Search

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2β1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.

The search for novel antimycobacterial drugs is a matter of urgency, since tuberculosis is still one of the top ten causes of death from a single infectious agent, killing more than 1.4 million people worldwide each year. Nine Amaryllidaceae alkaloids (AAs) of various structural types have been screened for their antimycobacterial activity. Unfortunately, all were considered inactive, and thus a pilot series of aromatic esters of galanthamine, 3- O -methylpancracine, vittatine and maritidine were synthesized to increase biological activity. The semisynthetic derivatives of AAs were screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra and two other mycobacterial strains ( M. aurum , M. smegmatis ) using a modified Microplate Alamar Blue Assay. The most active compounds were also studied for their in vitro hepatotoxicity on the hepatocellular carcinoma cell line HepG2. In general, the derivatization of the original AAs was associated with a significant increase in antimycobacterial activity. Several pilot derivatives were identified as compounds with micromolar MICs against M. tuberculosis H37Ra. Two derivatives of galanthamine, 1i and 1r , were selected for further structure optimalization to increase the selectivity index.

Pancracine, a montanine-type Amaryllidaceae alkaloid (AA), is one of the most potent compounds among natural isoquinolines. In previous studies, pancracine exhibited cytotoxic activity against diverse human cancer cell lines in vitro. However, further insight into the molecular mechanisms that underlie the cytotoxic effect of pancracine have not been reported and remain unknown. To fill this void, the cell proliferation and viability of cancer cells was explored using the Trypan Blue assay or by using the xCELLigence system. The impact on the cell cycle was determined by flow cytometry. Apoptosis was evaluated by Annexin V/PI and by quantifying the activity of caspases (-3/7, -8, and -9). Proteins triggering growth arrest or apoptosis were detected by Western blotting. Pancracine has strong antiproliferative activity on A549 cells, lasting up to 96 h, and antiproliferative and cytotoxic effects on MOLT-4 cells. The apoptosis-inducing activity of pancracine in MOLT-4 cells was evidenced by the significantly higher activity of caspases. This was transmitted through the upregulation of p53 phosphorylated on Ser392, p38 MAPK phosphorylated on Thr180/Tyr182, and upregulation of p27. The pancracine treatment negatively altered the proliferation of A549 cells as a consequence of an increase in G1-phase accumulation, associated with the downregulation of Rb phosphorylated on Ser807/811 and with the concomitant upregulation of p27 and downregulation of Akt phosphorylated on Thr308. This was the first study to glean a deeper mechanistic understanding of pancracine activity in vitro. Perturbation of the cell cycle and induction of apoptotic cell death were considered key mechanisms of pancracine action.

Plants of the Amaryllidaceae family are promising therapeutic tools for human diseases and have been used as alternative medicines. The specific secondary metabolites of this plant family, called Amaryllidaceae alkaloids (AA), have attracted considerable attention due to their interesting pharmacological activities. One of them, galantamine, is already used in the therapy of Alzheimer's disease as a long acting, selective, reversible inhibitor of acetylcholinesterase. One group of AA is the montanine-type, such as montanine, pancracine and others, which share a 5,11-methanomorphanthridine core. So far, only 14 montanine-type alkaloids have been isolated. Compared with other structural-types of AA, montanine-type alkaloids are predominantly present in plants in low concentrations, but some of them display promising biological properties, especially in vitro cytotoxic activity against different cancerous cell lines. The present review aims to summarize comprehensively the research that has been published on the Amaryllidaceae alkaloids of montanine-type.

Magnetic nanoparticles of ε-Fe 1.76 Ga 0.24 O 3 with the volume-weighted mean size of 17 nm were prepared by thermal treatment of a mesoporous silica template impregnated with metal nitrates and were coated with silica shell of four different thicknesses in the range 6-24 nm. The bare particles exhibited higher magnetization than the undoped compound, 22.4 Am 2 kg -1 at 300 K, and were characterized by blocked state with the coercivity of 1.2 T at 300 K, being thus the very opposite of superparamagnetic iron oxides. The relaxometric study of the silica-coated samples at 0.47 T revealed promising properties for MRI, specifically, transverse relaxivity of 89-168 s -1 mmol(f.u.) -1 L depending on the shell thickness was observed. We investigated the effects of the silica-coated nanoparticles on human A549 and MCF-7 cells. Cell viability, proliferation, cell cycle distribution, and the arrangement of actin cytoskeleton were assessed, as well as formation and maturation of focal adhesions. Our study revealed that high concentrations of silica-coated particles with larger shell thicknesses of 16-24 nm interfere with the actin cytoskeletal networks, inducing thus morphological changes. Consequently, the focal adhesion areas were significantly decreased, resulting in impaired cell adhesion., (© 2020 Wiley Periodicals, Inc.)

