Effect of the natural polymethoxylated flavone artemetin on lipid oxidation and its impact on cancer cell viability and lipids

Abstract

The biochemical class of the polymethoxylated flavonoids represents uncommon phenolic compounds in plants presenting a more marked lipophilic behavior due to the alkylation of its hydroxylic groups. As a polymethoxylated flavone, which concerns a different bioavailability, artemetin (ART) has been examined in vitro against lipid oxidation and its impact on cancer cells has been explored. Despite this flavone only exerted a slight protection against in vitro fatty acid and cholesterol oxidative degradation, ART significantly reduced viability and modulated lipid profile in cancer Hela cells at the dose range 10–50 μM after 72 h of incubation. It induced marked changes in the monounsaturated/saturated phospholipid class, significant decreased the levels of palmitic, oleic and palmitoleic acids, maybe involving an inhibitory effect on de novo lipogenesis and desaturation in cancer cells. Moreover, ART compromised normal mitochondrial function, inducing a noteworthy mitochondrial membrane polarization in cancer cells. A dose-dependent absorption of ART was evidenced in HeLa cell pellets (15.2% of the applied amount at 50 μM), coupled to a marked increase in membrane fluidity, as indicate by the dose-dependent fluorescent Nile Red staining (red emissions). Our results validate the ART role as modulatory agent on cancer cell physiology, especially impacting viability, lipid metabolism, cell fluidity, and mitochondrial potential.

Introduction

Flavonoids are a well-known group of polyphenolic compounds characterized by a benzo-γ-pyrone structure and occurring ubiquitously in plants and foods/beverages of natural origin [1,2]. These secondary metabolites have attracted considerable attention due to their numerous biological properties that include both pharmacological and alimentary aspects [[1], [2], [3], [4], [5], [6]] such protecting various cell types from oxidative stress via different mechanisms [[1], [2], [3]], quenching radicals, chelating different ions [1,2,7], inducing cytoprotective enzymes and antioxidant transcriptional genes [3], inhibiting cancer cell viability and proliferation, angiogenesis, and migration [2,4,8]. Not negligible, cell lipid metabolism has been suggested as another possible target of dietary natural flavonoids in cancer cells [8].

An important issue is the deep correlation between the direct antioxidant potency of flavonoids and their structure that involves the number and substitutions of hydroxyl groups (Fig. 1) strongly affecting not only their free radical scavenging activity [1], but their bioavailability in terms of absorption, distribution, metabolism, and excretion [6].

Generally, flavonoids are considered to penetrate the plasma membrane both by passive diffusion and by transporters [5,6] and properties such as molecular weight and lipophilicity, mainly established by number and type of substituents (hydroxyl, methoxyl, prenyl, and glycosyl groups), are basically involved in their absorption [5,6]. Moreover, numerous studies evidenced the ability of flavonoids to affect cell membrane fluidity and properties, interacting with the lipid membrane surface or inserting themselves into the lipid bilayer [5,9]. It has been shown that flavonoid structural properties as the partition coefficient (log P3), total number of H-bonds, and topological surface area (TPSA) [10] can reveal how these compounds penetrate cell membrane [5].

Considering all these aspects, we focused our attention on O-methylated flavone artemetin (5-hydroxy-3,6,7,3′,4′-pentamethoxyflavone) (ART, 1) (Fig. 1) already identified as an active compound in plants and dietary spices that enjoy a long application in traditional medicine, as Achillea millefolium L. [11,12], Artemisia absinthium [12,13], Vitex trifolia [14], and Artemisia argyi [15]. ART is an uncommon polymethoxylated lipophilic flavone (log P3 = 3.4, Table 1) [10] presenting only a free hydroxyl group (in the A ring, Fig. 1) involved in a hydrogen bond with the ketone moiety, structural characteristics that could lead to substantial different biological activity.

Anyway, several studies investigated the antioxidant [12,13,16], anti-hypertensive [11], anticancer [17], antiparasitic [13], and anti-inflammatory [14,15] properties of ART, highlighting some controversial results regarding its bioactivity [13,18,19].

In this manuscript we investigated the direct ART ability to prevent the oxidative degradation of cholesterol and phospholipids, essential components of biological membranes and lipoproteins [20], which degradation plays an essential role in the development of tissue damage associated with several pathological states (inflammation, cancer, atherosclerosis, and neurodegenerative diseases) [21,22]. Moreover, experiments were designed to evidence the impact of ART on lipid profile in cancer cells, with regard to the total fatty acid composition, phospholipids (PL) and free cholesterol (FC) levels. Lipid metabolism is considered a promising anticancer target and several antitumor drugs (lipophilic or amphiphilic molecules) act through the membranes by changing the fluidity, the general lipid membrane organization and structure, and inhibiting the expression of enzymes involved in lipid metabolism [[23], [24], [25], [26], [27]]. Changes in lipid profile in ART-treated cancer cells together with the investigation of the cell viability and the changes occurring on cell morphology, cytoplasmic membranes, and mitochondria membrane potential were investigated. Finally, the ART absorption in cancer HeLa cells was preliminary assessed. The bioactivity profile of ART was compared to that of eupatilin (EUP, 7-dihydroxy-3′,4′,6-trimethoxyflavone) (Fig. 1), a flavone analogue with antioxidant and anticancer activities, characterized by a less extent of methoxylation in the A and C rings than ART [8], in the prospective to underline structure activity relationship.