Single-Cell Kinetic Modeling of ß-Lapachone Metabolism in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) cells are highly heterogeneous in their metabolism and typically experience elevated reactive oxygen species (ROS) levels such as superoxide and hydrogen peroxide (H2O2) in the tumor microenvironment. Tumor cells survive under these chronic oxidative conditions by upregulating antioxidant systems. To investigate the heterogeneity of cellular responses to chemotherapeutic H2O2 generation in tumor and healthy tissue, we leveraged single-cell RNA-sequencing (scRNA-seq) data to perform redox systems-level simulations of quinone-cycling ß-lapachone treatment as a source of NQO1-dependent rapid superoxide and hydrogen peroxide (H2O2) production. Transcriptomic data from 10 HNSCC patient tumors was used to populate over 4000 single-cell antioxidant enzymatic network models of drug metabolism. The simulations reflected significant systems-level differences between the redox states of healthy and cancer cells, demonstrating in some patient samples a targetable cancer cell population or in others statistically indistinguishable effects between non-malignant and malignant cells. Subsequent multivariate analyses between healthy and malignant cellular models pointed to distinct contributors of redox responses between these phenotypes. This model framework provides a mechanistic basis for explaining mixed outcomes of NAD(P)H:quinone oxidoreductase 1 (NQO1)-bioactivatable therapeutics despite the tumor specificity of these drugs as defined by NQO1/catalase expression and highlights the role of alternate antioxidant components in dictating drug-induced oxidative stress.

NAD(P)H Quinone Oxidoreductase-1 Expression Promotes Self-Renewal and Therapeutic Resistance in Non-Small Cell Lung Cancer.

Identifying cellular drivers responsible for enhancing cancer cell resistance to therapeutics provides critical information for designing more effective drugs. Populations of slowly growing, self-renewing, chemo-resistant cells purportedly contribute to the development of therapeutic resistance in many solid tumors. In the current study, we implemented a tumor spheroid model to determine whether NAD(P)H quinone oxidoreductase-1 (NQO1) was requisite for self-renewal and promotion of the drug-resistant phenotype in non-small cell lung cancer (NSCLC). We found that stable depletion of NQO1 in A549 and H358 human NSCLC tumor models inhibits self-renewal capabilities, as demonstrated by a reduced ability to form primary, secondary, and tertiary spheroids. In contrast, the rescue of NQO1 expression restored the tumor cells' ability to form spheroids. Additionally, we discovered that NQO1 depletion renders cisplatin-refractory tumor spheroids highly susceptible to drug treatment. Together, these results suggest that NQO1 loss reduces the self-renewing capabilities of NSCLC cells and enhances their susceptibility to clinically relevant therapeutics. These findings describe a novel role for NQO1 and suggest that combining NQO1-inhibitors with conventional chemotherapeutics may enhance anti-tumor effects.

Targeting Base Excision Repair in Cancer: NQO1-Bioactivatable Drugs Improve Tumor Selectivity and Reduce Treatment Toxicity Through Radiosensitization of Human Cancer.

Ionizing radiation (IR) creates lethal DNA damage that can effectively kill tumor cells. However, the high dose required for a therapeutic outcome also damages healthy tissue. Thus, a therapeutic strategy with predictive biomarkers to enhance the beneficial effects of IR allowing a dose reduction without losing efficacy is highly desirable. NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in the majority of recalcitrant solid tumors in comparison with normal tissue. Studies have shown that NQO1 can bioactivate certain quinone molecules (e.g., ortho-naphthoquinone and ß-lapachone) to induce a futile redox cycle leading to the formation of oxidative DNA damage, hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), and catastrophic depletion of NAD+ and ATP, which culminates in cellular lethality via NAD+-Keresis. However, NQO1-bioactivatable drugs induce methemoglobinemia and hemolytic anemia at high doses. To circumvent this, NQO1-bioactivatable agents have been shown to synergize with PARP1 inhibitors, pyrimidine radiosensitizers, and IR. This therapeutic strategy allows for a reduction in the dose of the combined agents to decrease unwanted side effects by increasing tumor selectivity. In this review, we discuss the mechanisms of radiosensitization between NQO1-bioactivatable drugs and IR with a focus on the involvement of base excision repair (BER). This combination therapeutic strategy presents a unique tumor-selective and minimally toxic approach for targeting solid tumors that overexpress NQO1.

Y Chromosome LncRNA Are Involved in Radiation Response of Male Non-Small Cell Lung Cancer Cells.

