Mouse Mesenchymal Stem Cell Line Research Articles

Synthesis, comprehensive characterization and investigation of biological properties of newly synthesized Pt(II)-aminothiazole complexes: Combining experimental and computational approach

The synthesis of new platinum(II) complexes with 2-amino-6-methyl-benzothiazole and 2-amino-6-chlorobenzothiazole as ligands were reported and the proposed structure of the complexes were determined using elemental microanalysis, infrared, 1H and 13C NMR spectroscopy. To verify the suggested structures a computational approach utilizing Density Functional Theory was implemented. The results showed a strong correlation with experimental data, confirming the hypothesized structures of investigated compounds.The interactions of novel platinum(II) complexes with human serum albumin and calf thymus DNA were studied by fluorescence spectroscopy and UV-Vis absorption. The predominantly elevated values of the binding constant, Kb, and the Stern-Volmer quenching constant, KSV, stem from the effective attachment of complexes to both HSA and CT-DNA. To establish the mode of interaction, viscosity measurements were conducted. The results showed that complexes are not performing intercalation between the DNA bases, and which probably bind to minor/major grooves.In vitro cytotoxic effect of ligands and platinum(II) complexes was evaluated in the panel of cancer cell lines (mouse mammary carcinoma and colon cancer, and on human mammary carcinoma and colon cancer), and on non-tumor cells, mouse mesenchymal stem cell line using MTT assay. Platinum(II) complex with 2-amino-6-methyl-benzothiazole showed a very good cytotoxic activity mainly by inducing apoptosis.In vitro antimicrobial assay of ligands and complexes was tested against 11 microorganisms using microdilution method and the minimum inhibitory concentration and minimum microbicidal concentration were identified. All tested compounds exhibited moderate antifungal activity and significantly better action on S. aureus ATCC 25923.

Phenolic Compounds Isolated from Thlaspi Arvense and Their Effects on Osteoblast Differentiation in the Mouse Mesenchymal Stem Cell Line C3H10T1/2

Phenolic Compounds Isolated from <i>Thlaspi Arvense</i> and Their Effects on Osteoblast Differentiation in the Mouse Mesenchymal Stem Cell Line C3H10T1/2

Anti-osteoporosis effects of triterpenoids from the fruit of sea buckthorn (Hippophae rhamnoides) through the promotion of osteoblast differentiation in mesenchymal stem cells, C3H10T1/2.

In a previous study, we discovered that the ethanolic extract of sea buckthorn (Hippophae rhamnoides) fruits exhibited anti-osteoporosis effects both in vitro and in vivo. Through bioassay-guided fractionation, we identified the hexane fraction (HRH) as the active fraction, which was further fractionated using preparative HPLC. Among the resulting six fractions, HRHF4 showed significant activity. In the present study, we focused on the bioassay-guided isolation of bioactive compounds from the HRHF4 fraction. We successfully identified the active HRHF43 fraction, which led us to the isolation of potential bioactive compounds (1-6). The chemical structures of these compounds were determined using NMR data, LC-MS analysis, and HR-ESI-MS data as four triterpenes, ursolic acid (1), uvaol (2), oleanolic aldehyde (3), and ursolic aldehyde (4), together with two fatty acids, methyl linoleate (5) and ethyl oleate (6). To evaluate the efficacy of promoting osteoblast differentiation and the expression of mRNA biomarkers related to osteogenesis, we tested the isolated compounds in the mouse mesenchymal stem cell line, C3H10T1/2. Alkaline phosphate staining demonstrated that triterpenes (1-4) displayed osteogenic activity. Particularly noteworthy, ursolic aldehyde (4) exhibited the most potent effect, showing an 11.2-fold higher activity at a concentration of 10μg/mL compared to the negative control. Moreover, ursolic aldehyde (4) upregulated the gene expression of bone formation-related biomarkers, including Runx2, Osterix, Alp, and Osteopontin. These findings suggest that the fruit extract of H. rhamnoides may have potential as a nutraceutical for promoting bone health, with ursolic aldehyde (4) identified as an active constituent.

Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

Melatonin plays a synergistic rather than a major role during osteogenic differentiation via MT2 in mouse mesenchymal stem cell line C3H10T1/2.

Melatonin plays a synergistic rather than a major role during osteogenic differentiation via MT2 in mouse mesenchymal stem cell line C3H10T1/2.

