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There is a great need to track the selectivity of anticancer drug activity and to understand the mechanisms of associated biological activity. Here we focus our studies on the specific NQO1 bioactivatable drug, ß-lapachone, which is in several Phase I clinical trials to treat human non-small cell lung, pancreatic and breast cancers. Multi-electrode chips with electrochemically-active DNA monolayers are used to track anticancer drug activity in cellular lysates and correlate cell death activity with DNA damage. Cells were prepared from the triple-negative breast cancer (TNBC) cell line, MDA-MB-231 (231) to be proficient or deficient in expression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme, which is overexpressed in most solid cancers and lacking in control healthy cells. Cells were lysed and added to chips, and the impact of β-lapachone (β-lap), an NQO1-dependent DNA-damaging drug, was tracked with DNA electrochemical signal changes arising from drug-induced DNA damage. Electrochemical DNA devices showed a 3.7-fold difference in the electrochemical responses in NQO1+ over NQO1- cell lysates, as well as 10-20-fold selectivity to catalase and dicoumarol controls that deactivate DNA damaging pathways. Concentration-dependence studies revealed that 1.4 µM β-lap correlated with the onset of cell death from viability assays and the midpoint of DNA damage on the chip, and 2.5 µM β-lap correlated with the midpoint of cell death and the saturation of DNA damage on the chip. Results indicate that these devices could inform therapeutic decisions for cancer treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Many cancer treatments, such as those for managing recalcitrant tumors like pancreatic ductal adenocarcinoma, cause off-target toxicities in normal, healthy tissue, highlighting the need for more tumor-selective chemotherapies. β-Lapachone is bioactivated by NAD(P)H:quinone oxidoreductase 1 (NQO1). This enzyme exhibits elevated expression in most solid cancers and therefore is a potential cancer-specific target. β-Lapachone's therapeutic efficacy partially stems from the drug's induction of a futile NQO1-mediated redox cycle that causes high levels of superoxide and then peroxide formation, which damages DNA and causes hyperactivation of poly(ADP-ribose) polymerase, resulting in extensive NAD + /ATP depletion. However, the effects of this drug on energy metabolism due to NAD + depletion were never described. The futile redox cycle rapidly consumes O 2 , rendering standard assays of Krebs cycle turnover unusable. In this study, a multimodal analysis, including metabolic imaging using hyperpolarized pyruvate, points to reduced oxidative flux due to NAD + depletion after β-lapachone treatment of NQO1+ human pancreatic cancer cells. NAD + -sensitive pathways, such as glycolysis, flux through lactate dehydrogenase, and the citric acid cycle (as inferred by flux through pyruvate dehydrogenase), were down-regulated by β-lapachone treatment. Changes in flux through these pathways should generate biomarkers useful for in vivo dose responses of β-lapachone treatment in humans, avoiding toxic side effects. Targeting the enzymes in these pathways for therapeutic treatment may have the potential to synergize with β-lapachone treatment, creating unique NQO1-selective combinatorial therapies for specific cancers. These findings warrant future studies of intermediary metabolism in patients treated with β-lapachone., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Selenium-containing quinone-based 1,2,3-triazoles were synthesized using click chemistry, the copper catalyzed azide-alkyne 1,3-dipolar cycloaddition, and evaluated against six types of cancer cell lines: HL-60 (human promyelocytic leukemia cells), HCT-116 (human colon carcinoma cells), PC3 (human prostate cells), SF295 (human glioblastoma cells), MDA-MB-435 (melanoma cells) and OVCAR-8 (human ovarian carcinoma cells). Some compounds showed IC50 values < 0.3 μM. The cytotoxic potential of the quinones evaluated was also assayed using non-tumor cells, exemplified by peripheral blood mononuclear (PBMC), V79 and L929 cells. Mechanistic role for, Nad(p)h: Quinone Oxidoreductase 1 (NQO1) was also elucidated. These compounds could provide promising new lead derivatives for more potent anticancer drug development and delivery, and represent one of the most active classes of lapachones reported., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

