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

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.


Introduction
Therapeutic options used in patients with advanced renal cell carcinoma (RCC) were limited, but this has changed dramatically in the last few years [1][2][3][4][5]. Previously, traditional chemotherapy, hormonal therapy, or radiation was not effective in the treatment of advanced RCC, and immunotherapy provided only limited benefit. Improvements in our understanding of the molecular basis of RCC have led to the development of targeted agents tailored to inhibiting intracellular signal transduction pathways that drive angiogenesis, tumorigenesis, and progression. Two signaling pathways, namely Raf/MEK/ERK and phosphat-idylinositol 3-kinase (PI3K)/Akt/mammalian target of Rapamycin (mTOR) pathways, play central roles in many types of tumor cell proliferation, and can lead to aberrant signaling and uncontrolled proliferative diseases [6]. Therefore, we focused on these two pathways which were known to be upregulated in many types of cancer, including RCC.
BAY 43-9006 targeting Raf, as well as vascular endothelial growth factor (VEGF), and RAD001 and CCI-779 targeting mTOR are currently in widespread clinical use for patients with advanced RCC. Their common biological targets were von Hippel-Lindau (VHL)/hypoxia inducible factor/VEGF pathway to inhibit angiogenesis, and Raf/MEK/ERK and PI3K/Akt/mTOR pathways to

Cancer Medicine
Open Access inhibit cancer cell proliferation. Although these targeted agents have improved objective response rates and progression-free survival as compared with prior standard of care agents, advanced RCC remains an incurable disease and newer anticancer drugs are needed.
A novel cytotoxic peptide, Bisebromoamide, was isolated from the marine cyanobacterium Lyngbya species harvested in Okinawa, Japan, at our laboratory in 2009 [7,8]. This compound specifically inhibited the phosphorylation of ERK in platelet-derived growth factoractivated normal rat kidney cells. As the ERK pathway is upregulated in many types of cancers, we consider this extract from Lyngbya species to have the potential to inhibit RCC cell proliferation. We aimed to evaluate the direct antitumor effect and elucidate the potential mechanism of Bisebromoamide actions on human RCC cells.

Cell lines and cultures
The two renal cancer cell lines, 769-P and 786-O (purchased from American Type Culture Collection [ATCC], Rockville, MD), were cultured in RPMI 1640 medium (Invitrogen, Groningen, the Netherlands) with 10% fetal bovine serum and streptomycin. These cells were established from clear cell RCC [9]. Clear cell RCC represents 80-90% of all RCCs, and most of recent moleculartargeted drugs target clear cell RCC. About 70% of clear cell RCC features mutation or inactivation of the VHL tumor suppressor gene. As 769-P and 786-O cells have VHL mutation in each different mechanism [10], we selected the two renal cancer cell lines in our study.

Cell viability assay
For testing sensitivity to Bisebromoamide at different concentrations (0.1, 1, and 10 lmol/L), cells were seeded in flat-bottomed 96-well plates. After 24 h, the culture medium was replaced with medium containing the reagents and then incubated for another 48 or 72 h. Cell viability was determined employing an assay for watersoluble Tetrazolium (WST)-1 salts (Takara, Shiga, Japan). At the end of the incubation period, WST reagents were added to each well and incubated for 1 h. Cell viability was estimated colorimetrically by reading color intensity in a plate reader at 570 nm. Relative viability was calculated as a percent of the control. Each experiment was performed in triplicate.

Western blotting
Fifty micrograms of total protein was separated by SDSpolyacrylamide gel electrophoresis on 12.5% acrylamide gel and transferred to nitrocellulose membranes. Nonspecific binding was blocked in tris-buffered saline containing 5% nonfat dry milk before incubation with the primary antibodies. After washing, the blots were incubated with peroxidase-labeled secondary antibody (Dako Denmark A/S, Glostrup, Denmark). Signals were detected using enhanced chemiluminescence reagents with the ECL Pluse Western Blotting Detection System and then analyzed. Intensity was quantified using the LAS 3000 imaging system (Fujifilm, Tokyo, Japan).

Detection of apoptosis by flow cytometry
After treatment with Bisebromoamide (1 lmol/L) for 72 h, adherent and nonadherent cells were pooled and fixed. Breaks at the 3′-OH DNA end were detected using the TUNEL technique with an ApoTag ® Plus Fluorescein In Situ Apoptosis Detection Kit according to supplier's instructions (Chemicon, Temecula, CA). Fluorescein isothiocyanate-labeled cells were analyzed by flow cytometry using an Epics ® Altrae flow cytometer (Beckman Coulter, Fullerton, CA). The proportions of cells at different cell-cycle phases were assessed by the incorporation of BrdU/propidium iodide (PI) (Sigma) staining. After RCC cells had been exposed to the culture medium containing Bisebromoamide (1 lmol/L), the cells were stained with FITC-labeled BrdU and PI, and analyzed using a flow cytometer. Each experiment was performed in triplicate.

Statistical analysis
All values are expressed as means AE standard error (SE). Statistical analysis was performed by Student's t-test with P < 0.05 considered significant.

