Andrzej Poła

Differential interaction of Sophora isoflavonoids with lipid bilayers.

The mechanisms of some biological effects exerted by flavonoids (e.g. activity against lipid oxidation, multidrug resistance modulation) may involve their interactions with lipid bilayers. Due to variety of substituents attached to the flavonoid nucleus individual isoflavones significantly differ in their properties; in particular they may differently interact with membranes. For this reason we have investigated the interactions of different isoflavones with lipid bilayers. The influence of four plant isoflavones on the phase transitions of dipalmitoylphosphatidylcholine (DPPC) and on liposome aggregation was studied, using microcalorimetry and absorption measurements, respectively. We found that isoflavones substituted with one or two prenyl groups less effectively induce liposome aggregation than more polar ones, possessing no prenyl groups. For aggregation-promoting compounds, rather small differences in the influence on phosphatidylcholine, phosphatidylserine and phosphatidylinositol liposomes were recorded. On the other hand, the alteration of DPPC phase transitions by prenyl-substituted isoflavones was more pronounced than changes induced by non-prenyl ones. On the basis of observed effects we conclude that prenyl-substituted isoflavones penetrate deeper into the lipid bilayer while more polar ones act closer to the membrane surface. Comparing our results with biological tests it seems that interactions with the hydrophobic core of membranes are responsible for the activity of the studied isoflavones.

Neither lipophilicity nor membrane-perturbing potency of phenothiazine maleates correlate with the ability to inhibit P-glycoprotein transport activity

Although phenothiazines are known as multidrug resistance modifiers, the molecular mechanism of their activity remains unclear. Since phenothiazine molecules are amphiphilic, the interactions with membrane lipids may be related, at least partially, to their biological effects. Using the set of phenothiazine maleates differing in the type of phenothiazine ring substitution at position 2 and/or in the length of the alkyl bridge-connecting ring system and side chain group, we investigated if their ability to modulate the multidrug resistance of cancer cells correlated with model membrane perturbing potency. The influence exerted on lipid bilayers was determined by liposome/buffer partition coefficient measurements (using the absorption spectra second-derivative method), fluorescence spectroscopy and calorimetry. Biological effects were assessed by a flow cytometric functional test based on differential accumulation of fluorescent probe DiOC2(3) by parental and drug-resistant cells. We found that all phenothiazine maleates were incorporated into lipid bilayers and altered their biophysical properties. With only few exceptions, the extent of membrane perturbation induced by phenothiazine maleates correlated with their lipophilicity. Within the group of studied derivatives, the compounds substituted with CF3- at position 2 of phenothiazine ring were the most active membrane perturbants. No clear relation was found between effects exerted by phenothiazine maleates on model membranes and their ability to modulate P-glycoprotein transport activity.

Increased lipid peroxidation, apoptosis and selective cytotoxicity in colon cancer cell line LoVo and its doxorubicin-resistant subline LoVo/Dx in the presence of newly synthesized phenothiazine derivatives

Cancer cells often develop the resistance to pro-apoptotic signaling that makes them invulnerable to conventional treatment. Therapeutic strategies that make cancer cells enter the path of apoptosis are desirable due to the avoidance of inflammatory reaction that usually accompanies necrosis. In the present study phenothiazines (fluphenazine and four recently synthesized derivatives) were investigated in order to identify compounds with a potent anticancer activity. Since phenothiazines are known as multidrug resistance modulators the sensitive human colorectal adenocarcinoma cell line (LoVo) and its doxorubicin-resistant, ABCB1 overexpressing, subline (LoVo/Dx) have been employed as a model system. In studied cancer cells cytotoxic effect of the phenothiazine derivatives was accompanied by apoptosis and autophagy induction as well as by the increase of cellular lipid peroxidation and intracellular reactive oxygen species generation. Molecular modelling revealed that reactivity of phenothazines (manifested by their low energy gap) but not lipophilicity was positively correlated with their anticancer potency, pro-oxidant properties and apoptosis induction ability. Additionally, some of the studied compounds turned out to be more potent cytotoxic and pro-apoptotic agents in doxorubicin-resistant (LoVo/Dx) cells than in sensitive ones (LoVo). The hypothesis was assumed that studied phenothiazine derivatives induced apoptotic cell death by increasing the production of reactive oxygen species.