Therapeutic strategies by modulating oxygen stress in cancer and inflammation

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Abstract

Oxygen is the essential molecule for all aerobic organisms, and plays predominant role in ATP generation, namely, oxidative phosphorylation. During this process, reactive oxygen species (ROS) including superoxide anion (O2radical dot) and hydrogen peroxide (H2O2) are produced as by-products, while it seems indispensable for signal transduction pathways that regulate cell growth and reduction–oxidation (redox) status. However, during times of environmental stress ROS levels may increase dramatically, resulting in significant damage to cell structure and functions. This cumulated situation of ROS is known as oxidative stress, which may, however, be utilized for eradicating cancer cells.

It is well known that oxidative stress, namely over-production of ROS, involves in the initiation and progression of many diseases and disorders, including cardiovascular diseases, inflammation, ischemia–reperfusion (I/R) injury, viral pathogenesis, drug-induced tissue injury, hypertension, formation of drug resistant mutant, etc. Thus, it is reasonable to counter balance of ROS and to treat such ROS-related diseases by inhibiting ROS production. Such therapeutic strategies are described in this article, that includes polymeric superoxide dismutase (SOD) (e.g., pyran copolymer-SOD), xanthine oxidase (XO) inhibitor as we developed water soluble form of 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP), heme oxygenase-1 (HO-1) inducers (e.g., hemin and its polymeric form), and other antioxidants or radical scavengers (e.g., canolol).

On the contrary, because of its highly cytotoxic nature, ROS can also be used to kill cancer cells if one can modulate its generation selectively in cancer. To achieve this goal, a unique therapeutic strategy was developed named as “oxidation therapy”, by delivering cytotoxic ROS directly to the solid tumor, or alternatively inhibiting the antioxidative enzyme system, such as HO-1 in tumor. This anticancer strategy was examined by use of O2radical dot or H2O2-generating enzymes (i.e., XO and d-amino acid oxidase [DAO] respectively), and by discovering the inhibitor of HO-1 (i.e., zinc protoporphyrin [ZnPP] and its polymeric derivatives). Further for the objective of tumor targeting and thus reducing side effects, polymer conjugates or micellar drugs were prepared by use of poly(ethylene glycol) (PEG) or styrene maleic acid copolymer (SMA), which utilize EPR (enhanced permeability and retention) effect for tumor-selective delivery. These macromolecular drugs further showed superior pharmacokinetics including much longer in vivo half-life, particularly tumor targeted accumulation, and thus remarkable antitumor effects.

The present review concerns primarily our own works, in the direction of “Controlling oxidative stress: Therapeutic and delivery strategy” of this volume.

Introduction

Reactive oxygen species (ROS) is a collective term of oxygen-derived species, including not only the oxygen radicals (superoxide anion radical O2radical dot, hydroxyl radicals radical dotOH etc.) but also some non-radical derivatives of O2 (hydrogen peroxide H2O2, singlet oxygen, alkyl peroxide etc.). They are generally very small molecules and are highly reactive due to the presence of unpaired valence shell electrons. ROS is potentially hazardous and it can be a by-product of cellular metabolism. It also involves cell development, growth, survival, cell killing, aging, drug metabolism, pathogenesis of viral infection and development of cancer [1], [2]. During the production of ROS, molecular oxygen usually acts as electron acceptors to become oxygen free radicals. In aerobic life, where molecular oxygen is ubiquitous, ROS becomes the primary mediators of free radical reactions in cells, which are generated during the production of ATP by aerobic metabolism in mitochondria. The leakage of electrons from mitochondria, during the electron-transport steps of ATP production, generates ROS, e.g. O2radical dot. O2radical dot is converted by superoxide dismutase (SOD) to generate H2O2, from which further radical dotOH is generated in a reaction catalyzed by Fe2+ or Cu2+ ions. In addition to these ROS, oxides of nitrogen (radical dotNO, radical dotNO2) are also free radicals, and they are frequently called reactive nitrogen species (RNS), and play important roles in biology similar to ROS.

ROS is a group of highly reactive molecules, which can react quickly and damage various types of biomolecules, including proteins [3], DNA [4] and lipids [5]. Under physiological conditions, to overcome the potential toxicity of ROS, cells have evolved a series of antioxidative defense systems to counteract these highly dangerous and extremely reactive insults. These defense systems include intracellular SOD, catalase and glutathione peroxidase that eliminate O2radical dot and H2O2. There are also other enzymes or compounds that contribute to scavenging free radicals (e.g., heme oxygenase-1 (HO-1), ascorbate, tocopherol and glutathione) (Fig. 1), by which the cellular insults caused by ROS are reduced to a nontoxic level [6].

