γ-Tocotrienol-induced autophagy in malignant mammary cancer cells

Abstract

γ-Tocotrienol, a member of the vitamin E family of compounds, displays potent antiproliferative and cytotoxic effects in a variety of cancer cell types at treatment doses that have little or no effect on normal cell viability or growth. Autophagy is a tightly regulated lysosomal self-digested process that can either promote cell survival or programmed cell death, but the role of autophagy in mediating γ-tocotrienol-induced cytotoxicity in breast cancer is not presently completely understood. Mouse (+SA) and human (MCF-7 and MDA-MD-231) mammary tumor cells lines were exposed to 0-40 µmol/L γ-tocotrienol for a 24 h treatment period. γ-Tocotrienol treatment caused a relatively large increase in the accumulation of monodansylcadaverine (MDC)-labeled vacuoles, a marker of autophagosome formation, in all tumor cell lines. Results also showed that γ-tocotrienol treatment induced an increased conversion of microtubule-associated protein, 1A/1B-light chain 3, from its cytosolic form (LC3B-I) to its lipidated form (LC3B-II), increased Beclin-1 levels, and increased acridine orange staining as determined by flow cytometry analysis, providing further evidence of γ-tocotrienol-induced autophagy in these mammary cancer cell lines. In contrast, similar treatment with γ-tocotrienol was not found to increase autophagy marker expression in immortalized mouse (CL-S1) and human (MCF-10 A) normal mammary epithelial cell lines. Treatment with γ-tocotrienol also caused a reduction in PI3K/Akt/mTOR signaling and a corresponding increase in the Bax/Bcl-2 ratio, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) levels in these cancer cell lines, suggesting that γ-tocotrienol-induced autophagy may be involved in the initiation of apoptosis. In summary, these findings demonstrate that the cytotoxic effects of γ-tocotrienol are associated with the induction of autophagy in a mouse and human mammary cancer cells.

Role of Rac1/WAVE2 Signaling in Mediating the Inhibitory Effects of γ-Tocotrienol on Mammary Cancer Cell Migration and Invasion

“…34,35) This stock γ-tocotrienol solution was then used to prepare different concentrations of treatment media.…”

“…Membranes were then blocked and incubated with specific primary antibodies diluted 1 : 1000 to 1 : 5000 as described previously. 35) Membranes were then washed and incubated with their respective horseradish-peroxide conjugated secondary antibodies diluted 1 : 5000. Immunocytochemical Fluorescence Staining MDA-MB-231 and +SA cells were initially plated at 2×10 4 cells/ chamber in 8-chamber glass (BD Falcon, San Jose, CA, U.S.A.) and maintained in control media.…”

“…The next day, viable cell count was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as described previously. 34,35) Briefly, cells were incubated at 37°C for 3 h in control media containing 0.5 mg/mL MTT. Afterwards, media was removed and MTT crystals dissolved in dimethyl sulfoxide (DMSO; 100 µL/well), and optical density of each sample was measured at 570 nm on a microplate reader (SpectraCount, Packard BioScience Company, Meriden, CT, U.S.A.).…”

“…Afterwards, cells were washed with PBS, trypsin was used to isolate cells and lysates were collected for later assay. 35) The Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, U.S.A.) was used to determine protein concentration in each sample. Equal amounts were loaded into each well and then electrophoresis through sodium dodecyl sulfate (SDS)-polyacrylamide minigels was performed.…”

Anticancer Effects of γ-Tocotrienol Are Associated with a Suppression in Aerobic Glycolysis

“…10) γ-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer effects at treatment doses that have little or no effect on normal cell viability. [11][12][13][14][15][16] Previous studies have shown that γ-tocotrienol significantly decreases growth factor receptor mediated activation of the PI3K/Akt/mTOR pathway. 14,17,18) Recently, it has also been shown that exposure to tocotrienols decreases c-Myc protein levels, [18][19][20] and block compensatory increases in HIF-1α during hypoxic conditions in cancer cells.…”