Antiproliferative and Antimigratory Activity of Poly-gallic Acid in Cancer Cell Lines.

BACKGROUND/AIM: Enzyme-mediated grafting of poly (gallic acid) (PGAL) and L-arginine and a-L-lysine onto PGAL produces reactive oxygen species (ROS)-suppressor multiradical molecules with low cytotoxicity, high thermostability and water solubility with cancer treatment potential. This study examined the anticancer effects of these molecules in hepatic (HepG2, ATCC HB-8065), breast (MCF7, ATCC HTB-22), and prostate (PC-3, ATCC CRL-1435 and DU 145, ATCC HTB-81) cancer cell lines, as well as in fibroblasts from healthy human skin as control cells. MATERIALS AND METHODS: PGAL was synthesized by the oxidative polymerization of the naturally abundant GA using laccase from Trametes versicolor. Insertions of amino acids L-arginine and α-L-lysine on the PGAL chain were carried out by microwave. The cells of dermal fibroblast (Fb) were obtained from primary skin cultures and isolated from skin biopsies. The cancer cells lines of hepatic (HepG2), breast (MCF7), and prostate (PC-3, DU 145) were obtained from ATCC. The viability of the cancer cells and the primary culture was obtained by the MTT assay. Proliferation was demonstrated by crystal violet assay. Cell migration was determined by Wound healing assay. Finally, cell cycle analysis was carried out with cells. RESULTS: The results show that 200 µg/ml of PGAL cultured in vitro with prostate cancer cells decreased viability, proliferation, and migration, as well as arrested cells in the G1 and S phases of the cell cycle. In contrast, the dermal fibroblasts and the hepatic line remained unaffected. The random grafting of L-Arg and a-L-Lys onto the PGAL chain also decreased the viability of prostate cancer cells. CONCLUSION: PGAL and PGAL-grafted amino acids are potential adjuvants for prostate cancer treatment, with improved physicochemical characteristics compared to GA.

A modified animal-free serum technique for efficient isolation and proliferation of mesenchymal stem cells from Hoffa fat pad.

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

Effect of smoking on the redox status of knee osteoarthritis: A preliminary study.

Even though smoking has been scarcely studied in osteoarthritis (OA) etiology, it is considered a controversial risk factor for the disease. Exposure to tobacco smoke has been reported to promote oxidative stress (OS) as part of the damage mechanism. The aim of this study was to assess whether smoking increases cartilage damage through the generation of OS. Peripheral blood (PB) and synovial fluid (SF) samples from patients with OA were analyzed. The samples were stratified according to smoking habit, Kellgren-Lawrence score, pain, and cotinine concentrations in PB. Malondialdehyde (MDA), methylglyoxal (MGO), advanced protein oxidation products (APOPs), and myeloperoxidase (MPO) were assessed; the activity of antioxidant enzymes such as gamma-glutamyl transferase (GGT), glutathione S-transferase (GST) and catalase (CAT), as well as the activity of arginase, which favors the destruction of cartilage, was determined. When stratified by age, for individuals <60 years, the levels of MDA and APOPs and the activity of MPO and GST were higher, as well as antioxidant system activity in the smoking group (OA-S). A greater degree of pain in the OA-S group increased the concentrations of APOPs and arginase activity (P < 0.01 and P < 0.05, respectively). Arginase activity increased significantly with a higher degree of pain (P < 0.01). Active smoking can be an important risk factor for the development of OA by inducing systemic OS in young adults, in addition to reducing antioxidant enzymes in older adults and enhancing the degree of pain and loss of cartilage.

Antiphagocytic Properties of Polygallic Acid with Implications in Gouty Inflammation.

