Cell Proliferation Effects Research Articles

Modified Hank's Balanced Salt Solution as a Storage Medium for Avulsed Teeth: InVitro Assessment of Periodontal Fibroblast Viability.

The optimal storage medium for an avulsed tooth should preserve the viability of periodontal fibroblasts (PDLF) to the highest degree, facilitating the re-attachment of periodontal fibers and improving the prognosis of replantation. This study compared the effect of the PDLF viability in Hank's balanced salt solution (HBSS), supplemented culture medium, that is, Dulbecco's Modified Eagle Medium (DMEM), and four modified HBSS mixtures. Periodontal tissues were obtained from extracted human teeth and processed for PDLF culture. The cells were then exposed to six experimental media: (i) HBSS, (ii) HBSS and ascorbic acid (HBSS + Vit C), (iii) HBSS and platelet-derived growth factor (HBSS + PDGF), (iv) a mixture of HBSS, PDGF, and Vit C (HBSS + PDGF + Vit C), (v) HBSS and platelet lysate (HBSS + PL), and (vi) DMEM for 3, 6, 12, and 24 h. A MTT assay was performed to determine the cell viability. Vitamin C-containing media maintained PDLF viability significantly better than HBSS + PDGF and HBSS + PL at 3, 6, 12, and 24 h (p < 0.05). The percentages of viable PDLF at 3, 6, 12, and 24 h were significantly higher than 0 h for HBSS + Vit C, HBSS + PDGF + Vit C, HBSS + PL, and DMEM (p < 0.05). All experimental media were able to maintain PDLF viability (DMEM>HBSS+Vit C; HBSS+PDGF+Vit C>HBSS+PL>HBSS+PDGF; HBSS). Although DMEM had the highest cell proliferative effect, it is impractical to be used as a transport medium due to its cost, storage, and availability. The supplementation of Vit C yielded significant cell proliferative effects; hence, HBSS + Vit C can be a better alternative as a storage medium than HBSS.

Anti-fatigue effect of an enzymatically derived deer velvet extract through muscle damage recovery and improvement of antioxidant levels

Ethnopharmacological relevanceDeer velvet (DV) has been extensively used in traditional Oriental medicine to treat various diseases. Its pharmacological spectrum encompasses tonicity, longitudinal bone growth of adolescence, blood retention, hemopoiesis facilitation, enhancement of organ function, physical function improvement, and augmentation of physical vitality. Among its myriad effects, DV notably exhibits anti-fatigue properties; however, its specific mode of action is yet to be fully elucidated. Aim of the studyThis study was undertaken to investigate the anti-fatigue and exercise performance-enhancing effects of YC-1101(HENKIV®), an enzymatically derived DV extract, in C2C12 cell lines and forced swimming mouse models. Materials and methodsThe effect of YC-1101 on increasing cell growth and lowering lactate dehydrogenase (LDH) activity was assessed in C2C12. The antioxidative effects of YC-1101 and its mechanistic underpinnings were also evaluated in C2C12 cells. Moreover, mice were subjected to an exhaustive swimming test subsequent to 3 weeks of YC-1101 extract administration. Fatigue-associated biochemical parameters, such as LDH activity and lactate, superoxide dismutase, glutathione peroxidase, and malondialdehyde levels, were measured in serum and muscle tissues. ResultsYC-1101 significantly promoted myoblast growth and reduced LDH activity, indicative of a cell-proliferative effect. Notably, free radical scavenging assays and analysis of reactive oxygen species production and antioxidant-related mRNA expression corroborated the significant involvement of YC-1101 in antioxidation, an important mechanism in anti-fatigue processes. Furthermore, animal experiments demonstrated prolonged swimming endurance and inhibition of muscle LDH accumulation in forced swimming mouse models. Serum biochemical analysis further revealed significant modulation of the expression of anti-fatigue-related biomarkers. Various bioactive low-molecular-weight DV peptides were enriched in YC-1101 compared to YHC-BE-2038 (a DV extract without enzymatic digestion). The anti-fatigue effect of YC-1101 was significantly stronger than that of YHC-BE-2038. ConclusionsThese findings suggest the potential of YC-1101 as a nutraceutical adjunct in ameliorating fatigue, concurrently facilitating muscle damage recovery, and exerting antioxidant effects via the nuclear factor E2-related factor 2-Kelch-like ECH-associated protein-1 pathway. It can be assumed that the complex action of low-molecular-weight DV peptides produced during the enzymatic degradation process was effective, and the further research is needed.