As discussed above, the balance of ROS formation and antioxidative defense level is crucial for cell survival and growth, and it is very important for the cell to remove ROS properly for it to remain viable and maintain its vital function. Namely, normal cellular function will be altered depending on this balance, which will in turn affect the fate of the cell. For example, most aerobic bacteria have adequate antioxidative systems, including SOD and catalase, which can eliminate hazardous ROS from aerobic respiration or ambient oxygen-derived ROS, thus reduce the potential insults of ROS. When an antioxidative enzyme defective mutant (e.g. SOD (−)) is generated, the growth of the bacteria in aerobic condition becomes suppressed. However, its growth in anaerobic condition is normal [7]. So-called facultative bacteria can adjust their metabolism depending on oxygen pressure. Similar phenomenon could be found in cancer cells. Cancer cells, particularly in the center area of the tumor nodule or mass, where ambient oxygen pressure is low, i.e. hypoxic conditions, they adapt more like anaerobic bacteria, having low levels of mitochondrial oxidative phosphorylation. This has been well known as Warburg-effect for a long time [8]. Under such hypoxic conditions, they produce ATP mainly by glycolysis, or even fermentation of amino acids. Recently, it has also been reported that acetyl-CoA synthetase played important roles in producing ATP for tumor cells [9]. Namely, generation of ATP in the hypoxic tumor cells is not an oxygen-dependent phenomenon. Parallel with these facts, it is very intriguing and more important that cancer cells are more frequently deficient in most crucial antioxidative enzymes, such as catalase, glutathione peroxidase and SOD [10], [11], [12], [13]. This means a high vulnerability of tumor cells to ROS will be observed [14], [15]. In fact, many conventional anticancer drugs including vinblastine, doxorubicin, camptothecin, cisplatin and inostamycin, exhibit antitumor activity via ROS generation if not totally [16]. Accordingly, a unique antitumor strategy named “oxidation therapy” was developed by delivering excess oxidative stress into tumor cells or targetedly disrupting the antioxidative defense systems of tumor cells [17], [18], [19], [20].

With regard to ROS and cancer, however, it should be noted that the biological effects of ROS in cancer are multiple and non-linear. Namely high levels of oxidative stress exhibit cytotoxicity as mentioned above, inhibiting cell proliferation and leading to apoptotic/necrotic cell death; whereas low or intermediate levels of oxidative stress are most effective in DNA damage, causing mutation, inflammatory reaction and promoting proliferation of cells, and ultimately inducing carcinogenesis via initiation, progressing to cancer development [21]. In deed, convincing evidence indicated that ROS is an endogenous class of carcinogens by triggering the mutation of the cells [22], [23], [24]. Chronic infection of Helicobacter pylori, hepatitis C virus, human papilloma virus (HPV) and their carcinogenesis can be explained by this mechanism. Namely, pathological levels of ROS induce increased damage to DNA, which accompanies trigger of mutagenesis via DNA base-modifications and mismatch repair [21]. In addition, RNS including nitric oxide (NO) and its derivatives such as peroxynitrite (ONOO) play important roles in the development of cancer and viral infection. Maeda and other researchers found that during the course of viral and bacterial infections, production of NO is enhanced through up-regulation of inducible nitric oxide synthase (iNOS) at the site of infection or inflammation, production of O2radical dot in parallel manner is also known in the same time course either by xanthine oxidase (XO) activation or NADPH oxidation to NO generation [25], [26], [27], [28]. NO and O2radical dot, react immediately and more reactive derivative, ONOO is generated, which plays probably more important role in carcinogenesis [29], [30], [31], [32], and also metastasis by activating matrix metalloproteinases (MMPs) [33], [34]. We further reported the increased viral and bacterial mutation under such condition of extensive ONOO generation [35], [36], [37].

Moreover, oxidative stress can stimulate the expansion of mutated cell clones by modulating genes related to proliferation and triggering redox-responsive signaling cascades such as epidermal growth factor (EGF), tyrosine phosphorylation, protein kinase C (PKC) [38], and transcription factors that regulate inflammation and apoptosis including NF-κB, Activator protein 1 (AP-1) and NF-E2-related factor (Nrf2) [39], [40], [41]. In addition, apoptosis signal-regulating kinase 1 (ASK1), which is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, plays crucial roles in ROS (e.g., H2O2)-mediated cellular responses [42], [43]. Namely, ROS such as H2O2 activates ASK1, which subsequently actives both p38 and c-Jun N-terminal kinase (JNK) pathway, inducing a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis [42], [43], [44]. ASK family, which consists of ASK1 and newly characterized ASK2, may probably be involved in ROS-mediated carcinogenesis and other human diseases, via MAPK signaling cascades by triggering apoptosis and inflammation [44].

In regards to these signal transduction pathways, it has been reported by Sawa et al. more recently that 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) that is a NO dependent nitrated derivative of cGMP, plays an important role in signal transduction via the S-guanylation of a redox-sensor signaling protein Kelch-like ECH-associated protein 1 (Keap1) [45]. The S-guanylation accompanying inactivation of Keap1 by 8-nitro-cGMP will result in nuclear export of Nrf2 and activation of transcriptional activity of Nrf2, which subsequently induces the expression of antioxidant enzymes such as HO-1 [41] and regulates the progression of inflammation. Accordingly, 8-nitro-cGMP seems to be a second messenger of NO, which may thus be closely associated with ROS/RNS-induced inflammatory pathological process and the subsequent carcinogenesis.

