十七β羟脱氢酶抑制剂的乳腺癌细胞内转运及功能研究
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摘要
乳腺癌是世界发病率及死亡率最高的恶性肿瘤之一，在女性中发病率占据首位，是常见的威胁女性健康的恶性疾病。据统计，乳腺癌在我国发病率也是居于前位，发病率呈现上升并年轻化的趋势。在大多数乳腺癌患者中，体内活化雌激素水平增高是其发病及复发的主要诱因之一。体内激素分泌传统方式包括有内分泌、旁分泌及自分泌，而随着深入对局部及细胞的研究，Labrie等提出细胞内分泌概念，即局部合成的活性激素不分泌到细胞外或循环中，而是直接作用于自身细胞。其相比于传统内分泌机制，激素不会在体循环中代谢或降解，因此细胞内分泌在激素相关的肿瘤中起到直接的重要作用。这种细胞内分泌的方式不会产生分泌到血液及细胞外的激素，所以细胞内分泌的激素在代谢和生成在检测上有一定困难。因而研究催化产生雌激素的关键酶的活性和量可以起到替代的检测作用
在绝经后的妇女体内，卵巢停止分泌，活化雌激素（主要是雌二醇）主要依靠十七β-羟脱氢酶（17β-HSD）等催化生成。该还原过程中的酶主要包括十七β-羟脱氢酶一型，五型，七型及十二型，其中十七β-羟脱氢酶一型（17β-HSD1）和十七β-羟脱氢酶七型（17β-HSD7）可以同时将雌酮转化为雌二醇（E2）并将睾酮降解为雄-5-烯-3β，17β-二醇(ADIOL)。而目前研究广泛认同雌激素可以促进乳腺癌细胞增殖而雄激素可以抑制其增殖。所以我们认为17β-HSD1和17β-HSD7有可能成为乳腺癌治疗及诊断的靶点，并将在某些个体化的乳腺癌治疗中起到关键的作用。
我们的实验针对17β-HSD1和17β-HSD7两种酶的催化底物及生物学功能进行了初步研究，并且进一步研究了17β-HSD7在细胞内调节而导致细胞周期的改变的可能机制。我们发现低剂量的两种催化产物E2及ADIOL对于雌激素受体（ER）阳性的乳腺癌细胞的增殖都有促进作用，在两者合用时，其对增殖作用未表现出加和的加促进作用，并且在单独使用ADIOL时，随剂量的增加，对于细胞增殖表现为先促进后抑制。考虑是由于E2及ADIOL都可以与ER结合，因此可以促进乳腺细胞增殖；而前者有与ER较强的结合作用，而后者是一种雄激素代谢产物，也可以与雄激素受体结合，所以当两者联合应用是，E2与ER竞争性结合，而ADIOL与雄激素受体结合，表现出两者促增殖作用减弱；而单独应用ADIOL随剂量增加，雌激素受体饱和，其转而与雄激素受体结合，因而表现出先促进后抑制。对于17β-HSD7的细胞内调节而导致细胞周期改变，我们初步发现该酶可以使S期细胞增加，并且其表达量与细胞周期蛋白E1的表达呈现出正相关。
17β-HSD1是E1转化为E2的关键酶，并且E2是体内主要的活化雌激素，所以课题主要针对抑制17β-HSD1的酶活性，从而抑制乳腺癌细胞增殖以达到治疗乳腺癌的作用。抑制酶活性有两种主要方法，一种是针对该酶基因，是蛋白在转录水平被抑制；另外一种是针对蛋白质结构与功能设计其抑制剂，抑制酶的活性。两种手段都有文献报道，其中小分子干扰核糖核酸（siRNA）可以在细胞水平抑制乳腺癌细胞的增殖，可以抑制多种乳腺癌细胞系的增殖，并调节细胞增值周期。siRNA的作用确切并且专一，但是在复杂的人体内，小片段RNA没有办法在血液或者局部组织中长期稳定存在并发挥干扰作用，即使使用修饰过的RNA，也使得其成本增高并不能完全解决体内降解问题，这是siRNA成为人体用药重要障碍的原因之一。但是抑制剂是通过解析酶的结构和作用的结合位点而人工合成，在稳定性方面siRNA无法相比。随着人们对蛋白质结构与功能的了解，有多种17β-HSD1的抑制剂的实验报道，其中RB-225-50和EM-1745可以在纯化的酶中发挥高效抑制作用，但在细胞水平却没有表现出突出的抑制作用。
从这类抑制剂的结构上看，一边是类似甾体雌激素的疏水端，一边是类似腺嘌呤的亲水端，也就是该化合物为两亲性，而两亲性化合物难以穿透细胞双层磷脂膜。为解决这一问题，我们初步设计两种方案，一种是改变化合物结构，加入或改变化学集团使得两亲性质改变，另一种是使用脂质体包裹抑制剂，使脂质体成为载体。如改变抑制剂的结构，对于酶活性抑制作用可能要重新评估，实验量比较大，因此，我们选用脂质体作为载体进行进一步研究。
脂质体是人工制备的双层或多层磷脂双分子微球囊，作为药物载体具备有延长药效、改善耐受性、提高药效及降低副作用等优点，拥有广阔的应用前景。本实验预计将抑制剂包封入脂质体双层磷脂间，以达到载药的目的，并验证其对于酶活性的抑制作用。实验中我们尝试应用三嵌段共聚物（F-68）修饰脂质体改善脂质体稳定性，应用阳离子脂质体改善细胞对脂质体的摄取，以及尝试市面上出售的前脂质体，最终确定了卵磷脂、胆固醇以及十八胺为主要材料的脂质体。经过试验不同的成膜材料及比例，确定了膜材料成分比例，检测了一个月四摄氏度保存后脂质体的粒径稳定性。并用异硫氰酸荧光素标记脂质体膜成分，检测细胞对该成分脂质体的摄取情况。最终得到一种制备相对简便、可以稳定保存并且容易被多种类型乳腺癌细胞系摄取的脂质体。
本研究通过上述脂质体将抑制剂转运进入高表达17β-HSD1的细胞后，表现出与对照相比无明显差异的的抑制率，接下来的实验中证实该脂质体因为可以抑制17β-HSD1的活性而不适宜成为该类固醇代谢的酶抑制剂的载体。未来的实验中考虑使用材料成分较单一的胶束作为药物载体。但是该实验首次将纳米药物载体技术应用于17β-HSD家族的酶功能研究中，为该领域研究开辟了新方向。
