High ROS levels in tumor cells as a basis for cancer therapy
George Liao MPS – 2060442
Master’s Thesis Under supervision of Dr. Inge A.M. de Graaf Department of Pharmacokinetics,
Toxicology and Targeting Second assessment by Dr. Angela Casini Department of Pharmacokinetics, Toxicology and Targeting Oct 17 2014
Cancer is the result of aberrant regulation of growth and death in cells. Due to its persistent nature, current therapies are usually unselective and highly toxic. Thus, therapies may frequently result in severe adverse effects.
Reactive oxygen species are reactive molecules that play a role in cellular signaling and are formed as side products in physiological processes. Oxidative stress may result in carcinogenesis and further cancer physiology maintenance. Cancer cells are known to exhibit significantly elevated ROS levels. Many factors may contribute to this increase; regardless, cancer cells regulate their ROS levels rigorously as high levels may also be toxic to them.
Recent developments in cancer therapy have thus focused on finding ways to use this high ROS level in cancer to therapeutic advantages. In this essay, three distinct approaches to use ROS have been discussed for treatment options: namely, by generating additional ROS molecules; by impairing the antioxidant defenses in the cancer cells; or by developing prodrugs that are activated by ROS molecules.
The current ROS-‐generating compounds and antioxidant modulating compounds are cancer selective because it only pushes the ROS concentration over the toxic threshold in cancer cells; the prodrug approach may be truly selective because it requires the aberrant high ROS levels in cancer cells to activate it. All three approaches are still mostly in their infancy, yet many of these compound classes have shown promising results in preclinical studies. However, many factors such as clinical efficacy have yet to be examined for the majority of the compounds. A viable strategy could be to pair these ROS-‐modulating compounds with each other or with existing chemotherapeutics.
REACTIVE OXYGEN SPECIES IN CANCER 9
Elevated ROS levels 9
ROS levels beyond the threshold 11
REACTIVE OXYGEN SPECIES AS A POTENTIAL THERAPEUTIC 13
ROS Generation 13
Motexafin gadolinium 13
Hirsutanol A 15
Methyl 3-‐(4-‐nitrophenyl) propiolate 16
ROS generation by secondary mechanisms 17
Antioxidant modulation 18
Arsenic trioxide 18
Buthionine sulfoximine 19
β-‐Phenethyl isothiocyanate 22
Antioxidant modulation conclusion 23
Prodrug Approach 24
Boronic acids and esters 24
Multiple effectors 27
CONCLUSION AND PROSPECTS 28
Cancer is the collective name for the group of diseases that results from aberrant cell growth and proliferation. Specifically, cancerous cells are characterized by an enhanced rate of growth, loss of function and the potential to invade healthy tissue.1 Neoplasms, or more commonly tumors, are the result of aberrant growth of tissue.
However, not all neoplasms are cancerous; in order to be considered cancerous, the neoplasm has to be invasive and deleterious to its surrounding tissue.
As cancer is a whole spectrum of diseases, there is not one single causative agent, nor one single underlying mechanism. Even with the same cancer, diverse mechanisms can be the cause. However in all cases for cancer to develop, the cells have to acquire mutations that allow growth-‐regulation bypass and apoptosis evasion, both of which are normally under strict supervision.2
Key mutations can be inherited through genetics or acquired from external factors.
Many external factors are implicated with carcinogenesis, one of which are reactive oxygen species. These molecules are able to oxidize cellular macromolecules and DNA, potentially resulting in damage and mutations to proteins and genes that usually manage cell growth, proliferation or apoptosis.3 Cancer arises when repair mechanisms fail for these so-‐called oncogenes and tumor suppressor genes; for instance, resulting in constitutive activity of growth factor receptors or loss of function of the DNA-‐repairing and pro-‐apoptotic p53 protein.2 Research has determined that many cancer types show aberrant activity for the same proteins.2
Reactive oxygen species (ROS) are inherently formed during physiological processes, such as for the catalysis of nutrients in peroxisomes4 or extermination of pathogens by immune cells,3 but also as byproducts during mitochondrial oxidative phosphorylation,5 enzymatic protein folding6 or metabolism of xenobiotics.7 Additionally, ROS may also play redox signaling roles.8 These amounts of ROS are unlikely to cause cancer as, in addition to DNA repair mechanisms, healthy cells can tolerate ROS because of antioxidant systems to ‘disarm’ them.3 Still, a delicate balance exists between the formation and elimination of ROS in all cells.