It is beneficial to develop systems that reproduce complex reactions of biological systems while maintaining control over specific factors involved in such processes. We demonstrated a DNA device for following the repair of DNA damage produced by a redox-cycling anticancer drug, beta-lapachone (β-lap). These chips supported ß-lap-induced biological redox cycle and tracked subsequent DNA damage repair activity with redox-modified DNA monolayers on gold. We observed drug-specific changes in square wave voltammetry from these chips at therapeutic ß-lap concentrations of high statistical significance over drug-free control. We also demonstrated a high correlation of this change with the specific ß-lap-induced redox cycle using rational controls. The concentration dependence of ß-lap revealed significant signal changes at levels of high clinical significance as well as sensitivity to sub-lethal levels of ß-lap. Catalase, an enzyme decomposing peroxide, was found to suppress DNA damage at a NQO1/catalase ratio found in healthy cells, but was clearly overcome at a higher NQO1/catalase ratio consistent with cancer cells. We found that it was necessary to reproduce key features of the cellular environment to observe this activity. Thus, this chip-based platform enabled tracking of ß-lap-induced DNA damage repair when biological criteria were met, providing a unique synthetic platform for uncovering activity normally confined to inside cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lung cancer is one of the most lethal forms of cancer and current chemotherapeutic strategies lack broad specificity and efficacy. Recently, β-lapachone (β-lap) was shown to be highly efficacious in killing non-small cell lung cancer (NSCLC) cells regardless of their p53, cell cycle and caspase status. Pre-clinical and clinical use of β-lap (clinical form, ARQ501 or 761) is hampered by poor pharmacokinetics and toxicity due to hemolytic anemia. Here, we report the development and preclinical evaluation of β-lap prodrug nanotherapeutics consisting of diester derivatives of β-lap encapsulated in biocompatible and biodegradable poly(ethylene glycol)-b-poly(D,L-lactic acid) (PEG-b-PLA) micelles. Compared to the parent drug, diester derivatives of β-lap showed higher drug loading densities inside PEG-b-PLA micelles. After esterase treatment, micelle-delivered β-lap-dC3 and -dC6 prodrugs were converted to β-lap. Cytotoxicity assays using A549 and H596 lung cancer cells showed that both micelle formulations maintained, Nad(p)h: quinone oxidoreductase 1 (NQO1)-dependent cytotoxicity. However, antitumor efficacy study of β-lap-dC3 micelles against orthotopic A549 NSCLC xenograft-bearing mice showed significantly greater long-term survival over β-lap-dC6 micelles or β-lap-HPβCD complexes. Improved therapeutic efficacy of β-lap-dC3 micelles correlated with higher area under the concentration-time curves of β-lap in tumors, and enhanced pharmacodynamic endpoints (e.g., PARP1 hyperactivation, γH2AX, and ATP depletion). β-Lap-dC3 prodrug micelles provide a promising strategy for NQO1-targeted therapy of lung cancer with improved safety and antitumor efficacy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Superparamagnetic iron oxide nanoparticles (SPION) are an important and versatile nano- platform with broad biological applications. Despite extensive studies, the biological and pharmacological activities of SPION have not been exploited in therapeutic applications. Recently, β-lapachone (β-lap), a novel anticancer drug, has shown considerable cancer specificity by selectively increasing reactive oxygen species (ROS) stress in cancer cells. In this study, we report that pH-responsive SPION-micelles can synergize with β-lap for improved cancer therapy. These SPION-micelles selectively release iron ions inside cancer cells, which interact with hydrogen peroxide (H(2)O(2)) generated from β-lap in a tumor-specific, NQO1-dependent manner. Through Fenton reactions, these iron ions escalate the ROS stress in β-lap-exposed cancer cells, thereby greatly enhancing the therapeutic index of β-lap. More specifically, a 10-fold increase in ROS stress was detected in β-lap-exposed cells pretreated with SPION-micelles over those treated with β-lap alone, which also correlates with significantly increased cell death. Catalase treatment of cells or administration of an iron chelator can block the therapeutic synergy. Our data suggest that incorporation of SPION-micelles with ROS-generating drugs can potentially improve drug efficacy during cancer treatment, thereby provides a synergistic strategy to integrate imaging and therapeutic functions in the development of theranostic nanomedicine.

Agents, such as β-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than β-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion that stimulate Ca2+ -dependent programmed necrosis, unique to this new class of NQO1 "bioactivated" drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1- cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with β-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to β-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers.