Breast cancer is one of the world's highest morbidity and mortality tumor infemales. According to statistics, breast cancer is also one of the most commonmalignancies in China. The morbidity of this disease is increase and its incidencetrends to become younger in average age. The high estrogen level in the blood or localmaybe is one of the major reasons of the most patients with breast cancer and itsrecurrence. The traditional endocrine theory includes the endocrine, paracrine andexocrine ways. The discovery of cell biology and molecularbiology opens up a theoryfor studying endocrine function and offers a new concept, the intracrine, which is firstnamed by Labrie, etc. The theory here is that the activity hormone synthesis inside thecells and direct effects on their own but not secreted to the outside of the cells or theblood. Compared to traditional endocrine mechanism, the sex hormones will notmetabolism or break down in the systemic circulation in the process of intracrine andit play a key role in the hormone-dependent tumors. It is difficult to determine directlythe hormones because of the metabolic and endocrine hormone is an intracellularprocess. The activity and the amount of the key enzyme could substitute formeasurement the cell intracrine.
In postmenopausal women, the ovaries stop produce hormones, the production ofactivated sex hormone (17beta-estradiol) mainly depends on a group reduced17beta-hydroxysteroid dehydrogenases. The enzymes include17beta-hydroxysteroiddehydrogenases type1,17beta-hydroxysteroid dehydrogenases type5,17beta-hydroxysteroid dehydrogenases type7and17beta-hydroxysteroiddehydrogenases type12.17Beta-hydroxysteroid dehydrogenases type1and type7can both participate in synthetic estrogens from estrogen and degrade testosterone todihydrotestosterone. Recent research widely accepted that estrogen can promoteestrogen receptor positive breast cancer cells proliferation and androgen can have theopposite effect. So we think17beta-hydroxysteroid dehydrogenases type1and type7May become a target of treatment and diagnosis of breast cancer.
This paper studies the effect of cell proliferation by the catalytic substrates whichare the above two kinds of enzymes and focused on cell cycle regulation when17beta-hydroxysteroid dehydrogenases type7changes in breast cancer cells. We foundthat above enzyme influence the S phase of cancer cells and there is positivecorrelation between the expression of17beta-hydroxysteroid dehydrogenases type7and cyclin E1.