Disturbance in this balance will result in accumulation of ROS. The resulting state is then called oxidative stress. It is this oxidative stress that could initiate cancer by causing mutations; the high amounts of ROS could prove to be too overwhelming for the cellular repair mechanisms. Following cancer initiation, ROS levels have been determined to be constitutively elevated in cancer cells.9 This enables ROS to also play an important role in cancer promotion and further exacerbation;10 this topic has already been covered by many reviews, thus it lies beyond the scope of the current essay. Interestingly though, persistent high levels of these molecules can also be deleterious to cancer cells.11, 12 In order to survive and thrive under this heightened oxidative stress, various antioxidant systems were also discovered to be upregulated in cancer; however some physiological antioxidants were observed to be diminished.11, 13, 14
It can thus be concluded that the regulation of ROS in cancer is a very complex matter; this intricacy is why ROS implications in cancer are not fully clear. However, the given fact that a threshold for ROS level exists in cancer could prove to be a valuable anti-‐cancer strategy. By using the high levels to our advantage or further manipulating the balance, drugs could potentially be developed that are activated by high ROS levels (prodrugs)15 or induce cytotoxicity by generating additional ROS molecules.7, 16
At present, only few anti-‐cancer drugs are truly selectively targeting the neoplasms.
Most drugs, however, are non-‐selective and attempt to target cancer cells by exploiting their characteristic rapid growth by inhibiting cell division.17 As a result, healthy cells frequently sustain collateral damage, which is a significant problem associated with chemotherapy. It is thus of the utmost importance to develop novel, selective drugs to challenge the unmet medical need. As ROS levels differ significantly in healthy-‐ and cancer cells, drugs that make use of this altered physiology may potentially be used as targeted therapy.
An illustration summarizing the different effects of varying ROS levels is presented in Figure 1, as obtained from the review on ROS in cancer by Liou and Storz.18
The aim of this essay is to explore how the high ROS levels in cancer cells may be used to develop targeted cancer therapies. As ROS are inherently toxic molecules, cancer cells are thus at a heightened risk for ROS damage. In order to understand the possibilities of a treatment with ROS, a general overview of physiological ROS maintenance and functioning will first be given, followed by the discussion of the possible underlying mechanisms of the constitutive oxidative stress in cancer cells.
Finally, the compounds that utilize the high ROS levels as part of their mechanism of action will be reviewed.
Figure 1 The concentration of reactive oxygen species in a cell determines its effect. Low ROS levels are physiological as they are formed during biochemical processes and may take part in cell signaling. Elevated ROS concentrations result in oxidative stress, which could cause carcinogenesis, cancer promotion and exacerbation. Beyond a threshold, ROS becomes toxic, even to the tumor, and could be used in a new anti-‐cancer approach.
Reactive oxygen species
Reactive oxygen species is the collective name for oxygen-‐containing reactive molecules. Despite their toxicity, these molecules are ubiquitous in all living organisms. Figure 2 shows three of the most common ROS species, as well as how they are related to the reduction of oxygen to water.
Of the three ROS molecules, the hydroxyl radical is the most reactive. Due to its extremely short half-‐life (nanoseconds), HO• will react with any structure close to its site of formation.3 Hydrogen peroxide, on the other hand, is the least reactive of the three; however, in no way does that mean it’s the least harmful: due to its neutral charge and longer half-‐life (milliseconds), H2O2 has the ability to diffuse away from its source and even cross cellular membranes.3 It may then react directly with cellular targets, but it can also be reduced to the highly reactive HO• by transition metals in the Fenton reaction, as presented in Figure 3.3
Many antioxidant systems exist to attenuate ROS molecules; the three most common antioxidant enzymes and their reaction are shown Figure 4. Superoxide dismutase (SOD) converts 2 moles of O2•-‐ to the less reactive H2O2. To prevent conversion of H2O2 to HO•, the enzymes catalase and glutathione peroxidase (GS-‐Px) are responsible for its conversion to water and oxygen.3 Oxidative stress arises, with its associated toxicity, when a disturbance in the balance takes place by either an overproduction of ROS, or a depletion of the antioxidant defenses.
