Cytotoxic effects of two extracts from garlic (Allium sativum L.) cultivars on the human squamous carcinoma cell line SCC-15

AbstractGarlic (Allium sativum L., Alliaceae) has acquired a reputation as a therapeutic agent and herbal remedy to prevent and treat several pathologies. The aim of the present study was to determine the effect of two Allium sativum L. cultivars, Harnaś and Morado, on reactive oxygen species (ROS) production, viability and apoptotic process in human squamous carcinoma cell line SCC-15. The experiments were conducted on SCC-15 cell line exposed to increasing concentrations of garlic extracts of 0.062, 0.125, 0.250, 0.500 and 1.000mg/mL. After the experiments, ROS formation, caspase-3 activity and neutral red uptake were measured in the cells, and in a collected medium lactate dehydrogenase (LDH) release was measured. The Spanish cultivar Morado has demonstrated higher potential to stimulate ROS production in SCC-15 cells after a short time period (6h) than the Polish cultivar Harnaś. However, the Polish cultivar Harnaś manifested more prolonged potential to stimulate ROS production in SCC-15 cells. Both studied garlic extracts induced cytotoxicity on SCC-15 cell line which was probably ROS-dependent. We also determined that in SCC-15 cells high concentrations of studied extracts did not cause activation of caspase-3 which suggested caspase-independent or necrotic cell death

The Connective Tissue of the Arterial Wall in Pathogenesis of Atherosclerosis and Hypertension

Tkanka łączna pełni istotną funkcję regulacyjną w procesach naprawczych w ścianie naczyń tętniczych, w reakcji na bodźce uszkadzające. W przebiegu miażdżycy i nadciśnienia dochodzi do przyspieszenia degradacji włókien elastynowych, warunkujących właściwości sprężyste ściany naczyniowej. Stwierdza się wzrost aktywności enzymów elastolitycznych osocza oraz ściany naczyniowej, a także wzrost stężenia peptydów elastynopochodnych w osoczu. Uzyskane na podstawie badań eksperymentalnych dane wskazują na istotną rolę układu renina-angiotensyna (RA) w patogenezie miażdżycy i nadciśnienia tętniczego. Powstająca w końcowym etapie aktywacji układu RA angiotensyna II (ANG II) wywołuje proliferację i migrację komórek mięśni gładkich naczynia (SMC), pobudza syntezę substancji pozakomórkowej tkanki łącznej w ścianie tętniczej, aktywuje monocyty (makrofagi), zwiększa agregację płytek krwi i obniża aktywność fibrynolityczną, modyfikuje cząstki LDL oraz indukuje powstawanie blaszki miażdżycowej powikłanej. Badania eksperymentalne wykazały, że aktywacja układu RA wpływa na metabolizm tkanki łącznej w ścianie naczyniowej. Angiotensyna II pobudza SMC do syntezy kolagenów typu I i III, fibronektyny, tenascyny, trombospondyny oraz glikozaminoglikanów - siarczanu dermatanu i siarczanu chondroityny. Pod wpływem ANG II SMC syntetyzują tkankowy inhibitor metaloproteinaz typu I, co sprzyja gromadzeniu się tkanki łącznej w ścianie tętnicy.In atherosclerosis numerous qualitative and quantitative changes in connective tissue metabolism parameters in serum and aorta occur. The elastin turnover is accelerated due to increased activity of elastases in serum and arterial wall. The activity of protease inhibitors is decreased. Also, the collagen's content of arterial wall was shown to be higher. Connective tissue is an important factor in regulation of smooth muscle cells (SMC) proliferation and migration, immunological processes during atherosclerosis and interactions with blond cells. The renin-angiotensin system plays an important role in atherogenesis and hypertension. Angiotensin II (ANG II) was shown to stimulate the phenotypic modulation of SMC. It induces SMC proliferation and migration from media to subendothelial layer, enhances the action of other growth factors, induces the adhesion and migration of monocytes and macrophages roto vascular wali, activates platelets and reduces fibrinolytic activity, stimulates modifications ofLDL particles and foam cells accumulation. During vascular injury there is an enhanced activity oflocal reninangiotensin system. It leads to overproduction of ANG II, both in serum and arterial wali. ANG II stimulates SMC to oversynthesize the extracellular matrix components - collagens type I and III, fibronectin, tenascin, thrombospondin, osteopontin and glycosaminoglycans - chondroitin and dermatan sulfates. It was shown that SMC under influence of ANG II synthesize less elastin

