Nitric Oxide (Donor/Induced) in Chemosensitization

publications.

Preface

Benjamin Bonavida

The physiological roles mediated by nitric oxide (NO) and derivatives have been the subject of many studies spanning embryogenesis, differentiation, morphogenesis, homeostasis, immune systems, etc. NO has also been used as a therapeutic in heart diseases and also in erectile dysfunction; however, the role of NO in cancer has been controversial. For many years, several reports indicated its protumorigenic activity and other reports of its antitumorigenic activity. This contradicting effect of NO was, in part, resolved by findings demonstrating that low levels of NO are protumorigenic while high levels are antitumorigenic on one hand and the type of tumor tissues being examined on the other hand. The potential therapeutic effect of NO in cancer began to be explored by several scientists, and its focus was initiated by the First International Conference on NO and Cancer in Paris, France, in 2007. Subsequently, the second through the Fourth International Workshops on NO and Cancer took place in Paris, France (2007); Kyoto, Japan (2010); Kingston, Canada (2013); and Seville, Spain (2015). Of interest, the Fifth International Workshop was held in Bologna, Italy, in Mar. 2017. In addition, two published books on NO and cancer have been edited by the editor of this volume [1,2].

The present volume titled Nitric Oxide (Donor/Induced) in Chemosensitization is the first volume that has been focused to address the biochemical, molecular, and genetic approaches in which NO and/or derivatives are used as chemosensitizing agents. Several contributors in this volume have reviewed different aspects related to the sensitizing properties mediated by NO in different settings and models. This volume consists of 14 review chapters that cover a wide spectrum of relevant topics spanning from studies in the laboratory and in animals and potential translational applications in clinical studies. Briefly, the highlights of each chapter are briefly presented below.

The first chapter by Edwards et al. titled Nitric-Oxide-Mediated Chemosensitization: Gene Therapy Versus Exogenous Introduction of NO Donors describes the potential application of NO as a hypersensitizing agent and its use in combination with chemotherapy to reverse drug resistance. The authors review the application of NO donors in comparison with gene therapy approaches. The authors caution of the use of gene therapy with NO for cancer applications due to several issues related to safety, reliability, specificity, and efficacy and also due to the microenvironment complexity and how gene expression will be manifested. Nevertheless, the authors are optimistic for the future of gene therapy with iNOS. They also believe that exogenous NO donor therapeutics will be more adaptable for chemosensitization in the clinic.

The chapter by Bonavida titled Nitric-Oxide Donors Sensitize Resistant Cancer Cells to Apoptosis Induced by Chemotherapy: Molecular Mechanisms of Sensitization discusses various studies performed by his group and by his collaborators and by others on the underlying biochemical and molecular mechanisms by which NO sensitizes drug-resistant tumor cells to chemotherapy-induced apoptosis. The discussion is centered on the inhibition of the constitutive expression of the cell survival/antiapoptotic NF-κB pathway in the majority of cancers. Treatment with NO donors inhibits NF-κB by S-nitrosylation of p50 and p65. In addition, a dysregulated NF-κB/Snail/YY1/RKIP/PTEN loop was reported in many cancers and whose role is to regulate cell survival, cell growth, and resistance to cytotoxic drugs. The introduction of NO donors modifies this resistant loop and results in chemosensitization of drug-resistant cancer cells. It is suggested of the potential clinical application of NO donors, under suitable conditions, in combination with low subtoxic doses of chemotherapy for the treatment of drug-resistant cancer cells.

The chapter by Scincinski et al. titled RRx-001 Reset: Chemosensitization via NO-Mediated M1 Macrophage Repolarization reviews the novel compound NO donor RRX-001 and its use in various in vitro model systems and also used in phase 1 and phase 2 clinical studies. They focus on the chemo- and radiosensitization properties of RRX-001 and demonstrate its chemosensitizing activity and also its role in arming tumor-associated macrophages. They also suggest that this new compound has the potential of its use in cancer patients who no longer have any treatment modality.

The chapter by Garrido et al. titled Nitric Oxide and Nitric-Oxide Donors in Preclinical Studies of Breast and Prostate Cancer reviewed the role of NO in different therapeutic approaches for breast and prostate cancers. While the authors are convinced of the merit of using NO in these cancers, they are careful in suggesting additional studies to better understand the role of NO in the different stages of the disease and its environment during the process of cancer progression. They suggest that NO be considered as an adjuvant in cancer therapy. They also suggest the prospective significance of iNOS overexpression in breast cancer and melanoma cancer and contradict the potential chemosensitizing activity of NO. However, they propose a need to investigate every cancer setting and microenvironment in order to determine whether to use NO donors or NO inhibitors.