At present, we do more researches about17beta-hydroxysteroid dehydrogenasestype1than type7. Because17beta-hydroxysteroid dehydrogenases type1isconsidered as a key enzyme for the transformed from estrone into estradiol and ourmajor topic is aimed at the inhibitor of17beta-hydroxysteroid dehydrogenases type1.
There are two key methods to quell the enzyme activity and both methods arepublished now. One is to reduce gene expression and the transcription of protein willsuppressed. The other one is to use inhibitor which is designed by the protein structureinhibit the activity of the enzyme. Small interfering ribonucleic acid can reduce geneof17beta-hydroxysteroid dehydrogenases type1expression and inhibit proliferationof two breast cells lines in vitro. The effect of small interfering ribonucleic acid isexactly and specificity, however, the small snippets or even modification ofribonucleic acid can little stable exist in the blood or tissue of human body. Then thesynthetic inhibitor become the important way. With the knowledge development ofthe structure of17beta-hydroxysteroid dehydrogenases type1, there are many kindsof inhibitors be found. One of these kinds including RB-225-50and EM–1745, theyshowed more significantly inhibit17beta-hydroxysteroid dehydrogenases type1inthe purified protein of than in the cells.
Structurally, the inhibitor has both hydrophilic (adenine) and hydrophobic(steroid estrogen) parts is kind of amphiphilic polymer, so it hard to across cancer cellmembranes. In order to solve the above problem, we select liposome as the inhibitorcarrier to block the function of17beta-hydroxysteroid dehydrogenases type1.
Liposome is an artificial lipid microparticulate with the structure of the lipidbilayer. It has the following advantages as the inhibitor carrier to prolong drugduration, combat multi-drug resistance reduce toxicity and sustained-release in some degree. Our study is expected to encapsuled the inhibitor in liposome and deliver theinhibitor into cancer cells. Furthermore, the inhibitor block the function of theenzymes. In our research we try to use poly (ethylene oxide)-poly (propyleneoxide)-poly (ethylene oxide)（F-68）modified liposome, positively charged liposomeimprove intake liposome by cell and the pre-liposome already on the market. At lastwe determine the lecithin, cholesterol, and octadecylamine as the main materials ofliposome. This liposome with suitable particle size will have a large uptake when itlabelled with fluorescein isothiocyanate. Finally we get a kind of stable liposome withsimple way to prepare.
This study used the above liposome as a carrier of inhibitor of17β-HSD1deliver it into cells which is high level of17β-HSD1. And the rate of inhibitioncompared to the control was not significantly difference. The experiment confirmedthis kind of liposome was not suitable to be the carrier of the enzyme inhibitorsinsteroid metabolism. We may use micelle as a carrier develier the inhibitor of17β-HSD1. However，the experiment of nano drug carrier technology was used onthefunction of the family of17β-HSD for thefirst time, and it provides a newdirectionof research.