Purpose: Pancreatic cancer is the fourth leading cause of cancer-related deaths, in which the 5-year survival rate is less than 5%. Current standard of care therapies offer little selectivity and high toxicity. Novel, tumor-selective approaches are desperately needed. Although prior work suggested that β-lapachone (β-lap) could be used for the treatment of pancreatic cancers, the lack of knowledge of the compound's mechanism of action prevented optimal use of this agent., Experimental Design: We examined the role of NAD(P)H:quinone oxidoreductase-1 (NQO1) in β-lap-mediated antitumor activity, using a series of MIA PaCa-2 pancreatic cancer clones varying in NQO1 levels by stable shRNA knockdown. The antitumor efficacy of β-lap was determined using an optimal hydroxypropyl-β-cyclodextran (HPβ-CD) vehicle formulation in metastatic pancreatic cancer models., Results: β-Lap-mediated cell death required ∼90 enzymatic units of NQO1. Essential downstream mediators of lethality were as follows: (i) reactive oxygen species (ROS); (ii) single-strand DNA breaks induced by ROS; (iii) poly(ADP-ribose)polymerase-1 (PARP1) hyperactivation; (iv) dramatic NAD(+)/ATP depletion; and (v) programmed necrosis. We showed that 1 regimen of β-lap therapy (5 treatments every other day) efficaciously regressed and reduced human pancreatic tumor burden and dramatically extended the survival of athymic mice, using metastatic pancreatic cancer models., Conclusions: Because NQO1 enzyme activities are easily measured and commonly overexpressed (i.e., >70%) in pancreatic cancers 5- to 10-fold above normal tissue, strategies using β-lap to efficaciously treat pancreatic cancers are indicated. On the basis of optimal drug formulation and efficacious antitumor efficacy, such a therapy should be extremely safe and not accompanied with normal tissue toxicity or hemolytic anemia., (©2011 AACR.)

Purpose: beta-Lapachone (ARQ 501, a formulation of beta-lapachone complexed with hydroxypropyl-beta-cyclodextrin) is a novel anticancer agent with selectivity against prostate cancer cells overexpressing the NAD(P)H:quinone oxidoreductase-1 enzyme. Lack of solubility and an efficient drug delivery strategy limits this compound in clinical applications. In this study, we aimed to develop beta-lapachone-containing polymer implants (millirods) for direct implantation into prostate tumors to test the hypothesis that the combination of a tumor-specific anticancer agent with site-specific release of the agent will lead to significant antitumor efficacy., Experimental Design: Survival assays in vitro were used to test the killing effect of beta-lapachone in different prostate cancer cells. beta-Lapachone release kinetics from millirods was determined in vitro and in vivo. PC-3 prostate tumor xenografts in athymic nude mice were used for antitumor efficacy studies in vivo., Results: beta-Lapachone killed three different prostate cancer cell lines in an NAD(P)H:quinone oxidoreductase-1-dependent manner. Upon incorporation of solid-state inclusion complexes of beta-lapachone with hydroxypropyl-beta-cyclodextrin into poly(D,L-lactide-co-glycolide) millirods, beta-lapachone release kinetics in vivo showed a burst release of approximately 0.5 mg within 12 hours and a subsequently sustained release of the drug ( approximately 0.4 mg/kg/d) comparable with that observed in vitro. Antitumor efficacy studies showed significant tumor growth inhibition by beta-lapachone millirods compared with controls (P < 0.0001; n = 10 per group). Kaplan-Meier survival curves showed that tumor-bearing mice treated with beta-lapachone millirods survived nearly 2-fold longer than controls, without observable systemic toxicity., Conclusions: Intratumoral delivery of beta-lapachone using polymer millirods showed the promising therapeutic potential for human prostate tumors.

