Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization

Abstract

In the context of an organism, epithelial cells by nature are designed to be the defining barrier between self and the outside world. This is especially true for the epithelial cells that form the lining of the digestive tract, which absorb nutrients and serve as a barrier against harmful substances. These cells are constantly bathed by a complex mixture of endogenous (bile acids, mucus, microbial metabolites) and exogenous (food, nutrients, drugs) bioactive compounds. From a cell biology perspective, this type of exposure would directly impact the plasma membrane, which consists of a myriad of complex lipids and proteins. The plasma membrane not only functions as a barrier but also as the medium in which cellular signaling complexes form and function. This property is mediated by the organization of the plasma membrane, which is exquisitely temporally (nanoseconds to minutes) and spatially (nanometers to micrometers) regulated. Since numerous bioactive compounds found in the intestinal lumen can directly interact with lipid membranes, we hypothesize that the dynamic reshaping of plasma membrane organization underlies the chemoprotective effect of select membrane targeted dietary bioactives (MTDBs). This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Keywords: Cancer prevention; Dietary bioactives; Membrane order; Membrane therapy; n−3 PUFA.

Figure 1. Putative role of microbial metabolites of procyanidin based dietary bioactives as modulators of colonocyte membrane-dependent oncogene signaling and cancer risk

We hypothesize that select poorly digestible dietary-microbial derived bioactives can promote a chemoprotective cell membrane microenvironment in the colon.

Figure 2. Procyanidins modulate colonocyte plasma membrane organization

In order to determine if microbial metabolites of procyanidins directly modulate plasma membrane biophysical properties (membrane order), immortalized young adult mouse colonocyte (YAMC) cells expressing oncogenic HRasG12V were incubated with a membrane order sensitive dye Di-4-ANEPPDHQ (5 uM) for 30 min prior to generation of giant plasma membrane vesicles (GPMVs). GPMVs were incubated with, (A) (+)-Catechin, or (B) Procyanidin B2, for at least 30 min, followed by determination of GPMV generalized polarization (GP) by (C) imaged based flow cytometry using an Amnis FlowSight system. Emission wavelengths of 480-560 and 640–745 were used for ordered (Green) and disordered (Red) channels, respectively. (D) GP was defined as the integrated fluorescence intensity from the ordered channel minus that of the disordered channel normalized by the total intensity (sum of the two channels). Quantification of membrane order is represented as mean GP, normalized to the untreated control for at least 4000 individual vesicles from two separate experiments. Statistical significance between untreated control and treatments (*P<0.05) was determined using 1-way ANOVA and Dunnett’s multiple comparisons test. (E) To determine if microbe derived metabolites indirectly modify plasma membrane biophysical properties, membrane order was also determined in live YAMC-HRasG12V cells, where cytoskeletal influences contribute to membrane biophysical properties. Experiments were performed by confocal microscopy using a Zeiss 780 system, after incubation with compounds for at least 30 min. Emission wavelengths of 508–544 and 651–695 were used for ordered and disordered channels, respectively. Scale bar, 50 μM. (F) Quantification of membrane order is represented as mean GP normalized to the untreated control for at least 10 fields of view containing approximately 100 cells. Statistical significance between untreated control and treatments (*P<0.05) was determined using 1-way ANOVA and Dunnett’s multiple comparisons test. (G) To assess effects on cytoskeletal-membrane dependent macropinocytosis, YAMC-HRasG12V cells were serum starved (0.5% FBS) for 18 h, then incubated with a macropinocytosis inhibitor (EIPA) or procyanidin metabolites for 30 min prior to EGF (25 ng/mL) stimulation for 5 min in the presence of fluorescently (FITC) labeled dextran (70 kDa, 1 mg/ml). (H) Quantification of macropinocytosis, normalized to non-stimulated control, for at least 13,000 cells from two separate experiments. Statistical significance between EGF stimulated control and treatments (*P<0.05) was determined using 1-way ANOVA and Dunnett’s multiple comparisons test.

Figure 3

Proposed mechanism describing the role of MTDB’s as modulators of colonocyte membrane-dependent oncogene signaling and cancer risk. 1) Procyanidins remodel plasma membrane organization. 2) Membrane remodeling disrupts Ras nanocluster/dimerization. 3) MTDB’s suppress Ras nanocluster/dimerization and attenuate oncogenic signaling. 4) This results in a reduction in tumor initiation/growth.