Numerous studies have implicated changes in the Y chromosome in male cancers, yet few have investigated the biological importance of Y chromosome noncoding RNA. Here we identify a group of Y chromosome-expressed long noncoding RNA (lncRNA) that are involved in male non-small cell lung cancer (NSCLC) radiation sensitivity. Radiosensitive male NSCLC cell lines demonstrated a dose-dependent induction of linc-SPRY3-2/3/4 following irradiation, which was not observed in radioresistant male NSCLC cell lines. Cytogenetics revealed the loss of chromosome Y (LOY) in the radioresistant male NSCLC cell lines. Gain- and loss-of-function experiments indicated that linc-SPRY3-2/3/4 transcripts affect cell viability and apoptosis. Computational prediction of RNA binding proteins (RBP) motifs and UV-cross-linking and immunoprecipitation (CLIP) assays identified IGF2BP3, an RBP involved in mRNA stability, as a binding partner for linc-SPRY3-2/3/4 RNA. The presence of linc-SPRY3-2/3/4 reduced the half-life of known IGF2BP3 binding mRNA, such as the antiapoptotic HMGA2 mRNA, as well as the oncogenic c-MYC mRNA. Assessment of Y chromosome in NSCLC tissue microarrays and expression of linc-SPRY3-2/3/4 in NSCLC RNA-seq and microarray data revealed a negative correlation between the loss of the Y chromosome or linc-SPRY3-2/3/4 and overall survival. Thus, linc-SPRY3-2/3/4 expression and LOY could represent an important marker of radiotherapy in NSCLC. SIGNIFICANCE: This study describes previously unknown Y chromosome-expressed lncRNA regulators of radiation response in male NSCLC and show a correlation between loss of chromosome Y and radioresistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4046/F1.large.jpg.

NQO1-dependent, Tumor-selective Radiosensitization of Non-small Cell Lung Cancers.

PURPOSE: Development of tumor-specific therapies for the treatment of recalcitrant non-small cell lung cancers (NSCLC) is urgently needed. Here, we investigated the ability of ß-lapachone (ß-lap, ARQ761 in clinical form) to selectively potentiate the effects of ionizing radiation (IR, 1-3 Gy) in NSCLCs that overexpress NAD(P)H:Quinone Oxidoreductase 1 (NQO1). EXPERIMENTAL DESIGN: The mechanism of lethality of low-dose IR in combination with sublethal doses of ß-lap was evaluated in NSCLC lines in vitro and validated in subcutaneous and orthotopic xenograft models in vivo. Pharmacokinetics and pharmacodynamics (PK/PD) studies comparing single versus cotreatments were performed to validate therapeutic efficacy and mechanism of action. RESULTS: ß-Lap administration after IR treatment hyperactivated PARP, greatly lowered NAD+/ATP levels, and increased double-strand break (DSB) lesions over time in vitro. Radiosensitization of orthotopic, as well as subcutaneous, NSCLCs occurred with high apparent cures (>70%), even though 1/8 ß-lap doses reach subcutaneous versus orthotopic tumors. No methemoglobinemia or long-term toxicities were noted in any normal tissues, including mouse liver that expresses the highest level of NQO1 (∼12 units) of any normal tissue. PK/PD responses confirm that IR + ß-lap treatments hyperactivate PARP activity, greatly lower NAD+/ATP levels, and dramatically inhibit DSB repair in exposed NQO1+ cancer tissue, whereas low NQO1 levels and high levels of catalase in associated normal tissue were protective. CONCLUSIONS: Our data suggest that combination of sublethal doses of ß-lap and IR is a viable approach to selectively treat NQO1-overexpressing NSCLC and warrant a clinical trial using low-dose IR + ß-lap against patients with NQO1+ NSCLCs.

Leveraging an NQO1 Bioactivatable Drug for Tumor-Selective Use of Poly(ADP-ribose) Polymerase Inhibitors.

Therapeutic drugs that block DNA repair, including poly(ADP-ribose) polymerase (PARP) inhibitors, fail due to lack of tumor-selectivity. When PARP inhibitors and ß-lapachone are combined, synergistic antitumor activity results from sustained NAD(P)H levels that refuel NQO1-dependent futile redox drug recycling. Significant oxygen-consumption-rate/reactive oxygen species cause dramatic DNA lesion increases that are not repaired due to PARP inhibition. In NQO1+ cancers, such as non-small-cell lung, pancreatic, and breast cancers, cell death mechanism switches from PARP1 hyperactivation-mediated programmed necrosis with ß-lapachone monotherapy to synergistic tumor-selective, caspase-dependent apoptosis with PARP inhibitors and ß-lapachone. Synergistic antitumor efficacy and prolonged survival were noted in human orthotopic pancreatic and non-small-cell lung xenograft models, expanding use and efficacy of PARP inhibitors for human cancer therapy.

Depleting Tumor-NQO1 Potentiates Anoikis and Inhibits Growth of NSCLC.