Beta-lapachone (beta-lap) is a novel anticancer agent that is bioactivated by NADP(H): quinone oxidoreductase 1 (NQO1), an enzyme overexpressed in a variety of tumors. Despite its therapeutic promise, the poor aqueous solubility of beta-lap hinders its preclinical evaluation and clinical translation. Our objective was to develop beta-lap-containing poly(ethylene glycol)-block-poly(D,L-lactide) (PEG-PLA) polymer micelles for the treatment of NQO1-overexpressing tumors. Several micelle fabrication strategies were examined to maximize drug loading. A film sonication method yielded beta-lap micelles with relatively high loading density (4.7+/-1.0% to 6.5+/-1.0%) and optimal size (29.6+/-1.5 nm). Release studies in phosphate-buffered saline (pH 7.4) showed the time (t(1/2)) for 50% of drug release at 18 h. In vitro cytotoxicity assays were performed in NQO1-overexpressing (NQO1+) and NQO1-null (NQO1-) H596 lung, DU-145 prostate, and MDA-MB-231 breast cancer cells. Cytotoxicity data showed that after a 2 h incubation with beta-lap micelles, a marked increase in toxicity was shown in NQO1+ cells over NQO1- cells, resembling free drug both in efficacy and mechanism of cell death. In summary, these data demonstrate the potential of beta-lap micelles as an effective therapeutic strategy against NQO1-overexpressing tumor cells.

Commonly used antitumor agents, such as DNA topoisomerase I/II poisons, kill cancer cells by creating nonrepairable DNA double-strand breaks (DSBs). To repair DSBs, error-free homologous recombination (HR), and/or error-prone nonhomologous end joining (NHEJ) are activated. These processes involve the phosphatidylinositol 3'-kinase-related kinase family of serine/threonine enzymes: ataxia telangiectasia mutated (ATM), ATM- and Rad3-related for HR, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ. Alterations in these repair processes can cause drug/radiation resistance and increased genomic instability. beta-Lapachone (beta-lap; also known as ARQ 501), currently in phase II clinical trials for the treatment of pancreatic cancer, causes a novel caspase- and p53-independent cell death in cancer cells overexpressing NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 catalyzes a futile oxidoreduction of beta-lap leading to reactive oxygen species generation, DNA breaks, gamma-H2AX foci formation, and hyperactivation of poly(ADP-ribose) polymerase-1, which is required for cell death. Here, we report that beta-lap exposure results in NQO1-dependent activation of the MRE11-Rad50-Nbs-1 complex. In addition, ATM serine 1981, DNA-PKcs threonine 2609, and Chk1 serine 345 phosphorylation were noted; indicative of simultaneous HR and NHEJ activation. However, inhibition of NHEJ, but not HR, by genetic or chemical means potentiated beta-lap lethality. These studies give insight into the mechanism by which beta-lap radiosensitizes cancer cells and suggest that NHEJ is a potent target for enhancing the therapeutic efficacy of beta-lap alone or in combination with other agents in cancer cells that express elevated NQO1 levels.

Lung cancer is the number one cause of cancer-related deaths in the world. Patients treated with current chemotherapies for non-small-cell lung cancers (NSCLCs) have a survival rate of approximately 15% after 5 years. Novel approaches are needed to treat this disease. We show elevated NAD(P)H:quinone oxidoreductase-1 (NQO1) levels in tumors from NSCLC patients. beta-Lapachone, an effective chemotherapeutic and radiosensitizing agent, selectively killed NSCLC cells that expressed high levels of NQO1. Isogenic H596 NSCLC cells that lacked or expressed NQO1 along with A549 NSCLC cells treated with or without dicoumarol, were used to elucidate the mechanism of action and optimal therapeutic window of beta-lapachone. NSCLC cells were killed in an NQO1-dependent manner by beta-lapachone (LD50, approximately 4 microM) with a minimum 2-h exposure. Kinetically, beta-lapachone-induced cell death was characterized by the following: (i) dramatic reactive oxygen species (ROS) formation, eliciting extensive DNA damage; (ii) hyperactivation of poly(ADP-ribose)polymerase-1 (PARP-1); (iii) depletion of NAD+/ATP levels; and (iv) proteolytic cleavage of p53/PARP-1, indicating mu-calpain activation and apoptosis. Beta-lapachone-induced PARP-1 hyperactivation, nucleotide depletion, and apoptosis were blocked by 3-aminobenzamide, a PARP-1 inhibitor, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a Ca2+ chelator. NQO1- cells (H596, IMR-90) or dicoumarol-exposed NQO1+ A549 cells were resistant (LD50, >40 microM) to ROS formation and all cytotoxic effects of beta-lapachone. Our data indicate that the most efficacious strategy using beta-lapachone in chemotherapy was to deliver the drug in short pulses, greatly reducing cytotoxicity to NQO1- "normal" cells. beta-Lapachone killed cells in a tumorselective manner and is indicated for use against NQO1+ NSCLC cancers.