Polygallic acid (PGAL) has been used in vitro to protect synoviocytes from monosodium urate (MSU) crystals due to its anti-inflammatory properties. However, MSU crystals can also activate other cells of the synovial fluid (SF). We studied the impact of PGAL on the phagocytosis of MSU crystals, inflammation, and oxidative stress using an in vitro model with SF leukocytes and THP-1 monocyte cells. SF leukocytes were stimulated with PGAL and MSU crystals, proinflammatory cytokines and phagocytosis were assessed. In THP-1 cells, the effect of PGAL on the phagocytosis of MSU crystals and the levels of IL-1ß, IL-6, TNF-α, and reactive oxygen species (ROS) was evaluated. PGAL was added to THP-1 cultures 24 h before MSU crystal addition as a pre-treatment, and IL-1ß was measured. One-way ANOVA with Tukey's post hoc test was performed, and a P value < 0.05 was considered statistically significant. PGAL (100 µg/mL) decreased phagocytosis in SF leukocytes by 14% compared to cells exposed to crystals without PGAL. In THP-1 cells, 100 and 200 µg/mL PGAL reduced phagocytosis by 17% and 15%, respectively. In SF cells, there was a tendency to decrease IL-1ß and IL-6. In THP-1 cells, decreases in IL-1ß and TNF-α, as well as a slight decrease in ROS, were identified. PGAL pre-treatment resulted in a reduction of IL-1ß. PGAL inhibits MSU phagocytosis by exerting an anti-inflammatory effect on cells exposed to crystals. The use of PGAL before an acute attack of gout suggests an important protective factor to control the inflammation.

Osteogenic Potential of Monosodium Urate Crystals in Synovial Mesenchymal Stem Cells.

Background and Objectives: Deposits of monosodium urate (MSU) crystals due to increased levels of uric acid (UA) have been associated with bone formation and erosion, mainly in patients with chronic gout. The synovial membrane (SM) comprises several types of cells, including mesenchymal stem cells (SM-MSCs); however, it is unknown whether UA and MSU induce osteogenesis through SM-MSCs. Materials and Methods: Cultures of SM were immunotyped with CD44, CD69, CD90, CD166, CD105, CD34, and CD45 to identify MSCs. CD90+ cells were isolated by immunomagnetic separation (MACS), colony-forming units (CFU) were identified, and the cells were exposed to UA (3, 6.8, and 9 mg/dL) and MSU crystals (1, 5, and 10 µg/mL) for 3 weeks, and cellular morphological changes were evaluated. IL-1ß and IL-6 were determined by ELISA, mineralization was assessed by alizarin red, and the expression of Runx2 was assessed by Western blot. Results: Cells derived from SM and after immunomagnetic separation were positive for CD90 (53 ± 8%) and CD105 (52 ± 18%) antigens, with 53 ± 5 CFU identified. Long-term exposure to SM-MSCs by UA and MSU crystals did not cause morphological damage or affect cell viability, nor were indicators of inflammation detected. Mineralization was observed at doses of 6.8 mg/dL UA and 5 µg/mL MSU crystals; however, the differences were not significant with respect to the control. The highest dose of MSU crystals (10 µg/mL) induced significant Runx2 expression with respect to the control (1.4 times greater) and SM-MSCs cultured in the osteogenic medium. Conclusions: MSU crystals may modulate osteogenic differentiation of SM-MSCs through an increase in Runx2.

HLA-B27 may modulate the interaction between ERAP1 polymorphisms and smoking in ankylosing spondylitis patients.

BACKGROUND: Ankylosing spondylitis (AS) is an autoimmune disease that affects the enthesis and synovial membrane of the spine, the sacroiliac vertebrae and peripheral joints. Genetic susceptibility to AS is mainly due to the presence of the HLA-B*27 (B27) allele, and endoplasmic reticulum aminopeptidase-1 (ERAP-1) plays a key role in antigen processing and presentation to HLA class I molecules. Tobacco consumption is one of the main environmental factors involved in the pathogenesis of various diseases, including AS. The objective of the present study was to evaluate the association and the interactive effects of variants of the ERAP1 gene with smoking in modulating the risk of AS. METHODS AND RESULTS: A case-control study in the Mexican population. The association of two functional variants of the ERAP1 gene (rs30187 and rs27044) in patients with AS was analyzed by the allelic discrimination method using TaqMan probes. B27 was typified by PCR-SSP. The interaction between the variants of ERAP1 and B27 and smoking was assessed using the multifactorial dimensionality reduction (MDR) method. There was no significant association of the two variants of ERAP1 in the cases compared with the controls (P > 0.05); however, a strong interaction between the variants and smoking could be demonstrated, with entropy values of 4.97% for rs30187 and 5.13% for rs27044. In addition, these interaction effects were increased in patients carrying the B27 allele. CONCLUSIONS: The rs30187 and rs27044 variants of the ERAP1 gene appear to potentiate the effect of smoking in patients with AS carrying the B27 allele.

