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Objective To construct the eukaryotic expression vector of pyruvate kinase M1 (PKM1) gene labeled with pXJ-40-myc and detect its biological activity in ocular B16 melanoma cells.Methods Ocular B16 melanoma ceils were randomly divided into experimental and control group,and the experimental group was transfected with pXJ-40-myc-PKM1 plasmid and the control group was transfected with pXJ-40-myc plasmid.Then PKM1 gene was amplified by PCR with human liver cDNA library as the template.The recombinant plasmid pXJ-40-myc-PKM1 was identified by bacteria PCR and double enzyme digestion,followed by transfection of pXJ40-myc-PKM1 and pXJ-40-myc plasmid into B16 melanoma cells,and finally,the expression of PKM1 protein was verified by the Western blot,while wound healing assay was used to detect the effects of PKM1 on the migration of ocular melanoma ceils.Results The length of PKM1 gene was 1800bp,which was consistent with the expected size.Compared with the control group,the result of bacteria PCR was positive.The length of double enzyme digestion was 4000 bp and 1800 bp respectively.Western blot results showed that recombinant plasmld pXJ-40-myc-PKM1 was successfully expressed in ocular B16 melanoma cells.Compared with the control group,wound healing assay showed that recombinant plasmid could inhibit the migration of ocular B16 melanoma cells.Conclusion The eukaryotic expression vector of pXJ-40-myc-PKM1 is successfully constructed,which can suppress the migration of ocular B16 melanoma cells.
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Objective To construct the eukaryotic expression vector of human BRE1B labeled with pCMV-Tag-2B and detect its biological activity in melanoma cells preliminarily.Methods Ocular B16 melanoma cells were randomly divided into the experimental group,in which the cells were transfected with pCMV-Tag-2B-BRE1B and the control group,which was transfected with pCMV plasmid.The CDS coding region of human BRE1B gene was amplified by PCR using human mammary gland cDNA as a template for construction of the recombinant plasmid pCMV-Tag-2B-BRE1B.After transfected with pCMV-Tag-2B-BRE1B and pCMV plasmid in the experimental and control group,respectively,Western blot was applied to detect the expression of BRE1B protein,while cell counting kit-8 (CCK8) and colony assays were used to analyze the effects of recombinant plasmid pCMV-Tag-2B-BRE1B on the growth of B16 melanoma cells.Results The CDS coding sequence of human BRE1B gene was amplified by PCR successfully,which was equal to the expected size.Compared with the control group,the sequence from bacteria PCR was identified as positive,with the length of 4000 bp and 3050 bp by double enzyme digestion respectively.Moreover,the coding sequence of the human BRE1B gene was exactly the same as the inserted DNA sequence.Western blot results showed that the expression of recombinant plasmid pCMV-Tag-2B-BRE1B was successfully expressed in the experimental group,but there was no specific fragments in the control group.And cell counting kit-8 (CCK8) and colony assays showed that pCMV-Tag-2B-BRE1B recombinant plasmid could inhibit the growth of B16 melanoma cells.Conclusion The eukaryotic expression vector of pCMV-Tag-2B-BRE1B labeled with pCMV-Tag-2B is constructed successfully,and it has inhibitory effects on the growth of ocular B16 melanoma cells.
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In continuing to search new materials for skin whitening agent from natural resources, we have focused on plants used by Dayak tribe (native people in East Kalimantan, Indonesia) for skin care treatment. The ethanol extract of the leaves of Sonneratia caseolaris showed anti-melanogenesis activity in melanin biosynthesis assays using B16 melanoma cells. By activity-guided fractionation, luteolin-7-O--glucoside (compound 1) was isolated as an active compound. In melanin formation inhibition assay, luteolin-7-O--glucoside showed the inhibitory activity in a dose-dependent manner and reach 44% of inhibition at 223.2 μM with 92% of cells viability. The compound also showed potent anti-oxidative stress ability with a significant intracellular H2O2-scavenging activity in HaCaT cells. These results showed a validation of traditional use for skin care treatment by Dayak tribe in East Kalimantan.
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2-Hydroxytyrosol (2-HT), originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC50 value of 13.0 µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC50, 32.5 µmol/L) in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH)-stimulated melanin formation in intact B16 melanoma cells.