Two major bovine milk whey proteins induce distinct responses in IEC-6 intestinal cells

Abstract α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) are major whey proteins in bovine milk. We studied the effects of these molecules on the intestinal cell response by comparing the native form with the denatured form containing oligomers obtained by treatment with 2,2,2-trifluoroethanol (TFE). We previously reported that proteins in native and TFE-treated forms exhibited cell growth stimulation and cytotoxicity, respectively, in undifferentiated rat crypt IEC-6 and human colon Caco-2 cells. However, neither whey protein showed cytotoxicity even in the TFE-treated form in differentiated Caco-2 cells. Only undifferentiated immature intestinal cells can distinguish between these native and denatured proteins. Moreover, α-LA and β-LG exhibited different oligomer formation characteristics during the TFE treatment. In the present study, we compared the effects of native and TFE-treated whey proteins on IEC-6 cells in more detail. The native forms of both whey proteins exhibited cell proliferative effects in a concentration-dependent manner. For the TFE-treated forms, α-LA showed rapid and potent cytotoxicity, whereas β-LG altered cell responses depending on its concentration and exposure time; lower concentration/shorter exposure and higher concentration/longer exposure induced cell growth stimulation and cytotoxicity, respectively. Pre-treatment of the cell membrane with cholesterol suppressed the effects on the cell response only in TFE-treated β-LG (TFE-β-LG). In a preliminary examination using inhibitors of signal transduction, TFE-treated α-LA acted on the intrinsic apoptosis pathway via Bcl-2-associated X and p53, whereas the action of TFE-LG did not require this pathway. Tyrosine phosphorylation is necessary for the cell proliferation effect of both native whey proteins; however, native α-LA, but not native β-LG, also required activation of the pathway with selective epidermal growth factor receptor tyrosine kinase and Janus kinase 2/3. In summary, the two major bovine milk whey proteins induced similar yet discrete responses in undifferentiated intestinal cells. Even when oligomers are formed, β-LG may be much less hazardous to immature intestinal cells than α-LA.

Highly Efficient Organic Light–Emitting Diode Using DNA as Scattering Layer

AbstractHerein, an organic light–emitting diode (OLED) is fabricated with increased luminous efficiency and biocompatibility by topographic DNA film. In addition to the natural abundance and biocompatibility of DNA, its semi‐flexible characteristics facilitated the emergence of the liquid crystal phase, allowing the formation of a periodic wavy undulation structure by applying shear force. The resultant zigzag structure possesses sufficient periodicity and roughness, enhancing the effective scattering of emitted light. Furthermore, the DNA‐coated red OLED demonstrates cell proliferative effects and exhibits high electrical and thermal stability even in a bent state, suggesting its potential application in attachable OLED‐based light therapy.

SSTR2 Mediates the Inhibitory Effect of SST/CST on Lipolysis in Chicken Adipose Tissue.

Somatostatin shows an anti-lipolytic effect in both chickens and ducks. However, its molecular mediator remains to be identified. Here, we report that somatostatin type 2 receptor (SSTR2) is expressed at a high level in chicken adipose tissue. In cultured chicken adipose tissue, the inhibition of glucagon-stimulated lipolysis by somatostatin was blocked by an SSTR2 antagonist (CYN-154086), supporting an SSTR2-mediated anti-lipolytic effect. Furthermore, a significant pro-proliferative effect was detected in SST28-treated immortalized chicken preadipocytes (ICP-1), and this cell proliferative effect may be mediated through the MAPK/ERK signaling pathway activated by SSTR2. In summary, our results demonstrate that SSTR2 may regulate adipose tissue development by affecting the number and volume of adipocytes in chickens.