Zinc Oxide–Incorporated Chitosan–Poly(methacrylic Acid) Polyelectrolyte Complex as a Wound Healing Material

A novel type of porous films based on the ZnO-incorporated chitosan-poly(methacrylic acid) polyelectrolyte complex was developed as a wound healing material. The structure of porous films was established by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and energy dispersive X-ray (EDX) analysis. Scanning electron microscope (SEM) and porosity studies revealed that the pore size and porosity of the developed films increased with the increase in zinc oxide (ZnO) concentration. The porous films with maximum ZnO content exhibited improved water swelling degree (1400%), controlled biodegradation (12%) for 28 days, a porosity of 64%, and a tensile strength of 0.47 MPa. Moreover, these films presented antibacterial activity toward Staphylococcus aureus and Micrococcus sp. due to the existence of ZnO particles. Cytotoxicity studies demonstrated that the developed films had no cytotoxicity against the mouse mesenchymal stem (C3H10T1/2) cell line. These results reveal that ZnO-incorporated chitosan-poly(methacrylic acid) films could be used as an ideal material for wound healing application.

Effect of spheroid size on gene expression profiles of a mouse mesenchymal stem cell line in spheroid culture.

Mesenchymal stem cell (MSC)-based therapies offer potential for bone repair. MSC spheroid cultures may harbor enhanced therapeutic potential over MSC monolayers through increased secretion of trophic factors. However, the impact of spheroid size on trophic factor expression is unclear. We investigated the effect of spheroid size on trophic factor-related gene expression. KUM10, a murine MSC line was used. RNA-seq was used to screen the transcriptional profiles of MSC monolayer and spheroid cultures. Differentially expressed genes identified in RNA-seq were evaluated by q-PCR in cultures of 5 × 104 (S group), 5 × 105 (M group), 5 × 106 (L group) cells/well. Comparison of expression levels between KUM10 monolayer and spheroid cultures identified 2140 differentially expressed genes, of which 1047 were upregulated and 1093 were downregulated in KUM10 spheroids. Among these, 12 upregulated genes (Bmp2, Fgf9, Fgf18, Ngf, Pdgfa, Pdgfb, Tgfb1, Vegfa, Vegfc, Wnt4, Wnt5a, Wnt10a) were associated with secretory growth factors. Of these, expression of Fgf9, Fgf18, Vegfa and Vegfc was elevated in the L group, and Pdgfb and Tgfb1 was elevated in the S group. Spheroid size may impact trophic factor expression. Our results will be useful for future studies assessing the utility of MSC spheroids for treating bone injury.

Quality evaluation of cell spheroids for transplantation by monitoring oxygen consumption using an on-chip electrochemical device

Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.

Myostatin inhibits insulin-like growth factor 1-dependent citrate secretion and osteogenesis via nicotinamide adenine dinucleotide phosphate oxidase-4 in a mouse mesenchymal stem cell line.

Citrate is an indispensable component of bone. Reduced levels of citrate in bone and serum are reported in the elderly and in osteoporosis patients. Myostatin (Mstn) is implicated in skeletal homeostasis, but its effects on osteogenesis remain incompletely understood. Nox4 has critical roles in bone homeostasis. TGF-β/Mstn-associated Smad2/3 signaling has been linked to Nox4 expression. Insulin-like growth factor (IGF-1) has been shown to counteract many regulatory effects of Mstn. However, the crosstalk among Mstn, IGF-1, and Nox4 is not well understood; the interactive effects of those factors on citrate secretion, osteogenic differentiation, and bone remodeling remain unclear. In this study, we demonstrated that osteogenic differentiation induced an IGF-1-dependent upregulation of citrate secretion that was suppressed by Mstn. Inhibition of Nox4 prevented Mstn-induced reduction of citrate secretion. In addition, Mstn reduced bone nodule formation; these changes were prevented by Nox4 inhibition. Moreover, Mstn increased the ratio of RANKL to OPG mRNAs to favor osteoclast activation. These results indicate that Mstn negatively regulates osteogenesis by increasing levels of Nox4, which reduced IGF-1 expression, citrate secretion, and bone mineralization while also altering the RANKL to OPG ratio. These findings provide new and highly relevant insights into the osseous effects of myostatin.

