Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion
碩士 === 國立臺灣大學 === 毒理學研究所 === 90 === Tissue invasion is an important determinant of angiogenesis and metastasis of tumors and constitutes an attractive target for cancer therapy. Melittin, an amphiphilic peptide isolated from honeybee Apis mellifera, is known to provide strong anti-inflamm...
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ndltd-TW-090NTU012290112015-10-13T14:41:12Z http://ndltd.ncl.edu.tw/handle/60456253857479173348 Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion Melittin抑制鼷鼠黑色素瘤細胞B16F10的侵犯和轉移作用之研究 Che-Shung Lin 林哲雄 碩士 國立臺灣大學 毒理學研究所 90 Tissue invasion is an important determinant of angiogenesis and metastasis of tumors and constitutes an attractive target for cancer therapy. Melittin, an amphiphilic peptide isolated from honeybee Apis mellifera, is known to provide strong anti-inflammatory and antimicrobial effect. First, we tested the effect of melittin on the highly invasive murine melanoma B16F10 cells in vitro. Effect of melittin on membrane permeability of B16F10 cells was investigated by measuring the leakage of macromolecules and biochemical data. We found that melittin induced the release of glucose, K+, phosphorus, LDH, GOT, GPT, and increased Ca2+ influx. No significant influence of melittin on the contents of Na+, Cl-, Mg2+, amylase and creatine kinase. Functional activity assays of melittin was conducted by monitoring intracellular calcium fluxes in response to BAPTA/AM, EGTA and TG. B16F10 cells exhibit robust intracellular Ca2+ fluxes when stimulated with melittin. Pretreatment with BAPTA/AM, EGTA and TG of B16F10 cells delayed lytic time by melittin. In the high dose, melittin cause DNA fragmented, cell apoptosis and lysis. Melittin was estimated in inhibitive effect of B16F10 cell invasion. We have combined the principles of the Matrigel outgrowth and the Boyden chamber assays in a three-step screen for invasion inhibitors. Addition of melittin 0.3 or 1 μM, cells that failed to invade to Matrigel were 2.4 and 2.3 fold respectively compared with control, and cell invaded to Matrigel were 80.67 ±1.54 % and 72.67 ±1.33 % respectively compared with control. Second , we tested the effect of melittin on the B16F10 cells in vivo. We examined the effect of melittin on the inhibition of lung metastasis induced by murine melanoma cells B16F10 in mice. Various administrations (i.p.) of melittin were found effectively to inhibit the lung metastasis as seen by the reduction in the number of lung tumor nodules (14-86%). Similar circumstances were found that melittin decreased dissemination and proliferation of tumor nodules in subcutaneous and abdominal tumorigenesis assay. To study the effect of melittin on B16F10 tumor-induced angiogenesis, we implanted Matrigel with B16F10 cells to abdominal s.c. tissue of mice along the peritoneal midline. Hemoglobin content of matrigel was measured after 9 days inoculation. The results obtained showed that the mice treated with melittin 0.5 μg/g (i.p. once everyday for 5 consecutive days) decreased angiogenic potential of Matrigel. In addition, life span of metastatic tumor bearing mice treated with melittin were also found to be increased in all models of lung, subcutaneous and abdominal tumorigenesis assay. Because melittin itself possessed toxicity, so we tested hemolytic activity and acute toxicity of melittin in vivo. It reveals give dose mice were administrated melittin 0.6 μg/g effect in inhibiting the metastasis and angiogenesis but had no significant influence on body weight, erythrocyte lysis and biochemical data of serum compared with the control mice without melittin administration. In conclusion, we have demonstrated that melittin that has the potential for inhibiting tumor cell invasion and angiogenesis is attractive candidates as an anticancer agent. It merits for further study that in combination with the conventional cytotoxic chemotherapy agents, it may prove to be efficacious in controlling cancer progression. Shoei-Yn Lin-Shiau 蕭水銀 2002 學位論文 ; thesis 120 en_US |
