**4.5 Exploiting NQO1-bioactivatable drugs as radiosensitizers**

Cancer cells, tissues, and organs subjected to ionizing radiation experience a wide spectrum of DNA lesions including SSBs, DSBs, AP sites and DNA-protein crosslinks. One unrepaired DSB is lethal to the cell [21, 31]. Hence, NQO1-bioactivatable drugs, when combined with IR (**Figure 1**), synergistically kill cancer cells due to the combined effect of DNA damage and PARP1 hyperactivation [21, 32]. Sublethal doses of NQO1 drugs and IR combine to release massive amounts of ROS due to

#### **Figure 1.**

*Translational Research in Cancer*

apoptotic cell death response [14].

death [10, 11, 16, 20, 24–27].

**necrosis**

these agents can also be activated by other drug metabolizing enzymes [18]. Human

Cancer cells overexpressing NQO1 and exposed to NQO1-bioactivatable drugs, such as β-lapachone, DNQ or IB-DNQ, acquire extensive DNA lesions as evidenced by alkaline comet assays [11]. The unstable hydroquinone form of these NQO1 bioactivatable drugs reacts with two oxygen molecules spontaneously to regenerate the original compound [20]. This futile redox cycle consumes ~60 moles of NADPH to generate ~120 moles of ROS in ~2 min for β-lapachone, leading to the generation of permeable hydrogen peroxide (H2O2). This diffuses into the nucleus and causes massive oxidative stress and SSBs [16]. Initial DNA damage is mainly through the formation of altered bases, SSBs, and apurinic/apyrimidinic (AP) sites generated through incorporation of 8-oxo-deoxyguanine [21]. Ultimately, damage caused by H2O2 results in extensive SSBs and DSBs. These lesions lead to PARP hyperactivation that can be prevented by BAPTA-AM (chelates Ca2+), PARP inhibitors, or the NQO1 inhibitor dicoumarol, in NQO1+ cells. In contrast, cells deficient in NQO1 due to NQO1 polymorphisms, \*2[C609T] or \*3[C465T], are unaffected by exposure to NQO1-bioactivatable compounds [14], lacking the enzyme activity for redox cycling Hyperactivation of PARP rapidly degrades the increased NAD+ pools generated as a result of the oxidation of NADH in the futile cycle [11, 20, 22]. NAD+ loss is not seen in cells treated with PARP1 inhibitors; instead, cells exposed to PARP inhibitors in combination with NQO1-bioactivatable drugs undergo a synergistic

**4.3 Calcium release, DNA damage and μ-Calpain-dependent programmed** 

**4.4 NQO1-bioactivatable drugs lead to perturbations in metabolic pathways**

Treatment with NQO1-bioactivatable drugs causes wide-scale metabolic changes in the cell, which can be attributed to cell death overwhelming the cellular machinery. Altering key enzymes in NAD metabolism results in synergy with NQO1 bioactivatable drugs. NAMPT is an important source of reducing equivalents for redox balance in cancer cells. Pretreatment with FK866, a NAMPT inhibitor, leads to accelerated cell death due to decrease in NAD+/NADH levels and reduced doses

drugs is the release of calcium from the core endoplasmic reticulum (ER) stores, which is otherwise inert [11, 23]. This results in specific programmed necrosis referred to as NAD+ -Keresis. Pre-treatment, with the calcium chelator, BAPTA-AM, suppresses PARP hyperactivation and results in specific inhibition of NQO1-dependent cell death by NQO1-bioactivatable drugs. Extensive DNA damage along with Ca2+ release from the ER results in the hyperactivation of PARP1 in NQO1+ cancer cells. PARP1 hyperactivation rapidly degrades the NAD+ and causes concomitant ATP losses within 30–40 min of drug treatment. μ-Calpain activation is observed upon treatment with NQO1-bioactivatable drugs within 8–24 h [16, 24]. The multitude of damage caused by treatment with these drugs overwhelms DNA repair machinery and depletes the cells of the energy resources, culminating in cell

One of the key components in the cell death response by NQO1-bioactivatable

cancer cells overexpressing NQO1 have been shown to be sensitive to NQO1 bioactivatable drugs alone and in combination with PARP inhibitors, cisplatin, radiation, and NAMPT inhibitors both in cell culture and xenograft models [14, 19].

