Combined-modality therapy with chemotherapy and radiation therapy has gained increasing importance in the treatment of human solid tumors.[1,2] In addition to controlling potential or overt metastatic disease, a number of chemotherapeutic drugs also enhance the cytotoxic effect of ionizing radiation [3,4] and help locally control the primary disease. However, the efficacy and use of currently available chemoradiation regimens are limited by their toxicities to normal tissue. The development of more effective chemoradiation regimens using radiosensitizers, which can preferentially enhance the cytotoxicity of ionizing radiation in cancer cells, remains a major challenge in radiation oncology.
DNA topoisomerase I is a novel cellular target of a number of antineoplastic compounds, including the camptothecin derivatives[6-9] and the newly identified DNA minor groove-binding ligands (MGBLs) such as Hoechst 33342.[10,11] Drug interference with topoisomerase I-mediated cleavage rejoining of DNA strands is thought to be the common mechanism of action of these drugs.[6-11] Instead of direct inhibition of catalytic activity of topoisomerase I, topoisomerase I drugs produce their cytotoxic effects by converting an essential DNA topology-modifying activity into a DNA-breaking poison, which damages DNA through interactions with cellular processes such as DNA replication.[5,12-15] The presence of up-regulated levels of topoisomerase I in tumor cells compared to normal cells suggests a therapeutic advantage of topoisomerase I-targeting drugs selective against slow-growing as well as rapidly proliferating tumors.[16-18] Recent advances in the molecular mechanisms of cytotoxic action and radiosensitization of DNA topoisomerase I-targeting drugs offer a unique opportunity for developing more effective chemoradiation therapy against human cancers.
Only one type-I human DNA topoisomerase has been identified as a molecular target of anticancer drugs. The human topoisomerase I, encoded by a single-copy TOP1 gene on chromosome 20, is a monomeric 100 kDa protein. It relaxes both positively and negatively supercoiled DNA and requires no energy cofactor for its activity.[19-21] The activities of topoisomerase I are important for many aspects of DNA metabolism, including the initiation and elongation of RNA transcription, DNA replication, and the regulation of DNA supercoiling, which is essential for maintaining the stability of the genome.[19-21]
During a typical catalytic cycle, topoisomerase I cleaves the DNA backbone, allowing the passage of DNA strands, and then reseals the DNA backbone in two successive transesterification reactions. As illustrated in Figure 1, a key covalent topoisomerase I-DNA intermediate is formed between the tyrosine-723 residue of the enzyme and a 3'-phosphate at the break site during the transient DNA cleavage stage. The 5'-end of the broken DNA appears to be held by noncovalent interactions within the enzyme.[22,23] All the known topoisomerase I-targeting drugs damage DNA by trapping this key covalent reaction intermediate, called the topoisomerase I cleavable complex.
Camptothecin and its derivatives (Figure 2) are the best characterized human topoisomerase I-targeting drugs. Camptothecin was originally isolated from the tree Camptotheca acuminata. Its broad spectrum antitumor activity in animal models, especially against colon tumors, led to clinical trials in the early 1970s. However, trials were terminated due to observations of excessive toxicity with the ring-open form of camptothecin (camptothecin, sodium salt, NSC-100880). It is now clear that the ring-open form of camptothecin is inactive against its molecular target, topoisomerase I.
Among camptothecin derivatives, topotecan(Drug information on topotecan) is positively charged and irinotecan(Drug information on irinotecan) (CPT-11[Camptosar]) is a prodrug that generates its active metabolite SN-38 intracellularly via carboxyl esteration. Both topotecan and irinotecan demonstrated efficacy in clinical trials,[24,25] and in 1997 were approved by the US Food and Drug Administration (FDA) for the treatment of recurrent colon cancer and ovarian cancer, respectively. S-phase-specific cytotoxicity,[12,13,15] selective cytotoxicity against tumorigenic over nontumorigenic cells,[26,27] and the ability to overcome MDR1-mediated drug resistance,[28,29] are features of camptothecin derivatives that may contribute to their excellent anticancer activity.