In this detailed phytochemical study of Narcissus cv. Professor Einstein, we isolated 23 previously known Amaryllidaceae alkaloids ( 1 - 23 ) of several structural types and one previously undescribed alkaloid, 7-oxonorpluviine. The chemical structures were identified by various spectroscopic methods (GC-MS, LC-MS, 1D, and 2D NMR spectroscopy) and were compared with literature data. Alkaloids which had not previously been isolated and studied for cytotoxicity before and which were obtained in sufficient amounts were assayed for their cytotoxic activity on a panel of human cancer cell lines of different histotype. Above that, MRC-5 human fibroblasts were used as a control noncancerous cell line to determine the general toxicity of the tested compounds. The cytotoxicity of the tested alkaloids was evaluated using the WST-1 metabolic activity assay. The growth of all studied cancer cell lines was inhibited by pancracine (montanine-type alkaloid), with IC 50 values which were in the range of 2.20 to 5.15 µM., Competing Interests: The authors declare no conflict of interest.

Ovariectomized mice on a high fat diet represent a model of diet-induced obesity during estrogen deficiency. Here, we tested the hypothesis that sensitivity to centrally administered leptin in ovariectomized mice with diet-induced obesity could be restored by estrogen supplementation. Ovariectomized C57BL/6 female mice were fed either a standard or high fat diet until they were 27 weeks old. Ovariectomized mice on a high fat diet developed extreme obesity and hyperleptinemia and moderate hyperinsulinemia compared to those on a standard diet. For the last 4 weeks, 17beta-estradiol-3-benzoate or its vehicle was administered subcutaneously in a 4-day cyclic regimen. Finally, leptin or saline was injected into the third ventricle, and food intake and body weight were measured for 36 h. In ovariectomized mice fed a standard diet, the decrease in food intake and body weight was significant and was pronounced in 17beta-estradiol-3-benzoate-supplemented mice. The response to centrally injected leptin in ovariectomized mice on a high fat diet was insignificant, whereas in 17beta-estradiol-3-benzoate-supplemented mice, the effect was significant, particularly with respect to body weight. We showed for the first time that central insensitivity to leptin in ovariectomized diet-induced obese mice was restored with 17beta-estradiol-3-benzoate supplementation, which also attenuated most of the parameters of metabolic syndrome. Only circulating adiponectin, a peripheral insulin sensitivity marker, was lowered following 17beta-estradiol-3-benzoate administration in both high fat and standard diet-fed ovariectomized mice, despite of decreased or unchanged glycemia, respectively.

The hypothalamus plays an important role in food consumption, receiving information about energy balance via hormonal, metabolic, and neural inputs. Its neurons produce neuropeptides influencing energy balance. Especially important to regulation of food consumption are certain hypothalamic structures, including the arcuate (ARC) and ventromedial (VMN) nuclei and the lateral hypothalamic area (LHA). We determined the impact of cholecystokinin (CCK) and cocaine and amphetamine regulated transcript (CART) peptide, on activity of ARC and VMN neurons and hypocretin (Hcrt) synthesizing neurons in LHA. ARC is an integrative nucleus regulating food consumption, VMN is considered to be a satiety centre, and LHA a hunger sensing centre. After overnight fasting, male C57Bl/6 mice received intraperitoneal injection of (i.p.) saline (SAL) or CCK (4microg/kg) or intracerebroventricular injection of (i.c.v.) CART peptide (0.1microg/mice) or CCK (i.p.) followed by CART peptide (i.c.v.) 5min later. Sixty minutes later, the presence of Fos or Fos/Hcrt immunostaining indicated activity of ARC and VMN neurons, as well as of Hcrt cells in LHA. CCK alone did not influence neuronal activity in any of the nuclei studied. CART peptide stimulated neurons in ARC and VMN (p<0.01) but decreased Hcrt neuronal activity in LHA (p<0.05). Co-administration of both peptides synergistically stimulated ARC neurons (p<0.01) and asynergistically inhibited LHA Hcrt neurons (p<0.01). Results indicate that CCK may modify the effect of CART peptide and thus substantially influence activity of neurons in hypothalamic structures involved in regulation of food intake.