紫杉醇和喜树碱及其衍生物的抗侵袭转移机理研究
摘要
Taxol与CPT及其衍生物是临床上常用的抗肿瘤药物，对多种难治性实体瘤有效，如乳腺癌、卵巢癌、肺癌、结肠癌、黑色素瘤等。近年来发现这两者药物还有抑制肿瘤转移的作用。本文对其抗肿瘤侵袭转移的机理进行了比较研究，主要结果如下：
第一部分 紫杉醇(Taxol)和喜树碱(Camptothecin，CPT)及其衍生物的抗侵袭转移机理研究
一、Taxol与CPT类的抗粘附、抗侵袭、抑制Ⅳ型胶原酶分泌作用
研究表明，0.1nM、10nM Taxol对小鼠黑色素瘤B16F10细胞与FN的粘附抑制率分别为28％和42％(P＜0.05)；0.1nM、10nM Taxol对其与LN的粘附抑制率为32％和39％(P＜0.05)；0.1nM CPT对B16F10与FN的粘附率没有明显影响，10nM CPT对其与FN的粘附抑制率为37％(P＜0.05)；0.1nM CPT对B16F10与LN的粘附率没有明显影响，10nM CPT对其与LN的粘附抑制率为13％(P＜0.05)。说明Taxol和CPT都能抑制B16F10细胞与FN或LN的粘附作用，同样浓度下，Taxol的抑制粘附作用比CPT稍强。
在B16F10细胞迁移运动的实验中，对照组运动细胞百分率为48±0.17％，经0.01μM Taxol或CPT处理12h后B16F10运动细胞的百分率分别下降为22±0.17％和29±0.16％，与对照组相比有显著性差异(p＜0.05)(图7)。对照组细胞运动速率为0.29±0.05μm／min，Taxol或CPT处理12h后细胞运动速率分别为0.24±0.02μm／min、0.25±0.03μm／min，与对照组相比有显著性差别(p＜0.05)。说明Taxol和CPT都能抑制B16F10细胞的迁移运动能力。
在人纤维肉瘤HT1080细胞分泌Ⅳ型胶原酶的实验中，0.01μM、1μm Taxol作用24h后，对HT1080细胞分泌MMP9的抑制率分别为75.1％和97.6％，对MMP2的抑制率分别为9.4％和94.0％；0.01μM、1μM CPT作用24h后对MMP9的抑制率分别为73.9％和89.7％，对MMP2分泌的抑制率分别为14.7％和94.0％。说明Taxol和CPT都能抑制HT1080细胞分泌MMP2或MMP9。
在小鼠黑色素瘤高转移株B16BL6细胞的侵袭实验中，0.001μM、0.01μM、0.1μMTaxol的侵袭抑制率分别为25％、55％、77％，能够明显抑制B16BL6细胞侵袭；而0.001μM、0.01μM、0.1μM CPT对B16BL6细胞的侵袭能力没有明显抑制。
二、Taxol与CPT类的血管生成抑制作用
MTT法结果显示Taxol、Topotecan(TPT)、10-羟基喜树碱(10-hydroxycamptothecin，10-HCPT)都能明显抑制HUVEC的增殖，其IC_(50)值分别为3.4、4.5、6.5nM，而CPT-11与之相比，对HUVEC增殖的抑制作用较弱，其IC_(50)值为110nM。
Taxol, Camptothecin (CPT) and its analogues have been widely used as first-line anticancer drugs, especially for breast, ovary, lung, colon cancers or melanoms. In recent years the two drugs have been found to exhibit anti-metastasis effect. This article reports anti-metastasis mechanisms of taxol, CPT and its analogues.
