Lung cancer is the number one cause of cancer-related deaths in the world. Patients treated with current chemotherapies for non-smallcell lung cancers (NSCLCs) have a survival rate of Ϸ15% 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. ␤-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 ␤-lapachone. NSCLC cells were killed in an NQO1-dependent manner by ␤-lapachone (LD 50, Ϸ4 M) with a minimum 2-h exposure. Kinetically, ␤-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 -calpain activation and apoptosis. ␤-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 Ca 2؉ chelator. NQO1 ؊ cells (H596, IMR-90) or dicoumarol-exposed NQO1 ؉ A549 cells were resistant (LD50, >40 M) to ROS formation and all cytotoxic effects of ␤-lapachone. Our data indicate that the most efficacious strategy using ␤-lapachone in chemotherapy was to deliver the drug in short pulses, greatly reducing cytotoxicity to NQO1 ؊ ''normal'' cells. ␤-Lapachone killed cells in a tumorselective manner and is indicated for use against NQO1 ؉ NSCLC cancers.DNA repair inhibitor ͉ non-small-cell lung cancer ͉ NQO1 ͉ apoptosis ͉ -calpain cell death

Alterations in the initiation and regulation of caspase-mediated apoptosis are associated with an array of pathological disease states, including chemotherapy resistance in cancer (1). Therefore, elucidating mechanisms that initiate non-caspasemediated cell death are crucial for the development and use of novel anticancer agents.A growing number of chemotherapeutic approaches focus on targeting specific DNA repair enzymes. In particular, inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) 2 that sensitize cells to DNA-damaging agents are under extensive investigation (2). PARP-1 functions as a DNA damage sensor that responds to both single-and/or double-strand DNA breaks (SSBs, DSBs), facilitating DNA repair and cell survival. After binding to DNA breaks, PARP-1 converts ␤-NAD ϩ (NAD ϩ ) into polymers of branched or linear poly(ADP-ribose) units (PAR) and attaches them to various nuclear acceptor proteins, including XRCC1, histones, and PARP-1 for its autoregulation (3). However, in response to extensive DNA damage, PARP-1 can be hyperactivated, eliciting rapid cellular NAD ϩ and ATP pool depletion. PARP-1-mediated NAD ϩ and ATP losses have affects on mitochondrial function by decreasing the levels of pyruvate and NADH. Loss of mitochondrial membrane potential (MMP) ensues, causing caspase-independent cell death by as yet unknown mechanisms (3). PARP-1 hyperactivation was documented in the cellular response to trauma, such as ischemia-reperfusion, myocardial infarction, and reactive oxygen species (ROS)-induced injury (3). In each case, inhibition of PARP-1 was necessary for the long-term survival of damaged cells (4).␤-lapachone (␤-lap) elicits a unique cell death process in various human breast, lung, and prostate cancers that have elevated levels of the two-electron oxidoreductase, NAD(P)H: quinone oxidoreductase 1 (NQO1) (EC 1.6.99.2) (5). ␤-lap induces an NQO1-dependent form of cell death wherein PARP-1 and p53 proteolytic cleavage fragments were noted (6), concomitant with -calpain activation (7). ␤-lap-induced lethality and proteolysis were abrogated by dicoumarol (an NQO1 inhibitor), and were muted in cells deficient in NQO1

B-Lapachone, an o-naphthoquinone, induces a novel caspase-and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase1 (NQO1). NQO1reduces B-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1rendered cells resistant to B-lapachone. Thus, B-lapachone has great potential for the treatment of specific cancers with elevated NQO1levels (e.g., breast, non^small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of B-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1when initially diluted in water. In solution, however, they undergo hydrolytic conversion to B-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatographyelectrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversionof each derivative to B-lapachone. Once converted, B-lapachone derivatives caused NQO1-dependent, A-calpain-mediated cell death in human cancer cells identical to that caused by B-lapachone. Interestingly, coadministration of N-acetyl-L-cysteine prevented derivative-induced cytotoxicity but did not affect B-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of B-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-L-cysteine, preventing their conversion to B-lapachone. The use of B-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, beta-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.

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 DNAdependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ. Alterations in these repair processes can cause drug/ radiation resistance and increased genomic instability. BLapachone (B-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 B-lap leading to reactive oxygen species generation, DNA breaks, ;-H2AX foci formation, and hyperactivation of poly(ADP-ribose) polymerase-1, which is required for cell death. Here, we report that B-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 B-lap lethality. These studies give insight into the mechanism by which B-lap radiosensitizes cancer cells and suggest that NHEJ is a potent target for enhancing the therapeutic efficacy of B-lap alone or in combination with other agents in cancer cells that express elevated NQO1 levels. [Cancer Res 2007;67(14):6936-45]

Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.

Supplementary Figure 4 from Nonhomologous End Joining Is Essential for Cellular Resistance to the Novel Antitumor Agent, β-Lapachone

<div>Abstract<p>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: <i>ataxia telangiectasia</i> 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. β-Lapachone (β-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 β-lap leading to reactive oxygen species generation, DNA breaks, γ-H2AX foci formation, and hyperactivation of poly(ADP-ribose) polymerase-1, which is required for cell death. Here, we report that β-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 β-lap lethality. These studies give insight into the mechanism by which β-lap radiosensitizes cancer cells and suggest that NHEJ is a potent target for enhancing the therapeutic efficacy of β-lap alone or in combination with other agents in cancer cells that express elevated NQO1 levels. [Cancer Res 2007;67(14):6936–45]</p></div>