Elastin-derived peptide VGVAPG decreases differentiation of mouse embryo fibroblast (3T3-L1) cells into adipocytes

Elastin is a highly elastic protein present in connective tissue. As a result of protease activity, elastin hydrolysis occurs, and during this process, elastin-derived peptides (EDPs) are released. One of the constitutively repeating elastin and EDP building sequences is the hexapeptide VGVAPG. Therefore, the aim of our research was to define the effect of VGVAPG peptide on adipogenesis in a mouse 3T3-L1 cell line. 3T3-L1 cells were differentiated according to a previously described protocol and exposed to increasing concentrations of VGVAPG or VVGPGA peptide. The obtained results showed that VGVAPG peptide does not stimulate reactive oxygen species (ROS) production, caspase-1 activation, and 3T3-L1 cell proliferation. In the second part of the experiments, it was proved that VGVAPG peptide decreased lipid accumulation as measured by oil red O staining, which was confirmed by the profile of increased expression markers of undifferentiated preadipocytes. In our experiments, 10 nM VGVAPG added for differentiating to adipocytes increased the expression of Pref-1, serpin E1, and adiponectin as compared to rosiglitazone (PPARγ agonist)-treated group and simultaneously decreased the expression of VEGF and resistin as compared to the rosiglitazone-treated group. The obtained results show that VGVAPG peptide sustains 3T3 cells in undifferentiated state. Abbreviations: DMSO: dimethyl sulphoxide; EBP: elastin-binding protein; EDPs: elastin-derived peptides; FBS: foetal bovine serum; Glb1: gene for beta-galactosidase; LDL: low-density-lipoprotein; PAI-1 (Serpin E1): plasminogen activator inhibitor-1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor gamma; Pref-1: preadipocyte factor 1; ROS: reactive oxygen species; VEGF-A: vascular endothelial growth factor-A; VGVAPG: Val-Gly-Val-Ala-Pro-Gly; β-Gal: beta-galactosidase; ORO: oil red O; IBMX: 3-isobutyl-1-methylxanthine; H2DCFDA: 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate; DMEM: Dulbecco’s Modified Eagle’s Medium; VVGPGA: Val-Val-Gly-Pro-Gly-Ala

Relationship between Serum Lipids and Angiotensin Converting Enzyme Activity in Cholesterol-fed Rabbits

