Ilona Zaręba

Proapoptotic Activity of Propolis and Their Components on Human Tongue Squamous Cell Carcinoma Cell Line (CAL-27)

Propolis has been used since ancient times in folk medicine. It is a popular medicine possessing a broad spectrum of biological activities. This material is one of the richest sources of polyphenolic compounds such as flavonoids and phenolic acids. The ethanolic extract of propolis (EEP) evokes antibacterial, antiviral, antifungal and anticancer properties. Due to pharmacological properties it is used in the commercial production of nutritional supplements. In this study, gas chromatography coupled with mass spectrometry (GC-MS) was used to quantify main polyphenols in EEPs. The effect of EEPs, individual EEPs components (chrysin, galangin, pinocembrin, caffeic acid, p-coumaric acid, ferulic acid) and their mixture on viability of human tongue squamous cell carcinoma cell line (CAL-27) as well as the molecular mechanisms of the process were examined. The results of MTTs assay demonstrated that EEP, polyphenols and mixture of polyphenolic compounds were cytotoxic for CAL-27 cells in a dose dependent manner. The mechanism of cytotoxicity induced by these components undergoes through apoptosis as detected by flow cytometry. The ethanolic extracts of propolis activated caspases -3, -8, -9. Mixture of polyphenols was found as the most potent inducer of apoptosis thorough both intrinsic and extrinsic pathway. Therefore, we suggest that anticancer properties of propolis is related to synergistic activity of its main components.

Comparison of protective effect of ascorbic acid on redox and endocannabinoid systems interactions in in vitro cultured human skin fibroblasts exposed to UV radiation and hydrogen peroxide

The mechanisms of biological activity of commonly used natural compounds are constantly examined. Therefore, the aim of this study was to compare ascorbic acid efficacy in counteracting the consequences of UV and hydrogen peroxide treatment on lipid mediators and their regulative action on antioxidant abilities. Skin fibroblasts exposed to UVA and UVB irradiation, treated with hydrogen peroxide and ascorbic acid. The redox system was estimated through reactive oxygen species (ROS) generation (electron spin resonance spectrometer) and antioxidants level/activity (HPLC/spectrometry) which activity was evaluated by the level of phospholipid metabolites: 4-hydroxynonenal, malondialdehyde, 8-isoprostanes and endocannabinoids (GC/LC-MS) in the human skin fibroblasts. Protein and DNA oxidative modifications were also determined (LC). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), its activators and inhibitors as well as pro/anti-apoptotic proteins and endocannabinoid receptors was examined (Western blot) and collagen metabolism was evaluated by collagen biosynthesis and prolidase activity (spectrometry). UVA and UVB irradiation and hydrogen peroxide treatment enhanced activity of xanthine and NADPH oxidases resulting in ROS generation as well as diminution of antioxidant phospholipid protection (glutathione peroxidase-glutathione-vitamin E), what led to increased lipid peroxidation and decreased endocannabinoids level. Dysregulation of cannabinoid receptors expression and environment of transcription factor Nrf2 caused apoptosis induction. Ascorbic acid partially prevented ROS generation, antioxidant capacity diminution and endocannabinoid systems disturbances but only slightly protected macromolecules such as phospholipid, protein and DNA against oxidative modifications. However, ascorbic acid significantly prevented decrease in collagen type I biosynthesis. Ascorbic acid in similar degree prevents UV (UVA and UVB) and hydrogen peroxide-dependent redox imbalance. However, this antioxidant cannot efficiently protect cellular macromolecules and avert metabolic dysregulation leading to apoptosis.

Prolidase‐proline dehydrogenase/proline oxidase‐collagen biosynthesis axis as a potential interface of apoptosis/autophagy

