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

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 apoptotic cell death response [14].

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

One of the key components in the cell death response by NQO1-bioactivatable 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 death [10, 11, 16, 20, 24–27].

#### **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

**147**

**Figure 2.**

**Figure 1.**

*NQO1-Bioactivatable Therapeutics as Radiosensitizers for Cancer Treatment*

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

of NQO1-bioactivatable drugs [28]. NAMPT knockdown has also been shown to sensitize cancer cells to ROS induction through ionizing radiation [29, 30].

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

*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-Calpain-*

*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.*

Cancer cells, tissues, and organs subjected to ionizing radiation experience a wide

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

*mediated programmed necrosis known as NAD + -Keresis.*

of NQO1-bioactivatable drugs [28]. NAMPT knockdown has also been shown to sensitize cancer cells to ROS induction through ionizing radiation [29, 30].
