Elsevier

Oral Oncology

Volume 45, Issue 11, November 2009, Pages 998-1004
Oral Oncology

Sulforaphane induces G2–M arrest and apoptosis in high metastasis cell line of salivary gland adenoid cystic carcinoma

https://doi.org/10.1016/j.oraloncology.2009.05.641Get rights and content

Summary

New chemotherapeutic strategy should be investigated to enhance clinical management in salivary gland adenoid cystic carcinoma (ACC). Recently, sulforaphane (SFN), as a natural compound from cruciferous vegetables exhibits a potent anti-cancer activity in various tumor cells, but remains uncertain in ACC cells. The present study examined whether SFN suppresses proliferation and in ACC cells, if so, the possible molecular targets would be further investigated. Cell survives, apoptosis, cell cycle progression and molecular targets were identified by multiple detecting techniques, including trypan blue dye exclusion assay, electron microscopy, AO/EB staining, flow cytometry and immunoblotting in human lung high metastasis cell line of salivary gland adenoid cystic carcinoma (ACC-M). The results showed that 5–20 μM SFN suppressed proliferation and induced apoptosis of ACC-M cells in dose- and time-dependent manners. Cell cycle analysis demonstrated treatment of ACC-M cells with 20 μM SFN resulted in G2/M cell cycle arrest, which was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin B1 and cyclin-dependent kinase 1 (Cdk1). In terms of apoptosis, SFN increased the expression of Bax and decreased the level of Bcl-2 and subsequently triggered release of cytochrome c from mitochondria and activation of caspase-3, but Fas level and caspase-8 activity remained unchanged at all time points. Furthermore, levels of nuclear factor-κB (NF-κB) p65 in both of the cytoplasm and the nucleus have also been markedly suppressed by SFN in a time-dependent manner. Taken together, these results suggest SFN inhibits cell growth via inducing G2/M cell arrest and apoptosis in ACC-M cells. These events have been associated with SFN-regulated multiple targets involved in ACC-M cell proliferation. The present study provides an evidence for testing SFN efficacy in vivo and warranting future investigations to exam the clinical potential of SFN in ACC chemotherapy.

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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