Effects of Bisebromoamide on the viability of renal cancer cells
After treatment with Bisebromoamide (0.1, 1, and 10 lmol/L) for 48 and 72 h, cell viability was significantly decreased in both cell lines at 1 and 10 lmol/L ( Fig. 1A and B). A probit analysis of the dose-response functions showed the IC 50 value after 72 h to be 1.54 AE 0.16 lmol/L for 769-P cells and 2.09 AE 0.08 lmol/L for 786-O cells. Based on these results, we decided to treat both cell lines with 1 lmol/L for 72 h in the following experiments.

Induction of apoptosis by Bisebromoamide in RCC cell lines
To determine whether the treatment of cells with Bisebromoamide may lead to apoptotic cell death, flow cytometric TUNEL analysis was performed. After treatment with 1 lmol/L Bisebromoamide for 72 h, apoptosis was observed in both cell lines ( Fig. 2A and B).  Fig. 3A and B). Expressions of cleaved caspase-3 in the two RCC lines were evaluated using Western blotting ( Fig. 7A and B). After 24 h of incubation, expressions of cleaved caspase-3 were increased in both cell lines tested.   p-ERKs at 1 lmol/L, while having little effect on t-ERKs in both cell lines (data not shown). Time-response experiments showed that Bisebromoamide inhibited the phosphorylation of ERK and Akt in both cell lines tested, whereas there were no evident effects on the expressions of t-ERKs and t-Akt ( Fig. 4A and B). Similar effects were recognized with phosphorylation of mTOR (Ser2448 and Ser2481) and phosphorylation of p70 S6 (Thr389 and Thr421/Ser424) in both cell lines (Fig. 5A and B).
Bisebromoamide has little effect on the upstream kinases of ERK and Akt

Discussion
In this study, we have demonstrated that Bisebromoamide has a cytotoxic effect on human RCC lines, and cytotoxic activity was apoptotic rather than necrotic. In addition, we showed that this compound suppressed cell proliferation in both cell lines by inhibiting the PI3K/Akt/mTOR pathway as well as the Raf/MEK/ERK pathway. These results suggest that Bisebromoamide inhibits two main signaling pathways, both of which play central roles in tumor cell proliferation. On the other hand, this drug did not inhibit the phosphorylation and expression of EGFR, MEK, PI3K and PDK1, and upstream receptors of Raf/ MEK/ERK and PI3K/Akt/mTOR pathways. These results do not imply that Bisebromoamide would inhibit the expression or activation of EGFR. Rather, Bisebromoamide would inhibit the activation of ERK and Akt, not MEK, PI3K, and PDK1. Therefore, we consider that the target of Bisebromoamide would be, at least in this study, ERK and Akt in renal cancer cells. The Raf/MEK/ERK signal transduction pathway is present in all eukaryotic cells. This pivotal pathway relays extracellular signals to the nucleus via a cascade of specific phosphorylation events involving Raf, MEK, and ERK to regulate fundamental cellular processes, including proliferation, differentiation, and cell survival [11]. The Raf/MEK/ERK pathway contributes to inducing growth factors involved in cell proliferation, as well as influencing apoptotic pathways, allowing cells to respond with the    aggressive growth behavior characteristic of RCC [12]. And this pathway has important antiapoptotic effects in RCC, thereby providing an attractive target for intervention [13]. Accordingly, inhibiting this pathway might lead to induce apoptosis. Moreover, phosphorylation of ERK is an independent prognostic factor in RCC. The association between activation of mitogen-activated protein kinases (MAPKs) and the carcinogenesis of human RCCs was first reported by Oka et al. [14] in 1995. They reported that MAPK activation was detected more frequently in high-grade than in low-grade RCCs, suggesting that constitutive activation of the MAPK cascade may play an important role in the carcinogenesis of RCCs, and that increased activation of MAPK could be associated with higher malignant potential in tumors. Campbell et al. [15] showed phosphorylation of ERK to be an independent prognostic factor in RCC that was associated with advanced and aggressive pathological features and to predict the onset of metastasis in patients with localized disease. On the other hand, the PI3K/Akt/mTOR pathway regulates several normal cellular functions that are also critical for tumorigenesis, including cellular proliferation, growth, survival, and mobility. This pathway is constitutively activated in various human malignancies, including kidney, prostate, breast, thyroid cancer, and others [16], and activation of this pathway plays a critical role in tumor progression [17]. Akt, which activates the mTOR pathway, is a subfamily of the serine/threonine protein kinases and has been implicated as being crucial in controlling the balance between cell survival and apoptosis [18]. Horiguchi et al. [19] demonstrated elevated activation of Akt to have a significant association with higher grade metastatic disease and poor survival in RCC. These results support our observation that Bisebromoamide induces apoptosis of RCC lines by inhibiting phosphorylation of both ERK and Akt.
Bisebromoamide is a novel cytotoxic peptide which was shown to specifically inhibit the phosphorylation of ERK. However, in this study, we demonstrated that this new drug acts as a multitarget kinase inhibitor that may directly block tumor growth by inhibiting several other kinases such as ERK, Akt, and mTOR in human RCC lines.
Effective inhibitors specific for many of the key components of the Raf/MEK/ERK or PI3K/Akt/mTOR pathway have been developed. For example, CI-1040, an oral MEK inhibitor, shows antitumor activity in patients with advanced non-small-cell lung, breast, colon, and pancreatic cancer [20]. Sorafenib, or BAY 43-9006, which targets Raf/MEK/ERK pathway, produced significant tumor growth inhibition and a reduction in 786-O tumors [21].