**4.2 NQO1-dependent ROS formation and PARP hyperactivation**

**146**

*Radiation sensitization by NQO1 bioactivatable drugs: sublethal doses of β-lapachone when bioactivated by NQO1 release massive amounts of ROS, resulting in synergy with IR and increased programmed necrosis. NQO1 bioactivatable drugs in combination with IR show tremendous synergy even at low doses. The combined effect of DNA damage and PARP hyperactivation provides more lethality to a cancer cell whereas NQO1 provides the specificity. This leads to increased ROS, gH2AX formation, hyperactivation of PARP, massive NAD and ATP losses, prevention of DSB repair, perturbations in the metabolic pathways, and μ-Calpainmediated programmed necrosis known as NAD + -Keresis.*

#### **Figure 2.**

*Sublethal doses of IR and β-lap in NQO1+ LNCaP cells cause PARP-1 hyper-activation and dramatic ATP loss: A, LNCaP cells expressing or lacking NQO1 were treated with IR + β-lap and monitored for PAR formation—UT, untreated control for IR; V, vehicle; DMSO only. B, Synergistic ATP loss was noted after IR + β-lap compared to single treatments alone. Results are means ± SE for experiments performed three times in duplicate. Student's t-tests compared single to combined treatments. \*\*\*p < 0.001, \*\*p < 0.01.*

#### **Figure 3.**

*β-Lap inhibits DNA double strand break repair: A. log-phase A549 NSCLC cells were treated with or without β-lap (6 μM) and cell extracts prepared at various times during treatment to detect PAR-PARP formation, γ-H2AX (pS139), pS1981 ATM, total ATM (t-ATM) and α-tubulin steady-state levels by Western blot. A549 cells were also exposed or not to IR (8 Gy) and analyzed 1 h later. Mock, non-irradiated cells. DM, media alone. B. Graphical representation of data shown in Figure 2A. C. Representative images of A549 cells exposed or not to IR (2 Gy) alone, β-lap (3 μM, 2 h) alone, the combination [IR (2 Gy) + β-lap (3 μM, 2 h)], or the combination with DIC (50 μM, NQO1 inhibitor) and assessed for DSB breaks over time (0–120 min) using 53BP1 as the surrogate marker (in red). Cells were also stained for nuclear DNA using DAPI (in blue). Scale bar = 10 μm. D. Graphical representation of data presented in Figure 2C; \*\*\*\*p < 0.0001.*

#### **Figure 4.**

*β-Lap radiosensitizes subcutaneous A549-luc xenografts in athymic nude mice: A. subcutaneous A549-luc xenografts (400 mm3 ) were generated in athymic nude mice and then treated with or without IR (2 Gy) then immediately with or without β-lap (20 mg/kg) for 5 treatments every other day. Representative antitumor responses (at day 20 post-treatment) are demonstrated for β-lap alone, IR alone, and the IR + β-lap combination. B. Antitumor responses (tumor volumes, mm3 ) over time are shown for the treatments described in Figure 3A. C. Overall survival of animals treated as described in Figure 3A. D. PK values for plasma and subcutaneous vs. orthotopic A549-luc tumors in athymic nude mice. Note the significantly high levels of β-lap in orthotopic vs. subcutaneous A549 tumor tissue, whereas plasma levels were identical in both sets of mice.*

their synergy, resulting in PARP hyperactivation, loss of nucleotides and increased programmed necrosis (**Figures 2** and **3**), beyond the capabilities of the single agents (IR or NQO1-bioactivatable drug) alone. Head and neck cancers, PDA and NSCLC