Synovial membrane mesenchymal stem cells for cartilaginous tissues repair.

BACKGROUND: The present review is focused on general aspects of the synovial membrane as well as specialized aspects of its cellular constituents, particularly the composition and location of synovial membrane mesenchymal stem cells (S-MSCs). S-MSC multipotency properties are currently at the center of translational medicine for the repair of multiple joint tissues, such as articular cartilage and meniscus lesions. METHODS AND RESULTS: We reviewed the results of in vitro and in vivo research on the current clinical applications of S-MSCs, surface markers, cell culture techniques, regenerative properties, and immunomodulatory mechanisms of S-MSCs as well as the practical limitations of the last twenty-five years (1996 to 2021). CONCLUSIONS: Despite the poor interest in the development of new clinical trials for the application of S-MSCs in joint tissue repair, we found evidence to support the clinical use of S-MSCs for cartilage repair. S-MSCs can be considered a valuable therapy for the treatment of repairing joint lesions.

Correction to: Afatinib is active in osteosarcoma cell lines.

The title of the article is incorrectly published in the original article. The correct article title is "Afatinib is active in osteosarcoma cell lines".

Cherry extracts attenuate inflammation and oxidative stress triggered by monosodium urate crystals in THP-1 cells.

The microwaves-assisted extraction (MAE) for concentration of cherry phytochemicals has seen explored. Polyphenols from cherries, Prunus avium (L.) L., were extracted using a microwave oven at 2,450 MHz, 453 W for a period of 60 s (T60), and was compared versus an unprocessed MAE extract (T0). The extracts were analyzed for total polyphenols, total anthocyanins, and antioxidant capacity. THP-1 cells were stimulated with monosodium urate (MSU) crystals at 150 µg/ml for 24 hr. Cherry extracts were added to cultures concurrently with MSU or 3 hr before MSU addition as pretreatments. Reactive oxygen species (ROS), IL-1ß levels, and MSU crystal phagocytosis were evaluated. T60 extract showed a higher concentration of polyphenols, anthocyanins, and antioxidant activity than T0 extract. ROS were inhibited using the 1:800 and 1:1,600 (v:v) dilutions from both extracts, even used as pretreatments. IL-1ß levels and MSU crystal phagocytosis were reduced. Cherry is a source of polyphenolic compounds with antioxidant and anti-inflammatory activity. PRACTICAL APPLICATIONS: The cherries and a cherry extract obtained via MAE has benefits as a possible coadjuvant to conventional gout therapy due to attenuate the inflammation and the oxidative stress triggered by monosodium urate crystals in THP-1 cells, which mimic an acute episode of gout.

Afatinib is active in osteosarcoma in osteosarcoma cell lines.

PURPOSE: Osteosarcoma is the most common bone tumor, mainly affecting adolescents and young adults, and metastatic disease has poor outcomes with a dismal overall survival. Currently, chemotherapy is the standard of care with limited results, finding that new therapies could improve these outcomes. Preclinical and clinical studies have suggested a possible important role of ErbB pathway aberrations in osteosarcoma etiology. The present study shows the effect of afatinib, an irreversible ErbB family blocker in osteosarcoma cell lines. METHODS: Within a panel of human osteosarcoma cell lines, we addressed cell viability assay using afatinib at increasing concentrations. Motility was measured in wound-healing assays and invasion capacity was assessed in Transwell chamber assays. Finally, to monitor ErbB pathway modulation by afatinib and related compounds, we used Western blot analyses. RESULTS: Cell viability inhibition, as well as a reduction of motility and migration of osteosarcoma cell line were observed after treatment with afatinib. Likewise, in the HOS cell line, afatinib decreased phosphorylation of key components in the ErbB signaling pathway. CONCLUSIONS: Afatinib shows relevant antitumor effect in several osteosarcoma cell lines, as it causes a significant impact on cell viability, motility, and migration with a significant decrease in the activation of ErbB pathway activity.

Polydatin and Resveratrol Inhibit the Inflammatory Process Induced by Urate and Pyrophosphate Crystals in THP-1 Cells.