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Objective To investigate the antitumor effect of 17aα-D-homo-ethynylestradiol-3-acetae on the mice transplanted with melanoma (B16) tumor cells,and to explore the possible synergistic effect with irradiation.Methods IRM-2 mice transplanted with B16 cells were randomly classified into control group,irradiation group,17aα-D-homo-ethynylestradiol-3-acetae drug ( high dose,medium dose,low dose) groups,and drug and irradiation combination group.Mice in drug group and the combination group were intraperitoneally injected with 5,7.5,and 10 mg/kg drug for 7 days.Mice in the irradiation and combination group received 1 Gy total body irradiation at 4 d after drug injection and then once a day for 5 days.The tumor inhibition efficiency,the number of bone marrow cells,thymus indices,and spleen indices were evaluated.Results Tumor weights in each drug group were significantly lower than those of the control( t =4.58,9.07,6.67,P ＜ 0.05 ).Drug combinated with 137Csγ-rays enhanced the antitumor effect so that the tumor weights in the combination group were significantly decreased ( t =8.06,10.35,6.71,P ＜0.05 ) in comparison with the control groups.Moreover,the numbers of marrow nucleated cells,thymus index and spleen index in the drug group were higher than those in the control group ( t =2.64,3.80,2.84,P ＜ 0.05 ).Conclusions 17aα-D-homo-ethynylestrudiol-3-acetae can inhibit cell growth of B16 melanoma in mice and may also have radioprotective effect on the hematopoietic system and immune system of mice.
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Nuclear factor of activated T-cells (NFAT) proteins are, calcium-regulated transcription factors, key regulator of stimulation-dependent gene activation. In our microarray analysis for the genes expressed in human black and white hairs, NFAT2 was significantly upregulated in the white hair, compared to the black hair. The aim of this study was to investigate functional role of NFAT2 in melanogenesis. Western blot analysis was performed to investigate the expression of NFAT2 protein in B16 melanoma cells. Our data showed that NFAT2 expression was increased in the hypopigmented B16 cells, while tyrosinase and MITF expression was decreased. To investigate the potential role of NFAT2, the recombinant adenovirus expressing microRNA specific for NFAT2 was transduced into the cultured B16 melanoma cells. Consistently, inhibition of NFAT2 enhanced tyrosinase activity and melanin content. Moreover, cyclosporine A, which is known as a calcineurin inhibitor blocking NFAT activation, enhanced tyrosinase activity and melanin content. These data suggest that NFAT2 may play an important role in regulation of melanogenesis in melanocyte.
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Humans , Adenoviridae , Blotting, Western , Calcineurin , Cyclosporine , Down-Regulation , White People , Hair , Hydroquinones , Melanins , Melanocytes , Melanoma, Experimental , Microarray Analysis , MicroRNAs , Monophenol Monooxygenase , NFATC Transcription Factors , Proteins , T-Lymphocytes , Transcription Factors , Transcriptional ActivationABSTRACT
0.05),the survival time was prolonged(P
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We evaluated the anti-tumor activity of fermented grain extracts using a mouse tumor model. An experimental diet containing materials from fermented rice germ, wheat germ, hulled rice, soybean and seaweed (fermented materials, FM) was fed to 4-week-old female C57BL6 mice for 14 days prior to and 21 days following the subcutaneous implantation of B16 melanoma (5×10 <sup>5</sup> cells/mouse). FM retarded tumor growth and increased the duration of host survival. We further examined the anti-tumor activity of FM using the B16 metastasis model. An experimental diet containing FM was fed to C57BL6 mice for 14 days prior to and 21 days following B16 tail vein administration (5×10<sup>4</sup> cells/mouse). The decrease in observed metastasis in the lungs of mice treated with FM was also significant. In order to identify this anti-tumor activity of FM, NK-activity in the FM fed mice was evaluated. However, the values were comparable to the control mice. These results suggest that the fermented grain extracts induce anti tumor activity <i>in vivo</i>, although the mechanism of this activity is not yet clear.<br>
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Aim To study the inhibitory and apoptosis-inducing effects of aspisol on proliferation of B16 melanoma in vitro and in vivo. Methods The effect of aspisol on the proliferation of B16 cells was analyzed by MTT assay; its effect on cell apoptosis was measured by flow cytometry. The suspension of melanoma cells was injected subcutaneously into forelimb axillas of C57BL/6J mice to establish xenograft models. From the next day, the mice received intraperitoneal injection of aspisol in different concentrations once a day for 28 days; the mice received injection of dacarbazine (DTIC) were used as positive controls,and received injection of normal saline (NS) were used as negative controls. The inhibition rate of tumor growth was calculated .The apoptosis rate was detected by TUNEL assay. The expression of Survivin and C-erbB-2 was detected by immunohistochemistry. Results Aspisol inhibited the proliferation of B16 cells, with the inhibition rate up to (68.78?1.27)%, and induced the apoptosis with the highest apoptosis rate up to 15.8%.The inhibition rate of tumor growth was 15.0% in 200 mg?kg-1 aspisol group, 32.3% in 400 mg?kg-1 aspisol group, 49.4% in 800 mg?kg-1 aspisol group,and 51.4% in 40 mg?kg-1 DTIC group. Typical apoptotic morphologic changes were seen in the 4 groups. Survivin and C-erbB-2 expression was significantly lower in aspisol groups than in NS group. Conclusion Aspisol could inhibit proliferation and induce apoptosis of B16 melanoma cells in vitro and in vivo, which may be correlated to down-regulation of Survivin and C-erbB-2.
