Uptake of Flaxseed Dietary Linusorbs Modulates Regulatory Genes Including Induction of Heat Shock Proteins and Apoptosis

Abstract

Flaxseed (Linum usitatissimum L.) is gaining popularity as a superfood due to its health-promoting properties. Mature flax grain includes an array of biologically active cyclic peptides or linusorbs (LOs, also known as cyclolinopeptides) that are synthesized from three or more ribosome-derived precursors. Two flaxseed orbitides, [1-9-NαC]-linusorb B3 and [1-9-NαC]-linusorb B2, suppress immunity, induce apoptosis in a cell line derived from human epithelial cancer cells (Calu-3), and inhibit T-cell proliferation, but the mechanism of LO action is unknown. LO-induced changes in gene expression in both nematode cultures and human cancer cell lines indicate that LOs promoted apoptosis. Specific evidence of LO bioactivity included: (1) distribution of LOs throughout the organism after flaxseed consumption; (2) induction of heat shock protein (HSP) 70A, an indicator of stress; (3) induction of apoptosis in Calu-3 cells; and (4) modulation of regulatory genes (determined by microarray analysis). In specific cancer cells, LOs induced apoptosis as well as HSPs in nematodes. The uptake of LOs from dietary sources indicates that these compounds might be suitable as delivery platforms for a variety of biologically active molecules for cancer therapy.

Keywords: anti-cancer; apoptosis; cyclic peptide; flaxseed; heat shock protein; linusorb.

Figure 1

Chemical structures of (A) CsA and (B–D) LOs.

Figure 2

Localization of observed LOs (LO1OB1, LO1OB2, and LOB3) in (A) pig and (B) rainbow trout adipose, identified via direct injection on an Agilent 1100-series HPLC-ToF-MS.

Figure 3

Induction of HSP 70A mRNA expression in C. elegans by LOB3 at different concentrations. The different letters (a–d) represent significance at p < 0.01. All samples were normalized to GAPDH expression. An Unamended culture medium and DMSO were used as the control treatment and negative control, respectively. Mean HSP 70 values and SDs from three repetitions shown. Data were analyzed by one-way ANOVA followed by Duncan’s multiple range test. Values not sharing a common letter are significantly different (p < 0.01).

Figure 4

Gene expressions of (A) PUMA and (B) BCL2 after LO treatments. DMSO is a common negative control and is used as a solvent for dissolving LOs as LOs do not dissolve in water. CPT (positive control) is a cytotoxic quinoline alkaloid that inhibits the DNA enzyme topoisomerase I and has shown remarkable anti-cancer activity. The different letters (a–j) represent significance at p < 0.05.