Anti-Osteoporosis Effects of the Fruit of Sea Buckthorn (Hippophae rhamnoides) through Promotion of Osteogenic Differentiation in Ovariectomized Mice.

The fruit of Hippophae rhamnoides has been widely used for medicinal purposes because of its anti-inflammatory, antioxidant, antiplatelet, and antimicrobial effects. Since there are no clear reports on the therapeutic efficacy of H. rhamnoides in osteoporosis, this study aimed to confirm the potential use of H. rhamnoides for the treatment of osteoporosis through its osteogenic differentiation-promoting effect in ovariectomized mice. Through an in vitro study, we compared the effects of the EtOH extract of H. rhamnoides fruits (EHRF) on the differentiation of C3H10T1/2, a mouse mesenchymal stem cell line, into osteoblasts based on alkaline phosphatase (ALP) staining and the relative expression of osteogenesis-related mRNAs. The EHRF significantly stimulated the differentiation of mesenchymal stem cells into osteoblasts and showed 7.5 times (* p < 0.05) higher osteogenesis than in the untreated control. A solvent fractionation process of EHRF showed that the hexane-soluble fraction (HRH) showed 10.4 times (** p < 0.01) higher osteogenesis than in the untreated control. Among the subfractions derived from the active HRH by preparative HPLC fractionation, HRHF4 showed 7.5 times (* p < 0.05) higher osteogenesis than in the untreated naïve cells, and HRH and HRHF4 fractions showed 22.6 times (*** p < 0.001) stronger osteogenesis activity than in the negative control. Osteoporosis was induced by excision of both ovaries in 9-week-old female ICR mice for in vivo analysis, and two active fractions, HRH and HRHF4, were administered orally for three months. During the oral administration period, body weight was measured weekly, and bone mineral density (BMD) and body fat density were measured simultaneously using a DEXA machine once a month. In particular, during the in vivo study, the average BMD of the ovariectomized group decreased by 0.0009 g/cm2, whereas the average BMD of the HRH intake group increased by 0.0033 g/cm2 (* p < 0.05) and that of the HRHF4 intake group increased by 0.0059 g/cm2 (** p < 0.01). The HRH and HRHF4 intake groups significantly recovered the mRNA and protein expression of osteogenic genes, including ALP, Osteopontin, Runx2, and Osterix, in the osteoporosis mouse tibia. These findings suggest that the active fractions of H. rhamnoides fruit significantly promoted osteoblast differentiation in mesenchymal stem cells and increased osteogenic gene expression, resulting in an improvement in bone mineral density in the osteoporosis mouse model. Taken together, H. rhamnoides fruits are promising candidates for the prevention and treatment of osteoporosis.

GPX7 Facilitates BMSCs Osteoblastogenesis via ER Stress and mTOR Pathway.

Emerging evidence indicates extensive oxidative stress is a consequence of obesity which impairs bone formation. Glutathione peroxidase 7 (GPX7) is a conserved endoplasmic reticulum (ER) retention protein, lacking of which causes accumulation of reactive oxygen species (ROS) and promotes adipogenesis. Since the imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cell (BMSC) leads to severe bone diseases such as osteoporosis, it is critical to investigate the potential protective role of Gpx7 in osteogenesis. Here, we provide evidence that deficiency of Gpx7 reduces osteogenesis, but increases adipogenesis in both human BMSCs (hBMSCs) and mouse mesenchymal stem cell line. Interestingly, further studies indicate this defect can be alleviated by the ER stress antagonist, but not the ROS inhibitor, unveiling an unexpected finding that, unlike adipogenesis, lacking of Gpx7 inhibits osteogenesis mediating by induced ER stress instead of enhanced ROS. Furthermore, the mTOR signalling pathway is found down‐regulation during osteogenic differentiation in Gpx7‐deficient condition, which can be rescued by relief of ER stress. Taken together, for the first time we identify a novel function of Gpx7 in BMSCs’ osteogenic differentiation and indicate that Gpx7 may protect against osteoporotic deficits in humans through ER stress and mTOR pathway interplay.

Ginkwanghols A and B, osteogenic coumaric acid-aliphatic alcohol hybrids from the leaves of Ginkgo biloba.