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碩士 === 國立臺灣大學 === 毒理學研究所 === 90 === Tissue invasion is an important determinant of angiogenesis and metastasis of tumors and constitutes an attractive target for cancer therapy. Melittin, an amphiphilic peptide isolated from honeybee Apis mellifera, is known to provide strong anti-inflammatory and antimicrobial effect. First, we tested the effect of melittin on the highly invasive murine melanoma B16F10 cells in vitro. Effect of melittin on membrane permeability of B16F10 cells was investigated by measuring the leakage of macromolecules and biochemical data. We found that melittin induced the release of glucose, K+, phosphorus, LDH, GOT, GPT, and increased Ca2+ influx. No significant influence of melittin on the contents of Na+, Cl-, Mg2+, amylase and creatine kinase. Functional activity assays of melittin was conducted by monitoring intracellular calcium fluxes in response to BAPTA/AM, EGTA and TG. B16F10 cells exhibit robust intracellular Ca2+ fluxes when stimulated with melittin. Pretreatment with BAPTA/AM, EGTA and TG of B16F10 cells delayed lytic time by melittin. In the high dose, melittin cause DNA fragmented, cell apoptosis and lysis. Melittin was estimated in inhibitive effect of B16F10 cell invasion. We have combined the principles of the Matrigel outgrowth and the Boyden chamber assays in a three-step screen for invasion inhibitors. Addition of melittin 0.3 or 1 μM, cells that failed to invade to Matrigel were 2.4 and 2.3 fold respectively compared with control, and cell invaded to Matrigel were 80.67 ±1.54 % and 72.67 ±1.33 % respectively compared with control.
Second , we tested the effect of melittin on the B16F10 cells in vivo. We examined the effect of melittin on the inhibition of lung metastasis induced by murine melanoma cells B16F10 in mice. Various administrations (i.p.) of melittin were found effectively to inhibit the lung metastasis as seen by the reduction in the number of lung tumor nodules (14-86%). Similar circumstances were found that melittin decreased dissemination and proliferation of tumor nodules in subcutaneous and abdominal tumorigenesis assay. To study the effect of melittin on B16F10 tumor-induced angiogenesis, we implanted Matrigel with B16F10 cells to abdominal s.c. tissue of mice along the peritoneal midline. Hemoglobin content of matrigel was measured after 9 days inoculation. The results obtained showed that the mice treated with melittin 0.5 μg/g (i.p. once everyday for 5 consecutive days) decreased angiogenic potential of Matrigel. In addition, life span of metastatic tumor bearing mice treated with melittin were also found to be increased in all models of lung, subcutaneous and abdominal tumorigenesis assay. Because melittin itself possessed toxicity, so we tested hemolytic activity and acute toxicity of melittin in vivo. It reveals give dose mice were administrated melittin 0.6 μg/g effect in inhibiting the metastasis and angiogenesis but had no significant influence on body weight, erythrocyte lysis and biochemical data of serum compared with the control mice without melittin administration.
In conclusion, we have demonstrated that melittin that has the potential for inhibiting tumor cell invasion and angiogenesis is attractive candidates as an anticancer agent. It merits for further study that in combination with the conventional cytotoxic chemotherapy agents, it may prove to be efficacious in controlling cancer progression.
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author2 |
Shoei-Yn Lin-Shiau |
author_facet |
Shoei-Yn Lin-Shiau Che-Shung Lin 林哲雄 |
author |
Che-Shung Lin 林哲雄 |
spellingShingle |
Che-Shung Lin 林哲雄 Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion |
author_sort |
Che-Shung Lin |
title |
Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion |
title_short |
Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion |
title_full |
Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion |
title_fullStr |
Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion |
title_full_unstemmed |
Inhibitory effect of melittin of murine melanoma cell B16F10 on metastasis and invasion |
title_sort |
inhibitory effect of melittin of murine melanoma cell b16f10 on metastasis and invasion |
publishDate |
2002 |
url |
http://ndltd.ncl.edu.tw/handle/60456253857479173348 |
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