The chapter by Ali et al. titled Evaluation of the Impact of Nitric Oxide on Resistance to Platinum-Based Chemotherapeutics reviewed the role of NO in tumor cells that are resistant to platinum-based chemotherapeutics. Platinum drugs are widely used in the treatments of various types of cancer and seldom used as monotherapy. Many patients are initially resistant or develop resistance to combination therapies. However, there are now agents to either sensitize or synergize with conventional chemotherapeutic drugs to reverse resistance. The authors discuss the underlying mechanism of the chemosensitizing effect mediated by NO. They also caution on the potential side effects of systemic NO administration and suggested that the combination of NO with nanoparticles may reduce some side effects. However, these NO nanosphere complexes must be designed and calibrated for each clinical administration.

The chapter by Plenchette et al. titled Nitric Oxide and Platinum Derivative-Based Regimens for Cancer Treatment: From Preclinical Studies to Clinical Trials reviews the literature on the therapeutic application of NO donors in combination with platinum compounds in both preclinical and clinical settings. They also reviewed the use of NO donors as sensitizing agents for other therapies including checkpoint inhibitor monoclonal antibodies. They also suggest that NTG as an NO donor is a good candidate for the use in combination therapy.

The chapter by Kashfi and Esmaili titled NO-H2S-Releasing Chimeras as a Multifaceted Approach to Cancer Therapy reviews the roles of nitric oxide and hydrogen sulfide donors as chemosensitizing agents in experimental model systems, both in vitro and in mice. The authors describe various anticancer compounds that incorporate both NO and H2S in complexes and focused on the recent H2S-releasing pharmaceuticals in their anticancer activities. They also discuss the minimum toxicity mediated by the NO-H2S complexes. Clearly, the authors suggest the potential clinical application of these compounds in the clinic.

The chapter by Vanini et al. titled Nitric-Oxide-Based Anticancer Therapeutics: The New Technologies of the Nanoparticles discusses the limitations of applying NO donors as chemosensitizing agents due to their short half-lives, the poor concentrations that reach the cancer cells, and also that they are highly nonspecific. They suggest that the application of nanoparticle-based delivery systems of NO and targeting them to the cancer cells will result in minimal side effects and overriding the above limitations by the NO donors. They also discuss and update the potential therapeutic applications of the combination of NO donors and chemotherapeutic drugs that are incorporated in nanoparticles.

Imran and Huerta's chapter titled, Nitric Oxide in Rectal Cancer: From Mice to Patients reviews the role of NO in rectal cancer in both preclinical and clinical settings. They discuss their findings in experimental rectal mouse models and their findings that the NO donor DETANOATE was a potent radiosensitizer. In addition, they report the findings in patients who were administered NO via NTG transdermal patches and report that this administration was safe when administered in combination with conventional treatments. Their findings have established the rationale for a phase 2 clinical trial.

Sen et al. chapter titled Chemoprotective and Chemosensitizing Effects of Nitric Oxide and Other Biologically Active Gases in Breast Cancer Chemotherapy: Potential Implications reviewed the literature on the various properties and effects of endogenous NO produced in breast cancer cells and breast tumors as well as the NO role in cancer homeostasis. They discuss how endogenous NO and H2S may cross talk and, thus, may reveal potential novel interventions. They also discuss in the chapter the relationship between tumor-associated macrophages and NO.

The chapter by Manke et al. titled Role of Nitric Oxide in Cancer Stem Cell Regulation and Metastasis discusses the role of NO in the regulation of its effect on the cell physiology and its role in metastases. They discuss the oncogenic properties of NO in various types of cancers. They also discuss the molecular mechanisms by which cancer stem cells regulate tumor growth invasiveness and metastases. They also point out that NO regulates tumor metastases by affecting cancer stem cells regulated by proteins such as caveolin-1. Thus, their study suggests the potential use of NO inhibitors to prevent CSC-mediated tumor growth.

The chapter by Lo Bello et al. titled Nitric Oxide Interacting with Glutathione Transferases reviewed the interaction of NO with glutathione transferases in the presence of GSH and iron. The authors emphasize the importance of delineating the underlying mechanisms that are involved in the metabolism of NO present at high concentrations in the cells in order to initiate cellular signaling involved in its anticancer activity.

