A novel hydrazide compound exerts anti-metastatic effect against breast cancer

BACKGROUND: There are currently a number of barriers hindering the successful treatment of breast cancer, including the metastatic spread of cancer cells. In looking for new anticancer agents, we reported two novel hydrazide derivatives with anti-cancer activity in human breast cancer cells. The current study aims to explore the therapeutic potential of the most effective one, N'-((5-nitrothiophen-2-yl)methylene)-2-(phenylthio)benzohydrazide (compound B), on metastatic breast cancer, which is resistant to available chemotherapeutics. METHODS: 4T1 mammary carcinoma cells were inoculated into the fat pad mammary of 5-7-week-old female BALB/c mice and then the effective compound was intraperitoneally administered for 4 weeks. Proliferation index and angiogenesis in tumor and lung tissues were examined with immunohistochemistry. In vitro assessments were also carried out to evaluate the effect of the compound on invasion of MDA-MB-231 cells. RESULTS: Our results demonstrated that this effective derivative significantly inhibited invasion of MDA-MB-231 cells in vitro as shown by Matrigel assay and quantitative real-time method for MMP-9 expression after 48 h of treatment. Daily administration of the compound suppressed the growth of primary tumor and its metastasis to lung, which was confirmed by H&E experiment at a dose of 1 mg/kg in a well-known metastatic model of 4T1 breast cancer in syngeneic BALB/c mice. These outcomes were supported by the immunohistochemical examinations of the tumor and lung tissues of mice. Tumors and lungs in mice treated with the effective compound showed a reduced proliferation index and a smaller microvessel density compared to the control. CONCLUSION: This study highlights an anti-metastatic role for a novel hydrazide derivative in both in vitro and in vivo models of breast cancer.

Morphological observation of bone marrow mesenchymal stem cells under matrigel three dimensional culture conditions / Observación morfológica de células madre mesenquimales de médula ósea en condiciones de cultivo tridimensional con matrigel

SUMMARY: Matrigel is a basement membrane matrix extracted from the EHS mouse tumor containing extracellular matrix protein, its main components are laminin, type IV collagen, nestin, heparin sulfate, growth factor and matrix metalloproteinase.At room temperature, Matrigel polymerized to form a three dimensional matrix with biological activity. It can simulate the structure, composition, physical properties and functions of the cell basement membrane in vivo, which is beneficial to the culture and differentiation of the cells in vitro, and can be used for the study of cell morphology, biochemical function, migration, infection and gene expression. In this study, Matrigel three-dimensional culture model of bone marrow mesenchymal stem cells(BMSCs) was established, and its morphology, proliferation and survival were observed. BMSCs were isolated and cultured with whole bone marrow adherence method. The Second generation BMSCs with good growth condition were selected and mixed with Matrigel to form cell gel complexes. The morphology and proliferation of mesenchymal stem cells were observed by phase contrast microscope and HE staining,Live/Dead staining was used to evaluate the cell activity.Phase contrast microscopy showed that BMSCs were reticulated in Matrigel and proliferated well, After 7 days, the matrix gel gradually became soft and collapsed, a few cell reticular crosslinking growth was seen at 14 days; HE staining showed that the cytoplasm of the cells was larger on the fourth day and the cells were elongated and cross-linked on the seventh day; Live/dead staining showed that most cells showed green fluorescence with the prolongation of culture time, on the first, 4 and 7 days, the activity of bone marrow mesenchymal stem cells in Matrigel gradually increased, and the percentages were 92.57 %, 95.54 % and 97.37 %, respectively. Matrigel three-dimensional culture system can maintain the morphology, function and proliferation ability of bone marrow mesenchymal stem cells.