Purpose: The purpose of the present study was to evaluate the efficacy of mild hyperthermia to potentiate the anticancer effects of beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) by up-regulating NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells., Experimental Design: Effects of beta-lapachone alone or in combination with mild heating on the clonogenic survival of FSaII fibrosarcoma cells of C3H mice and A549 human lung tumor cells in vitro was determined. Effects of heating on the NQO1 level in the cancer cells in vitro were assessed using Western blot analysis for NQO1 expression, biochemical determination of NQO1 activity, and immunofluorescence microscopy for NQO1 expression. Growth of FSaII tumors in the hind legs of C3H mice was determined after treating the host mice with i.p. injection of 45 mg/kg beta-lapachone followed by heating the tumors at 42 degrees C for 1 hour every other day for four times., Results: Incubation of FSaII tumor cells and A549 tumor cells with beta-lapachone at 37 degrees C reduced clonogenic survival of the cells in dose-dependent and incubation time-dependent manner. NQO1 level in the cancer cells in vitro increased within 1 hour after heating at 42 degrees C for 1 hour and remained elevated for >72 hours. The clonogenic cell death caused by beta-lapachone increased in parallel with the increase in NQO1 levels in heated cells. Heating FSaII tumors in the legs of C3H mice enhanced the effect of i.p.-injected beta-lapachone in suppressing tumor growth., Conclusion: We observed for the first time that mild heat shock up-regulates NQO1 in tumor cells. The heat-induced up-regulation of NQO1 enhanced the anticancer effects of beta-lapachone in vitro and in vivo.

Deficiencies in DNA mismatch repair (MMR) have been found in both hereditary cancer (i.e., hereditary nonpolyposis colorectal cancer) and sporadic cancers of various tissues. In addition to its primary roles in the correction of DNA replication errors and suppression of recombination, research in the last 10 years has shown that MMR is involved in many other processes, such as interaction with other DNA repair pathways, cell cycle checkpoint regulation, and apoptosis. Indeed, a cell's MMR status can influence its response to a wide variety of chemotherapeutic agents, such as temozolomide (and many other methylating agents), 6-thioguanine, cisplatin, ionizing radiation, etoposide, and 5-fluorouracil. For this reason, identification of a tumor's MMR deficiency (as indicated by the presence of microsatellite instability) is being utilized more and more as a prognostic indicator in the clinic. Here, we describe the basic mechanisms of MMR and apoptosis and investigate the literature examining the influence of MMR status on the apoptotic response following treatment with various therapeutic agents. Furthermore, using isogenic MMR-deficient (HCT116) and MMR-proficient (HCT116 3-6) cells, we demonstrate that there is no enhanced apoptosis in MMR-proficient cells following treatment with 5-fluoro-2'-deoxyuridine. In fact, apoptosis accounts for only a small portion of the induced cell death response.

Despite innovations in imaging, surgery, and radiation therapy, local failure remains the principle clinical problem in most CNS malignancies. To date, chemotherapy has not major impact in the treatment of most adult CNS tumors. The inroads made by chemotherapy in pediatric CNS malignancies suggest that novel drugs, or drug combinations, may improve therapy. Topoisomerase I (Topo I) inhibitors are a relatively new group of chemotherapy drugs with a novel mechanism of action. Drugs in this group currently undergoing clinical trials are the Camptothecin analogues Topotecan, CPT-11, and 9-aminocamptothecin. There is substantial preclinical and some clinical evidence to suggest that these drugs could be useful in the treatment of CNS malignancies. Preclinical studies with the water soluble Topo I inhibitor, Topotecan, demonstrate antineoplastic activity in a variety of CNS malignancies. In addition, Topotecan has good CNS penetration in primates, and recent preliminary phase I and II clinical trials of Topotecan in pediatric and adult CNS malignancies have been promising. In this paper, we describe the unique mechanism of action, antineoplastic activity, and radiosensitizing properties of Topo I inhibitors. We present the first report demonstrating potentiation of radiation lethality by Topotecan in a human glioma (D54) cell line. The dose enhancement ratio was 3.2 at 10% survival. Thus, there is evidence to suggest that Topo I inhibitors may be beneficial in the treatment of CNS neoplasms on the basis of their antineoplastic activity alone, as well as their radiosensitizing effects. Two clinical trials which utilize concurrent Topotecan and radiation in the treatment of pediatric and adult CNS malignancies are discussed.

