Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA 02215, USA.
BMC Cancer. 2010 Sep 13;10:487. doi: 10.1186/1471-2407-10-487.
Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37).
To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase.
The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 "factory" cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing.
The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products.
使用前体药物（如环磷酰胺[CPA]）的基于细胞色素 P450 的自杀基因疗法用于癌症，可以直接增加抗肿瘤活性，并通过旁观者杀伤机制增加。鉴于目前使用的技术难以在体内实现 100%有效的基因传递，旁观者细胞杀伤对于这种治疗策略的临床成功至关重要。先前的研究表明，泛半胱天冬酶抑制剂 p35 显著增加了稳定表达 P450 酶 CYP2B6 的肿瘤细胞中 CPA 诱导的旁观者杀伤（Schwartz 等人，（2002）癌症研究。62：6928-37）。
为了进一步开发这种方法，我们构建并表征了一种复制缺陷型腺病毒，Adeno-2B6/p35，它表达 p35 与 CYP2B6 及其电子转移伴侣 P450 还原酶结合。
Adeno-2B6/p35 感染的肿瘤细胞中 p35 的表达抑制了半胱天冬酶的激活，延迟了产生活性 CPA 代谢物的 CYP2B6“工厂”细胞的死亡，并与缺乏 p35 相比，增加了旁观者肿瘤细胞杀伤。感染 Adeno-2B6/p35 的肿瘤细胞容易被顺铂和多柔比星杀死，表明 p35 表达与获得普遍耐药性无关。最后，当使用复制能力强的腺病毒 ONYX-017 作为辅助病毒促进 Adeno-2B6/p35 的共同复制和传播并进一步增加 CPA 诱导的旁观者细胞杀伤时，p35 不会抑制病毒释放。
将 p35 引入基因治疗方案构成了增加细胞色素 P450 基因治疗旁观者杀伤的有效方法。这种策略也可用于增强其他旁观者细胞毒性疗法，包括涉及产生肿瘤细胞毒性蛋白产物的疗法。