RESUMEN: Matrigel es una matriz de membrana basal extraída del tumor de ratón EHS que contiene proteína de matriz extracelular. Los componentes principales son laminina, el colágeno tipo IV, nestina, sulfato de heparina, factor de crecimiento y metaloproteinasa de matriz. A temperatura ambiente, Matrigel se polimerizó para formar una matriz tridimensional. Es posible simular la estructura, la composición, las propiedades físicas y las funciones de la membrana basal celular in vivo, lo que es beneficioso para el cultivo y la diferenciación de las células in vitro, y se puede utilizar para el estudio de la morfología celular, la función bioquímica, la migración, infección y expresión génica. En este estudio, se estableció el modelo de cultivo tridimensional Matrigel de células madre mesenquimales de médula ósea (BMSC), y se observó su morfología, proliferación y supervivencia. Las BMSC fueron aisladas y cultivadas con el método de adherencia de la médula ósea completa. Se seleccionaron las BMSC de segunda generación con buenas condiciones de crecimiento y se mezclaron con Matrigel para formar complejos de gel de células. La morfología y la proliferación de las células madre mesenquimales se observaron con microscopio de contraste de fase y se tiñó con Hematoxilina-Eosina (HE); para evaluar la actividad celular se usó la tinción Live/Dead. La microscopía de contraste mostró que las BMSC se reticularon en Matrigel y proliferaron bien. Después de 7 días, se observó que el gel de matriz gradualmente se volvió blando y colapsó, y se visualizó un cruce transversal de algunas células reticulares a los 14 días. La tinción mostró que la mayoría de las células mostraron una fluorescencia verde con la prolongación del tiempo de cultivo; en los primeros 4 y 7 días, la actividad de las células madre mesenquimales de la médula ósea en Matrigel aumentó gradualmente y los porcentajes fueron de 92,57 %, 95,54 % y 97,37 %, respectivamente. El sistema de cultivo tridimensional de Matrigel puede mantener la morfología, la función y la capacidad de proliferación de las células madre mesenquimales de la médula ósea.

Effect of Matrigel on the establishment of human osteosarcoma animal model / 肿瘤研究与临床

Objective To investigate the effects of cell suspension including Matrigel, normal culture medium and phosphate buffer saline (PBS) on the xenograft model establishment of human osteosarcoma. The function of Matrigel on regulating human osteosarcoma cell differentiation and proliferation was analyzed. Methods Twenty-four BALB/c-nu/nu nude mice were randomly divided into three groups with 8 animals in every group: Matrigel and RPMI 1640 suspension (group M), RPMI 1640 culture medium (group R), PBS (group P). Human osteosarcoma cell-SaOS-2 was suspended in the three groups respectively. 1×106/ml equivalent cell counts were injected into the back of each anesthetized nude mouse (400 μl per mouse). Xenograft tumors were measured at regular intervals and the tumor volume was calculated. After 5 weeks of inoculation, the tumor parts were dissected. Paraffin-embedded sections from xenograft tumor tissues were fixed in 4 % paraformaldehyde and pathological study was made after paraffin embedding and cutting under microscope by HE stains. Results Eight nude mice formed tumor lumps at 0 day in group M and were gradually increased over time. Xenograft tumors of group R and group P disappeared in 2-4 days and some appeared again after 1 week with an increase of tumor size. After 5 weeks, the tumor volume in the group M was significantly larger than that in the group P and group R [(3 185 ± 488), (598 ± 189), (512 ± 109) mm3 respectively,F=85.7,P<0.001].After 5 weeks,tumor body was dissected.The tumor weight in the group M was significantly larger than that in the group P and group R[tumor weight:(2.22 ± 0.18),(1.48 ± 0.13),(1.47 ± 0.17) g respectively, F= 37.07, P< 0.001]. There was no difference between group R and group P in tumor volume and weight(P>0.05).Histopathological analysis showed that cells in the group M could keep original degrees of pathological differentiation in osteosarcoma cells. Besides, cells suspension of culture medium or PBS in the group P and group R were poorly differentiated. Conclusions Matrigel can promote high tumor growth rate and good uniformity of human osteosarcoma cells in experimental animals. The histological state is similar to original structure,which conforms to the occurrence and development of human osteosarcoma.