Prolidase is a cytosolic imidodipeptidase that specifically splits imidodipeptides with C‐terminal proline or hydroxyproline. The enzyme plays an important role in the recycling of proline from imidodipeptides for resynthesis of collagen and other proline‐containing proteins. The mechanism of prolidase‐dependent regulation of collagen biosynthesis was found at both transcriptional and post‐transcriptional level. The increase in the enzyme activity is due to its phosphorylation on serine/threonine residues. Prolidase‐dependent transcriptional regulation of collagen biosynthesis was found at the level of NF‐κB, known inhibitor of type I collagen gene expression. Proline dehydrogenase/proline oxidase (PRODH/POX) is flavin‐dependent enzyme associated with the inner mitochondrial membrane. The enzyme catalyzes conversion of proline into Δ1‐pyrroline‐5‐carboxylate (P5C), during which reactive oxygen species (ROS) are produced, inducing intrinsic and extrinsic apoptotic pathways. Alternatively, under low glucose stress, PRODH/POX activation produces ATP for energy supply and survival. Of special interest is that PRODH/POX gene is induced by P53 and peroxisome proliferator‐activated gamma receptor (PPARγ). Among down‐regulators of PRODH/POX is an oncogenic transcription factor c‐MYC and miR‐23b*. On the other hand, PRODH/POX suppresses HIF‐1α transcriptional activity, the MAPK pathway, cyclooxygenase‐2, epidermal growth factor receptor and Wnt/b‐catenin signaling. PRODH/POX expression is often down‐regulated in various tumors, limiting mitochondrial proline utilization to P5C. It is accompanied by increased cytoplasmic level of proline. Proline availability for PRODH/POX‐dependent ATP or ROS generation depends on activity of prolidase and utilization of proline in process of collagen biosynthesis. Therefore, Prolidase‐PRODH/POX‐Collagen Biosynthesis axis may represent potential interface that regulate apoptosis and survival.

Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

Introduction The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes. Materials and methods Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2). Results Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take place at the level of collagen biosynthesis, since no effect of ethanol and HA was found on process of collagen degradation by MMP-2 and MMP-9. Conclusion This study provides evidence that ethanol impairs collagen metabolism in human skin fibroblasts, leading to a significant decrease in the amount of produced protein. This mechanism probably is due to downregulation of prolidase activity, expression of β1 integrin and IGF-IR receptors, and the signaling pathway proteins induced by these receptors.

Methylparaben‐induced decrease in collagen production and viability of cultured human dermal fibroblasts

Parabens owing to their many advantageous properties are widely applied in cosmetics, food products and pharmaceuticals. However, recent research results have shown that they possess the ability to accumulate in the human body and exert many adverse effects. In this study, the impact of methylparaben (MP) as the most frequently used preservative in cosmetics, on human dermal fibroblasts and collagen production was evaluated. In cells treated with 0.01, 0.03 and 0.05% MP a dose‐dependent decrease in collagen biosynthesis was revealed, which was positively correlated with the activity of prolidase responsible for the recovery of proline. Consequently, the concentration of total collagen secreted into the medium was markedly diminished. A similar reduction in expression of the major skin collagen type I at both the protein and mRNA level as well as collagen type III and VI at the mRNA level was also detected. The decrease in the collagen level may result not only from the reduced synthesis but also increased degradation owing to MP‐induced activation of pro‐MMP‐2 (72 kDa). The increase in activity of MMP‐2 (66 kDa) was accompanied by a reduction in the inhibitory activity of TIMP‐2. In addition, an inhibitory effect of MP on cell survival and proliferation was revealed in this study. The increased expression and nuclear translocation of caspase‐3 as well as increased Bax and decreased Bcl‐2 expression may suggest MP‐induced cell apoptosis. In summary, we have provided new data on the adverse effects of methylparaben on human dermal fibroblasts and the main structural protein of the skin. Further studies on the mechanisms responsible for its action are in progress. Copyright

Differential effect of platelet-rich plasma fractions on β1-integrin signaling, collagen biosynthesis, and prolidase activity in human skin fibroblasts

The study was conducted to evaluate the effects of platelet-rich plasma (PRP), supernatant of PRP (SPRP) obtained by centrifugation, and supernatant of activated PRP (SActi-PRP) obtained by Ca2+ solution-treated PRP on collagen biosynthesis, prolidase activity, and β1-integrin signaling in cultured human skin fibroblasts. Incubation of fibroblasts with 5% PRP for 24 h contributed to ~5-fold increase in collagen biosynthesis compared to the control. In the cells treated with 5% of SPRP or SActi-PRP, collagen biosynthesis showed a 3-fold increase of the control. PRP, SPRP, and SActi-PRP stimulated prolidase activity similar to collagen biosynthesis. Collagen biosynthesis and prolidase activity are regulated by β1-integrin receptor signaling. Incubation of fibroblasts with PRP for 24 h contributed to a dose-dependent increase in the expression of β1-integrin receptor, while SActi-PRP increased the process to a much lower extent. SPRP had no effect on the β1-integrin receptor expression. All the studied fractions of blood increased the expression of FAK as well as the expression of phosphorylated MAP-kinases. However, PRP was found to be the most effective stimulator of expression of these particular kinases. These studies suggest that a complex of factors, including growth factors, adhesion molecules, and prolidase contained in PRP, all evoke growth and collagen-promoting activities in human dermal fibroblasts.

