Abstract 4442: Novel mechanism of gamma-tocotrienol-induced apoptosis in breast cancer cells: endoplasmic reticulum stress-dependent increase in death receptor signaling pathways

Abstract

Abstract Tocotrienols are naturally occurring vitamin E compounds found in the seed endosperms of monocots and enriched in palm oil. Tocotrienols have gained substantial attention for their distinct functions in health and disease including their neuroprotective, cholesterol lowering and anti-cancer properties. Our study focused on investigating the mechanisms involved in anti-cancer actions by tocotrienols using the BALB/c 66cl-4-GFP syngeneic mouse mammary tumor model and human MCF-7 and MDA-MB-231 breast cancer cells in cell culture. In the 66cl-4-GFP implanted mammary tumor model, dietary delivery of gamma-tocotrienol (250 mg/kg diet) inhibited tumor growth (41% inhibition compared to the control diet). Immunohistochemical analyses of tumor sections from gamma-tocotrienol treated mice showed decreased proliferation (Ki-67) and increased apoptosis (TUNEL). Gamma-tocotrienol inhibited colony formation by 66cl-4 as well as human MCF-7 and MDA-MB-231 breast cancer cells. Anti-proliferative effects of gamma-tocotrienol were determined to be derived primarily from treatment-induced apoptosis. Mechanistic studies using human breast cancer cells, showed gamma-tocotrienol to cleave caspases 8, 9, and 3, suggesting the involvement of death receptor signaling. Western blot and RT-PCR analyses showed that death receptor 5 (DR5) was upregulated at both the protein level as well as mRNA level after gamma-tocotrienol treatment. Silencing of DR5 using small interfering RNA (siRNA) attenuated gamma-tocotrienol induced apoptosis. Studies investigated C/EBP homologous protein (CHOP), a transcription factor that serves as an indicator for endoplasmic reticulum (ER) stress. In our study, CHOP expression was highly induced by gamma-tocotrienol treatment, and shown to directly bind to the promoter region of DR5 gene by chromatin immunoprecipitation analyses. Silencing CHOP using siRNA partially blocked gamma-tocotrienol induced apoptosis and DR5 upregulation. Functional knock-down of JNK and p38MAPK using siRNA suppressed the upregulation of CHOP as well as DR5, suggesting upregulation of CHOP-mediated DR5 expression by gamma-tocotrienol treatment is mediated by JNK and p38 MAPK. Additional analyses showed that not only gamma-tocotrienol but also δ-tocotrienol and the tocotrienol-rich fraction of palm oil increased the level of CHOP, Grp78, ATF-4, and Xbp-1 splicing, all of which are recognized biomarkers of ER stress. In conclusion, tocotrienols trigger apoptosis by inducing ER stress which leads to JNK and p38 MAPK-dependent CHOP and DR5 upregulation. Research was supported by The Clayton Foundation for Research, NIH Grant CA59739 and NIEHS Center Grant ES007784. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4442.