Part I Metastasis inhibition of Taxol, CPT and its analogues
1. Anti-adhesion, anti-invasion, inhibitory effect of metalloproteinase secretion of Taxol and CPT
The results showed that inhibition rate of 0.1nM,10 nM Taxol on B16F10 adhesion to FN were 28 %和 42 % (P<0.05) , the inhibition rate of B16F10 adhesion to LN were 32%和 39% (P<0.05) . 0.1 nM CPT had no significant effect on B16F10 to FN, but the inhibition effect of 10nM CPT to FN and LN were 37 % and 13%) respectively. The results showed that both Taxol and CPT had the anti-adhesion effect of B16F10 to FN and LN, at the same concentrations Taxol showed higher inhibitory effect than CPT.
By testing the anti-migration effect of Taxol and CPT on B16F10 cells, the percentage of migration cells in the control group were 48 + 0.17 %, after treatment with 0.01μM Taxol or CPT for 12 h, the percentage of migration cells were reduced to 22±0.17 % and 29±0.16 % (p<0.05). The migration velocities in the control group were 0.29 ±0.05 μm/min, while after treatment with 10nM Taxol or CPT for 12 h, the migration velocities were reduced to 0.24±0.02 μm/min,0.25±0.03 μm/min (p<0.05). The results showed both Taxol and CPT had anti-migration effect of B16F10 cells.
The gelatin zymography of metalloproteinase secretion of HT1080 cells demonstrated that after 0.0lμM, 1μM Taxol treatment for 24 h, the inhibition rate of MMP9 secrection were 75.1 % and 97.6 %, inhibition rate of MMP2 secretion were 9.4 % and 94.0 % . Besides, after 0.01 μM, 1μM CPT treatment for 24 h, the inhibition effect of MMP9 secrection were 73.9 % and 89.7 %, inhibition effect of MMP2 secretion were 14.7 % and 94.0 %. The results showed that Taxol and CPT could inhibit the MMP2 and MMP9 secretion of HT1080 cells.
In the transwell chamber experiment, the anti-invasive effect of 0.001μM, 0.01μM and 0.1μM Taxol on B16BL6 cells were 25%, 55% and 77 % seperately, it showed that Taxol conld inhibit the B16BL6 invasion significantly. But 0.00lμM, 0.01μM and 0.1μM CPT had no anti-invasive effect on B16BL6 cells.
2. The anti-angiogenesis effect of Taxol, CPT and its analogues
MTT method showed that Taxol, TPT, 10-HCPT had a strong anti-proliferation effect on
第一部分 紫杉醇(Taxol)和喜树碱(Camptothecin，CPT)及其衍生物的抗侵袭转移机理研究
一、Taxol与CPT类的抗粘附、抗侵袭、抑制Ⅳ型胶原酶分泌作用
研究表明，0.1nM、10nM Taxol对小鼠黑色素瘤B16F10细胞与FN的粘附抑制率分别为28％和42％(P＜0.05)；0.1nM、10nM Taxol对其与LN的粘附抑制率为32％和39％(P＜0.05)；0.1nM CPT对B16F10与FN的粘附率没有明显影响，10nM CPT对其与FN的粘附抑制率为37％(P＜0.05)；0.1nM CPT对B16F10与LN的粘附率没有明显影响，10nM CPT对其与LN的粘附抑制率为13％(P＜0.05)。说明Taxol和CPT都能抑制B16F10细胞与FN或LN的粘附作用，同样浓度下，Taxol的抑制粘附作用比CPT稍强。
在B16F10细胞迁移运动的实验中，对照组运动细胞百分率为48±0.17％，经0.01μM Taxol或CPT处理12h后B16F10运动细胞的百分率分别下降为22±0.17％和29±0.16％，与对照组相比有显著性差异(p＜0.05)(图7)。对照组细胞运动速率为0.29±0.05μm／min，Taxol或CPT处理12h后细胞运动速率分别为0.24±0.02μm／min、0.25±0.03μm／min，与对照组相比有显著性差别(p＜0.05)。说明Taxol和CPT都能抑制B16F10细胞的迁移运动能力。
在人纤维肉瘤HT1080细胞分泌Ⅳ型胶原酶的实验中，0.01μM、1μm Taxol作用24h后，对HT1080细胞分泌MMP9的抑制率分别为75.1％和97.6％，对MMP2的抑制率分别为9.4％和94.0％；0.01μM、1μM CPT作用24h后对MMP9的抑制率分别为73.9％和89.7％，对MMP2分泌的抑制率分别为14.7％和94.0％。说明Taxol和CPT都能抑制HT1080细胞分泌MMP2或MMP9。