Lnc RNA LOC102163816 Promotes Proliferation of Porcine Follicular Granulosa Cells Via miR-455-3p/PTK2B/PI3K/AKT Pathway.

The proliferation and differentiation of granulosa cells (GCs) is a crucial process in follicular development. However, the molecular regulatory mechanism of follicular proliferation and differentiation of GCs needs further research. Studies have reported that follicular fluid exosomes are involved in regulation of proliferation of GCs, but the specific mechanism is unclear. This study demonstrated that LOC102163816 is upregulated in porcine GCs treated with follicular fluid exosomes. Further study defined LOC102163816 to be a novel long noncoding RNA that is highly homologous to human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and enriched in porcine follicular fluid exosomes. We have speculated that LOC102163816 might have a cell-proliferative effect similar to that of MALAT1. We found that overexpression of LOC102163816 promoted transition from the G1 phase to the S phase of the cell cycle, thereby promoting proliferation of GCs. To explore the specific mechanism underlying this promotion of proliferation, miRNA sequencing was performed after overexpression of LOC102163816. Our results showed that LOC102163816 sponged miR-455-3p, promoting expression of protein tyrosine kinase 2 beta (PTK2B), thereby activating the PI3K/AKT signaling pathway to regulate proliferation of porcine follicular GCs. These findings provide useful insights into follicular development.

Anticarcinogenic potential of the mushroom polysaccharide lentinan on gastric and colon cancer cells: Antiproliferative, antitumorigenic, Mu-2-related death-inducing gene, MUDENG ramifications

Mushrooms have been known as the elixirs of health, they are well known from ages immemorial for their nutritional and medicinal properties immunoceutical and immunomodulatory properties. Lentinula edodes (“shiitake”) is a well-accomplished mushroom for its biological and medicinal attributes of huge significance. Lentinan is a bioactive polysaccharide that is extracted from shiitake mushrooms. In the present study, standard commercially available lentinan polysaccharide was used to demonstrate its anticancer activity. The results confirmed the anti-cancer cell proliferative effect of lentinan on colon and gastric cancer cell lines through the inhibition of G2/M phase in the cell cycle. Lentinan also could attenuate wound healing and colony formation abilities and migration in AGS and HT-29 cells, demonstrating its tumorigenesis potential. Lentinan could increase phospho-p38 and decrease phopho-ERK1/2, Mu-2-related death-inducing gene (MuD) protein. Besides, lentinan also induced reactive oxygen species (ROS) production which was directly involved in cell death. In this study, various aspects of the anticarcinogenic activity of lentinan including the MuD influence have been demonstrated. This is the first report evidencing cross-talk by lentinan and MuD protein confirming the anti-apoptotic function of lentinan.

Moracin E and M isolated from Morus alba Linné induced the skeletal muscle cell proliferation via PI3K-Akt-mTOR signaling pathway

Twigs of Morus alba have been used in traditional medicine to treat muscle-related symptoms such as aches, numbness, and stiffness. Despite its clinical use in traditional medicine, its active compounds and mode of action have not yet been investigated. Therefore, we aimed to isolate the compounds from the twigs of M. alba and deduce active compounds, key gene targets, and mechanism of action against sarcopenia using network pharmacology analysis. Using various isolation techniques and spectroscopic methods, 43 phytochemicals, including 3 new flavonoids, were isolated and performed network pharmacology analysis. According to the computational-assistant analysis, 28 compounds, 9 genes, and the PI3K-Akt-mTOR signaling pathway were deduced as expected active compounds (EAC), key targets, and the main signaling pathway. To verify the predicted results, the cell proliferation activities of the EAC were evaluated. Especially, moracin E and M significantly increased by 130% (p < 0.001) and 57% (p < 0.05), respectively, which have more than 2- and 1.5-fold stronger effects compared to the control. Furthermore, both increased the expression level of proteins involved in the PI3K-Akt-mTOR signaling pathway and myogenic proteins, including myogenin and MyoD. This study demonstrated that moracin E and M exhibit cell proliferative effects on skeletal muscle cells through the PI3K-Akt-mTOR signaling pathway.