Ginkgo biloba (Ginkgoaceae), commonly known as "ginkgo", is called a living fossil, and it has been cultivated early in human history for various uses in traditional medicine and as a source of food. As part of ongoing research to explore the chemical diversity and biologically active compounds from natural resources, two new coumaric acid-aliphatic alcohol hybrids, ginkwanghols A (1) and B (2) were isolated from the leaves of G. biloba. The coumaric acid-aliphatic alcohol hybrids of natural products have rarely been reported. The structures of the new compounds were determined by extensive NMR spectroscopic analysis, HRESI-MS, and quantum chemical ECD calculations, and by comparing the experimental HRESI-MS/MS spectrum of chemically transformed compound 1a with the predicted HRESI-MS/MS spectra proposed from CFM-ID 3.0, a software tool for MS/MS spectral prediction and MS-based compound identification. Ginkwanghols A (1) and B (2) increased alkaline phosphatase (ALP) production in C3H10T1/2, a mouse mesenchymal stem cell line, in a dose-dependent manner. In addition, ginkwanghols A and B mediated the promotion of osteogenic differentiation as indicated by the induction of the mRNA expression of the osteogenic markers ALP and osteopontin (OPN).

Atomic Layer Deposition of Bioactive TiO2 Thin Films on Polyetheretherketone for Orthopedic Implants.

TiO2 thin films were deposited on the orthopedic implant material polyetheretherketone (PEEK) by plasma enhanced atomic layer deposition (PEALD) and characterized for their ability to enhance the osseointegrative properties. PEALD was chosen for film deposition to circumvent drawbacks present in line-of-sight deposition techniques, which require technically complex setups for a homogeneous coating thickness. Film conformality was analyzed on silicon 3D test structures and PEEK with micron-scale surface roughness. Wettability and surface energy were determined through contact angle measurements; film roughness and crystallinity were determined by atomic force microscopy and X-ray diffraction, respectively. Adhesion properties of TiO2 on PEEK were determined with tensile strength tests. Cell tests were performed with the mouse mesenchymal tumor stem cell line ST-2. TiO2-coated PEEK disks were used as substrates for cell proliferation tests and long-term differentiation tests. After 28 days of cultivation, a mineralized bone matrix was observed. Furthermore, the collagen I and osteocalcin content were determined. The results reveal that the osteogenic properties of the TiO2 thin film are comparable to those of hydroxyapatite, and thus bioactive properties of PEEK implants are improved by TiO2 thin films deposited with PEALD.

MiR17-92 cluster drives white adipose tissue browning.

White adipose tissue (WAT) browning may have beneficial effects for treating metabolic syndrome. miRNA are important regulators of the differentiation, development, and function of brown and beige adipocytes. Here, we found that the cold-inducible miRNA17-92 cluster is enriched in brown adipose tissue (BAT) compared with WAT. Overexpression of the miR17-92 cluster in C3H10T1/2 cells, a mouse mesenchymal stem cell line, enhanced the thermogenic capacity of adipocytes. Furthermore, we observed a significant reduction in adiposity in adipose tissue-specific miR17-92 cluster transgenic (TG) mice. This finding is partly explained by dramatic increases in white fat browning and energy expenditure. Interestingly, the miR17-92 cluster stimulated WAT browning without altering BAT activity in mice. In addition, when we removed the intrascapular BAT (iBAT), the TG mice could maintain their body temperature well under cold exposure. At the molecular level, we found that the miR17-92 cluster targets Rb1, a beige cell repressor in WAT. The present study reveals a critical role for the miR17-92 cluster in regulating WAT browning. These results may be helpful for better understanding the function of beige fat, which could compensate for the lack of BAT in humans, and may open new avenues for combatting metabolic syndrome.

Ergostane-Type Steroids from Korean Wild Mushroom Xerula furfuracea that Control Adipocyte and Osteoblast Differentiation.