**149**

**Acknowledgements**

A. Boothman.

*NQO1-Bioactivatable Therapeutics as Radiosensitizers for Cancer Treatment*

have been shown to be sensitive to nontoxic doses of β-lapachone when combined with IR [21, 32]. Using NQO1-bioactivatable drugs as radiosensitizers leads to increases in ROS, γH2AX formation, hyperactivation of PARP1, massive NAD+ and ATP losses, inhibition of DSB repair, perturbation in carbon flux pathways and μ-Calpain mediated programmed necrosis known as NAD + -Keresis. The cell death responses observed are independent of any oncogenic drivers [21, 31–33]. This lethal combination between radiation therapy and NQO1-bioactivatable drugs prolongs long-term survival and promotes enhanced tumor shrinkage at non-toxic doses of each agent (IR and Drug, **Figure 4**). Thus, combining NQO1-bioactivatable drugs with radiation therapy, should be a long-standing treatment modality for tumors

**5.1 Advantages of NQO1-bioactivatable drugs vs. other radiosensitizers**

The major advantage of using NQO1-bioactivatable drugs as radiosensitizers is the tumor selectivity afforded by the drugs themselves. Synergy is afforded by a number of tumor-selective responses to the drugs. First, the dependence of the drugs on NQO1 levels is perfect for the specific treatment of various difficult-totreat human cancers, including non-small cell lung, pancreatic, breast, prostate, and head and neck cancers. Tumor selectivity requires approximately 100 units of enzyme activity, whereas lower levels of NQO1 results in mild metabolomic alterations used for the treatment of metabolic syndromes [34]. Second, the minimum time of exposure of 30–120 min fits the pharmacokinetics of the drug. It should be noted that all studies thus far indicate that the drugs have to be available immediately after or at the same time as exposure with IR. Pre-treatment prior to IR is ineffective. Third, synergy between NQO1-bioactivatable drugs and IR occurs due to PARP1 hyperactivation causing massive NAD+ and ATP loss, preventing repair of the DNA damage created by IR. NQO1-bioactivatable drugs are highly specific to tumors, causing little normal tissue toxicity, which is unaffected by IR treatment [14, 16, 20, 25, 31]. Preclinical in vivo data suggest that radiosensitization trials with NQO1-bioactivatable drugs are warranted for non-small cell lung, pancreatic,

A clinical trial of radiation sensitization effects of the new drug, isobutyldeoxynyboquione (IB-DNQ ), against non-small cell lung (NSCLC) and/or pancreatic adenocarcinomas (PDAC) is warranted. These cancers are almost uniformly NOQ1 over-expressive and they have routinely low levels of catalase [14]. We have developed CLIA assessments of NQO1 status and enzymatic levels for these studies. The pharmacokinetics of IB-DNQ in these cancers, particularly in NSCLC and PDAC cancers, is relatively short at about 6 h, but long enough for sensitization of tumors to the NQO1-bioactivatable drug + IR. Biomarker and DSB repair kinetics are ongoing in our laboratory in preparation for these radiosensitization studies.

This work was supported by NIH/NCI R01s - CA210489 and CA224493 to David

*DOI: http://dx.doi.org/10.5772/intechopen.90205*

breast prostate, and head and neck cancers.

**5.2 Future directions for NQO1-bioactivatable drugs**

overexpressing NQO1.

**5. Discussion**

#### *NQO1-Bioactivatable Therapeutics as Radiosensitizers for Cancer Treatment DOI: http://dx.doi.org/10.5772/intechopen.90205*

have been shown to be sensitive to nontoxic doses of β-lapachone when combined with IR [21, 32]. Using NQO1-bioactivatable drugs as radiosensitizers leads to increases in ROS, γH2AX formation, hyperactivation of PARP1, massive NAD+ and ATP losses, inhibition of DSB repair, perturbation in carbon flux pathways and μ-Calpain mediated programmed necrosis known as NAD + -Keresis. The cell death responses observed are independent of any oncogenic drivers [21, 31–33]. This lethal combination between radiation therapy and NQO1-bioactivatable drugs prolongs long-term survival and promotes enhanced tumor shrinkage at non-toxic doses of each agent (IR and Drug, **Figure 4**). Thus, combining NQO1-bioactivatable drugs with radiation therapy, should be a long-standing treatment modality for tumors overexpressing NQO1.