Comparison among 3 kinds of culture substrates of odontogenic induced pluripotent stem cells / 重庆医学

Objective To comparatively study the characteristics of 3 kinds of culture substrates of human odontogenic induced pluripotent stem cells(iPSCs).Methods The human odontogenic iPSCs were cultured by 3 kinds of substrates:mouse embryonic fibroblasts(MEF),matrigel and recombinant human vitronectin(VTN-N).The iPSCs growth situation was compared among three groups.Results The preparation time of these 3 kinds of substrates was 14,3,1 hlespectively,and,the difference was statistically significant (P＜0.05).The iPSCs reprogramming time was (30± 1.6),(26 ± 2.1),(27 ± 1.4) d,lespectively,wht that in the MEF group significantly higer than in other two groups (P＜0.05).The reprogramming efficiencies were 0.3 ％ ± 0.03 ％,0.56 ％ ± 0.08 ％,0.7 ％ ± 0.02 ％ respectively (P＜ 0.05).Three kinds of substrate could better support iPSCs growth and make them to maintain un-differentiation status.Conclusion with no heterologous animal components,and the adrantaga of simple pleparation,oonfrollable standard and shorter gramming time is easy to prepare,the standard is controllable and the reprogramming time is shorter,which is an ideal substrate for supporting iPSCs growth.

Hepatogenic Differentiation Capacity of Human Wharton’s Jelly Mesenchymal Stem Cell in a Co-culturing System with Endothelial Cells in Matrigel/collagen Scaffold in the Presence of Fetal Liver Extract

BACKGROUND: Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) isolated from medical waste product can be considered as an accessible source of cells in regenerative medicine. Stem cell-derived hepatocytes have poor function and need appropriate niche to reconstruct the liver structure. Therefore, we attempted to find a novel approach in differentiating HWJMSCs into functional hepatic cells using 3D culture conditions and liver extract that recapitulates vital stage in liver development. MATERIALS AND METHODS: HWJMSCs were extracted from human Wharton’s jelly, characterized by flow cytometry, and differentiated towards osteogenic and adipogenic lineages. HWJMSCs were co-cultured with HUVECs in 3D matrigel/collagen scaffolds in the presence of fetal liver extract for 14 days. The expression of specific liver genes were evaluated by lectins, PAS and immunocytochemistry. RESULTS: According to flow cytometry data, isolated cells from HWJMSCs were shown to express MSC markers. HWJMSCs co-cultured with HUVECs in matrigel/collagen scaffold with extract expressed albumin, lectins UEA and PNA. Immunohistochemistry of the cells in matrigel/collagen scaffold with or without extract exhibited a positive reaction for CK19. CONCLUSIONS: Co-culturing of the HWJMSC/HUVEC in 3D matrigel/collagen scaffold is bimimicary of in vivo cell condition. The results showed that administration of the liver extract in 3D matrigel/collagen culture of HWJMSC/HUVEC can induce hepatocyte marker expression.

Establishment of a mouse model of orthotopic Lewis lung cancer / 中国实验动物学报

Objective To establish a mouse model of orthotopic Lewis lung carcinoma using Matrigel, to evaluate the tumor growth and metastasis, and to provide a more stable mouse model of orthotopic lung cancer, which is more similar to human lung cancer.Methods Logarithmic phase of cultured Lewis lung cancer cells were suspended in Matrigel, vac-cinated into the left lung of inbred C57BL/6 mice.Five mice were killed on the 4th, 7th, 10th, 13th, and 16th days, re-spectively, and to observe the median survival, tumor formation rate, tumor growth, and metastasis.Pathological changes of the mouse lung, liver, kidney and spleen were examined.Results In 5 mice killed on the 7th postoperative day, small tumor nodules were observed on the lung in three mice and no tumor was visible by gross inspection in the other two mice, but small tumor nodules were observed under the microscope.For all the mice killed on the 10th postoperative day, tumors were visible to the naked eye on the lung of all the five mice.On the 13th day, orthotopic tumor was observed on the lung with bloody pleural effusion and pleural metastasis in all the five mice.On the 25th day, in addition to the pleural metasta-sis, one mouse had pericardial metastasis and renal metastasis.The survival periods of the 5 mice were 17 d, 20 d, 22 d, 22 d, and 25 d, respectively, with a median survival period of 21.2 d (17-25 d), and the tumor formation rate was 100%.Conclusions Mouse models of orthotopic Lewis lung carcinoma is successfully established using injection of tumor cells suspended in Matrigel.This model is more similar to the growth of human lung cancer, with good stability, high tumor formation rate and characteristics of distant metastasis, therefore, is worthy of further application.