As part of our current work to discover structurally and/or biologically novel compounds from Korean wild mushrooms, we isolated five ergostane-type steroids (1-5) from the fruiting bodies of Xerula furfuracea via repeated column chromatographic separations and HPLC purification. The chemical structures of the isolated steroids were shown to be (22E,24R)-24-methylcholesta-4,22- diene-3,6-dione (1), ergosta-7,22-diene-3β,5α,6β-triol (2), ergosta-7,22-diene-3β,5α,6β,9α-tetraol (3), (22E,24R)-5α,8α-epidioxyergosta-6,22-diene-3β-ol-3-O-β-D-glucopyranoside (4), and (22E,24R)-5α,8α-epidioxyergosta-6,9,22-triene-3β-ol-3-O-β-D-glucopyranoside (5) based on comparison of the data regarding their spectroscopic and physical properties with those of previous studies. Notably, this is the first report on the presence of the identified steroids (1-5) in this mushroom. We tested compounds 1-5 to determine their effects on adipogenesis and osteogenesis in the mouse mesenchymal stem cell line C3H10T1/2 and found that compounds 4 and 5 suppressed the differentiation of stem cells into adipocytes. Notably, in addition to its suppressive effect on adipogenesis, compound 5 was also shown to promote the osteogenic differentiation of stem cells. These findings demonstrate that the bioactive compounds isolated might be effective for the treatment of menopause-associated syndromes, such as osteoporosis and obesity, as the isolated compounds were shown to suppress adipogenesis and/or promote osteogenesis of stem cells.

SUN-543 Myostatin Inhibits IGF1-Dependent Citrate Production during Osteogenesis in a Mouse Mesenchymal Stem Cell Line

Bone formation and osteoblast activation require glucose. Citrate is a key intermediate in the tricarboxylic acid (TCA) cycle that is the major energy-yielding metabolic pathway in cells. Citrate homeostasis, therefore, is critically important for glucose metabolism and energy production. In addition, citrate is an indispensable component of bone. The vast amount of citrate with its carboxylate groups provides a huge capacity for calcium binding on apatite that stabilizes the size of nanocrystal of bone. Reduced citrate levels in bone and serum are reported in elderly compared to young subjects, as well as in osteoporotic patients. IGF1, a growth factor that elicits downstream signaling events similar to insulin and also controls TCA activity, is essential in bone matrix mineralization. However, the source of citrate in bone and what roles that IGF1 plays in regulating citrate metabolism and deposition during osteogenesis are largely unknown. Myostatin (Mstn), a muscle cell-secreted cytokine, is involved in regulating energy metabolism, however, its effect on osteogenesis and bone formation are not well delineated. In this study, the interactive effect of Mstn and IGF1 on citrate production during osteogenesis was examined in C3H10T1/2 mouse mesenchymal stem cells (MSC). MSCs cultured in osteogenic medium induced high levels of citrate secretion with the peak (5.6-fold) was observed at a later stage (14-21d) of osteogenic differentiation. The levels of IGF1 mRNA and protein were also upregulated with a similar pattern, but of reduced magnitude than that of citrate secretion. In contrast, cells treated with Mstn showed a significant inhibition in both citrate secretion and IGF1 expression during the same period of osteogenesis. Alizarin Red staining (ARs) that was used to confirm the biological effect of Mstn showed a significant decrease in ARs in Mstn-treated cells compared to that of control cells. In addition, alkaline phosphatase (ALP) staining revealed a dramatical decrease in ALP expression and activity. These results suggest that inhibited citrate levels by Mstn led to defects in osteogenic differentiation and mineralization. To determine whether IGF1 plays role in these processes, an anti-IGF1 neutralizing antibody that inhibits IGF1 expression was employed. MSCs treated with IGF1 antibody showed a 35% reduction in citrate secretion compared to that of control cells. Moreover, the addition of exogenous IGF1 partially reversed the inhibitory effect of Mstn on citrate secretion, indicating that increased levels of citrate during osteogenesis is partially IGF1-dependent and that Mstn-inhibited citrate production is, at least in part, mediated by reduced IGF1 levels. These results imply a novel effect of Mstn on modulating osteogenesis of bone marrow MSCs by preventing IGF1-dependent citrate production that leads to defects in osteogenic differentiation and mineralization.

Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation

C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes (Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S, the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers (Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation.