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10000 u,SP2:6 000 u0.05),the melanogenesis and tyrosinase activity were inhibited remarkably(P
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Objectives: To establish melanoma models of B16 cell line in ICR mice. Methods: Inoculating B16 Melanoma cells in ICR mice's feet, back, abdomen and caudal vein, to establish transplanted and metastatic melanoma models. We observed the morphologic characteristics and examined the pathological parameters of melanoma. We analyzed the relations between the number of B16 cells inoculated and the genesis of tumors, the influence of tumor growth on mice's behavior,and growth curve of the melanoma. Results:Six days later, sesamoid, linear and nodal even kaulifloweroid melanoma were seen gradually in ICR mice's feet, back, abdomen and lung (anatomid) after inoculating. The bearing tumor rates have a positive relation to the number of inoculated B16 cells. The rates of bearing tumor was near 100%, after inoculating with 5?10 6 B16 cells. Conclusions: The B16 melanoma cell line derived from C57/B16 mice(black ) has high rates of bearing tumor in ICR mice (white). The B16 melanoma models in ICR mice are ideal tumor models that have the characteristics of easy established,easy observing and easy obtaining.
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BACKGROUND: Plant extracts have been widely used as important therapeutic drugs for many centuries all over the world. There have been many reports that natural products have various kinds of biological activities such as anti-allergy, anti-inflammatory and antimicrobial activities. Recently, the screening for the efficacy and safety of natural products has been extensively performed. OBJECTIVE: This study was carried out to find a beneficial plant extract possessing excellent antioxidative and anti-melanogenic activities. We have found that the leaf of Robinia pseudo-acacia L. has active substances which are involved in those activities. METHODS: To confirm the antioxidative activity of the extract obtained from the leaves of Robinia pseudo-acacia L., scavenging ability of the extract on DPPH free radicals and its inhibitory effects on lipid autoxidation and peroxidation were investigated. In addition, inhibitory effects of the extract on mushroom tyrosinase as well as melanin biosynthesis in cultured B16 melanoma cells were evaluated. RESULTS: The acacia extract showed not only powerful antioxidative activity but also antimelanogenic acitivity as strong as that of arbutin which is a well known inhibitor of melanogenesis. CONCLUSION: These resulis suggest that the extract from the leaves of Robinia pseudo-acacia L. could be used as a 4ghtening and antioxidative agent for the skin.
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Acacia , Agaricales , Arbutin , Biological Products , Free Radicals , Mass Screening , Melanins , Melanoma, Experimental , Monophenol Monooxygenase , Plant Extracts , Plants , Robinia , SkinABSTRACT
Objective: Study on the growth character of B16 melanoma cell which can express angiostatin. Methods: Angiostatin gene was constructed from human plasminogen cDNA by deletion mutation. A B16 melanoma cell clone named BAG28 which stably expresses angiostatin was established by introducing gene into it. Results: BAG28 in vitro had no changes in proliferation rate and the ability of clone formation in soft agar. Study in vitro showed that the tumor weight had reduced about 87% ( P
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Interferon y (IFN-?) could enhance the metastatic potential of a variety of tumors via increasing expression of MHC class I molecules or other unidentified mechanisms. It has been demonstrated that transfection of T-cell costimulatory molecule B7 gene into tumor cells could increase tumor immunogenicity and render them susceptible to immune attack. In this study, we investigate whether transfection of B7-1 gene could antagonize IFN-?-induced tumor metastasis. Lipofectamine-mediated transfection was used to introduce murine B7-1 (mB7-1) gene or neo gene into a low-metastatic variant of B16 melanoma cell line. The transfectants and parent B16 cells were treated with 100U/ml of IFN-? for 36 h before being subjected to experimental metastasis assay. The expression of MHC class I and class II molecules on the cells was analysed by flow cytometry. Pre-treatment of parent B16 cells and mock gene-transfected B16 cells (B16-neo) with IFN-? significantly increased their lung-metastizing capacity, while the same IFN-? treatment of B16 cells transfected with mB7-1 gene (B16-B7-1) showed no increase in the number of lung metastatic nodules as compared with control cells. Almost equally elevated expression of MHC class I (H-2Kb and H-2Db) molecules was found on the surface of B16, B16-neo and B16-B7-1 cells. However, a much higher expression of class II (I-Ab) molecule on B16-B7-1 cells than that on B16 and B16-neo cells was observed. These results demonstrate that transfection of B7-1 gene into B16 melanoma could reduce its IFN-?-induced metastasis and that the elevated MHC class II expression on B7-1-expressing cells might play a role in the B7-1 -associated reduction of metastasis.
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The effect of low dose radiation on tumor metastasis in mice was investigated by using the models of B16 melanoma bloodtract pulmonary metastasis and Lewis lung carcinoma. The results were reported as follows: (1) The number of B16 melanoma pulmonary metastatic colonies of the mice pre-received different doses(25,50,75,100mGy) of whole body irradiation were lower than that of unirradiated control group mice (P
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Objective:To study the effects of different molecular weight of collagen polypeptides from Apostichopus japonicus (A1:6 000U