The chapter by Weidensteiner titled Application of MRI to Study the Role of Nitric Oxide in Cancer Therapy discussed the application of noninvasive imaging methods to study the function and the effect of NO and NO donors in vivo as anticancer agents. She discusses the superior application using MRI as an imaging technique. While MRI has been used in many settings, its use in NO was limited. She lists the MRI as a superior method to monitor different strategies of NO therapy.

The chapter by Peñarando et al. titled "S-Nitrosothiol Metabolism in Cancer and Therapeutic Implications" reviews the complexity of NO-mediated effects in the cells and its various chemical reactions that may lead to various phenotypic and genetic changes in the cancer cells. They point out the difficulty of translating preclinical data from the bench to the bedside. They report the identification and the characterization of enzyme-mediated processes that are responsible for targeting the S-nitrosylation or the denitrosylation of proteins. Such modifications might be exploited for various antitumor therapeutic approaches and their potential as prognostic biomarkers.

It is clear from the above that this volume has provided various mechanisms underlying potential therapeutic effects of NO and also its pitfalls. Clearly, the excellent contributors have provided several suggestions and means toward the future application of NO as a general chemosensitizing agent in the reversal of drug-resistant cancers.

The editor acknowledges the significant assistance in the preparation and editing of the contents of this volume and acknowledges Arah Cho, Kevin Li, and Leah Moyal. Their assistance was valuable without which this volume would not have been completed in time.

References

[1] Bonavida B. Nitric oxide (NO) and cancer: prognosis, prevention, and therapy. In: Bonavida B., ed. Cancer drug discovery and development. New York: Springer; 2010.

[2] Bonavida B., ed. Nitric oxide and cancer: pathogenesis. New York: Springer; 2015:1–308.

Chapter 1

Nitric-Oxide-Mediated Chemosensitization: Gene Therapy Versus Exogenous Introduction of NO Donors

Melissa Edwards*; Ilham Alshiraihi*; Christian Schmidt†; Joachim Storsberg†; Mark A. Brown*,‡    * Colorado State University, Fort Collins, CO, United States

† Fraunhofer-Institute for Applied Polymer Research (IAP), Potsdam-Golm, Germany

‡ Colorado School of Public Health, Fort Collins, CO, United States

Abstract

Growing resistance of cancer cells to cytotoxic agents combined with dose-limiting toxicity continue to limit the full potential of chemotherapeutics in the clinical management of tumors. To respond to this impediment, researchers are increasingly turning to prospective adjuvant therapeutics for their ability to hypersensitize resistant malignant cells and, thereby, restore chemosensitivity. Among these adjuvants, nitric oxide has repeatedly been proved to serve as an effective hypersensitizing agent. Herein, we consider the applications of gene therapy versus nitric oxide donors for the delivery of chemosensitizing concentrations of nitric oxide.

Keywords

Chemosensitization; Gene therapy; iNOS; Nitric oxide donor; Prodrug

Abbreviations

DETA/NO diethylenetriamine/nitric oxide

DISC death-inducing signaling complex

GAPDH glyceraldehyde-3-phosphate dehydrogenase

HO-1 heme oxygenase-1

iNOS inducible nitric oxide synthase

NF-κβ nuclear factor-kappa B

NO nitric oxide

NOS nitric oxide synthase

SNO S-nitrosothiol

TNF tumor necrosis factor

Introduction

The combination of chemotherapeutic resistance and dose-limiting toxicity severely restricts the potential impacts of chemotherapeutics in the management of malignant neoplasias [1–4]. These constraints have motivated a precipitous increase in studies related to adjuvant therapies devised to restore the tumor-directed cytotoxicity of chemotherapeutics. Among them, nitric oxide (NO) has been shown to induce devastating chemosensitization of refractory tumors [5–10]. Thus, the stage is set for the full-scale development of a therapeutic that capitalizes upon the hypersensitizing effects of NO. This begs the question: what mode of delivery—gene therapy or introduction of exogenous NO donors—is more likely to provide the greatest benefit and the least risk? To answer this question, we will begin with an overview of the molecular mechanisms of NO-induced cytotoxicity in tumors.

Some of the earliest studies on NO, as it relates to tumor biology, illustrated that it is a powerful inducer of macrophage-mediated cytotoxicity [11,12]. Since then, additional studies have reported the involvement of NO in the regulation of other tumor-targeting immunological mechanisms [13–15]. Similarly, NO interaction with superoxide anions has been linked with the induction of apoptosis in malignant cells as the result of the formation of peroxynitrite and its subsequent impacts on mitochondrial membrane permeability, leading to the release of cytochrome c oxidase [16,17].