Evaluation of Turkish propolis for its chemical composition, antioxidant capacity, anti-proliferative effect on several human breast cancer cell lines and proliferative effect on fibroblasts and mouse mesenchymal stem cell line

Propolis is an extremely complex resinous natural compound collected by honey bees from various plant sources and exhibits pharmacological and biological properties attributed to the presence of polyphenols. This study examined the total phenolic and flavonoid contents as well as the total antioxidant capacity using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), cupric-reducing antioxidant capacity (CUPRAC), and ferric-reducing antioxidant power (FRAP) methods. Turkish propolis has very high total phenolic (314.36 ± 3.65 mg GAE/g propolis) and total flavonoid contents (522.71 ± 11.45 mg QE/g propolis). The highest antioxidant capacity value was obtained by the CUPRAC method (1184.94 ± 63.27 mg TE/g propolis). Phenolic profile of Turkish propolis was also determined by high performance liquid chromatography with photodiode array detection (HPLC-PDA) method. The main phenolic compounds identified in Turkish propolis was flavonoids including pinocembrin > chrysin > galangin > pinobanksin > pinostrobin and phenolic acids including caffeic acid > p-coumaric acid > ferrulic acid > t-cinnamic acid. In the present work, anti-proliferative and proliferative effects of propolis extracts were also investigated on two different breast cancer cell lines; MDA-MB-231, UACC-3199 and on two normal cell lines; fibroblasts and mouse mesenchymal stem cell lines. According to the XTT results, Turkish propolis sample showed significant anti-proliferative effect on MDA-MB-231 and UACC breast cancer cell lines. Interestingly, Turkish propolis sample had proliferative effect on both fibroblasts and mouse mesenchymal stem cells. These results suggest that Turkish propolis can be considered as a potent agent on breast cancer treatment for further investigations.Evaluación del propóleos turco según su composición química, capacidad antioxidante, efecto antiproliferativo en varias líneas celulares de cáncer de mama humano y efecto proliferativo en fibroblastos y en la línea de células madre mesenquimales de ratones.El propóleos es un compuesto natural resinoso extremadamente complejo recogido por las abejas melíferas a partir de diversas fuentes vegetales, y presenta propiedades farmacológicas y biológicas atribuidas a la presencia de polifenoles. Este estudio examinó el contenido total de fenoles y flavonoides, así como la capacidad antioxidante total utilizando los métodos 2,2′-azino-bis(ácido 2,2′-etilbenzotiazolina-6-sulfónico (ABTS), 2,2-difenil-1-picrilhidrazilo (DPPH), capacidad antioxidante reductora de cúpricos (CUPRAC por sus siglas en inglés) y poder antioxidante reductor de férricos (FRAP por sus siglas en inglés). El propóleos turco tiene un contenido fenólico total muy alto (314,36 ± 3,65 mg GAE/g de propóleos) y un contenido flavonoide total (522,71 ± 11,45 mg QE/g de propóleos). El valor más alto de capacidad antioxidante se obtuvo con el método CUPRAC (1184.94 ± 63.27 mg TE/g propóleo). El perfil fenólico del propóleos turco también se determinó por cromatografía líquida de alta resolución con el método de detección de matriz de fotodiodos (HPLC-PDA). Los principales compuestos fenólicos identificados en el propóleos turco fueron los flavonoides, incluyendo pinocembrina > crisina > galangina > pinobanksina > pinostrobina y ácidos fenólicos, incluyendo ácido cafeico > ácido p-cumárico > ácido férrico > ácido t-cinámico. En el presente trabajo, también se investigó el efecto antiproliferativo y proliferativo de los extractos de propóleos en dos líneas celulares diferentes de cáncer de mama; MDA-MB-231, UACC-3199 y en dos líneas celulares normales; fibroblastos y líneas de células madre mesenquimales de ratones. Según los resultados del XTT, la muestra de propóleos turcos mostró un efecto antiproliferativo significativo en las líneas celulares de cáncer de mama MDA-MB-231 y UACC. Curiosamente, la muestra de propóleos turcos tuvo un efecto proliferativo tanto en los fibroblastos como en las células madre mesenquimales de ratones. Estos resultados sugieren que el propóleos turco puede ser considerado como un agente potente en el tratamiento del cáncer de mama para investigaciones posteriores.

Abstract 4128: Loss of p53 and Rb enhances ligand-dependent hedgehog signaling in development and cancer

Abstract The evolutionary conserved Hedgehog (Hh) signaling pathway plays fundamental morphogenic and mitogenic roles in tissue development and homeostasis, as well as in the initiation and progression of various cancers. Signaling is driven by the binding of Sonic hedgehog (Shh) ligand to the receptor Patched (Ptch1), to initiate primary cilia dependent Smoothened (Smo)-Gli activation. Although mutations in Hh pathway components are described in some cancers, the majority of Hh tumors exhibit ligand-dependent Hh signaling, where Hh ligand is produced by the stroma and/or tumor cells to maintain pathway activation and self-renewal. Here, we have utilized various Hh-dependent developmental models to better understand the tumorigenic mechanisms of Hh ligand-dependent signaling in cancer. During cerebellar development, Shh is secreted from cerebellar purkinje cells for rapid expansion of the cerebellar granule cell precursor (GCP) population in the external granule layer (EGL). Inactivation of Shh in purkinje cells (Pcp2Cre;Shhfl/fl) leads to reduced GCP proliferation, premature eradication of the EGL and reduced cerebellar size. In contrast, Shh overexpression by purkinje cells (Pcp2Cre;ShhTg) results in increased GCP expansion, a sustained EGL and increased cerebellar size. Indeed, signs of remnant EGL persist into adulthood, similar to the pre-neoplastic lesions observed in Ptch1+/- mice, believed to be the origin of SHH medulloblastoma. During skeletal development, Indian hedgehog secreted from pre-hypertrophic chondroblasts is required for osteoblast progenitor specification from multipotent mesenchymal stem cells. Sustained Hh signaling in osteoblast precursors (OsxCre;SmoM2 or OsxCre;ShhTg) results in reduced ossification of the calvaria, maxilla and mandible, reduced long bone width, and reduced expression of mature osteoblast markers. Together, these models implicate sustained Hh expression in the maintenance of an immature cell state at the expense of differentiation. Moreover, in a mouse model of osteosarcoma, inactivation of Smo (OsxCre;p53fl/fl;Rbfl/fl;Smofl/fl) abolished the malignant phenotype of OsxCre;p53fl/fl;Rbfl/fl mice, leading to benign osteoid osteoma consistent with osteoblast differentiation. Increased Hh signaling in cancers with prevalent p53 and Rb mutations led us to investigate a role for p53 and Rb in Hh ligand-dependent signaling. Genetic inactivation of p53 and/or Rb in the developing mouse neural tube resulted in expansion of the Hh-dependent Nkx2.2 ventral domain, consistent with a Hh gain of function phenotype. Similarly, siRNA knockdown of p53 and/or Rb in mouse mesenchymal stem cell line C3H10T1/2 markedly increased Hh-dependent osteoblast specification in response to Hh ligand. Taken together, we propose that p53 and Rb mutations enhance ligand-dependent Hh pathway activation that maintains cells in a primitive state and promotes tumorigenesis. Citation Format: Catherine R. Cochrane, Anette Szczepny, Samantha W. Jayasekara, Vijesh Vaghjiani, D. Watkins, Jason E. Cain. Loss of p53 and Rb enhances ligand-dependent hedgehog signaling in development and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4128.

LC/MS-based Analysis of Bioactive Compounds from the Bark of Betula platyphylla var. japonica and Their Effects on Regulation of Adipocyte and Osteoblast Differentiation

Betula platyphylla var. japonica (Betulaceae), also known as Asian white birch, is an endemic medicinal tree, the bark of which has been used in Chinese traditional medicine for the treatment of various inflammatory diseases. In our continuing search for bioactive compounds from Korean natural resources, a phytochemical investigation of the bark of B. platyphylla var. japonica led to the isolation of 7-oxo-β-sitosterol (1) and soyacerebroside I (2) from its ethanol extract as main components by liquid chromatography (LC)/mass spectrometry (MS)-based analysis. The structures of isolates were identified by comparison of 1H and 13C nuclear magnetic resonance spectroscopic data and physical data with the previously reported values and LC/MS analyses. To the best of our knowledge, this is the first study to demonstrate that the isolated compounds, 7-oxo-β- sitosterol and soyacerebroside I, were isolated in B. platyphylla var. japonica. We examined the effects of the isolates on the regulation of adipocytes and osteoblast differentiation. These isolates (1 and 2) produced fewer lipid droplets compared to the untreated negative control in Oil Red O staining of the mouse mesenchymal stem cell line without altering the amount of alkaline phosphatase staining. The results demonstrated that both compounds showed marginal inhibitory effects on adipocyte differentiation but did not affect osteoblast differentiation.