Karolina Szewczyk

Overview of Metabolism and Bioavailability Enhancement of Polyphenols

A proper diet is one of major factors contributing to good health and is directly related to general condition of the organism. Phenolic compounds are abundant in foods and beverages (fresh and processed fruits and vegetables, leguminous plants, cereals, herbs, spices, tea, coffee, wine, beer) and their pleiotropic biological activities result in numerous health beneficial effects. On the other hand, high reactivity and very large diversity in terms of structure and molecular weight renders polyphenols one of the most difficult groups of compounds to investigate, as evidenced by ambiguous and sometimes contradictory results of many studies. Furthermore, phenolics undergo metabolic transformations, which significantly change their biological activities. Here, we discuss some aspects of metabolism and absorption of phenolic compounds. On the basis of information reported in the literature as well as in summaries of clinical trials and patent applications, we also give an overview of strategies for enhancing their bioavailability.

Synergistic interactions between anticancer chemotherapeutics and phenolic compounds and anticancer synergy between polyphenols.

Chemoprevention has recently gained a new dimension due to the possibility of studying the mechanisms of action of chemopreventive agents at the molecular level. Many compounds have been proved to inhibit early stages of carcinogenesis in experimental models. These compounds include both recognized drugs (such as tamoxifen and nonsteroidal anti-inflammatory drugs) and natural constituents of edible and therapeutic plants, particularly polyphenols. Phenolics are characterized by high structural diversity and, consequently, a very broad spectrum of biological activities. They are increasingly looked upon as a valuable alternative or a support for synthetic drugs, as evidenced by a growing number of clinical trials regarding the use of phenolic compounds and polyphenol-rich extracts in chemoprevention and therapy. In the present work, we discuss the effectiveness of natural polyphenols as cancer preventive and therapeutic agents resulting from their synergy with synthetic or semisynthetic anticancer drugs as well as with other phenolic compounds of plant origin.

Flavanols from Japanese Quince (Chaenomeles Japonica) Fruit Inhibit Human Prostate and Breast Cancer Cell Line Invasiveness and Cause Favorable Changes in Bax/Bcl-2 mRNA Ratio

Polyphenols are natural compounds of high structural diversity which translates into a very wide spectrum of biological activities, including chemoprevention. Here we report that a Japanese quince fruit flavanol preparation (JQFFP) caused favorable changes in Bax/Bcl-2 mRNA ratio, which rendered normal and cancer cells more resistant and more sensitive, respectively, to apoptosis. DU145 human prostate cancer cells were characterized by the most advantageous Bax/Bcl-2 ratio. The growth and invasiveness of MDA-MB-231 human breast cancer cells were strongly suppressed by JQFFP, which was accompanied with a decrease in MMP-9 activity and stimulation of TIMP-1 expression. Importantly, JQFFP did not decrease normal human prostate PNT1A cell number, whereas Bax/Bcl-2 ratio decreased which implies increased resistance to apoptosis. In conclusion, JQFFP exhibited a potent antiproliferative effect against cancer cells, inhibited their invasiveness, and decreased expression level of several genes involved in apoptosis, angiogenesis, and metastasis.

Flavanols from evening primrose (Oenothera paradoxa) defatted seeds inhibit prostate cells invasiveness and cause changes in Bcl-2/Bax mRNA ratio.

In this study, we assessed the influence of an evening primrose flavanol preparation (EPFP) on proliferation and invasiveness of human prostate cancer cells (DU 145) and immortalized prostate epithelial cells (PNT1A). We report for the first time that EPFP reduces DU 145 cell proliferation (IC50 = 97 μM GAE for 72 h incubation) and invasiveness (by 24% versus control at 75 μM GAE). EPFP strongly inhibited PNT1A invasiveness in a concentration-dependent manner (by 67% versus control at 75 μM GAE) and did not cause a reduction in their proliferation. Furthermore, EPFP inhibited the activities of MMP-2 and MMP-9 secreted to culture medium by PNT1A cells by 84% and 34% versus control at 100 μM GAE, respectively. In the case of DU 145, MMP-9 activity at 100 μM GAE was reduced by 37% versus control. Moreover, the evening primrose seed flavanols suppressed the expression of selected genes (MMP-1, MMP-9, MMP-14, c-Fos, c-Jun, and VEGF) and also caused favorable changes in Bcl-2/Bax mRNA ratio which render DU 145 cells more sensitive to apoptosis-triggering agents. An additional confirmation of the proapoptotic activity of EPFP toward DU 145 was visualization of characteristic apoptotic bodies by DAPI staining. In conclusion, this study suggests that EPFP may increase apoptosis and reduce angiogenesis of prostate cancer cells.