Ethyl acetate fraction of Osmanthus fragrans var. aurantiacus and its triterpenoids suppress proliferation and survival of colorectal cancer cells by inhibiting NF-κB and COX2

Ethnopharmacological relevanceColorectal cancer (CRC) remains a significant global health concern, and targeting inflammation has emerged as a promising approach for its prevention and treatment. Medicinal plants and phytochemicals have garnered attention for their potential efficacy against inflammation with minimal toxicity. Osmanthus fragrans var. aurantiacus Makino (O. fragrans) has a history of traditional use in Korea and China in treating various inflammation-related conditions, but its potential use for CRC has not been uncovered. Aim of the studyThis study aims to explore the potential anti-proliferative and pro-apoptotic properties of O. fragrans, focusing on its impact on CRC treatment. By investigating O. fragrans, we aim to uncover its anti-proliferative and apoptotic effects in human CRC cells, potentially paving the way for effective and well-tolerated therapeutic strategies for CRC patients. Materials and methodsEthanol (EtOH) extracts of O. fragrans leaf and flower, along with specific fractions (n-hexane, ethyl acetate (EtOAc), n-butanol, and the aqueous residue) were evaluated for their anti-proliferative effects in human CRC cells using MTT assays, and compared to normal colon cells. Mechanistic insights and chemical profiling were obtained through flow cytometry, colorimetric assays, western blotting, and molecular docking, and high-performance liquid chromatography (HPLC) system. ResultsBoth flower and leaf EtOH extracts of O. fragrans exhibited significant anti-proliferative effects in human CRC cells, with the leaf extract demonstrating higher potency. The EtOAc fraction from the leaf extract displayed the strongest anti-CRC cell proliferative effects while no cytotoxic effects in normal colon cells. Chemical profiling of these fractions identified triterpenoids as significant components in the EtOAc fractions. The leaf EtOAc fraction caused cell cycle arrest and apoptosis, accompanied by elevating intracellular reactive oxygen species and mitochondrial dysfunction in CRC cells. Additionally, it inhibited NF-κB and ERK1/2 signaling, leading to reduced COX2 expression. Notably, two triterpenoids isolated from the leaf EtOAc fraction, maslinic acid and corosolic acid, displayed potent anti-cancer activity in CRC cells without affecting normal colon cells. Corosolic acid exhibited a strong binding affinity to COX2 and reduced its expression, supporting its role in the anti-inflammatory and anti-cancer effects. ConclusionsOur findings suggest that O. fragrans, particularly its triterpenoid-rich EtOAc fraction, holds promise as a novel therapeutic agent for CRC prevention and therapy. These results provide valuable insights into the potential application of O. fragrans and its bioactive compounds in combating CRC.

Anti-inflammatory effect of polydeoxyribonucleotides (PDRN) extracted from red alga (Porphyra sp.) (Ps-PDRN) in RAW 264.7 macrophages stimulated with Escherichia coli lipopolysaccharides: A comparative study with commercial PDRN.