Proline oxidase silencing induces proline-dependent pro-survival pathways in MCF-7 cells

Proline degradation by proline dehydrogenase/proline oxidase (PRODH/POX) contributes to apoptosis or autophagy. The identification of specific pathway of apoptosis/survival regulation is the aim of this study. We generated knocked-down PRODH/POX MCF-7 breast cancer cells (MCF-7shPRODH/POX). PRODH/POX silencing did not affect cell viability. However, it contributed to decrease in DNA and collagen biosynthesis, increase in prolidase activity and intracellular proline concentration as well as increase in the expression of iNOS, NF-κB, mTOR, HIF-1α, COX-2, AMPK, Atg7 and Beclin-1 in MCF-7shPRODH/POX cells. In these cells, glycyl-proline (GlyPro, substrate for prolidase) further inhibited DNA and collagen biosynthesis, maintained high prolidase activity, intracellular concentration of proline and up-regulated HIF-1α, AMPK, Atg7 and Beclin-1, compared to GlyPro-treated MCF-7 cells. In MCF-7 cells, GlyPro increased collagen biosynthesis, concentration of proline and expression of caspase-3, cleaved caspases -3 and -9, iNOS, NF-κB, COX-2 and AMPKβ. PRODH/POX knock-down contributed to pro-survival autophagy pathways in MCF-7 cells and GlyPro-derived proline augmented this process. However, GlyPro induced apoptosis in PRODH/POX-expressing MCF-7 cells as detected by up-regulation of active caspases -3 and -9. The data suggest that PRODH/POX silencing induces autophagy in MCF-7 cells and GlyPro-derived proline supports this process.

Constituents of Propolis: Chrysin, Caffeic Acid, p-Coumaric Acid, and Ferulic Acid Induce PRODH/POX-Dependent Apoptosis in Human Tongue Squamous Cell Carcinoma Cell (CAL-27)

Propolis evokes several therapeutic properties, including anticancer activity. These activities are attributed to the action of polyphenols. Previously it has been demonstrated, that one of the most abundant polyphenolic compounds in ethanolic extracts of propolis are chrysin, caffeic acid, p-coumaric acid, and ferulic acid. Although their pro-apoptotic activity on human tongue squamous cell carcinoma cells (CAL-27) was established previously, the detailed mechanism of this process remains unclear. Considering the crucial role of proline metabolism and proline dehydrogenase/proline oxidase (PRODH/POX) in the regulation of cancer cell survival/apoptosis, we studied these processes in polyphenol-treated CAL-27 cells. All studied polyphenols evoked anti-proliferative activity, accompanied by increased PRODH/POX, P53, active caspases-3 and -9 expressions and decreased collagen biosynthesis, prolidase activity and proline concentration in CAL-27 cells. These data suggest that polyphenols of propolis induce PRODH/POX-dependent apoptosis through up-regulation of mitochondrial proline degradation and down-regulation of proline utilization for collagen biosynthesis.

The possible pre -and post -UVA radiation protective effect of amaranth oil on human skin fibroblast cells

Background: The health effects of Amaranth Oil (AO) are attributed to its specific chemical composition. That makes it an outstanding natural product for the prevention and treatment of ultraviolet (UV) irradiation-related pathologies such as sunburn, photoaging, photoimmunosuppression, and photocarcinogenesis. Most of the studies are taken on animal model, and there is a lack of research on the endogenous effect of AO on fibroblast level, where UVA takes it harmful place. Objective: The aim of this study was evaluation if AO can protect or abolish UVA exposure effect on human skin fibroblast. Materials and Methods: The 0.1% AO, 0.25% AO, and 0.5% AO concentration and irradiation for 15 min under UVA-emitting lamp were studied in various condition. In all experiments, the mean values for six assays ± standard deviations were calculated. Results: Pretreatment with various concentrations of AO was tested. The highest concentration of AO where cell survival was observed was 0.5%. Cytotoxicity assays provided evidence for pre- and post-UVA protective effect of 0.1% AO among three tested concentrations. The results also provide evidence that UVA has inhibitory effect on collagen biosynthesis in confluent skin fibroblast, but presence of 0.1% AO abolishes pre- and post-UVA effect comparing to other used AO concentration. The assessment results on DNA biosynthesis show the significant abolished post-UVA effect when 0.1% and 0.5% of AO were added. Conclusion: AO gives pre- and post-UVA protection in low concentration. This provides the evidence for using it not as a main protective factor against UV but as one of the combined components in cosmetic formulation. Abbreviations used: AO: Amaranth Oil.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX–Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways.