Sulforaphane induces G2–M arrest and apoptosis in high metastasis cell line of salivary gland adenoid cystic carcinoma
Introduction
Salivary gland adenoid cystic carcinoma (ACC) is a high malignant carcinoma, which accounts for around 10% of all salivary gland neoplasms and approximately 3∼5% of all head and neck malignancies.1 It has a tendency for a prolonged clinical course, with local recurrences and distant metastases sometimes occurring many years after presentation. After surgery and radiation therapy, the disease-specific survival at 10 years for patients with ACCs remains to be 29–40%.2 Chemotherapy is essential to prevent lung metastases and prolong longevity, but the resistance of malignant carcinoma to the chemotherapy medicines has been one of the major obstacles to successful anti-cancer therapy. Chemotherapeutic agents, such as doxorubicin, paclitaxel, 5-fluorouracil clearly have some degree of activity in this disease, but responses are only in the range of 15–30%, and response duration seems to be generally short, usually 5–13 months, suggesting that most deaths from ACCs are caused by lung metastases correlated with resistance to chemotherapy.3
Epidemiologic studies have indicated an inverse correlation between the dietary intake of fruits and vegetables and the risk of various types of cancers, including ACCs.[4], [5] Several studies have documented the cancer-preventive activity of a significant number of isothiocyanates (ITCs), the majority of which occur in plants, especially in cruciferous vegetables. The most characterized ITC is sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane, SFN].[6], [7] SFN has received a great deal of attention because of its ability to simultaneously modulate multiple cellular targets involved in cancer development. Evidences are accumulating to show that SFN suppresses proliferation in cultured cancer cells by causing apoptosis and/or cell cycle arrest.[8], [9] Despite compelling epidemiological evidence for the protective effects of cruciferous vegetables against various malignant tumors, activity of SFN against ACCs has not been examined. In addition, an understanding of mechanism by which SFN inhibits proliferation in ACCs cells is critical for its future clinical trial. A most resent study indicates that SFN induces cell type-specific apoptosis in human breast cancer cell lines. For instance, activation of apoptosis by SFN in MDA-MB-231 cells seemed to be initiated through induction of Fas ligand, which resulted in activation of caspase-8, caspase-3, and poly(ADPribose) polymerase, whereas apoptosis in the other breast cancer cell lines was initiated by decreased Bcl-2 expression, release of cytochrome c into the cytosol, activation of caspase-9 and caspase-3, but not caspase-8, and poly(ADP-ribose) polymerase cleavage.9 It seems that mechanism of SFN-induced cell death has been associated with cell type. Therefore, it is necessary to elucidate the molecular targeting that involved in anti-proliferation of SFN in ACCs cells.
In brief, this study aimed to evaluate the growth inhibitory effects of SFN on ACC-M, a human lung high metastatic cell line of ACCs, and also explore its molecular targets involved in regulation of cell cycle and apoptosis. The results of this study may also provide evidence for future treatment of ACCs patients, i.e. whether it is appropriate to supplement SFN to chemotherapy regimens.
Section snippets
Reagents and antibodies
Sulforaphane (SFN, >95.6%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). SFN was prepared as 20 mM stock solutions in dimethyl sulfoxide (DMSO, Sigma) and stored at −20 °C. For each experiment, the reagents were diluted with cell culture medium to the concentrations indicated, with a final DMSO concentration of 1% (v/v). Antibodies against cyclin B1, cyclin-dependent kinase 1(CdK1), Fas, Bcl-2, Bax, and cytochrome c were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against
SFN suppressed proliferations of cultured ACC-M cells
Treatment of ACC-M cells with SFN at doses up to 40 μM for 48 h inhibited cell proliferation. SFN treatment markedly reduced the number of cells attached to the plating surface while slightly increase the number of cells floating in culture media within 48 h. However, SFN-inhibited cell proliferation reached plateau at the concentration of 20 μM SFN (Fig. 1A). This dose was chosen as the maximum concentration for all the following assays. Similarly, cells were treated with 5 or 20 μM SFN for 24, 48
Discussion
In this study, we observed that SFN, as a naturally occurring phytochemical compound in cruciferous vegetables such as watercress, exerts potent anti-proliferative effects on ACC-M cells in vitro. We found that SFN exerts activity against proliferation of ACC-M cells by arresting cells in the G2/M phase of the cell cycle and causing apoptosis. One point has been addressed that growth suppressed concentration of SFN (5, 20, and 40 μM) in the present study was consistent with SFN concentrations
Conflict of interest statement
None declared.
Acknowledgement
This project was supported by the Postdoctoral Science Foundation of Heilongjiang Province (LRD06-185), China.
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