Resveratol (RES) and its natural precursor polydatin (PD) are polyphenols that may display a broad variety of beneficial effects including anti-inflammatory properties. This study aimed to investigate the role of RES and PD in the inflammatory process induced by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals in vitro. A monocytic cell line (THP-1) was primed for 3 hours with phorbol myristate acetate (100 ng/mL) and stimulated with synthetic MSU (0.05 mg/mL) and CPP (0.025 mg/mL) crystals. RES and PD were added to cultures concurrently with the crystals, or as 2-hour pretreatment. The effect of the two polyphenols was evaluated on intracellular and extracellular IL-1ß levels, NACHT-LRRPYD-containing protein-3 (NLRP3) inflammasome expression, reactive oxygen species (ROS) and nitric oxide (NO) production, and the assessment of crystal phagocytosis. RES and PD strongly inhibited IL-1ß induced by crystals after cell pretreatment. Cell pretreatment was effective also in reducing IL-1 mRNA expression while no effect was observed on NLRP3 gene expression. RES and PD had no effect on crystal phagocytosis when used as pretreatment. Both polyphenols were significantly effective in inhibiting ROS and NO production. Our results demonstrated that RES and PD are effective in inhibiting crystal-induced inflammation. Data obtained after cell pretreatment allow us to hypothesize that these polyphenols act on specific signaling pathways, preventing inflammation.

Impact of the gene-gene interactions related to the HIF-1α signaling pathway with the knee osteoarthritis development.

INTRODUCTION/OBJECTIVES: Articular cartilage is the target tissue of osteoarthritis (OA), and because it lacks capillary networks, the microenvironment is hypoxic. Hypoxia inducible factor-1 alpha (HIF-1α) regulates the homeostasis of this tissue. The aim of this study was to investigate whether genetic polymorphisms of the HIF-1α signaling pathway are involved in the development of knee OA. METHOD: We performed a case-control association study and genotyped 134 knee OA patients and 267 healthy controls. All participants were genotyped in order to evaluate 42 SNPs from 22 genes involved in the HIF-1α signaling pathway using the OpenArray technology. Gene-gene interactions (epistasis) were analyzed using the multifactor dimensionality reduction (MDR) method. RESULTS: The MDR analysis showed epistasis between AKT2 (rs8100018) and IGF1 (rs2288377), AKT2 (rs8100018) and IGF1 (rs35767), IGF1 (rs35767) and COL2A1 (rs1793953), and between GSK3B (rs6438552) and IGF1 (rs35767) polymorphisms, with information gain values of 21.24%, 8.37%, 9.93%, and 5.73%, respectively. Additionally, our model allowed us to identify high- and low-risk genotypes among COL2A1 rs1793953, GSK3B rs6438552, AKT2 rs8100018, and IGF1 rs35767 polymorphisms. CONCLUSIONS: Knowing the interactions of these polymorphisms involved in HIF-1α signaling pathway could provide a new diagnostic support tool to identify individuals at high risk of developing knee OA.

Epistasis between ADIPOQ rs1501299 and PON1 rs662 polymorphisms is potentially associated with the development of knee osteoarthritis.

Overweight produces oxidative stress (OS) on the articular cartilage, with the subsequent risk of developing knee osteoarthritis (OA). Associations between genetic polymorphisms related to OS and OA have been reported, but it is currently unknown whether there exist interactions among them that affect OA development. To identify and evaluate interactions between multiple SNPs related to OS in Mexican knee OA patients. Ninety-two knee OA patients were included in the study, which were compared to 147 healthy controls. Nine variants of six genes (PEPD, AGER, IL6, ADIPOQ, PON1, and CA6) related to OS were genotyped in both study groups through the OpenArray system. Epistasis was analyzed with the multifactor dimensionality reduction (MDR) method. The MDR analysis revealed a significant interaction (p = 0.0107) between polymorphisms rs1501299 (ADIPOQ) and rs662 (PON1), with an entropy value of 9.84%; in addition, high and low risk genotypes were identified between these two polymorphisms. The effect of the interaction between rs1501299 (ADIPOQ) and rs662 (PON1) polymorphisms seems to play an important role in OA pathogenesis; so the epistasis analysis may provide an excellent tool for identifying individuals at high risk for developing OA.

Anti-inflammatory effects of polyphenols in arthritis.