Effects of mesenchymal stem cells treated with BMP-2 and VEGF on regeneration of large bone defects / 동물의과학연구지

This study evaluated the possibility of clinical application using matrigel-based bioceramic/polymer scaffolds treated with bone morphogenetic protein, angiogenic factor, and mesenchymal stem cells (MSCs) for new bone formation. In the in vitro study, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) containing matrigel, which is a basement membrane gel, was injected into HA/PCL scaffolds to estimate the release rates of growth factors. In the in vivo study, BMP-2, VEGF, and MSCs with matrigel-based scaffolds were implanted into rat femoral segmental defects, and new bone formation was evaluated at 4 and 8 weeks. In the results, the release rates of BMP-2 and VEGF explosively increased by day 5. For the in vivo study results, radiological evaluation revealed that the matrigel-based HA/PCL scaffolds with BMP-2 and VEGF grafted (M+B+V) and matrigel-based HA/PCL scaffolds with BMP-2, VEGF, and MSC grafted (MSC) groups showed increased bone volume and bone mineral density. Moreover, in the histological evaluation, large new bone formation was observed in the M+B+V group, and high cellularity in the scaffold was observed in the MSC group. In conclusion, grafted matrigel-based HA/PCL scaffolds with BMP-2, angiogenic factor, and MSCs increased new bone formation, and in clinical cases, it may be effective and useful to enhance healing of delayed fractures.

The effect of angiogenic factors with HA/PCL scaffold containing matrigel in a rat calvarial defect model / 동물의과학연구지

The current study was conducted in order to investigate bone formation using matrigel and angiogenic factors with HA and poly epsilon-caprolactone (HA/PCL) in a rat calvarial defect model. Calvarial defect formation was surgically created in Sprague Dawley rats (n=36). Rats in the control group (CD group, n=6) did not receive a graft. The HA/PCL scaffold was grafted with matrigel (M-HA/PCL group, n=6) or without matrigel (HA/PCL group, n=6); and 100 ng of vascular endothelial growth factor with HA/PCL scaffold containing matrigel (VEGF100 group, n=6), 100 ng (PDGF100 group, n=6) and 300 ng (PDGF300 group, n=6) of PDGF with HA/PCL scaffold containing matrigel were grafted in calvarial defects, respectively. Four weeks after surgery, bone formation was evaluated with micro computed tomography (micro CT) scanning, and histologically. According to the results, bone mineral density was significantly increased in the VEGF100, PDGF100, and PDGF300 groups compared to the HA/PCL group, in which angiogenic factors were not applied. In histological evaluation, more new bone formation around scaffolds was observed in the PDGF100 and the PDGF300 groups, compared with the VEGF100 group. Thus, the results indicate that HA/PCL containing matrigel with VEGF and PDGF is an effective grafting material for enhancement of bone formation in critical-sized bone defects. Especially, due to its price and capacity for bone formation, PDGF may be more effective than VEGF.

Isolation and Characterization of Smooth Muscle Cells from Rat Corpus Cavernosum Tissue for the Study of Erectile Dysfunction