在绝经后的妇女体内，卵巢停止分泌，活化雌激素（主要是雌二醇）主要依靠十七β-羟脱氢酶（17β-HSD）等催化生成。该还原过程中的酶主要包括十七β-羟脱氢酶一型，五型，七型及十二型，其中十七β-羟脱氢酶一型（17β-HSD1）和十七β-羟脱氢酶七型（17β-HSD7）可以同时将雌酮转化为雌二醇（E2）并将睾酮降解为雄-5-烯-3β，17β-二醇(ADIOL)。而目前研究广泛认同雌激素可以促进乳腺癌细胞增殖而雄激素可以抑制其增殖。所以我们认为17β-HSD1和17β-HSD7有可能成为乳腺癌治疗及诊断的靶点，并将在某些个体化的乳腺癌治疗中起到关键的作用。
我们的实验针对17β-HSD1和17β-HSD7两种酶的催化底物及生物学功能进行了初步研究，并且进一步研究了17β-HSD7在细胞内调节而导致细胞周期的改变的可能机制。我们发现低剂量的两种催化产物E2及ADIOL对于雌激素受体（ER）阳性的乳腺癌细胞的增殖都有促进作用，在两者合用时，其对增殖作用未表现出加和的加促进作用，并且在单独使用ADIOL时，随剂量的增加，对于细胞增殖表现为先促进后抑制。考虑是由于E2及ADIOL都可以与ER结合，因此可以促进乳腺细胞增殖；而前者有与ER较强的结合作用，而后者是一种雄激素代谢产物，也可以与雄激素受体结合，所以当两者联合应用是，E2与ER竞争性结合，而ADIOL与雄激素受体结合，表现出两者促增殖作用减弱；而单独应用ADIOL随剂量增加，雌激素受体饱和，其转而与雄激素受体结合，因而表现出先促进后抑制。对于17β-HSD7的细胞内调节而导致细胞周期改变，我们初步发现该酶可以使S期细胞增加，并且其表达量与细胞周期蛋白E1的表达呈现出正相关。
17β-HSD1是E1转化为E2的关键酶，并且E2是体内主要的活化雌激素，所以课题主要针对抑制17β-HSD1的酶活性，从而抑制乳腺癌细胞增殖以达到治疗乳腺癌的作用。抑制酶活性有两种主要方法，一种是针对该酶基因，是蛋白在转录水平被抑制；另外一种是针对蛋白质结构与功能设计其抑制剂，抑制酶的活性。两种手段都有文献报道，其中小分子干扰核糖核酸（siRNA）可以在细胞水平抑制乳腺癌细胞的增殖，可以抑制多种乳腺癌细胞系的增殖，并调节细胞增值周期。siRNA的作用确切并且专一，但是在复杂的人体内，小片段RNA没有办法在血液或者局部组织中长期稳定存在并发挥干扰作用，即使使用修饰过的RNA，也使得其成本增高并不能完全解决体内降解问题，这是siRNA成为人体用药重要障碍的原因之一。但是抑制剂是通过解析酶的结构和作用的结合位点而人工合成，在稳定性方面siRNA无法相比。随着人们对蛋白质结构与功能的了解，有多种17β-HSD1的抑制剂的实验报道，其中RB-225-50和EM-1745可以在纯化的酶中发挥高效抑制作用，但在细胞水平却没有表现出突出的抑制作用。
从这类抑制剂的结构上看，一边是类似甾体雌激素的疏水端，一边是类似腺嘌呤的亲水端，也就是该化合物为两亲性，而两亲性化合物难以穿透细胞双层磷脂膜。为解决这一问题，我们初步设计两种方案，一种是改变化合物结构，加入或改变化学集团使得两亲性质改变，另一种是使用脂质体包裹抑制剂，使脂质体成为载体。如改变抑制剂的结构，对于酶活性抑制作用可能要重新评估，实验量比较大，因此，我们选用脂质体作为载体进行进一步研究。
脂质体是人工制备的双层或多层磷脂双分子微球囊，作为药物载体具备有延长药效、改善耐受性、提高药效及降低副作用等优点，拥有广阔的应用前景。本实验预计将抑制剂包封入脂质体双层磷脂间，以达到载药的目的，并验证其对于酶活性的抑制作用。实验中我们尝试应用三嵌段共聚物（F-68）修饰脂质体改善脂质体稳定性，应用阳离子脂质体改善细胞对脂质体的摄取，以及尝试市面上出售的前脂质体，最终确定了卵磷脂、胆固醇以及十八胺为主要材料的脂质体。经过试验不同的成膜材料及比例，确定了膜材料成分比例，检测了一个月四摄氏度保存后脂质体的粒径稳定性。并用异硫氰酸荧光素标记脂质体膜成分，检测细胞对该成分脂质体的摄取情况。最终得到一种制备相对简便、可以稳定保存并且容易被多种类型乳腺癌细胞系摄取的脂质体。
本研究通过上述脂质体将抑制剂转运进入高表达17β-HSD1的细胞后，表现出与对照相比无明显差异的的抑制率，接下来的实验中证实该脂质体因为可以抑制17β-HSD1的活性而不适宜成为该类固醇代谢的酶抑制剂的载体。未来的实验中考虑使用材料成分较单一的胶束作为药物载体。但是该实验首次将纳米药物载体技术应用于17β-HSD家族的酶功能研究中，为该领域研究开辟了新方向。
Breast cancer is one of the world's highest morbidity and mortality tumor infemales. According to statistics, breast cancer is also one of the most commonmalignancies in China. The morbidity of this disease is increase and its incidencetrends to become younger in average age. The high estrogen level in the blood or localmaybe is one of the major reasons of the most patients with breast cancer and itsrecurrence. The traditional endocrine theory includes the endocrine, paracrine andexocrine ways. The discovery of cell biology and molecularbiology opens up a theoryfor studying endocrine function and offers a new concept, the intracrine, which is firstnamed by Labrie, etc. The theory here is that the activity hormone synthesis inside thecells and direct effects on their own but not secreted to the outside of the cells or theblood. Compared to traditional endocrine mechanism, the sex hormones will notmetabolism or break down in the systemic circulation in the process of intracrine andit play a key role in the hormone-dependent tumors. It is difficult to determine directlythe hormones because of the metabolic and endocrine hormone is an intracellularprocess. The activity and the amount of the key enzyme could substitute formeasurement the cell intracrine.