Wstęp: Zahamowanie układu renina-angiotensyna zmniejsza rozwój ogniska miażdżycowego w różnych doświadczalnych modelach miażdżycy tętnic. W niektórych badaniach eksperymentalnych obserwowano poprawę parametrów gospodarki lipidowej po zastosowaniu inhibitorów enzymu konwertującego angiotensynę. Brak jest danych na temat powiązania metabolizmu lipidów z aktywnością układu renina-angiotensyna. Metody: Celem pracy było określenie aktywności układu renina-angiotensyna w doświadczalnej hipercholesterolemii oraz korelacji pomiędzy parametrami układu lipidowego i aktywnością enzymu konwertującego angiotensynę (ACE). Do badań wykorzystano 20 królików rasy nowozelandzkiej, samców, które podzielono na 2 równoliczne grupy: grupa kontrolna (K) otrzymywała paszę standardową, a grupa badana (B) otrzymywała paszę o 1-procentowej zawartości cholesterolu. Po 6 miesiącach we krwi oznaczono stężenie cholesterolu całkowitego, frakcji LDL, HDL, trójglicerydów, fosfolipidów oraz aktywność ACE. Wyniki: We wszystkich przypadkach analizowanych parametrów gospodarki lipidowej stężenie w grupie zwierząt skarmianych paszą bogatocholesterolową było znamiennie wyższe. Aktywność ACE w surowicy krwi badanych zwierząt była wyższa o 15% niż w grupie kontrolnej (p < 0,05). Zaobserwowaliśmy istotną korelację pomiędzy aktywnością ACE a stężeniem trójglicerydów (r = 0,67, p < 0,04) i cholesterolu frakcji HDL (r = -0,89, p < 0,001). Wnioski: Obserwowany w warunkach hipercholesterolemii wzrost aktywności ACE w surowicy krwi może być jednym z mechanizmów jej patogennego działania. Uzyskane w naszych badaniach wyniki sugerują związek między układem renina-angiotensyna a metabolizmem lipidów i jednocześnie wskazują, że wpływ inhibitorów ACE na parametry lipidowe może wynikać z zahamowania aktywności ACE w surowicy krwi.Background Blockade of renin-angiotensin system reduces the development of atherosclerotic plaque in different animal models. In some experimental studies improvement of lipids metabolism was also observed after treatment with ACE-inhibitors. There is still little known about the mechanism of ACE-inhibitors action on lipids metabolism in hypercholesterolemic animals. Methods The aim of the study was to assess the renin-angiotensin system activity in experimentally-induced hypercholesterolemia and the possible relationships between lipids parameters and ACE activity. Twenty male New-Zealand rabbits were divided into two groups with the following dietary conditions: standard diet (n = 10) and 1% cholesterol diet (n = 10). After six months of experiment the blood was collected for analysis of ACE activity and lipids. Total, LDL and HDL cholesterol, triglycerides, phospholipids and ACE activity was measured enzymatically. Results At the end of the experiment there were no correlations between ACE activity and serum lipids in normolipidemic group. When compared with the control group, in cholesterol-fed rabbits ACE activity was increased (p < 0.05). We observed a positive correlation between ACE activity and serum triglycerides (r = 0.6%, p < 0.04) and inverse correlation between ACE activity and HDL cholesterol (r = -0.89, p < 0.001) in hypercholesterolemic rabbits. There was no correlation between ACE activity and any other lipids parameters in this group. Conclusions We have shown that standard diet supplemented with 1% cholesterol leads to increased activation of ACE and that there is a correlation between this activity and triglycerides and HDL cholesterol metabolism. This may be in part responsible for the observed changes in lipids metabolism after ACE-inhibitors in hyperlipidemic animals and then be one of the antiatherosclerotic properties of ACE-inhibitors

Cytotoxic effects of two extracts from garlic (Allium sativum L.) cultivars on the human squamous carcinoma cell line SCC-15

Garlic (Allium sativum L., Alliaceae) has acquired a reputation as a therapeutic agent and herbal remedy to prevent and treat several pathologies. The aim of the present study was to determine the effect of two Allium sativum L. cultivars, Harnaś and Morado, on reactive oxygen species (ROS) production, viability and apoptotic process in human squamous carcinoma cell line SCC-15. The experiments were conducted on SCC-15 cell line exposed to increasing concentrations of garlic extracts of 0.062, 0.125, 0.250, 0.500 and 1.000 mg/mL. After the experiments, ROS formation, caspase-3 activity and neutral red uptake were measured in the cells, and in a collected medium lactate dehydrogenase (LDH) release was measured. The Spanish cultivar Morado has demonstrated higher potential to stimulate ROS production in SCC-15 cells after a short time period (6 h) than the Polish cultivar Harnaś. However, the Polish cultivar Harnaś manifested more prolonged potential to stimulate ROS production in SCC-15 cells. Both studied garlic extracts induced cytotoxicity on SCC-15 cell line which was probably ROS-dependent. We also determined that in SCC-15 cells high concentrations of studied extracts did not cause activation of caspase-3 which suggested caspase-independent or necrotic cell death. Keywords: Garlic, SCC-15, Allium sativum, Cytotoxicity, ROS, Apoptosi