UNLABELLED: The fundamental role that NAD(P)H/quinone oxidoreductase 1 (NQO1) plays, in normal cells, as a cytoprotective enzyme guarding against stress induced by reactive oxygen species (ROS) is well documented. However, what is not known is whether the observed overexpression of NQO1 in neoplastic cells contributes to their survival. The current study discovered that depleting NQO1 expression in A549 and H292 lung adenocarcinoma cells caused an increase in ROS formation, inhibited anchorage-independent growth, increased anoikis sensitization, and decreased three-dimensional tumor spheroid invasion. These in vivo data further implicate tumor-NQO1 expression in a protumor survival role, because its depletion suppressed cell proliferation and decreased lung tumor xenograft growth. Finally, these data reveal an exploitable link between tumor-NQO1 expression and the survival of lung tumors because NQO1 depletion significantly decreased the percentage of ALDH((high)) cancer cells within the tumor population. IMPLICATIONS: Loss of tumor-NQO1 expression inhibits tumor growth and suggests that novel therapeutics directed at tumor-NQO1 may have clinical benefit.

Review of poly (ADP-ribose) polymerase (PARP) mechanisms of action and rationale for targeting in cancer and other diseases.

Poly (ADP-ribose) polymerases (PARPs) are a family of related enzymes that share the ability to catalyze the transfer of ADP-ribose to target proteins. PARPs play an important role in various cellular processes, including modulation of chromatin structure, transcription, replication, recombination, and DNA repair. The role of PARP proteins in DNA repair is of particular interest, in view of the finding that certain tumors defective in homologous recombination mechanisms, may rely on PARP-mediated DNA repair for survival, and are sensitive to its inhibition. PARP inhibitors may also increase tumor sensitivity to DNA-damaging agents. Clinical trials of PARP inhibitors are investigating the utility of these approaches in cancer. The hyperactivation of PARP has also been shown to result in a specific programmed cell death pathway involving NAD+/ATP depletion, mu-calpain activation, loss of mitochondrial membrane potential, and the release of apoptosis inducing factor. Hyperactivation of the PARP pathway may be exploited to selectively kill cancer cells. Other PARP forms, including tankyrase 1 (PARP 5a), which plays an important role in enhancing telomere elongation by telomerase, have been found to be potential targets in cancer therapy. The PARP pathway and its inhibition thus offers a number of opportunities for therapeutic intervention in both cancer and other disease states.

Tumor-selective, futile redox cycle-induced bystander effects elicited by NQO1 bioactivatable radiosensitizing drugs in triple-negative breast cancers.

AIMS: ß-Lapachone (ß-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 ß-lap treatment to elicit bystander effects, DNA damage, and cell death in neighboring NQO1(low) cells. RESULTS: ß-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 ß-lap treatment. ß-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 ß-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 (ß-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.

Catalase abrogates ß-lapachone-induced PARP1 hyperactivation-directed programmed necrosis in NQO1-positive breast cancers.

Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. ß-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 ß-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 ß-lapachone in 2 minutes. ß-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 ß-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. ß-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 ß-lapachone and other NQO1 bioactivatable drugs.

Superparamagnetic iron oxide nanoparticles: amplifying ROS stress to improve anticancer drug efficacy.

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, ß-lapachone (ß-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 ß-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 ß-lap in a tumor-specific, NQO1-dependent manner. Through Fenton reactions, these iron ions escalate the ROS stress in ß-lap-exposed cancer cells, thereby greatly enhancing the therapeutic index of ß-lap. More specifically, a 10-fold increase in ROS stress was detected in ß-lap-exposed cells pretreated with SPION-micelles over those treated with ß-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.

Hemoglobin-based oxygen carrier mitigates transfusion-mediated pancreas cancer progression.

BACKGROUND: Perioperative blood transfusion in pancreatic cancer patients is linked to decreased survival; however, a causal mechanism has not been determined. Previously we have shown that the plasma fraction of stored packed red blood cells (pRBCs) promotes pancreas cancer progression and associated morbidity. We hypothesize these untoward effects will be mitigated by use of a hemoglobin-based oxygen carrier (HBOC). METHODS: Cytokines and growth factors were measured in the plasma fraction from stored pRBCs and in an HBOC via cytokine array followed by formal enzyme-linked immunosorbent assay (ELISA). In an immunocompetent murine model, pancreas cancer progression was determined in vivo by bioluminescence, tumor weight, and number of metastases. RESULTS: Elevated levels of epidermal growth factor (EGF), platelet-derived growth factor BB (PDGF-BB), and regulated upon activation, normal T cell expressed and secreted (RANTES) were present in the plasma fraction of stored pRBCs, but were not found in the HBOC. Intravenous delivery of plasma fraction to mice with pancreatic cancer resulted in increased bioluminescence activity compared with mice that received HBOC. Metastatic events and pancreatic primary tumor weights were significantly higher in animals receiving plasma fraction from stored pRBCs compared with animals receiving HBOC. CONCLUSIONS: Intravenous receipt of the acellular plasma fraction of stored pRBCs promotes pancreatic cancer progression in an immunocompetent mouse model. These untoward events are mitigated by use of an HBOC.