Procyanidins from Evening Primrose (Oenothera paradoxa) Defatted Seeds Inhibit Invasiveness of Breast Cancer Cells and Modulate the Expression of Selected Genes Involved in Angiogenesis, Metastasis, and Apoptosis

There is a growing interest in plant polyphenols (including flavanols) that exhibit pleiotropic biological activities such as antiinflammatory, antioxidant, and anticancer effects. Here, we report for the first time the inhibition of MDA-MB-231 breast cancer cell viability and invasiveness by an evening primrose flavanol preparation (EPFP). We observed a decrease in MDA-MB-231 viability of 50% vs. a control after 72 h of incubation with EPFP at a concentration of 58 μM gallic acid equivalents (GAE) and an inhibition of their invasiveness of 65% vs. a control at 75 μM GAE after 48 h of incubation. EPFP caused a 10-fold reduction in matrix metalloproteinase-9 (MMP-9) activity at 100 μM GAE. Furthermore, through modulation of mRNA expression, EPFP reduced the expression levels of the following proteins: antiapoptotic Bcl-2, angiogenic vascular endothelial growth factor (VEGF), and 2 transcription factors (c-Jun, c-Fos). Moreover, analysis by flow cytometry revealed that EPFP induced apoptosis in MDA-MB-231 cells. In conclusion, our data shows that EPFP inhibits cell viability by increasing apoptosis and decreases cell invasiveness by decreasing angiogenesis.

Comparison of Anti‐Invasive Activity of Parthenolide and 3‐Isopropyl‐2‐Methyl‐4‐Methyleneisoxazolidin‐5‐One (MZ‐6) – A New Compound with α‐Methylene‐γ‐Lactone Motif – on Two Breast Cancer Cell Lines

The biological activities of parthenolide, a sesquiterpene lactone isolated from feverfew, have been attributed to the presence of the α‐methylene‐γ‐lactone skeleton. The lactone skeleton can react via the Michael type addition with sulfhydryl groups of enzymes and other functional proteins, interfering with key biological processes in the cell. In the present study, we describe an efficient method of preparation of 3‐isopropyl‐2‐methyl‐4‐methyleneisoxazolidin‐5‐one (MZ‐6), a synthetic compound with α‐methylene‐γ‐lactone ring, as in parthenolide, additionally modified by introduction of a nitrogen atom. Furthermore, we investigated the cytotoxic activity and anti‐metastatic potential of MZ‐6 in comparison with parthenolide. Both compounds showed considerable cytotoxicity against breast cancer MCF‐7 and MDA‐MB‐231 adenocarcinoma cells in vitro and were then evaluated for their anti‐metastatic potential. The experimental results showed that MZ‐6 and parthenolide suppressed, to a similar degree, migration of MCF‐7, but not more aggressive MDA‐MB‐231 cells. In both cell lines, tested compounds down‐regulated mRNA and protein levels of metalloproteinase‐9 and urokinase plasminogen activator, the key proteases involved in the degradation of extracellular matrix and dissemination of cancer cells. The obtained results indicate that simple analogs of α‐methylene‐γ‐lactones can be good substitutes for more complex structures isolated from plants.

Anticancer Activity of New Synthetic α‐Methylene‐δ‐Lactones on Two Breast Cancer Cell Lines

Natural products are important leads in drug discovery. The search for effective plant‐derived anticancer agents or their synthetic analogues has continued to be of interest to biologists and chemists for a long time. In this report, cytotoxicity and anticancer activity of new synthetic α‐methylene‐δ‐lactones was tested against two breast cancer cell lines, invasive, hormone‐independent MDA‐MB‐231 and hormone‐dependent MCF‐7. Cytotoxicity was examined using MTT assay. The ability to induce apoptosis and changes in mitochondrial membrane potential was studied by flow cytometry. The expression levels of pro‐ and anti‐apoptotic genes were determined by quantitative real‐time PCR. Cancer cell migration and invasion were assessed by wound healing and Matrigel assays. Additionally, secretion of proteins associated with invasiveness, metalloproteinase‐9 (MMP‐9) and urokinase plasminogen activator (uPA) was investigated using commercial ELISA kits and MMP‐9 activity by gelatin zymography. A natural sesquiterpene lactone, parthenolide, was used as a positive control. Screening results showed all four analogues to be highly cytotoxic. The most potent compound of the series, 1‐isopropyl‐2‐methylene‐1,2‐dihydrobenzochromen‐3‐one, designated DL‐3, which reduced the number of viable MDA‐MB‐231 and MCF‐7 cells with the IC50 values of 5.3 μM and 3.54 μM, respectively, was selected for further research. DL‐3 activated the intrinsic pathway of apoptosis, associated with the loss of mitochondrial membrane potential and changes in Bax/Bcl‐2 ratio. DL‐3 also inhibited the movement of both types of breast cancer cells. Suppression of cell migration and invasion was the result of the decreased secretion of enzymes responsible for the degradation of the extracellular matrix, MMP‐9 and uPA. These findings show that the synthetic α‐methylene‐δ‐lactone, DL‐3, displays potential to be further explored in the development of new anticancer agents.

