Advantageous/Unfavorable Effect Of Quercetin On The Membranes Of SK-N-SH Neuroblastoma Cells Part 1

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Abstract: Quercetin is a polyphenolic compound, the effects of which raise scientists' doubt. The results of many experiments show that it has anti-cancer, anti-inflammatory, and antioxidant properties, while other studies indicate its pro-oxidative and cytotoxic action. This compound can react with reactive oxygen species, and due to its chemical properties, it can be found in the hydrophobic-hydrophilic area of cells. These features of quercetin indicate that its action in cells will be associated with the modification of membranes and its participation in maintaining the redox balance. Therefore, this study distinguishes these two mechanisms and determines whether they are important for cell function. We check:(1)Whether the selected concentrations of quercetin are cytotoxic and destructive for SK-N-SH cell membranes (MTT, LDH, MDA tests) in situations with and without the applied oxidative stress; (2) what is the level of changes in the structural/mechanical properties of the lipid part of the membranes of these cells due to the presence of polyphenol molecules; and (3) whether the antioxidative action of quercetin protects the membrane against its modification. Our results show that changes in the stiffness/elasticity of the lipid part of the membrane constitute the decisive mechanism of action of quercetin, potentially influencing cellular processes whose initial stages are associated with membranes (e.g., reception of signals from the environment, transport).

Keywords: quercetin; neuroblastoma cells; cell membrane; Langmuir monolayer; oxidative stress

1. Introduction

Quercetin (3,3',4',5,7-pentahydroxy flavone) is a bioactive polyphenol occurring in plants [1-3] and is the most abundant flavonoid in the human diet [4].

It exhibits a wide range of properties—among anti-inflammatory [5] and immunomodulatory [6]. Literature data indicate that it may be a potential anticancer factor [7,8]. It owes its action to the ability to modify the course of intracellular signaling pathways[9,10] and to antioxidant properties that have been demonstrated, among others, on PC12 cells [11,12]and human neuronal SH-SY5Y cells [13].

This compound is an antiproliferative agent, inducing programmed cell death in a number of tumor lines, for example, in HL-60 and NB-4 cells from acute myelogenous leukemia [14,15] and K-562-chronic myelogenous leukemia [16]. It was also shown that quercetin inhibits the propagation of various types of cancers, such as in the lung, liver, breast, and colon [17-20]. Vijayababu et al.[21]found that it induces apoptosis of prostatic carcinoma cells(PC-3), as well as inhibits invasion, migration, and signaling molecules involved in cell survival and proliferation of this type of cell [22].

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However, the action of quercetin is not clear. There are also data indicating high cytotoxicity of this compound [23-25]. Research shows that quercetin, depending on the concentration, location in the cell, and the source of free radicals, may show a pro-oxidative effect. High doses generated oxidative stress in human lymphocytes [26].

Such dualistic action of quercetin may be related to the fact that as an antioxidant it may become an oxidation product. The result of this reaction is the formation of a semiquinone radical which oxidizes to quercetin quinone [2]. This compound can react with thiol groups becoming toxic to cells [26].

Another possibility for beneficial/unfavorable action on cells is the effect of this compound on membranes. Due to the chemical nature, and the presence of aromatic rings in the molecule, it can locate itself in hydrophobic cell compartments, thus changing its physicochemical properties [27,28]. Mechanical and structural properties of the lipid part of membranes are very important for the course of all processes related to membranes, i.e., transport, signaling, and stimuli conduction. These processes are mainly carried out by proteins anchored in membranes. polyphenols benefits Changes in the stiffness or flexibility of the membrane may block the possibility of adopting the appropriate conformation of individual protein domains, which may consequently disrupt or inhibit the process carried out by a given protein. It is, therefore, likely that changing the membrane composition by introducing even a small amount of quercetin between lipid molecules can significantly change their mobility.

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In view of existing literature ambiguity as to the effects of quercetin on cells, this research evaluates the anti or pro-oxidative action of quercetin and checks the influence of this compound on the functioning of cell membranes, including the determination of the structural modification of the lipid part of these membranes. saline cistanche This goal was achieved by examining the effect of different concentrations of quercetin on the survival of SK-N-SH cells and the level of damage to their membranes. The potential antioxidant activity of this compound was checked by initiating oxidative stress to cells after prior incubation with quercetin. standardized cistanche The action at the membrane level was tested in model systems giving unambiguous information about:(1)The structural modification of the lipid monolayer of composition reflecting the cell membrane(SK-N-SH) caused by the presence of various amounts of quercetin, and (2) the protective abilities of the tested flavonoid against peroxidation of unsaturated fatty acids in the lipids that build the cell membrane (SK-N-SH).

2. Results

2.1.Natioe Cell Membranes

The effect of quercetin on human SK-N-SH cells was investigated using the MTT assay, which determines mitochondrial activity.

SK-N-SH were exposed for 24 h to various concentrations of quercetin (3.1, 6.25, 12.5, 25,50, 100, and 200 uM). Cell treatment with lower concentrations of quercetin did not show any significant effect. In contrast, the addition of quercetin at a concentration of 200 uM caused a slight increase in the number of viable cells(11% compared to control)(Figure la).