Polydeoxyribonucleotide (PDRN) is a DNA-derived drug extracted from the sperm cells of Oncorhynchus mykiss or O. keta. PDRN exhibits wound healing and anti-inflammatory activities by activating adenosine A2A receptor and salvage pathways. However, commercial PDRN products (e.g., Placentex, Rejuvenex, and HiDr) have limitations as they are exclusively extracted O. mykiss and O. keta, which are expensive and can only be used as extraction sources during a specific period when their sperm cells are activated. Therefore, this study aimed to extract PDRN from Porphyra sp. (Ps-PDRN) and investigate whether it has anti-inflammatory activity through a comparative study with commercial product. The results indicated that Ps-PDRN had an anti-inflammatory effect on Escherichia coli lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. It inhibited nitric oxide production and inducible nitric oxygen synthase protein expression by suppressing phosphorylation of p38 and ERK, without cytotoxicity. Furthermore, Ps-PDRN promoted cell proliferation and collagen production in human dermal fibroblast. In conclusion, our study confirms that Ps-PDRN exhibits both anti-inflammatory and cell proliferative effects. These results indicated that Ps-PDRN has the potential as a bioactive drug for tissue engineering.

Protein Adsorption, Calcium-Binding Ability, and Biocompatibility of Silver Nanoparticle-Loaded Polyvinyl Alcohol (PVA) Hydrogels Using Bone Marrow-Derived Mesenchymal Stem Cells.

Several approaches have evolved to facilitate the exploration of hydrogel systems in biomedical research. In this sense, poly(vinyl alcohol) (PVA) has been widely used in hydrogel (HG) fabrication for several therapeutic applications. The biological properties of PVA hydrogels (PVA-HGs) are highly dependent on their interaction with protein receptors and extracellular matrix (mainly calcium) deposition, for which there is not enough evidence from existing research yet. Thus, for the first time, the functional properties, like protein and mineral interactions, related to the proliferation of mesenchymal stem cells (MSCs) by silver nanoparticle (AgNP)-loaded PVA hydrogels (AgNPs-PVA-HGs) were investigated in the present study. The UV absorption spectrum and TEM microscopic results showed a maximum absorbance of synthesized AgNPs at 409 nm, with an average particle size of 14.5 ± 2.5 nm, respectively. The functional properties, such as the calcium-binding and the protein adsorption of PVA-HG, were accelerated by incorporating AgNPs; however, the swelling properties of the HGs were reduced by AgNPs, which might be due to the masking of the free functional groups (hydroxyl groups of PVA) by AgNPs. SEM images showed the presence of AgNPs with a more porous structure in the HGs. The proliferative effect of MSCs increased over culture time from day 1 to day 7, and the cell proliferative effect was upregulated by HGs with more pronounced AgNPs-PVA-HG. In addition, both HGs did not produce any significant cytotoxicity in the MSCs. The histological (bright light and H&E staining) and fluorescence microscopic images showed the presence of a cytoskeleton and the fibrillar structure of the MSCs, and the cells adhered more firmly to all HGs. More fibrillar bipolar and dense fibrillar structures were seen in the day 1 and day 7 cultures, respectively. Interestingly, the MSCs cultured on AgNPs-PVA-HG produced extracellular matrix deposition on day 7. Accordingly, the present results proved the biocompatibility of AgNPs-PVA-HG as a suitable system for culturing mammalian stem cells for regenerative tissue applications.

WXJ-202, a novel Ribociclib derivative, exerts antitumor effects against breast cancer through CDK4/6.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are key regulatory proteins in the cell division and proliferative cycle in humans. They are overactive in many malignant tumors, particularly in triple-negative breast cancer (TNBC). Inhibition of CDK4/6 targets can have anti-tumor effects. Here, we designed and synthesized a novel derivative of Ribociclib that could affect CDK4/6, named WXJ-202. This study aimed to investigate the effects of compound WXJ-202 on proliferation, apoptosis, and cell cycle arrest in human breast cancer cell lines and their molecular mechanisms. We assayed cell viability with methyl thiazolyl tetrazolium (MTT) assay. Clone formation, migration, and invasion ability were assayed by clone formation assay, wound healing assay, and transwell invasion assay. The effect of compound WXJ-202 on apoptosis and cell cycle was detected by flow cytometry analysis. Western blotting was performed to detect the expression of proteins related to the CDK4/6-Rb-E2F pathway. The anti-cancer effects were studied in vivo transplantation tumor models. WXJ-202 was shown to inhibit cell proliferation, colony formation, migration, and invasion, as well as induce apoptosis and cycle arrest in breast cancer cells. The levels of proteins related to the CDK4/6-Rb-E2F pathway, such as CDK4, CDK6, and p-Rb, were decreased. Finally, studies had shown that compound WXJ-202 exhibited significant anti-tumor activity in transplantation tumor models. In this research, the compound WXJ-202 was shown to have better anti-tumor cell proliferative effects and could be used as a potential candidate against TNBC tumors.