O2 e O2 e H2O2 e HO e H2O Oxygen Superoxide anion
2 + 2H+ Superoxide Dismutase H2O2 + O2 H2O2 + RH2 Catalase R + 2 H2O
2 H2O2 2 H2O + O2 2 GSH + H2O2 Glutathione Peroxidase GS–SG + 2 H2O
Figure 2 The stepwise reduction of oxygen to water yields 3 reactive oxygen species.3
Figure 3 The Fenton reaction involves the reduction of H2O2 to the highly reactive HO• (encircled) and a hydroxyl ion by a transition metal. O2–• may drive the reaction by reducing the oxidized metals.
H2O2 HO + HO
Figure 4 The schemes of the reactions catalyzed by the three major antioxidant enzymes.
Superoxide dismutase catalyzes the conversion of 2 moles of O2–• to H2O2 and oxygen. Catalase is able to further reduce H2O2 to water; in peroxisomes, the enzyme oxidizes other molecules simultaneously with the reduction of peroxide. When H2O2 accumulates, catalase can switch to reducing 2 moles of peroxide at the same time. Glutathione peroxidase is also able to reduce H2O2 to water by oxidizing the scavenger glutathione (GSH).
Traditionally, ROS have always been associated with oxidative stress and its potential to cause damage to cellular structures. However recently, ROS molecules have been implicated with physiological signaling in the cell.8 Several downstream transduction pathways of growth factors and cytokines seem to involve ROS production. The specific mechanisms are still unclear, but one important hypothesis is that ROS may play a role in the oxidative modification of downstream proteins, for instance kinases or transcription factors.8
Under physiological conditions, the main source of ROS production is the peroxisome as H2O2 are generated to oxidize and catabolize nutrients by catalases.4 However, when large amounts of H2O2 accumulate in peroxisomes, these molecules may escape by diffusion and interact with other cellular structures.4
The ER is the major site for mRNA translation and post-‐translational modifications such as disulfide bond formation. The protein that is responsible for bond formation incidentally forms H2O2 in the process as a side product.3, 6
NADPH oxidase, which is primarily located on the cell membranes of immune cells, produces O2–• to exterminate invading pathogens.19 The O2–• molecules are released at once during respiratory burst, but as ROS molecules are non selective, this release has the potential to damage other cells as well; hence the association of inflammation with cancer. Other isoforms of NADPH oxidase are present on non-‐
immune cells and may be involved with the production of ROS for signaling purposes.8
Redox active enzymes, such as the CYP enzymes, are important for the metabolism of xenobiotics. However, electrons may leak out in the process and create ROS molecules; this process is called uncoupling.3 Additionally, depending on the compound, CYP enzymes may induce ROS promotion through redox cycling: the compound is first reduced by CYP, after which the electron is passed on to oxygen, thus making it a substrate for CYP again and so on.3
The electron transport chain (ETC), located on the mitochondrial inner membrane, is a very important site of ROS generation;5 in addition to physiological ROS production, it is also the main site for exogenous induction.3 The ETC is an intricate network of proteins that is responsible for the ATP-‐generating oxidative phosphorylation. As the name suggests, electrons are transported over the network from donating cofactors to oxygen molecules at the end to form water. This process of electron transport is prone to leakage however, which means that oxygen may be prematurely reduced, resulting in O2–•.5 Escaped ROS molecules can then interact with structures of the ETC, mitochondrial macromolecules (such as mitochondrial DNA (mtDNA)) or even other structures within the cell. Due to the potential for large amounts of O2–• to form, mitochondria have their own SOD isoform to further reduce the molecules; the mitochondrial SOD binds manganese and is thus called MnSOD.5
ROS can thus be generated at many different sites; luckily, under physiological conditions, the antioxidant systems are amply capable to neutralize the ROS molecules before irreparable damage can be inflicted. However, if the homeostasis is affected, ROS may accumulate and potentially initiate cancer by either directly damaging the genome on oncogene or tumor suppressor gene positions or indirectly by interacting with the redox-‐susceptible signaling pathways.