The study also investigated the effect of varying concentrations of hydrogen peroxide (1-5 mM) on the cells after 3 h contact. It was observed that the lowest tested concentration caused a slight decrease in cell viability (by about 10% compared to the control), while higher concentrations were toxic to SK-N-SH cells. In the presence of H2O2 at3 and 5 mM concentrations in the culture medium, a significant(of approximately 54%)decrease in cell viability was noted compared to cells not treated with H2O2 (Figure 1b).

To determine the protective effect of quercetin against H2O, toxicity, cells were treated with various concentrations of quercetin for 24 h followed by 3 h contact of the sample with hydrogen peroxide of 3 and 5 mM. It was observed that quercetin of 3.1-50 uM concentrations caused a slight, where can i buy cistanche about9%, increase in the number of viable cells, as compared to cells treated with H, O, alone. In contrast, for SK-N-SH cells preincubated in a medium containing higher quercetin concentrations(100 and 200 uM) and then contacted with H2O2, cell viability increased by approximately 18 and 33% (Figure 1c).

The next stage of the work was to check to what extent quercetin and H, O, affect the membranes of the tested cells. For this purpose, the lactate dehydrogenase(LDH) test was used to assess the level of cell membrane damage, and the malondialdehyde(MDA)level was measured to evaluate the degree of lipid peroxidation. The SK-N-SH cells were treated for 24 h with selected concentrations of quercetin（6.25;25;100;200 μM）， and then the hydrogen peroxide (3-5 mM) was added for another 3 h.

Figure 1. The cell viability was determined by MTT assay. SK-N-SH cells were exposed for 24 h to various concentrations of quercetin (3.1-200 uM). The percentage of cell viability was referred to the untreated control cells (a); cells contacted for 3 h with various concentrations of H2O2 (mM) (b), SK-N-SH cells preincubated for 24 h with different concentrations of quercetin and then contacted for 3 h with H, O2. The percentage of cell viability was related to cells treated only with H,O2 (c). Values represent the mean ± SD of three to six independent experiments. Different letters indicate significant differences (p ≤0.05).

As shown in Table 1 the presence of quercetin in the medium used for 24 h treatment of SK-N-SH cells did not cause any significant change in LDH release.

Pretreatment with a quercetin concentration of 6.25 uM did not influence MDA content, while the higher doses of quercetin （25; 100 and 200 μM）induced an increase in lipid peroxidation by about 12%(Figure 2a).

Treatment of the cells with both quercetin and hydrogen peroxide did not affect LDH release in comparison to cells treated with hydrogen peroxide alone (Figure 2b).

Figure 2. The effect of quercetin on SK-N-SH cells exposed to H2O was measured by the degree of lipid peroxidation (concentration of MDA)(a) and LDH release (b). The cells were pretreated for 24 h with selected quercetin concentrations(6.25; 25;100; 200 uM), followed by3 h of contact with H2O2(3-5 mM). Anti aging cistanche Percentage of LDH leakage was referred to values obtained for cells treated with H2O, only(b). Values represent the mean± of three to six independent experiments. Different letters indicate significant differences (p ≤0.05).

2.2. Model Membranes

2.2.1.The Influence of Quercetin on the Mechanical Properties of Membranes

To test the effect of quercetin on the membranes of neuroblastoma cells, lipid mono-lavers (of a composition reflecting the lipid part of the membranes of the SK-N-SH cell line)were formed. Monolayers were made on pure buffer pH 7.4(control) and on a buffer containing quercetin at concentrations of 6.25， 12.5，25， and 50 μM. For such monolayers， the dependencies of surface pressure （元） on the area per single molecule in the monolayer (A) were measured (Figure 3).

The course of (π-A) isotherms obtained for the lipid mixture mimicking composition characteristic of the neuroblastoma membrane is characterized by a monotonic course, without noticeable phase transitions. The presence of quercetin clearly modifies this relationship, causing a shift in the curves towards higher A values. This tendency is directly proportional to the concentration of quercetin in the buffer solution. The dependence of the static Cs-Icompression modulus on rt was calculated based on the isotherms (inset in Figure 3).

Based on the obtained isotherms, the values of physicochemical parameters characterizing the tested monolayers were determined (Table2).

Figure 3. Surface pressure isotherms and corresponding compressibility modulus (inset) of the lipid model of neuroblastoma layers spread on the subphase contained the indicated concentrations of quercetin dissolved in buffer.

Table 2. Surface parameters of monolayers(Alim-the limiting area per one molecule; πcoll-collapse pressure; Cs-1-maximal values of compression modulus) were calculated for monolayers of the model of neuroblastoma membrane. Data represent the mean from three experiments ±SD. Different letters indicate significant differences (p ≤0.05).

With increasing quercetin amount, Alim(minimal area per molecule in the densely packed layer) increased proportionally to its concentration. For the highest level of quercetin, this increase was as large as 17.5% compared to the control. The remaining parameters, ie., πcoll, and Cs-I(surface pressure at the collapse point of the layer and static compression modulus) were less sensitive to the quercetin content in the subphase. The changes in Scroll ranged from 3-5%, and for Cs- from 0.2 to 8.8% compared to the control.

This article is extracted from Molecules 2021, 26, 4945. https://doi.org/10.3390/molecules26164945 https://www.mdpi.com/journal/molecules