One of the central mechanisms of NO in cellular signaling is mediated by posttranslational modifications to important regulatory proteins [18,19]. The S-nitrosylation reaction produces S-nitrosothiol (SNO) following the reversible addition of a nitroso moiety to the reactive thiol of cysteine residues [20–22]. S-Nitrosylation depends upon the redox state at the reaction site. Selectivity for the sulfhydryl group of thiols is governed by protein-protein interactions and colocalization with NOS [23]. Cellular SNO concentration is ultimately regulated by denitrosylases such as thioredoxin [24], GSNO reductase [25], and xanthine oxidoreductase [26]. A range of protein targets have been reported for which the S-nitrosylation modification governs processes associated with DNA repair, apoptosis, and transcription [27–29]. Among them, the Fas receptor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NF-κB, and Hdm2 represent some of the most promising pathways in the clinical management of cancer.

The Fas receptor (Fig. 1A), involved in signaling of apoptosis, is a part of the tumor necrosis factor (TNF) superfamily. Fas ligand binding to the receptor results in caspase activation and establishment of the death-inducing signaling complex (DISC) [43,44]. Although malignant cells normally express Fas, the binding of FasL often fails to activate apoptosis mechanisms in these cells, thereby preventing the formation of DISC [44,45]. However, S-nitrosylation of a cytoplasmic cysteine residue on Fas increases its ability to recruit lipid rafts that have been shown to induce formation of the DISC complex and to reestablish sensitivity to the apoptotic cascade associated with Fas [30–33].

Fig. 1 (A) Structural representation of FAS/CD95 transmembrane domain: S-nitrosylation of a cytoplasmic cysteine residue on Fas induces formation of the DISC complex and facilitates apoptosis [ 30 – 33 ]. (B) Structural representation of Hdm2: S-nitrosylation of Hdm2 hinders its association with p53, thereby allowing repair of critically damaged DNA or the destruction of malignant cells [34 , 35] . (C) Structural representation of GAPDH: S-nitrosylation of GAPDH leads to cytotoxicity [ 36 – 38 ]. (D) Structural representation of NF-κβ: S-nitrosylation of NF-κB hinders DNA binding and inhibits upregulation of antiapoptotic genes [39 , 40] . S-Nitrosylation of IκB blocks nuclear translocation of NF-κB and inhibits upregulation of antiapoptotic genes [41 , 42] .

Hdm2 (Fig. 1B), is a potent regulatory protein for the activity of p53 [46]. In the absence of critical DNA damage, the attachment of Hdm2 to p53 is repressive by virtue of its hindrance to the transactivation domain of p53 [47]. Likewise, Hdm2, which is often aberrantly expressed at excessive levels in human malignancies [48], has the capacity to inhibit the expression of p53 [47]. However, S-nitrosylation of Hdm2 precludes its association with p53, thereby allowing repair of critically damaged DNA and/or the destruction of potentially malignant cells [34,35].

GAPDH (Fig. 1C) has a range of important cellular functions [49] related to glycolysis [50], regulation of gene expression [51], axoplasmic transport [52], and shuttling of vesicles from the endoplasmic reticulum to the Golgi complex [53]. It is also subject to modification via S-nitrosylation at a cysteine residue in its catalytic domain [32,49,54,55]. In response to cellular stress, this modification facilitates the binding of GAPDH to the ubiquitin ligase, Siah1 [36,56]. The resulting complex is translocated into the nucleus and initiates the degradation of Siah1 substrates [56,57]. Those degradation products create a cytotoxic environment leading to cell death [36]. Mitochondrial GAPDH is, likewise, involved in the initiation of cellular death by arresting ATP production following stress-induced spikes in NO [32,37,38].

The transcription factor, nuclear factor-kappa B (NF-κB) (Fig. 1D), is chiefly involved in inflammatory pathways, cell survival signaling, and cell proliferation [58–60]. In the absence of cellular stress, NF-κB inhibits apoptosis by initiating the expression of antiapoptotic sequences. However, in response to cell damage, cytoplasmic IκB interactions with NF-κB prevent nuclear translocation, thereby preventing its activation of antiapoptotic genes [61]. Thus, activation of NF-κB requires phosphorylation of IκB that, in turn, hinders its interaction with NF-κB [61]. S-Nitrosylation of NF-κB occurs at a residue involved in DNA binding, thereby inhibiting its upregulation of antiapoptotic genes [39,40]. Likewise, S-nitrosylation of IκB blocks the phosphorylation of that protein allowing for cytoplasmic sequestration of NF-κB [41,42].

Gene Therapeutic Expression of