F10
Coagulation factor X precursor (EC 3.4.21.6) (Stuart factor) (Stuart-Prower factor) [Contains: Factor X light chain; Factor X heavy chain; Activated factor Xa heavy chain]
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Ultraviolet light exposure generates, in human tissues, radical species, which represent the main cause of photo-aging, DNA damage and skin cancer onset. On the other hand, Mediterranean plants, being continuously subjected to high solar radiation levels, are naturally adapted to take on this type of abiotic stress, thanks to the production of antioxidant secondary metabolites. For these reasons, several plant extracts were documented to be excellent antineoplastic drugs. We investigated the potential antitumor activity of the flower extract obtained by Spartium junceum L., a Mediterranean shrub, correlating it with the plant metabolic profile. After selecting the best extraction method to obtain as more secondary metabolites as possible from S. junceum flowers, we characterized the extract metabolic content. Then, by in vitro analyses, the antioxidant profile and the antineoplastic activity on B16-F10 murine melanoma cell of our extract were investigated. Spectrophotometric assays, HPLC-DAD and GC-MS analyses provided us information about flower extract composition and antioxidant activity. MTT assay and Trypan Blue exclusion test were performed to assess the extract toxicity and the viability, after treatments, of B16-F10 cancer cells and of C2C12 murine myoblasts. In vitro experiments (i.e. cytofluorimetry, protein analysis and qPCR) allowed us to analyze the effect of the plant extract on B16-F10 cell redox state, melanogenesis and cell cycle. Senescence induction was investigated by using a specific kit. We observed that the hydroalcoholic extract of S. junceum flowers (HFE) strongly inhibited B16-F10 murine melanoma cell proliferation, while just a feeble effect was observed on C2C12 murine myoblasts. Moreover, we found that HFE exerted a pro-oxidant activity on melanoma cells, inhibited melanogenesis and caused cell cycle arrest in G2/M phase, inducing senescence. These anti-cancer properties of HFE could be related to the rich metabolic profile of the extract that we characterized by HPLC-DAD and GC-MS analyses. This evidence suggests that S. junceum phytocomplex can be used as a selective, nontoxic, economic and easily available anticancer drug.
MeSH Terms
- Animals
- Antioxidants
- Cell Line, Tumor
- Cell Proliferation
- Cellular Senescence
- Flowers
- Melanoma, Experimental
- Mice
- Plant Extracts
- Secondary Metabolism
- Spartium
Keywords
- Antioxidant
- Cancer
- Proliferation
- Secondary metabolite
- Senescence
- Spanish broom
The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9 /- mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9 /- mice developed spontaneous corneal neovascularization. beta-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9 /- mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1.
MeSH Terms
- ADAM Proteins
- ADAMTS9 Protein
- Aging
- Animals
- Biocatalysis
- Cell Movement
- Corneal Neovascularization
- Embryo, Mammalian
- Endothelial Cells
- Enzyme Activation
- Gene Knockdown Techniques
- Humans
- Mice
- Mice, Inbred C57BL
- Microvessels
- Neoplasm Transplantation
- Neoplasms
- Neovascularization, Pathologic
- Organ Specificity
- Phosphorylation
- RNA, Messenger
- RNA, Small Interfering
- Receptors, Vascular Endothelial Growth Factor
- Thrombospondin 1
- Thrombospondins
- Vascular Endothelial Growth Factor A
Platycodon grandiflorum is a traditional oriental herbal medicine that is known for its immunostimulatory and anti-tumor effects. This study examined the anti-metastatic activities of an aqueous extract from the root of P. grandiflorum (Changkil: CK) using in vitro and in vivo metastasis assays. CK inhibited the invasion of B16-F10 melanoma cells through a reconstituted basement membrane (Matrigel)-coated filter, and strongly inhibited the adhesion of B16-F10 melanoma cell to extracellular matrices such as Matrigel, fibronectin and laminin substrates. CK also inhibited an experimentally induced lung cancer and prolonged the survival time in vivo. In addition, CK augmented NK cell activity. These results show that CK can reduce the extent of a lung metastasis of B16-F10 melanoma cells by inhibiting the adhesion of tumor cells to the basement membrane possibly and activating NK cells.
