/ERK pathway were analyzed by Western blotting. After Bisebromoamide treatment for 48 and 72 h, cell viability was significantly decreased in both cell lines at 1 and 10 μmol/L. After treatment with 1 μmol/L Bisebromoamide for 72 h, apoptosis and the increased percentage of cells in the sub-G1 phase were observed in both cell lines. Bisebromoamide inhibited the phosphorylation of ERK and Akt in both cell lines tested. Similar effects were demonstrated for phosphorylation of mTOR and p70 S6. Bisebromoamide is a promising potential agent against RCC due to its ability to inhibit both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways.

Keywords: Akt; Bisebromoamide; ERK; apoptosis; mTOR; renal cell carcinoma.

Figure 1

Effects of Bisebromoamide on the viability of renal cancer cells (A and B). Cells were treated with various concentrations of Bisebromoamide, and cell viability was assessed by water-soluble Tetrazolium (WST)-1 assay. Bisebromoamide dose-dependently (1–10 μmol/L) and time-dependently (48–72 h) inhibited proliferation of all cell lines. Bars represent SE. The single asterisk indicates P < 0.05 as compared with untreated cells. Data shown are the averages of three experiments per cell line.

Figure 2

Detection of apoptosis by flow cytometric TUNEL analyses (A and B). Two renal cancer cell lines were untreated or treated with 1 μmol/L Bisebromoamide for 72 h. The cells were then stained using the TUNEL method to identify apoptotic cells. The cells with log fluorescence intensity >101 were considered positive for apoptosis.

Figure 3

Cell-cycle analysis by flow cytometric propidium iodide analysis (A and B). A sub-G1 peak indicative of apoptosis was obtained in both cell lines treated with 1 μmol/L Bisebromoamide for 72 h.

Figure 4

Effect of Bisebromoamide on the activation of ERKs and Akt in two renal cancer cell lines (A and B). Cells were treated with 1 μmol/L Bisebromoamide for 24 h. Changes in the expressions of ERKs and Akt were analyzed by Western blotting. Bisebromoamide downregulated the expression of p-ERKs as well as that of p-Akt in both cell lines. There were no effects on the expressions of t-ERKs and t-Akt.

Figure 5

Effect of Bisebromoamide on the activations of mammalian target of Rapamycin (mTOR) and p70 S6 kinase in two renal cell carcinoma lines (A and B). Cells were treated with 1 μmol/L Bisebromoamide for 24 h. Changes in the expressions of mTOR and p70 S6 kinase were analyzed by Western blotting. Bisebromoamide downregulated the expression of p-mTOR (Ser2448 and Ser2481) as well as that of p-p70 S6 kinases (Thr389 and Thr421/Ser424), whereas there were no effects on the expressions of t-mTOR and t-p70 S6 kinase.

Figure 6

Effect of Bisebromoamide on the activations of MEK and PDK1 in two renal cell carcinoma lines (A and B). Cells were treated with 1 μmol/L Bisebromoamide for 24 h. There was little effect on the phosphorylation of MEK and PDK1 in both cell lines.

Figure 7

Effect of Bisebromoamide on the activations of epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), and cleaved caspase-3 in two renal cell carcinoma lines (A and B). Cells were treated with 1 μmol/L Bisebromoamide for 24 h. There was little effect on the phosphorylation of EGFR and PI3K in both cell lines. Cleaved caspase-3 was upregulated after 12-h incubation in both 769-P and 786-O cells.