PURPOSE: Primary culture of the cavernous smooth muscle cells from corpus cavernous tissues is known to be difficult, mainly because of contamination with fibroblasts. We applied a new method for better isolation of rat penile smooth muscle cells (RPSMCs) from rat corpus cavernosum tissue for reliable ex vivo research on erectile dysfunction. MATERIALS AND METHODS: With the use of 8-week-old adult male Sprague-Dawley rats, ex vivo migrations of rat cavernous tissue were measured by penis and aortic ring assay by use of a Matrigel-based D-valine-modified culture method. The expression of alpha-smooth muscle actin (alpha-SMA) and platelet/endothelial cell adhesion molecule (PECAM)-1 in the RPSMCs was determined by standard immunofluorescent staining and immunoblotting. The expression patterns of phosphodiesterase (PDE) family mRNA in RPSMCs were compared with patterns in rat aortic smooth muscle cells (RASMCs) by use of quantitative real-time reverse transcription polymerase chain reaction. RESULTS: Immunocytochemical staining showed greater alpha-SMA-positive and PCAM-1-negative fluorescence. Moreover, whereas the expression of alpha-SMA was detected in the RPSMCs, that of PECAM-1 was not. The levels of PDE1A, PDE1B, PDE1C, PDE2A, PDE3A, PDE4A, PDE4B, PDE4C, PDE4D, and PDE5A mRNA in the RPSMCs were about 3.2-, 4.4-, 3.4-, 29.0-, 3.5-, 2.8-, 2.9-, 6.1-, 45.0-, and 6.0-fold the corresponding expression in RASMCs. CONCLUSIONS: We developed a two-stage tissue culture method utilizing a Matrigel-based sprouting culture system to facilitate stromal cell sprouting and an adherent culture system using D-valine to eliminate the contamination of fibroblasts into the smooth muscle cells.

Effects of Netrin-1 on angiogenesis in vitro and in vivo / 中国综合临床

Objective To investigate the effects of Netrin-1 on angiogenesis in vitro and in vivo. Methods We performed in vitro rat aortic ring assay and in vivo Matrigel plug assay to determine the effect of Netrin-1 on angiogenesis. Results 10 μg/L, 50 μg/L and 100 μg/L Netrin-1 stimulated microvessel sprouting from the adventitia of aortic rings and the effect reached maximum at 50 μg/L The in vivo Matrigel plug assay showed orange color change if Nestrin-1 was positive; and CD34 immunofluorescent staining showed vascular structures in the Matrigel plug with hemachrome ( 53.4 ± 7. 3 ), which was significantly higher than control ( 5. 8 ± 0. 9 )Conclusions Netrin-1 can induce angiogenesis in vitro and in vivo.

Effect of conjunction matrigel with MFP implantation on the tumorigenesis, proliferation, apoptosis and metastasis of breast cancer cells with different expression of Her2 / 中国病理生理杂志

AIM: To detect the effect of conjunction matrigel with mammary fad pat(MFP)implantation on the tumorigenesis, proliferation, apoptosis and metastasis of Her2 positive and negative breast cancer model. METHODS: The Her2 positive BT 474 and Her2 negative MDA-MB 231 breast cancer cells were injected into MFP of nude mice with or without matrigel to establish breast cancer model. The tumor volume was measured every 3 d. Followed up for 30 d after implantation, the nude mice were killed and the tumors and associated organs were dissected for pathological sectioning and staining with hematoxylin and eosin. The time of tumor formation and the tumorigenesis were determined after implantation. The tumor volume and metastasis rate were calculated and compared with each other. The proliferation and apoptosis of Her2 positive and negative tumors were also determined. RESULTS: Matrigel and MFP implantation technology shortened the time of tumorigenesis significantly(P<0.01). The tumorigenesis rate of BT 474 and MDA-MB 231 breast cancer cells did not show any different(P>0.05). The metastasis rate of MDA-MB 231 breast cancer cells were improved from 25.0% to 37.5%(P<0.05). CONCLUSION: Matrigel and MFP implantation can be combined to shorten the time of tumor formation by two kinds of breast cancer cells, and improve the metastasis of Her2 negative MDA-MB 231 cells. Using matrigel does not show any effect of proliferation and apoptosis on Her2 positive and negative breast cancer cells.