MeSH Terms
- Animals
- Antineoplastic Agents
- Cell Adhesion
- Cell Line, Tumor
- Cell Survival
- Dose-Response Relationship, Drug
- Drug Screening Assays, Antitumor
- Killer Cells, Natural
- Longevity
- Lung Neoplasms
- Lymphocyte Activation
- Male
- Melanoma, Experimental
- Mice
- Neoplasm Invasiveness
- Neoplasm Metastasis
- Plant Extracts
- Platycodon
Despite the overall successful application of the tet-system to regulate gene expression in vitro and in vivo, nothing is known so far about the long-term stability of this system in transgenic mice. In this study, mice of generation F2, F3, F4, or F10 of two independent tTA(CMV) transgenic lines were bred with NZL-2 mice containing a tTA-responsive bidirectional promoter that allows the simultaneous expression of two reporter genes encoding luciferase and beta-galactosidase. Analysis of the expression of transgenes in double transgenic mice revealed a dramatic reduction of tTA transactivator mRNA over time. As a consequence, the expression of both reporter genes was gradually reduced from generation to generation in tissues of embryonic and adult NZL-2/tTA(CMV) mice. Luciferase activity in NZL-2/tTA(CMV)(F10) mice was reduced 8-10-fold compared to NZL-2/ tTA(CMV)(F2) mice, and beta-galactosidase expression was no longer detectable. In summary, we describe the long-term instability of the tet-system in our NZL-2/tTA(CMV) double transgenic mice. The molecular basis of this observation and experimental tools to overcome this limitation need to be addressed in future.
MeSH Terms
- Aging
- Animals
- Blotting, Southern
- Crosses, Genetic
- Embryo, Mammalian
- Female
- Gene Expression Profiling
- Gene Expression Regulation
- Genes, Reporter
- Luciferases
- Male
- Mice
- Mice, Transgenic
- Promoter Regions, Genetic
- RNA, Messenger
- Tetracycline
- Time Factors
- Transgenes
- beta-Galactosidase
CD44, the transmembrane adhesion molecule, is expressed in the fetal brain and supposed to mediate neuroglial, interactions. We evaluated the expression and distribution of CD44 in the developing human retina. Four developing human eyes were evaluated at 6, 10, 16, and 21 weeks of gestation, as well as the eyes of one infant and four adults. Frozen sections were immunohistochemically stained with monoclonal antibodies to three human CD44 clones (BU52, F10-44-2, and DF1485) and to vimentin, and antiserum to glial fibrillary acidic protein (GFAP). Specimens were evaluated by light and electron microscopy. Positive immunostaining for CD44 was first detected at 21 weeks of gestation in the longitudinal fibers that extended from the inner to the outer limiting membrane and around capillary vessels with the simultaneous expression of vimentin and GFAP. Immunoelectron microscopy demonstrated the presence of CD44 on the surface of Müller cells and astrocytes. CD44 was faintly seen in the Müller cells in the periphery and definitely present in the astrocytes in the infant and adult retinas. CD44 was expressed in Müller cells at a late stage of fetal development and in the fetal, infant, and adult astrocytes, which suggests that it is important in the morphogenesis and homeostasis of the neural retina.
MeSH Terms
- Adult
- Aged
- Aging
- Antibodies, Monoclonal
- Child, Preschool
- Embryonic and Fetal Development
- Gestational Age
- Humans
- Hyaluronan Receptors
- Immunohistochemistry
- Infant
- Microscopy, Immunoelectron
- Middle Aged
- Retina
Cancer incidence increases with age, but the growth and spread of tumors is often slow and prolonged in the elderly. Transplantable murine tumors grow and spread less readily in older mice and can be used as models to study the effect of host age. Neovascularization is crucial for the growth of solid tumors, and alterations in the host vascular response may underlie the changes in tumor growth occurring with age. We have used transplantable tumor cells and tumors, and tumor extracts, to better understand differences in the biology of tumor growth and vascularization as a function of host age. Englebreth-Holm-Swarm (EHS) carcinoma and B16-F10 melanoma cells were injected into C57BL mice of different ages. Tumor growth, histology, and cellular DNA synthesis were compared. Vascularization was determined using basic fibroblast growth factor and an in vivo angiogenesis assay. EHS tumor extracts were assayed for biologic activity in vitro using human endothelial cells and in vivo using mouse EHS-BAM carcinoma and human TSU-Pr1 prostate carcinoma cells. EHS tumors formed larger tumors in young than in old C57BL mice. Rapid tumor growth resumed upon transfer of tumor tissue from old animals into young animals. The rate of DNA synthesis of tumor tissue from old animals in organ culture was lower than in tissue from young animals. Histologically, tumors grown in old animals exhibited a threefold higher ratio of extracellular matrix to tumor cells than those grown in young animals. Tumors from adult animals exhibited numerous small blood vessels; those from old animals contained fewer, much larger vessels. Similar results were observed in young mice fed a reduced-calorie diet. Young animals elicited a greater and more rapid angiogenic response than old animals. Extracts of tumors grown in old animals failed to support endothelial cell differentiation in culture. Tumor cells injected together with such old extracts showed reduced tumor growth in nude mice. The rate of growth and morphology of the EHS tumor were altered with age, partly due to a reduced capacity to vascularize the tumors because of a lack of angiogenic factors or the presence of host inhibitors. Alterations in host factors detected in this tumor model may underlie a variety of age-dependent changes that could influence tumor growth and the repair and regeneration of normal tissue. Reducing the vascularization of tumors represents a potential target to reduce their growth and progression.