In postmenopausal women, the ovaries stop produce hormones, the production ofactivated sex hormone (17beta-estradiol) mainly depends on a group reduced17beta-hydroxysteroid dehydrogenases. The enzymes include17beta-hydroxysteroiddehydrogenases type1,17beta-hydroxysteroid dehydrogenases type5,17beta-hydroxysteroid dehydrogenases type7and17beta-hydroxysteroiddehydrogenases type12.17Beta-hydroxysteroid dehydrogenases type1and type7can both participate in synthetic estrogens from estrogen and degrade testosterone todihydrotestosterone. Recent research widely accepted that estrogen can promoteestrogen receptor positive breast cancer cells proliferation and androgen can have theopposite effect. So we think17beta-hydroxysteroid dehydrogenases type1and type7May become a target of treatment and diagnosis of breast cancer.
This paper studies the effect of cell proliferation by the catalytic substrates whichare the above two kinds of enzymes and focused on cell cycle regulation when17beta-hydroxysteroid dehydrogenases type7changes in breast cancer cells. We foundthat above enzyme influence the S phase of cancer cells and there is positivecorrelation between the expression of17beta-hydroxysteroid dehydrogenases type7and cyclin E1.
At present, we do more researches about17beta-hydroxysteroid dehydrogenasestype1than type7. Because17beta-hydroxysteroid dehydrogenases type1isconsidered as a key enzyme for the transformed from estrone into estradiol and ourmajor topic is aimed at the inhibitor of17beta-hydroxysteroid dehydrogenases type1.
There are two key methods to quell the enzyme activity and both methods arepublished now. One is to reduce gene expression and the transcription of protein willsuppressed. The other one is to use inhibitor which is designed by the protein structureinhibit the activity of the enzyme. Small interfering ribonucleic acid can reduce geneof17beta-hydroxysteroid dehydrogenases type1expression and inhibit proliferationof two breast cells lines in vitro. The effect of small interfering ribonucleic acid isexactly and specificity, however, the small snippets or even modification ofribonucleic acid can little stable exist in the blood or tissue of human body. Then thesynthetic inhibitor become the important way. With the knowledge development ofthe structure of17beta-hydroxysteroid dehydrogenases type1, there are many kindsof inhibitors be found. One of these kinds including RB-225-50and EM–1745, theyshowed more significantly inhibit17beta-hydroxysteroid dehydrogenases type1inthe purified protein of than in the cells.
Structurally, the inhibitor has both hydrophilic (adenine) and hydrophobic(steroid estrogen) parts is kind of amphiphilic polymer, so it hard to across cancer cellmembranes. In order to solve the above problem, we select liposome as the inhibitorcarrier to block the function of17beta-hydroxysteroid dehydrogenases type1.
Liposome is an artificial lipid microparticulate with the structure of the lipidbilayer. It has the following advantages as the inhibitor carrier to prolong drugduration, combat multi-drug resistance reduce toxicity and sustained-release in some degree. Our study is expected to encapsuled the inhibitor in liposome and deliver theinhibitor into cancer cells. Furthermore, the inhibitor block the function of theenzymes. In our research we try to use poly (ethylene oxide)-poly (propyleneoxide)-poly (ethylene oxide)（F-68）modified liposome, positively charged liposomeimprove intake liposome by cell and the pre-liposome already on the market. At lastwe determine the lecithin, cholesterol, and octadecylamine as the main materials ofliposome. This liposome with suitable particle size will have a large uptake when itlabelled with fluorescein isothiocyanate. Finally we get a kind of stable liposome withsimple way to prepare.
This study used the above liposome as a carrier of inhibitor of17β-HSD1deliver it into cells which is high level of17β-HSD1. And the rate of inhibitioncompared to the control was not significantly difference. The experiment confirmedthis kind of liposome was not suitable to be the carrier of the enzyme inhibitorsinsteroid metabolism. We may use micelle as a carrier develier the inhibitor of17β-HSD1. However，the experiment of nano drug carrier technology was used onthefunction of the family of17β-HSD for thefirst time, and it provides a newdirectionof research.
引文
[1] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics.2009[J].CA Cancer J Clin,2009,59(4):225-49.
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