Characterization and evaluation of Bletilla striata polysaccharide/konjac glucomannan blend hydrogel for wound healing.

Bletilla striata polysaccharide (BSP) is effective for wound healing and has important applications in health care. A series of blend hydrogels was designed with BSP and konjac glucomannan (KGM) in this study to overcome the deficient mechanical performance caused by the excessive dissolution of BSP without affecting its physiological activity. The interplay between them, as well as the effects of KGM concentration on the physical properties and microstructures of hydrogels, were also explored. It was proved that the frame of the hydrogel was primarily formed by KGM. BSP was dispersed uniformly and linked to KGM through hydrogen bonding, which effectively improved the physical properties, such as increasing the water-holding capacity, improving the swelling degree, and enhancing the mechanical properties. Blend hydrogel BK2-2 (containing 1.0% BSP and 1.0% KGM, w/v) was found to be the optimal formulation based on the thermal stability and microstructure, which was used for further research. In vitro experiments revealed the L929 cell proliferative effects of the blend hydrogel, and no difference was found with BSP sponge extract after 72 h of exposure. In vivo animal studies indicated that the BK2-2 accelerated wound healing compared with the control group; however, no difference was found with dressings only made of BSP. These results demonstrated that KGM improved the physical properties of BSP-based material without negatively affecting its physiological properties. Also, the BSP/KGM blend hydrogel had good comprehensive properties and is expected to be used as a wound healing material in the future.

An Up-to-Date Review of Biomaterials Application in Wound Management.

Whether they are caused by trauma, illness, or surgery, wounds may occur throughout anyone’s life. Some injuries’ complexity and healing difficulty pose important challenges in the medical field, demanding novel approaches in wound management. A highly researched possibility is applying biomaterials in various forms, ranging from thin protective films, foams, and hydrogels to scaffolds and textiles enriched with drugs and nanoparticles. The synergy of biocompatibility and cell proliferative effects of these materials is reflected in a more rapid wound healing rate and improved structural and functional properties of the newly grown tissue. This paper aims to present the biomaterial dressings and scaffolds suitable for wound management application, reviewing the most recent studies in the field.

The apoptotic and proliferative effects of tulathromycin and gamithromycin on bovinetracheal epithelial cell culture

Gamithromycin and tulathromycin are commonly used in the treatment of bovine respiratory bacterial diseases. The current work was undertaken to establish the apoptotic, necrotic, and cytotoxic effects of these antibiotics in the target animal. Cells with apoptosis and necrosis were determined by dual staining method, cytotoxic effects were determined by MTT assay, cell proliferative effects were examined by XCelligence real-time cell analysis system (RTCA-SP). The comparison between gamithromycin and tulathromycin concentrations on tracheal cells in terms of % cell viability was found to be significantly different. While the cell viability percentage of gamithromycin was higher at 150 μg/mL, 180 μg/mL, and 240 μg/mL than tulathromycin, and at 2 μg/mL, 4 μg/mL, 10 μg/mL, 20 μg/mL, and 50 μg/mL concentrations tulathromycin cell viability was higher than gamithromycin (p < 0.05). When the staining method data were evaluated, the difference between the results of % apoptotic index at 20 μg/mL concentration was significant and it was found that gamithromycin had more apoptotic effect than tulathromycin (p < 0.05). It was seen that tulathromycin and gamithromycin applied on tracheal epithelial cells at concentrations of 2 and 10 ?g/mL increased the viability depending on time. The increase in epithelial cell proliferation of gamithromycin and tulathromycin due to time shows that these antibiotics can maintain longterm prophylactic treatment against diseases.