An NQO1 substrate with potent antitumor activity that selectively kills by PARP1-induced programmed necrosis.

Agents, such as ß-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 ß-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 ß-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to ß-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.

Modulating endogenous NQO1 levels identifies key regulatory mechanisms of action of ß-lapachone for pancreatic cancer therapy.

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 ß-lapachone (ß-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 ß-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 ß-lap was determined using an optimal hydroxypropyl-ß-cyclodextran (HPß-CD) vehicle formulation in metastatic pancreatic cancer models. RESULTS: ß-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 ß-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 ß-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.

Prostate cancer radiosensitization through poly(ADP-Ribose) polymerase-1 hyperactivation.

The clinical experimental agent, ß-lapachone (ß-lap; Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether ß-lap may warrant clinical evaluation as a radiosensitizer. ß-Lap killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species, irreversible DNA single-strand breaks (SSB), poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, NAD(+)/ATP depletion, and µ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), ß-lap radiosensitized NQO1(+) prostate cancer cells under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSB level, γ-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of µ-calpain-induced programmed cell death. Radiosensitization by ß-lap was blocked by the NQO1 inhibitor dicoumarol or the PARP-1 inhibitor DPQ. In a mouse xenograft model of prostate cancer, ß-lap synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ∼60% of human prostate tumors evaluated relative to adjacent normal tissue, where ß-lap might be efficacious alone or in combination with radiation. Our findings offer a rationale for the clinical utilization of ß-lap (Arq 501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation.

Beta-lapachone micellar nanotherapeutics for non-small cell lung cancer therapy.

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.

Dual phosphoinositide 3-kinase/mammalian target of rapamycin blockade is an effective radiosensitizing strategy for the treatment of non-small cell lung cancer harboring K-RAS mutations.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC often harbors oncogenic K-RAS mutations that lead to the aberrant activation of several intracellular networks including the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Oncogenic K-RAS predicts poor prognosis and resistance to treatment with ionizing radiation (IR). Oncogenic K-Ras expression in the respiratory epithelium is sufficient to initiate NSCLC tumorigenesis, which requires the catalytic subunit of PI3K. Thus, effective inhibition of the PI3K signaling should lead to significant antitumor effects. However, therapy with rapamycin analogues has yielded disappointing results due in part to compensatory up-regulation of AKT. We hypothesized that dual PI3K/mTOR blockade would overcome these limitations. We tested this hypothesis with BEZ235, a novel dual PI3K/mTOR inhibitor that has recently entered clinical development. We found that BEZ235 induces a striking antiproliferative effect both in transgenic mice with oncogenic K-RAS-induced NSCLC and in NSCLC cell lines expressing oncogenic K-RAS. We determined that treatment with BEZ235 was not sufficient to induce apoptosis. However, we found that dual PI3K/mTOR blockade effectively sensitizes NSCLC expressing oncogenic K-RAS to the proapoptotic effects of IR both in vitro and in vivo. We conclude that dual PI3K/mTOR blockade in combination with IR may benefit patients with NSCLC expressing oncogenic K-RAS. These findings may have general applicability in cancer therapy, because aberrant activation of PI3K occurs frequently in human cancer.

In vivo off-resonance saturation magnetic resonance imaging of alphavbeta3-targeted superparamagnetic nanoparticles.

Magnetic resonance imaging is a powerful clinical imaging technique that allows for noninvasive tomographic visualization of anatomic structures with high spatial resolution and soft tissue contrast. However, its application in molecular imaging of cancer has been limited by the lack of sensitivity and detection accuracy in depicting the biochemical expression of these diseases. Here, we combine an ultrasensitive design of superparamagnetic polymeric micelles (SPPM) and an off-resonance saturation (ORS) method to enhance the imaging efficacy of tumor biomarkers in vivo. SPPM nanoparticles encoded with cyclic(RGDfK) were able to target the alpha(v)beta(3)-expressing microvasculature in A549 non-small cell lung tumor xenografts in mice. ORS greatly improved tumor detection accuracy over the conventional T(2)*-weighted method by its ability to turn "ON" the contrast of SPPM. This combination of ORS imaging with a tumor vasculature-targeted, ultrasensitive SPPM design offers new opportunities in molecular imaging of cancer.

Intratumoral delivery of beta-lapachone via polymer implants for prostate cancer therapy.

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.

Nonhomologous end joining is essential for cellular resistance to the novel antitumor agent, beta-lapachone.

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.