Procyanidins From Japanese Quince (Chaenomeles Japonica) Fruit Induce Apoptosis in Human Colon Cancer Caco-2 Cells in a Degree of Polymerization-Dependent Manner

Plant proanthocyanidins, including procyanidins, display various biological activities. Here we report an inhibition of human colon cancer Caco-2 cell growth by the extract from Japanese quince fruit and the procyanidin-rich fractions of the extract. We observed that the amount of apoptotic Caco-2 cells increased by 52.1% vs. control after 72-h incubation with 50 μg extract/mL, as assessed by flow cytometry and image cytometry. Under the same experimental conditions the corresponding values for human colon cancer HT-29 cells and for rat normal intestinal IEC-6 cells were 5.0% and 8.1%, respectively. The extract fractions enriched with higher oligomers exhibited the highest proapoptotic activity. In conclusion, the Japanese quince procyanidins exhibited proapoptotic activity in Caco-2 cells within a submilimolar concentration range.

Influence of polyphenol extract from evening primrose (Oenothera paradoxa) seeds on human prostate and breast cancer cell lines.

There is growing interest in plant polyphenols which exhibit pleiotropic biological activities, including anti-inflammatory, antioxidant, and anticancer effects. The objective of our study was to evaluate the influence of an evening primrose extract (EPE) from defatted seeds on viability and invasiveness of three human cell lines: PNT1A (normal prostate cells), DU145 (prostate cancer cells) and MDA-MB-231 (breast cancer cells). The results revealed that after 72 h of incubation the tested extract reduced the viability of DU 145 and MDA-MB-231 with IC50 equal to 14.5 μg/mL for both cell lines. In contrast, EPE did not inhibit the viability of normal prostate cells. Furthermore, EPE reduced PNT1A and MDA-MB-231 cell invasiveness; at the concentration of 21.75 μg/mL the suppression of invasion reached 92% and 47%, respectively (versus control). Additionally, zymographic analysis revealed that after 48 h of incubation EPE inhibited metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) activities in a dose-dependent manner. For PNT1A the activities of MMP-2 and MMP-9 decreased 4- and 2-fold, respectively, at EPE concentration of 29 μg/mL. In the case of MDA-MB-231 and DU 145 the decrease in MMP-9 activity at EPE concentration of 29 μg/mL was 5.5-fold and almost 1.9-fold, respectively. In conclusion, this study suggests that EPE may exhibit antimigratory, anti-invasive and antimetastatic potential towards prostate and breast cancer cell lines.

Analysis of changes in expression of IL-4/IL-13/STAT6 pathway and correlation with the selected clinical parameters in patients with atopic asthma

Introduction: Asthma is associated with activation of interleukin-4 (IL-4)/interleukin-13 (IL-13)/signal transducer and activator of transcription factor-6(STAT6) inflammatory response via overexpression of all pathway components: IL-4, IL-13, and STAT6. Objectives: To evaluate the association of IL-4, IL-13, and STAT6 expression and immunoexpression with atopic asthma development. Patients and methods: Fifty patients with atopic asthma and 20 healthy controls were enrolled into the study. Relative gene expression was analyzed by qPCR method. Immunoexpression was assessed by ELISA method. Results: The expression levels of IL-4, IL-13, and STAT6 were higher in patients compared to the controls, but a statistically significant difference was observed only for IL-13 (P = 0.03). In immunoexpression analysis, a statistically significant difference between patients and controls was found for IgE (P = 0.03). Significant positive correlations in the patient group were found between IL-13 gene expression and total level of serum IgE (rho = 0.230, P = 0.033), STAT6 gene/STAT6 protein and total level of serum IgE (STAT6: rho = 0.077, P = 0.038; STAT6: rho = 0.049, P = 0.042), IL-4, and STAT6 expression (rho = 0.098, P = 0.048). Any significant correlations were found between expression/immunoexpression levels of the studied genes and clinical classification, clinical features, or lung function parameters. Conclusions: Our data support the role of Th2 cytokines (IL-4, IL-13) and STAT6 in Th1/Th2 imbalance and highlight the etiological relationship between IL-4/IL-13/STAT6 signaling and atopy and asthma.