MeSH Terms
- Aging
- Animals
- Carcinoma
- Energy Intake
- Melanoma, Experimental
- Mice
- Mice, Inbred BALB C
- Mice, Inbred C57BL
- Neovascularization, Pathologic
- Tumor Cells, Cultured
The growth characteristics and colonization potential of a transplantable melanoma administered to young (3 mo) and old (24 mo) C57BL/6 mice were investigated. After sc injection of B16-F10 melanoma cells, tumor growth was slower, and final tumor volume was less in the older mice. Furthermore, after iv injection of B16-F1 melanoma cells, the number of pulmonary colonies was also less, and the survival was greater in the older mice. These findings indicate an age advantage in this experimental tumor model that may be attributed to either physical or immunologic factors.
MeSH Terms
- Aging
- Animals
- Male
- Melanoma
- Mice
- Mice, Inbred C57BL
- Neoplasm Transplantation
An attempt has been made to determine (a) whether aging plays an important role in resistance against metastasis and (b) whether dithiothreitol, an effective in vitro mitogenic potentiator of splenic cells of young and old mice, can modulate the occurrence of pulmonary metastasis. B16-F10 melanoma cells were injected into the outer ear of young and old female C57BL/6 mice; and the growth of the primary tumor, the palpable size of the cervical lymph node, and the number of lung metastases were then determined at various intervals. The ear was amputated when the primary tumor reached 4 mm in mean diameter. The following results were obtained. (a) The growth rate of the primary tumor in young mice is comparable to that in old mice. (b) Enlargement of the cervical lymph node occurs earlier in old than in young mice. (c) Old mice are more vulnerable to pulmonary metastases, but small metastasized pulmonary colonies are more prominent in old than in young mice. (d) Dithiothreitol (100 micrograms) injected every 2 days after the inoculation of tumor cells is effective in reducing the incidence of pulmonary metastases in old mice.
MeSH Terms
- Aging
- Animals
- Antibody Formation
- Cell Division
- Cell Line
- Cytotoxicity, Immunologic
- Female
- Immunity, Cellular
- Immunity, Innate
- Kinetics
- Lung Neoplasms
- Melanoma
- Mice
- Mice, Inbred C57BL
- Neoplasm Metastasis
Descendants of rats possessing lower responsiveness to the removal of the parathyroid gland [4] were examined for the aging process of the hypothalamic-pituitary-ovarian axis. The first generation rats of these descendants were born to mothers parathyroidectomized (Px) on the fifth day of gestation and subsequent generation rats were developed by brother-sister mating without any special treatment. More than 50% of the eighth to tenth generation (F8-F10) offsprings of the Px-rats showed regular 4-day estrous cycles at 15-16 months of age, while nearly 80% of normal F8-F10 rats developed persistent estrus at 13-14 months of age. In 14-15 month-old Px-offspring rats the LH and FSH surges occurred at 1630-1730 h of proestrus to a similar extent as those shown in 3-4 month-old normal rats. The release of LH and FSH following a single injection of luteinizing hormone-releasing hormone (LHRH) in 13 month-old Px-offspring rats was nearly normal, reaching a maximal level at 15 min as in young adult rats. In 13 month-old normal rats, serum LH measured after an injection of LHRH increased progressively until 60 min. The ovaries of the Px-offspring rats were heavier than those of age-matched normal rats and included well-developed corpora lutea and follicles in several sizes. The results suggest a delay in the aging process of the hypothalamic-pituitary-ovarian axis of the Px-offspring rats.
MeSH Terms
- Aging
- Animals
- Calcium
- Estradiol
- Estrus
- Female
- Follicle Stimulating Hormone
- Gonadotropin-Releasing Hormone
- Hypothalamo-Hypophyseal System
- Luteinizing Hormone
- Ovary
- Parathyroid Glands
- Pregnancy
- Progesterone
- Rats
An attempt has been made to determine whether changes in the host environment due to ageing or sex differences can influence the metastatic mode of malignant tumours. B16-F10 melanoma cells with a high pulmonary metastasizing potential were injected into the outer ear of C57BL/6 mice; the growth of primary tumour and metastasis of the cervical node were assessed every two days, and the number of lung compared; and in the second young male and female mice were used. The rate of growth of primary tumours was found to be comparable between young and ole mice. Metastasis in the cervical lymph node occurred earlier in old than in young mice. Lung metastases occurred earlier in old than in young mice, but their growth was slower in old than in young mice. Metastasis in the cervical node occurred earlier in males than in females. More lung metastases were found in male than in female mice.
MeSH Terms
- Aging
- Animals
- Female
- Lung Neoplasms
- Lymph Nodes
- Lymphatic Metastasis
- Male
- Melanoma
- Mice
- Mice, Inbred C57BL
- Neoplasm Metastasis
- Sex Factors