A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA. hy926 endothelial cell line

Angiogenesis, the development of new capillary vessels, has a host of clinical manifestations. The identification of agents that increase or decrease angiogenesis is of great pharmaceutical interest. Classically, in vitro angiogenesis utilizes human umbilical vein endothelial cells (HUVEC) grown in matrigel. This valid and simple method has the drawbacks that each cell population is distinct and the constraint of obtaining primary source material. Herein we utilize the established EA.hy926 endothelial cell line as our model for in vitro angiogenesis and present a novel formula to quantify endothelial cell remodeling to identify pro- and anti-angiogenic agents. Furthermore, our technique details the procedures to identify and quantify compounds that have the capacity to generate pro- or anti-angiogenic factors when given to non-endothelial cells, which we define herein as angiogenic potential. In conclusion, we propose a novel formula that we are confident accurately reflects the degree of in vitro angiogenesis allowing the quantification of prospective angiogenic compounds.

Morphological study on the effects of Matrigel on the human lutienized granulosa cells in vitro / 대한해부학회지

This study investigated the effect of Matrigel on the differentiation and proliferation of luteinized granulosa cells in vitro. Human luteinized granulosa cells (LGC), obtained after the oocyte retrieval from the IVF process were cultured either on plastic or on Matrigel. Phase-contrast microscopy showed that LGC were flat and attached on the surface of poly-D-lysine while they formed three dimensional cell aggregates when cultured on Matrigel. Bromodeoxiuridine (BrdU) labeling followed by immunocytochemistry for BrdU and 3b-hydroxysteroid dehydrogenase (HSD) demon-strated that BrdU-labeld cells were shown in both of the 3b-HSD positive and negative granulosa cells when cells were cultured on Matrigel and on poly-D-lysine. Progesterone secretion was increased until culture day 4, and then slightly decreased. The difference in progesterone secretion between the cells cultured on Matrigel and those on poly-D-lysine was increased, as the culture day was increased. The results suggested that Matrigel provides a better culture environment for LCG and maintains proliferation of LCG.

The Effect of reconstituted basement membrane on the expression of the anterior pituitary hormones of the rat / 대한해부학회지

This study was performed to investigate the effect of Matrigel, a reconstituted basement membrane, on the expression of the anterior pituitary hormones in culture. Rat pituitary cells cultured for 6 days on Matrigel showed 3-dimensional, lobular structures with connecting cells while those on plastic showed flat, polygonal cells forming a monolayer. Western blot analysis showed that prolactin (PRL) content in the anterior pituitary cells was higher compared to those cultured on plastic. In comparison, TSH expression was not increased in cultures on Matrigel. The total cell number and the proportion of fibroblasts was decreased. These results suggested that Matrigel is a useful culture substrate for the enhanced expression of PRL but not for TSH. Further studies are needed in order to find a useful culture substrate for TSH cells.

Observation of Endothelial Cell Differentiation by 3D Cell culture system: Angiogenesis inhibition of Thrombospondin-1 / 대한해부학회지

Angiogenesis plays a fundamental role in development of circulation system, reorganization of reproductive system, wound healing. Pathological angiogenesis is deeply involved in a variety of diseases, particularly solid tumor growth and metastasis. However, it is not easy to study the mechanism of angiogenesis because endothelial cells proceed complex differentiation by interaction with extracellular matrix proteins and growth factors. However, human umbilical vein endothelial cells (HUVEC) form polygonal networks of capillary-like tubes in 3D Matrigel cultures. Differentiation of endothelial cells will be observed accurately by application of videomicroscopy. Thrombospondin-1 is secreted by a wide variety of cells including endothelial cells and is incorporated into their matrix. Thrombospondin-1 can modulate differentiation of endothelial cells by increasing cell-cell interactions as well as cell-substrate interactions. The current study was undertaken to determine which mechanism is involved in inhibition of angiogenesis by Thrombospondin-1. They was secreted from HUVEC during the process of angiogenesis in 3D Matrigel culture. When applied to endothelial cells attachment to the surface of Matrigel was not decreased, but spreading was decreased. In addition, bigger clusters was formed by enhancement of cell to cell binding by Thrombospondin-1. They inhibit cord and tube formation of HUVEC by inhibition of migration. These results suggest that Thrombospondin-1 inhibits angiogenesis by blocking differentiation of endothelial cells to motile phenotype in 3D Matrigel culture.