Search for Reversing Agents of Multidrug-Resistance in Tumor Cells from Marine Organisms

在我们对海洋生物活性物质的研究过程中，我们集中精力寻找肿瘤细胞多药耐药（MDR）的逆转剂。本研究采用两种耐药表皮样癌细胞系，即过表达P-糖蛋白（P-gp）的KB-C2细胞和过表达多药耐药相关蛋白（MRP）的KB-CV 60细胞进行筛选。我们发现，血松碱克服了KB-C2细胞的MDR。Araguspongines是从一种冲绳海绵Xestospongia sp.中分离得到的，其结构为2，9-二取代1-氧喹里嗪啶的大环二聚体。在10 μg/ml浓度下，三七碱D能完全抑制KB-C2细胞的多药耐药。Araguspongine D的立体异构体Araguspongines B和E也能完全逆转KB-C2细胞的MDR。由龙蒿碱E合成的5，5 ′-N-二甲基衍生物和2，2 ′-二醇均无逆转活性。我们从软珊瑚Sinularia sp.中分离到西松烷型二萜化合物，其在10 μg/ml浓度下可完全抑制KB-C2细胞的多药耐药。这种西松烷类化合物似乎是一种人工产物，它是由已知的天然西松烷通过加入n-BuOH而产生的。合成了一系列以醇取代正丁酯的西松膜衍生物，发现这些衍生物的可逆活性与醇的碳数有关。Agosterol A是从海绵中分离得到的一种新的多羟基甾醇乙酸酯，具有多药耐药逆转作用。3μM浓度的Agosterol A可完全逆转KB-C2细胞对秋水仙碱的耐药性和KB-CV 60细胞对长春新碱的耐药性。在3 μM浓度下，Agosterol A使KB-C2和KB-CV 60细胞中长春新碱的蓄积恢复至与亲本KB 3-1细胞相同的水平。

In the course of our study of bioactive substances from marine organisms, we focused on a search for reversing agents of multidrug resistance (MDR) in tumor cells. We used two kinds of MDR human epidermoid carcinoma cell lines, KB-C2 cells overexpressing P-glycoprotein (P-gp) and KB-CV60 cells overexpressing multidrug resistance associated protein (MRP) for the screening assay. We found that araguspongines conquered MDR in KB-C2 cells. Araguspongines were isolated from an Okinawan marine sponge of Xestospongia sp. and characterized as macrocyclic dimer of 2,9-disubstituted 1-oxaquinolizidine. Araguspongine D, which is a major component among araguspongines, completely conquered MDR in KB-C2 cells at 10 μg/ml. Araguspongines B and E, which were stereoisomers of araguspongine D, also completely reversed MDR in KB-C2 cells. 5,5'-N-Dimethyl derivative and 2,2'-diol synthesized from araguspongine E did not show reversing activity. We isolated cembrane-type diterpenes reversing MDR in KB-C2 cells from a soft coral of Sinularia sp. This cembranoid completely conquered MDR in KB-C2 cells at 10 μg/ml. This cembranoid is seemed to be an artifact compound, which was produced from known natural cembranolide by addition of n-BuOH. We synthesized a series of cembrane derivatives replaced the n-butyl ester with various alcohols and found that the reversing activity of those derivatives were related to the carbon number of alcohol. Agosterol A, a novel polyhydroxylated sterol acetate, was isolated from a marine sponge of Spongia sp. as a reversing agent of MDR. Agosterol A perfectly reversed resistance to colchitine in KB-C2 cells and resistance to vincristine in KB-CV60 cells at 3μM concentration. Agosterol A recovered the accumulation of vincristine in KB-C2 and KB-CV60 cells to the level equal to that of parental KB 3-1 cells at 3 μM concentration.