在小鼠黑色素瘤高转移株B16BL6细胞的侵袭实验中，0.001μM、0.01μM、0.1μMTaxol的侵袭抑制率分别为25％、55％、77％，能够明显抑制B16BL6细胞侵袭；而0.001μM、0.01μM、0.1μM CPT对B16BL6细胞的侵袭能力没有明显抑制。
二、Taxol与CPT类的血管生成抑制作用
MTT法结果显示Taxol、Topotecan(TPT)、10-羟基喜树碱(10-hydroxycamptothecin，10-HCPT)都能明显抑制HUVEC的增殖，其IC_(50)值分别为3.4、4.5、6.5nM，而CPT-11与之相比，对HUVEC增殖的抑制作用较弱，其IC_(50)值为110nM。
Taxol, Camptothecin (CPT) and its analogues have been widely used as first-line anticancer drugs, especially for breast, ovary, lung, colon cancers or melanoms. In recent years the two drugs have been found to exhibit anti-metastasis effect. This article reports anti-metastasis mechanisms of taxol, CPT and its analogues.
Part I Metastasis inhibition of Taxol, CPT and its analogues
1. Anti-adhesion, anti-invasion, inhibitory effect of metalloproteinase secretion of Taxol and CPT
The results showed that inhibition rate of 0.1nM,10 nM Taxol on B16F10 adhesion to FN were 28 %和 42 % (P<0.05) , the inhibition rate of B16F10 adhesion to LN were 32%和 39% (P<0.05) . 0.1 nM CPT had no significant effect on B16F10 to FN, but the inhibition effect of 10nM CPT to FN and LN were 37 % and 13%) respectively. The results showed that both Taxol and CPT had the anti-adhesion effect of B16F10 to FN and LN, at the same concentrations Taxol showed higher inhibitory effect than CPT.
By testing the anti-migration effect of Taxol and CPT on B16F10 cells, the percentage of migration cells in the control group were 48 + 0.17 %, after treatment with 0.01μM Taxol or CPT for 12 h, the percentage of migration cells were reduced to 22±0.17 % and 29±0.16 % (p<0.05). The migration velocities in the control group were 0.29 ±0.05 μm/min, while after treatment with 10nM Taxol or CPT for 12 h, the migration velocities were reduced to 0.24±0.02 μm/min,0.25±0.03 μm/min (p<0.05). The results showed both Taxol and CPT had anti-migration effect of B16F10 cells.
The gelatin zymography of metalloproteinase secretion of HT1080 cells demonstrated that after 0.0lμM, 1μM Taxol treatment for 24 h, the inhibition rate of MMP9 secrection were 75.1 % and 97.6 %, inhibition rate of MMP2 secretion were 9.4 % and 94.0 % . Besides, after 0.01 μM, 1μM CPT treatment for 24 h, the inhibition effect of MMP9 secrection were 73.9 % and 89.7 %, inhibition effect of MMP2 secretion were 14.7 % and 94.0 %. The results showed that Taxol and CPT could inhibit the MMP2 and MMP9 secretion of HT1080 cells.
In the transwell chamber experiment, the anti-invasive effect of 0.001μM, 0.01μM and 0.1μM Taxol on B16BL6 cells were 25%, 55% and 77 % seperately, it showed that Taxol conld inhibit the B16BL6 invasion significantly. But 0.00lμM, 0.01μM and 0.1μM CPT had no anti-invasive effect on B16BL6 cells.
2. The anti-angiogenesis effect of Taxol, CPT and its analogues
MTT method showed that Taxol, TPT, 10-HCPT had a strong anti-proliferation effect on
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