Our current understanding of the cytotoxic mechanism of camptothecin is demonstrated by the fork collision model (Figure 3), which was proposed based on studies both in cultured cells and in cell-free extracts. Upon binding of topoisomerase I to DNA, two different reaction intermediates, the noncleavable complex and the cleavable complex, are formed. In a relaxation reaction in the absence of drugs, the cleavable complex and the noncleavable complex are at equilibrium.
By inhibiting the rejoining step, topoisomerase I drugs perturb this equilibrium by trapping a reversible topoisomerase I-camptothecin-DNA ternary reaction intermediate, the topoisomerase I cleavable complex. The perturbed equilibrium can be rapidly reversed by removing drug molecules from the medium. Studies using cell synchronization techniques and specific inhibitors of DNA polymerases indicate the involvement of active DNA synthesis in the induction of the highly S-phase-specific camptothecin cytotoxicity.[30,31] It is currently hypothesized that the collision between the replication machinery and the drug-trapped topoisomerase I cleavable complex leads to eventual G2-phase cell-cycle arrest and cell death. A similar cytotoxic mechanism has been proposed for some newly identified topoisomerase I-targeting drugs, including the MGBLs Hoechst 33342 and Hoechst 33258.
Understanding the mechanism of interaction between topoisomerase I-targeting drugs and ionizing radiation is a prerequisite toward successful use of their combination in cancer treatment. Controversial early studies using cultured cells and human xenografts suggested that camptothecin derivatives modulate the cytotoxic effects of ionizing radiation.[32-39] To answer key questions, such as whether camptothecin derivatives are radiosensitizers and whether DNA topoisomerase I is involved in such radiosensitization, we conducted clonogenic survival assays using cultured mammalian cells. We found that drug incubation with camptothecin derivatives radiosensitized log-phased human MCF-7 breast cancer cells in a schedule-dependent manner (Table 1). The radiosensitization effect was observed when the cells were exposed to drug treatment before or concurrent with radiation treatment, but not after radiation treatment (Table 1). The implication based on this observation is that camptothecin derivatives should be administered before or concurrently with radiation during chemoradiation clinical trials to optimize the radiosensitization effect.
Camptothecin derivatives interact with DNA topoisomerase I in a stereo-specific fashion. For example, assayed by the ability to induce topoisomerase I-mediated DNA cleavage, the 20(S)-stereoisomer of 10,11-methylenedioxycamptothecin is 10,000-fold more active than its 20(R)-isomer (see Figure 4A). This pair of camptothecin derivatives was used to investigate the role of DNA topoisomerase I in mediating radiosensitization. As shown in Figure 4B, only the 20(S)-10,11-methylenedioxycamptothecin radiosensitized human breast cancer MCF-7 cells. The prerequisite role of such an intact stereo-specific interaction in the induction of radiosensitization was further supported by the observation that the mutant topoisomerase I-containing DC3F cells were relatively resistant to radiosensitization.[40-42]
DNA Repair Inhibition
Some investigators have suggested DNA repair inhibition as a mechanism of radiosensitization by camptothecin derivatives. If this theory is correct, a larger radiosensitizing effect should be observed in cells that are growth inhibited (G0/G1 cells) to maximize their repair function for potentially lethal damages. We found that camptothecin only minimally enhanced the cytotoxic effect of radiation in plateau phase cells, which were arrested by growing to confluency, as well as in synchronized G1-phase cells obtained by mitotic shake-off technique. This finding may indicate a possible therapeutic advantage of camptothecin derivatives to radiosensitize actively proliferating cancer cells selectively.
The molecular mechanism of radiosensitization of DNA topoisomerase I-targeting drugs remains to be defined. Based on available information, a plausible mechanism of radiosensitization of topoisomerase I drugs is proposed (Figure 5). It is possible that induction of radiosensitization is originally initiated by the topoisomerase I-trapped cleavable complex. This drug-stabilized cleavable complex, with a concealed single-strand DNA break, may be viewed as a potentially sublethal DNA damage. Interaction with as yet undefined cellular processes such as DNA replication, RNA transcription, and DNA repair may transform such potentially sublethal DNA damage into sublethal DNA damage. It is plausible that such sublethal DNA damage could then be converted into lethal DNA damage with the addition of radiation-induced DNA damage.