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Unlabelled: Ionizing radiation (IR) is a key therapeutic regimen for many head and neck cancers (HNC). However, the 5-year overall survival rate for locally advanced HNCs is approximately 50% and better therapeutic efficacy is needed., Nad(p)h: quinone oxidoreductase 1 (NQO1) is overexpressed in many cancers, and β-lapachone (β-lap), a unique NQO1 bioactivatable drug, exploits this enzyme to release massive reactive oxygen species (ROS) that synergize with IR to kill by programmed necrosis. β-Lap represents a novel therapeutic opportunity in HNC leading to tumor-selective lethality that will enhance the efficacy of IR. Immunohistochemical staining and Western blot assays were used to assess the expression levels of NQO1 in HNC cells and tumors. Forty-five percent of endogenous HNCs expressed elevated NQO1 levels. In addition, multiple HNC cell lines and tumors demonstrated elevated levels of NQO1 expression and activity and were tested for anticancer lethality and radiosensitization by β-lap using long-term survival assays. The combination of nontoxic β-lap doses and IR significantly enhanced NQO1-dependent tumor cell lethality, increased ROS, TUNEL-positive cells, DNA damage, NAD(+), and ATP consumption, and resulted in significant antitumor efficacy and prolonged survival in two xenograft murine HNC models, demonstrating β-lap radiosensitization of HNCs through a NQO1-dependent mechanism. This translational study offers a potential biomarker-driven strategy using NQO1 expression to select tumors susceptible to β-lap-induced radiosensitization. Mol Cancer Ther; 15(7); 1757-67. ©2016 AACR., Competing Interests: of Potential Conflicts of Interest: No potential conflicts of interest were disclosed., (©2016 American Association for Cancer Research.)

Aims: β-Lapachone (β-lap), a novel radiosensitizer with potent antitumor efficacy alone, selectively kills solid cancers that over-express, Nad(p)h: quinone oxidoreductase 1 (NQO1). Since breast or other solid cancers have heterogeneous NQO1 expression, therapies that reduce the resistance (e.g., NQO1(low)) of tumor cells will have significant clinical advantages. We tested whether NQO1-proficient (NQO1(+)) cells generated sufficient hydrogen peroxide (H2O2) after β-lap treatment to elicit bystander effects, DNA damage, and cell death in neighboring NQO1(low) cells., Results: β-Lap showed NQO1-dependent efficacy against two triple-negative breast cancer (TNBC) xenografts. NQO1 expression variations in human breast cancer patient samples were noted, where ~60% cancers over-expressed NQO1, with little or no expression in associated normal tissue. Differential DNA damage and lethality were noted in NQO1(+) versus NQO1-deficient (NQO1(-)) TNBC cells and xenografts after β-lap treatment. β-Lap-treated NQO1(+) cells died by programmed necrosis, whereas co-cultured NQO1(-) TNBC cells exhibited DNA damage and caspase-dependent apoptosis. NQO1 inhibition (dicoumarol) or H2O2 scavenging (catalase [CAT]) blocked all responses. Only NQO1(-) cells neighboring NQO1(+) TNBC cells responded to β-lap in vitro, and bystander effects correlated well with H2O2 diffusion. Bystander effects in NQO1(-) cells in vivo within mixed 50:50 co-cultured xenografts were dramatic and depended on NQO1(+) cells. However, normal human cells in vitro or in vivo did not show bystander effects, due to elevated endogenous CAT levels. Innovation and Conclusions: NQO1-dependent bystander effects elicited by NQO1 bioactivatable drugs (β-lap or deoxynyboquinone [DNQ]) likely contribute to their efficacies, killing NQO1(+) solid cancer cells and eliminating surrounding heterogeneous NQO1(low) cancer cells. Normal cells/tissue are protected by low NQO1:CAT ratios.

Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. β-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of β-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ∼120 moles of superoxide were formed per mole of β-lapachone in 2 minutes. β-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of β-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. β-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of β-lapachone and other NQO1 bioactivatable drugs., (©2013 AACR.)

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.)

Lung cancer is the leading cause of cancer-related deaths with current chemotherapies lacking adequate specificity and efficacy. Beta-lapachone (beta-lap) is a novel anticancer drug that is bioactivated by NAD(P)H:quinone oxidoreductase 1, an enzyme found specifically overexpressed in non-small cell lung cancer (NSCLC). Herein, we report a nanotherapeutic strategy that targets NSCLC tumors in two ways: (a) pharmacodynamically through the use of a bioactivatable agent, beta-lap, and (b) pharmacokinetically by using a biocompatible nanocarrier, polymeric micelles, to achieve drug stability, bioavailability, and targeted delivery. Beta-lap micelles produced by a film sonication technique were small ( approximately 30 nm), displayed core-shell architecture, and possessed favorable release kinetics. Pharmacokinetic analyses in mice bearing subcutaneous A549 lung tumors showed prolonged blood circulation (t(1/2), approximately 28 h) and increased accumulation in tumors. Antitumor efficacy analyses in mice bearing subcutaneous A549 lung tumors and orthotopic Lewis lung carcinoma models showed significant tumor growth delay and increased survival. In summary, we have established a clinically viable beta-lap nanomedicine platform with enhanced safety, pharmacokinetics, and antitumor efficacy for the specific treatment of NSCLC tumors., ((c)2010 AACR.)

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.

beta-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces beta-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 NQO1 rendered cells resistant to beta-lapachone. Thus, beta-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non-small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of beta-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to beta-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to beta-lapachone. Once converted, beta-lapachone derivatives caused NQO1-dependent, mu-calpain-mediated cell death in human cancer cells identical to that caused by beta-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect beta-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of beta-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to beta-lapachone. The use of beta-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.

Purpose: To reveal the interaction between beta-lapachone (beta-lap) and ionizing radiation (IR) in causing clonogenic death in cancer cells and to elucidate the potential usefulness of beta-lap treatment in combination with radiotherapy of cancer., Methods and Materials: FSaII tumor cells of C3H mice were used. The cytotoxicity of beta-lap alone or in combination with IR in vitro was determined using clonogenic survival assay method. The IR-induced changes in the expression and the enzymatic activity of NAD(P)H:quinone oxidoreductase (NQO1), a mediator of beta-lap cytotoxicity, were elucidated and the relationship between the NQO1 level and the sensitivity of cells to beta-lap was investigated. The combined effect of IR and beta-lap to suppress tumor growth was studied using FSaII tumors grown subcutaneously in the thigh of C3H mice., Results: beta-Lap caused clonogenic death of FSaII tumor cells in vitro in a dose- and time-dependent manner. When cells were treated first with beta-lap and then exposed to IR in vitro, the resultant cell death was only additive. On the contrary, exposing cells to IR at 2.5 Gy first and then treating the cells with beta-lap killed the cells in a synergistic manner. Importantly, the 2.5 Gy cells were sensitive to beta-lap as long as 10 h after irradiation, which was long after the sublethal radiation damage was repaired. Irradiation of FSaII cells in vitro with 2.5 Gy significantly increased the expression and enzymatic activity of NQO1. The growth delay of FSaII tumors caused by an intraperitoneal injection of beta-lap in combination with 20 Gy irradiation of tumor was significantly greater than that caused by beta-lap or 20 Gy irradiation alone., Conclusion: The sensitivity of cells to beta-lap is dependent on NQO1 activity. IR caused a long-lasting increase in NQO1 activity in cancer cells, thereby sensitizing cells to beta-lap and treatment of experimental mouse tumors with IR and beta-lap suppressed tumor growth in a synergistic manner. The combination of beta-lap and radiotherapy is a potentially effective regimen for the treatment of human cancer.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells., (Copyright 2001 Academic Press.)