Vanillin extracted from proso and barnyard millets induces cell cycle inhibition and apoptotic cell death in MCF-7 cell line.

Consuming whole grain food has been motivated due to numerous health benefits arising from their bioactive components. This study aims to study whether the active compound extracted from Proso and Barnyard millets inhibits cell proliferation and induces apoptotic cell death in MCF-7 cell line. Cell proliferative effect was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using MCF-7 cell line. Cytotoxicity was determined by release of lactate dehydrogenase (LDH) enzyme from cells. Apoptotic morphological changes in MCF-7 cells were observe under fluorescence microscope using double staining of Hoeschst 33342/propidium iodide (PI). Induction of apoptosis was analyzed using Annexin V-fluorescein isothiocyanate/PI through flow cytometry. In this study, cell proliferative effect of the bioactive compounds from proso millet (Compound 1) and barnyard millet (Compound 2) was evaluated using MCF-7 cell line. Both the compounds significantly inhibited the proliferation of MCF-7 cells after treated with 250 μg/ml and 1000 μg/ml concentration for 48 h. Cytotoxic activity of compounds was assessed by the release of LDH showed that these extracted compounds were not toxic to the cells. Apoptosis was confirmed by Hoechst 33,342/PI dual-staining, Annexin V-FTIC/PI staining, and flow cytometry results of cell cycle analysis shows that there was a significant cell arrest in the G0/G1 phase and increased the apoptotic cells in sub-G0 phase in a dose-dependent manner. This study suggests that the extracted vanillin compound from these millets have effectively induced apoptotic cell death in breast cancer cell line.

Analysis of Estrogenic Activity in Maryland Coastal Bays Using the MCF-7 Cell Proliferation Assay.

Contaminants of Emerging Concern (CECs) with estrogenic or estrogenic-like activity have been increasingly detected in aquatic environments and have been an issue of global concern due to their potential negative effects on wildlife and human health. This study used the MCF-7 cell proliferation assay (E-Screen) to assess the estrogenic activity profiles in Maryland Coastal Bays (MCBs), a eutrophic system of estuaries impacted by human activities. Estrogenic activity was observed in all study sites tested. Water samples from MCBs increased MCF-7 cell proliferation above the negative control from 2.1-fold at site 8, located in Sinepuxent Bay close to the Ocean City Inlet, to 6.3-fold at site 6, located in Newport Bay. The proliferative effects of the sediment samples over the negative control ranged from 1.9-fold at the Assateague Island National Seashore site to 7.7-fold at the Public Landing site. Moreover, elevated cell proliferation (p < 0.05) was observed when cells were co-exposed with 17ß-Estradiol (E2), while reduction in cell proliferation was observed when cells were co-exposed with the antagonist ICI 182, 780 suggesting that cell proliferative effects were primarily mediated by the estrogen receptor (ER). These results suggest the occurrence of some estrogenic or hormonal-like compounds in the MCBs and are consistent with our previous findings based on vitellogenin analyses.