Accordingly, it is critical to control the amount of ROS carefully in so-named oxidation therapy, because insufficient induction of ROS will inversely lead to increased growth of tumors.

Besides cancer, ROS is known to be implicated in many diseases, because of its high reactivity and cytotoxicity. Under pathological conditions such as acute and chronic infection and inflammation, overproduction of these highly reactive metabolites will induce lethal damage to cellular integrity and survival [46], [47], [48], resulting in reversible or irreversible tissue injury. These pathological changes, namely ROS-related diseases and disorders include microbial infections, inflammation, atherosclerosis, diabetes, ischemia–reperfusion (I/R) injury, neurologic disorders, Parkinson disease, hypertension, anticancer drug-induced tissue injury, smoking-related diseases, and aging [20], [46], [47], [48], [49], [50], [51], [52]. Thus, it is reasonable to develop therapeutics for these ROS-related diseases by suppressing ROS generation. Indeed, this therapeutic rationale has been challenged by many research groups, with the use of various antioxidative agents of low molecular weight nature as well as enzymes, such as SOD and catalase. Further, inhibitors of ROS generating enzyme XO, or inducers of antioxidative enzyme HO-1 were being considered along this line [46], [27], [53], [54], [55], [56], [57], [58], [59], [60].

These therapeutics, however, are opposite in mechanism to the anticancer strategy of ROS-dependent cytotoxicity as described above. Although it seems to be controversial, it was found experimentally practical if one can control the production of ROS at the local pathological site, i.e. inducing the ROS generation in cancer tissue selectively. For the treatment of inflammation and other ROS-related diseases, suppression or elimination of ROS production can be a remedy. Our approaches toward these goals are discussed below.

One crucial problem to be solved for anticancer strategy by ROS is how to target the ROS production in cancer tissues selectively; otherwise unexpected or systemic side effects will occur. Of great importance, this problem could be effectively solved by enhanced permeability and retention (EPR)-effect using macromolecular drugs, polymeric micelles or nanoparticles [61], [62], [63], [64] (Fig. 2). EPR effect is a universal phenomenon seen in solid tumor for macromolecular drugs in that the macromolecular or polymeric drugs will accumulate and remain selectively in solid tumor tissues, and also inflammatory tissues due to anatomical characteristics and pathophysiological reaction of these tissues. Namely, vascular mediatory factors such as bradykinin and NO play a key role in extravasation [62], [63], [64] (Table 1). Thus EPR effect is now becoming a gold standard for anticancer drug design. More recent reviews of EPR effect are seen in Refs [64], [65].

In addition to the tumor tropic mechanism by EPR effect, macromolecular therapeutics (e.g., PEGylated proteins) will offer better pharmacokinetics (i.e., longer plasma half-life), which is thus used for various other diseases such as inflammation [64], [65], [66]. The macromolecule-based ROS-mediated therapy will thus be warranted for the clinical application.

In this review, the therapeutic rationales of ROS will be discussed, and some candidate agents, especially those with high therapeutic response (e.g., PEGylated d-amino acid oxidase [DAO], PEGylated zinc protoporphyrin [ZnPP]) based on the EPR effect are introduced below.

Section snippets

Therapeutic strategies by inhibiting ROS generation: the use of antioxidative agents

Antioxidative agents include small ROS scavengers, inhibitors of ROS generating enzymes, as well as antioxidative enzymes. The first antioxidative agent applied in clinic is 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186, edaravone, Radicut®), a small molecular ROS scavenger, which was approved for acute brain infarction in Japan, 2001 [67], [68]. In our group, we focused on inhibitors of XO (O2radical dot generating enzyme), as well as SOD and recently HO-1, which will be discussed in details below.

Oxystress inducing anticancer therapy: oxidation therapy

As discussed above, because of the toxic nature of ROS and insufficient antioxidant enzymes in cancer cells, overproduction of ROS will eliminate unwanted cancer cell. Based on this principle, a unique “oxidation therapy” was introduced [14], [15], [17], [18], [19], [20]. One way to achieve this anticancer therapy is to deliver ROS generating enzymes to tumor tissues directly, such as glucose oxidase, XO and DAO, where more selective delivery to tumor tissue can be accomplished by EPR effect

Conclusions

ROS, a group of highly reactive molecules, exhibits vital role in aerobic organisms as the indispensable factors of signal transduction pathway to regulate cell growth and drug metabolism [1], [2], and it is like double-edge sword. Under physiological conditions, damages are reduced by cellular antioxidative defenses (e.g., SOD, catalase, HO-1) and repair mechanisms [6]. However, under pathological circumstances such as inflammation [46], [47], [48], [49], over-burden of ROS will lead to

Acknowledgement

The works included in this paper are supported in part by Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 20590049, No. 20015045 and No. S0801085).

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    This review is part of the Advanced Drug Delivery Reviews theme issue on “Controlling Oxidative Stress: Therapeutic and Delivery Strategies”.

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