Purpose: Topotecan (TPT) is a water-soluble Topoisomerase I (Topo I) inhibitor with reported antineoplastic activity against a variety of solid tumors (including nonsmall cell lung, small cell lung, ovarian, breast, esophageal, and head and neck primaries) and leukemias. We sought to determine: (1) if TPT enhanced the lethal effects of ionizing radiation: and (b) the biological and biochemical characteristics of the enhancement., Methods and Materials: Quiescent human radioresistant melanoma (U1-Mel) cells were x-irradiated (1-12 Gy) and exposed to various TPT concentrations (0.1-300 microM) either before (for 4 h), during, or after (for 4 h) radiation. Survival was determined via colony forming assays and normalized to correct for drug cytotoxicity. The effects of TPT on radiation-related potential lethal damage repair (PLDR) and sublethal damage repair (SLDR) were measured. A modification of the SDS-KCl assay was used to quantify DNA-Topo I complexes., Results: Enhanced radiation lethality by TPT was observed using quiescent U1-Mel cells. The sensitizer enhancement ratio (SER) after a 4 h postirradiation exposure of 4 microM TPT was 1.6 at 10% survival. The effect was: (A) dependent on drug concentration, with lethality enhancement and minimal drug lethality alone in the 2-10 microM range for a 4 h posttreatment; (b) dependent on timing, with enhancement observed only when drug was present at the time of, or shortly after, radiation; and (c) irreversible, with inhibition of PLDR and SLDR. Exposure to TPT during or after radiation substantially elevated DNA-Topo I complexes (four- to tenfold) over control levels and complex formation correlated to some degree with loss of survival., Conclusions: TPT enhanced radiation lethality in vitro at low drug concentrations that are clinically feasible. The rationale and design of an ongoing Phase I trial which utilizes concurrent TPT and radiation is discussed.

Purpose: Preclinical studies have demonstrated differences in potency, solubility, and tumor specific activity among the camptothecin (CPT) analogues. 9-Aminocamptothecin (9-AC) has demonstrated greater potency in animal studies than other clinically available Topoisomerase I (Topo I) inhibitors. We sought to determine: (a) if 9-AC enhanced the lethal effects of ionizing radiation to a greater extent than other Topo I inhibitors; and (b) the biological and biochemical characteristics of the enhancement., Methods and Materials: Quiescent radioresistant human melanoma (U1-Mel) cells were x-irradiated (1-7 Gy) and exposed to various concentrations of 9-AC (0.1-100 microM), either before (for 4 h), during, or after (for 4 h) irradiation. Survival was determined via colony forming assays and normalized to correct for drug cytotoxicity. The effect of 9-AC on radiation-related potential lethal damage repair PLDR) (PLDR) was also measured. A modification of the SDS-KCl assay was used to quantify DNA-Topo I complexes., Results: Enhancement of radiation lethality was observed using confluent U1-Mel cells. The sensitizer enhancement ratio (SER) after a 4 h postirradiation exposure of 10 microM 9-AC was 2.5 at 10% survival. Toxicity from the drug alone was greater than topotecan (TPT), but less than CPT. The radiation synergy effect was: (a) dependent on drug concentration (> or = 2 microM); (b) dependent on timing, with enhancement present only when the drug was present at the time of, or shortly after, radiation; and (c) irreversible, with inhibition of PLDR. Exposure to 9-AC during or after irradiation substantially elevated the number of DNA-Topo I complexes (four- to tenfold) over control levels and correlated with enhanced loss of survival., Conclusion: 9-Aminocamptothecin enhanced radiation lethality in vitro at low drug concentrations with characteristics similar to other Topo I inhibitors. A greater SER, but greater lethality with the drug alone, was obtained in comparison to TPT. The clinical implications of these findings remain unexplored.