Andrographolide promotes proliferative and osteogenic potentials of human placenta-derived mesenchymal stem cells through the activation of Wnt/\u03b2-catenin signaling

IntroductionThe in vitro expansion and differentiation of mesenchymal stem cells derived from bone marrow (BM-hMSCs) are considered as potential therapeutic tools for clinical applications in bone tissue engineering and regenerative medicine. However, invasive sampling and reduction in number and proliferative capacity with age are the major limitations of BM-hMSCs. Recently, human placenta-derived MSCs (PL-hMSCs) obtained by a non-invasive procedure have attracted much interest. Attempts to increase the potential of PL-hMSCs would be an important paradigm in regenerative medicine. Herein, we examined the proliferative and osteogenic effect of andrographolide (AP) on PL-hMSCs.MethodsMesenchymal stem cells were isolated from full-term normal human placentas and were characterized before using. Cell cytotoxicity and proliferative effect of AP were examined by MTT and BrdU assay, respectively. The non-toxicity concentrations of AP were further assessed for osteogenic effect determined by alkaline phosphatase (ALP) expression and activity, alizarin red staining, and osteoblast-specific gene expressions. Screening of genes involved in osteogenic differentiation-related pathways modulated by AP was explored by a NanoString nCounter analysis.ResultsPL-hMSCs generated in this study met the MSC criteria set by the International Society of Cellular Therapy. The non-cytotoxic concentrations of AP on PL-hMSCs are up to 10 μM. The compound increased PL-hMSC proliferation concomitant with increases in Wnt/β-catenin level and activity. It also enhanced osteogenic differentiation in association with osteoblast-specific mRNA expression. Further, AP promoted bone formation and increased bone structural protein level, osteocalcin, in osteoblastic cells. Gene screening analysis showed the upregulation of genes related to Wnt/β-catenin, TGFβ/BMP, SMAD, and FGF signaling pathways.ConclusionWe demonstrated, for the first time, the potential role of AP in promoting proliferation, osteogenic differentiation, and osteoblast bone formation of PL-hMSCs. This study suggests that AP may be an effective novel agent for the improvement of PL-hMSCs and stem cell-based therapy for bone regeneration.

Proteomics study on the effect of combined treatment of electrical pulses and tomato lipophilic extract in the downregulation of proliferating cell nuclear antigen in triple-negative breast cancer cell

Objective: To analyze the effect of electrical pulses (EP) with tomato lipophilic extract (TLE) on human, triple negative breast cancer (TNBC) cell (MDA-MB-231) and human epithelial, non-cancerous breast cell (MCF-10A) and to gain insights into the mechanisms of action. Methods: We prepared TLE using the solvent extraction method. We identified the constituents of TLE using LC-MS analysis. TLE with the dose of 250 μg/ml was combined with 800 V/cm, 100 μs, eight electrical pulses to treat cancerous MDA-MB-231 and non-cancerous MCF-10A breast cells, and their survivability was studied. We studied the proteins responsible for TLE's anticancer activity in TNBC cells using label-free, quantitative proteomics study. To understand the relationship between changes protein and transcription level, we did a real-time quantitative PCR (RT-qPCR) study. We detected Hydrogen Peroxide (H2O2) levels of both untreated and TLE/EP treated cells to determine how oxidative stress regulates various intracellular pathways. Results: TLE comprises fatty acids that heighten MDA-MB-231 cell proliferation. However, electrical pulses application nullifies these cell proliferative effects of fatty acids, as evident by the significant MDA-MB-231 cell viability reduction for TLE+EP treatment. Tomato extract, electrical pulses, and combinational treatment did not affect normal breast MCF-10A cells. A total of 1877 proteins were identified from the proteomics study. Conclusion: Applying EP in combination with TLE suppresses and reverses back the proliferative effects caused by TLE. EP and TLE+EP treatments increase hydrogen peroxide reactive oxygen species production in TNBC to inflict cell death due to excessive oxidative stress. Proteins, such as PCNA, LDHB, and ENO1, were downregulated in EP treatment, and it was confirmed with the mRNA levels by qPCR results. Our findings render innovative perceptions into the mechanisms of electrical pulses with TLE. Significance: Our research serves as a technological platform capable of identifying the set of proteins responsible for minimizing TNBC cell proliferation when treated with TLE+EP.

Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

BackgroundGrowth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms.ResultsWe present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents.ConclusionsWe demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.