Ferguson P J, Fisher M H, Stephenson J, Li D H, Zhou B S, Cheng Y C
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599-7365.
Cancer Res. 1988 Nov 1;48(21):5956-64.
The alkaloid derivative 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene)-beta-D-glucopyranoside (etoposide, VP-16) is believed to exert cytotoxicity by causing double-stranded DNA breaks through interruption of the breaking-resealing reaction of topoisomerase II (topo II). Thus it was conceivable that cells could become resistant to VP-16 by a decrease in topo II enzyme level, since this would lead to fewer DNA breaks. As well, given the structure of VP-16, it was also possible that a pleiotropic mechanism of resistance could decrease sensitivity to this drug. To study these possibilities, a series of VP-16-resistant human KB cell lines was established by stepwise selection. The concentrations of VP-16 required to inhibit cell proliferation by 50% in the parent line and KB/1c, KB/7d, KB/20a, and KB/40a lines were, respectively, 0.16, 4.7, 24, 31, and 47 microM. These cell lines expressed cross-resistance to 4'-(9-acridinylamino)methanesulfon-m-anisidide, doxorubicin, vincristine, and methotrexate, although the pattern of relative drug sensitivity was quite different from that of pleiotropic resistant cell lines reported elsewhere. The resistance to vincristine and methotrexate did not increase above the level of the KB/1c cells, and resistance to VP-16, doxorubicin, and especially vincristine was unstable in VP-16-resistant cells cultured in the absence of drug. Although the drug resistance marker Mr 180,000 glycoprotein could not be detected in any of our cell lines, cellular accumulation of [3H]VP-16 was reduced 50-75% in the resistant lines compared with parent KB. With increasing VP-16 resistance, the level of topo II protein, detected by antibody staining, decreased at each step of selection, concomitant with a general decrease in topo II unknotting activity. Sensitivity of the topo II unknotting assay to inhibition by VP-16 was the same for the parent and all resistant cell lines. The level of topo I activity and enzyme increased slightly in the resistant cells. Thus, these cell lines are resistant to VP-16 by virtue of at least two mechanisms: (a) reduced levels of topo II, which confers cross-resistance to other compounds which are topo II-dependent cytotoxic agents; and (b) reduced accumulation of drug, which is likely also responsible for vincristine and methotrexate resistance. However, the possible existence of other mechanisms of resistance cannot be ruled out.
生物碱衍生物4'-去甲基表鬼臼毒素9-(4,6-O-亚乙基)-β-D-吡喃葡萄糖苷（依托泊苷，VP-16）被认为通过干扰拓扑异构酶II（拓扑II）的断裂-重新封闭反应导致双链DNA断裂，从而发挥细胞毒性作用。因此可以推测，细胞可能通过降低拓扑II酶水平而对VP-16产生耐药性，因为这会导致较少的DNA断裂。此外，鉴于VP-16的结构，也有可能存在一种多效性耐药机制会降低对该药物的敏感性。为了研究这些可能性，通过逐步筛选建立了一系列对VP-16耐药的人KB细胞系。在亲代细胞系以及KB/1c、KB/7d、KB/20a和KB/40a细胞系中，抑制细胞增殖50%所需的VP-16浓度分别为0.16、4.7、24、31和47微摩尔。这些细胞系对4'-(9-吖啶基氨基)甲磺酰间茴香胺、阿霉素、长春新碱和甲氨蝶呤表现出交叉耐药性，尽管相对药物敏感性模式与其他地方报道的多效性耐药细胞系有很大不同。对长春新碱和甲氨蝶呤的耐药性未超过KB/1c细胞的水平，并且在无药物培养的VP-16耐药细胞中，对VP-16、阿霉素，尤其是长春新碱的耐药性不稳定。尽管在我们的任何细胞系中都未检测到耐药标记物180,000道尔顿糖蛋白，但与亲代KB细胞相比，耐药细胞系中[3H]VP-16的细胞内蓄积减少了50 - 75%。随着对VP-16耐药性的增加，通过抗体染色检测到的拓扑II蛋白水平在每次筛选步骤中均下降，同时拓扑II解结活性总体降低。拓扑II解结试验对VP-16抑制的敏感性在亲代细胞系和所有耐药细胞系中相同。耐药细胞中拓扑I活性和酶水平略有增加。因此，这些细胞系对VP-16产生耐药性至少有两种机制：(a) 拓扑II水平降低，这赋予了对其他依赖拓扑II的细胞毒性化合物的交叉耐药性；(b) 药物蓄积减少，这可能也是长春新碱和甲氨蝶呤耐药的原因。然而，不能排除其他耐药机制的可能存在。