Polyphenols have been extensively investigated with regard to their antioxidant, anti-inflammatory, and immunomodulant properties in many inflammatory chronic conditions. The aim of this review is to summarise how these compounds can modulate the inflammatory pathways which characterise the most prevalent arthropathies including osteoarthritis, rheumatoid arthritis and crystal-induced arthritis. Among polyphenols, epigallocatechin gallate, carnosol, hydroxytyrosol, curcumin, resveratrol, kaempferol and genistein have been the most widely investigated in arthritis. The most important results of the studies outlined in this article show how polyphenolic compounds are able to inhibit the expression and the release of a number of pro-inflammatory mediators and proteolytic enzymes, the activity of different transcriptional factors and the production of reactive oxygen species in vitro. Studies on animal models of rheumatoid arthritis, osteoarthritis and gout show interesting results in terms of reduced tissue damage, restored cartilage homeostasis, and decreased levels of uric acid, respectively. Despite the multiple protective effects of polyphenols, there are no dietary recommendations for patients affected by rheumatic diseases. Future studies, including intervention trials, should be conducted to determine the relevance of polyphenols consumption or supplementation in arthritis. © 2017 Society of Chemical Industry.

The Overexpression of NALP3 Inflammasome in Knee Osteoarthritis Is Associated with Synovial Membrane Prolidase and NADPH Oxidase 2.

Osteoarthritis is characterized by the presence of proinflammatory cytokines and reactive oxygen species. We aimed to clarify the role of prooxidant enzyme content at the synovial membrane level and how it correlates with the inflammatory process in patients with knee osteoarthritis (KOA). In synovial membranes from KOA patients and control group, we analyzed the protein content of prooxidant enzymes such as Nox2, xanthine oxidase (XO), and prolidase as well as the proinflammatory NALP3. Results show that protein content of prolidase and Nox2 increased 4.8- and 8.4-fold, respectively, and XO showed an increasing trend, while the NALP3 inflammasome increased 5.4-fold with respect to control group. Levels of prolidase and XO had a positive correlation between the levels of NALP3 and Nox2. By principal component analysis the protein expression pattern by study groups was evaluated. Three clusters were identified; protein expression patterns were higher for clusters two (prolidase) and three (XO and Nox2) between KOA patients and controls. Data suggest that prooxidant enzymes increase in synovial membrane of KOA patients and may contribute to the inflammatory state and degradation of the articular cartilage.

Polymorphic variation of hypoxia inducible factor-1 A (HIF1A) gene might contribute to the development of knee osteoarthritis: a pilot study.

BACKGROUND: Osteoarthritis (OA) is a multifactorial degenerative condition of the whole joint with a complex pathogenesis whose development and progression is significantly mediated by interactions between the joint cartilage and articular tissues, particularly, proinflammatory mediators and oxidative stress, which results in cartilage deterioration and subchondral bone destruction. HIF-1 alpha regulates oxygen homeostasis in hypoxic tissues such as joint cartilage; efficiency of transcriptional activity of the HIF1A gene is strongly influenced by the presence of polymorphic variants. Given the loss of articular cartilage and with intention to restore damaged tissue, WISP-1 participates in the development of subchondral bone; further, its expression is highly increased in chondrocytes of OA patients. The aim of this study was to evaluate gene frequencies of HIF1A and WISP1 polymorphisms in Mexican patients suffering from knee OA. METHODS: We determined HIF1A rs11549465 (P582S), rs11549467 (A588T), and rs2057482 (C191T), and WISP1 rs2929970 (A2364G) polymorphisms in 70 Mexican patients with knee OA and compare them to those present in 66 ethnically matched healthy controls. Genotyping for these polymorphisms was performed by Real-Time PCR using TaqMan probes. RESULTS: Gene frequencies exhibited a significant increase of the CC genotype of rs11549465 polymorphism in knee OA patients as compared with those present in controls (P = 0.003 OR = 5.7, 95% CI = 1.7-21.6); CT genotype and T allele showed decreased frequency in the knee OA group vs. the controls (P = 0.003 OR = 0.2, CI = 0.05-0.6; and P = 0.004 OR = 0.2, CI = 0.05-0.65, respectively). Allele frequencies of the other polymorphic variants were similar in both patients and controls. CONCLUSIONS: These results suggest that the presence of the rs11549465 SNP (HIF1A) plays a role protective in the loss of articular cartilage in our population, and offers the possibility to further study the molecular mechanisms within cartilage and subchondral bone.