Reactive oxygen species in cancer
Following cancer initiation, research has determined that basal ROS levels are constitutively elevated in cancer cells.9 The exact mechanism for this shift in ROS balance is still unclear, as many factors could potentially contribute. Thus, the factors that can induce ROS molecules in general will be presented in this chapter, followed by a discussion on how cancer cells may adapt to survive under oxidative stress.
Elevated ROS levels
As cancer is a diverse disease with many forms of expression, many mechanisms could therefore contribute to the elevated basal ROS levels. The originating location of the neoplasm is also important as differentiated cells have different expressions of cellular constituents and cells may thus function differently even as cancer cells.
As previously described, the mitochondria are an important source of ROS. Under normal circumstances, the electron-‐leakage from the ETC is easily manageable by antioxidants. However, this balance can be seriously affected following mitochondrial damage; ROS formation may namely be significantly increased when the ETC or mitochondrial DNA become damaged.20 Direct damage to structures of the ETC could result in decreased efficiency of electron transport, which may cause an increased leakage of electrons and thus an increased premature oxygen reduction. Damage to the mitochondrial DNA results in an impaired ETC as well, as 13 of the 100 ETC-‐proteins are encoded by mtDNA.20 Due to the convenient proximity of the mitochondrial structures to the site of ROS production, ROS is thus an important factor for its own amplification.
In parallel with the altered ROS levels, research has determined that cancer cells may also have aberrant levels and regulation of antioxidant systems, which could potentially contribute to the shift in ROS regulation towards higher levels.21 Although one must keep in mind that this is highly cancer specific as cellular constituents in cancer, including antioxidants, depend on the healthy cell’s physiological function.
However, in their review, Oberley and Oberley have noticed that cancers involving the lungs, kidney and prostate often show diminished levels of catalase and GS-‐Px, which will result in the accumulation of H2O2.21 On the other hand, the mitochondrial MnSOD is diminished in many cancer forms, causing accumulation of O2–•.21 Aberrant regulation of antioxidants can thus potentially contribute to accumulation of ROS, however this aspect is highly cancer specific and the size of its role differs.
One of the main characteristics of cancer is the uninhibited proliferative power of the cells. Oncogenes, such as Ras, Bcr-‐Abl and c-‐Myc, are fundamental for this aspect as many cancer forms express overactivity of their encoded proteins, which have functions associated with inducing cell growth and division.2 Oncogene activity of the aforementioned Ras,22 Bcr-‐Abl23 and c-‐Myc24 have all been associated with ROS production;8 thus in the case of cancer with hyperactive oncogenes, significant amounts of ROS may be produced during normal cancer functioning. As with
physiological signaling involving ROS, the exact reason for the pair-‐wise production of ROS with oncogene activation is also unclear. The ROS molecules could potentially promote cancer growth by causing additional genomic instability; or, it could simply perform its physiological function of oxidative modification of downstream proteins, including transcription factors.
As a cancer cell grows rapidly and excessively, eventually even beyond its normal tissue structures, it may end up outgrowing its physiological blood supply. As a result, the blood vessels may not be able to supply all areas of the neoplasm with sufficient oxygen; hypoxia could thus become a problem in these areas. H2O2 has been implicated with the signaling for angiogenesis by stabilizing the transcription factor hypoxia-‐inducible factor-‐1 (HIF-‐1).25 These ROS molecules originate from the mitochondria and are induced under hypoxic conditions: under low oxygen conditions, the ETC is impaired which may cause more electrons to leak prematurely than normal.26 The ‘leaked’ electrons will reduce the scarce amounts of oxygen available to form O2–•, which is subsequently reduced by MnSOD to H2O2.25 As hypoxia is a common condition in tumor cells, it may very well be one of the contributors to the elevated ROS levels.
Recently, a group of researchers, led by El Sayed, have hypothesized that the Warburg effect may be a contributing factor to the high basal ROS levels.27 The Warburg effect is the phenomenon that cancer cells show a high level of pyruvate fermentation rather than oxidative metabolism through the Krebs cycle, regardless of the level of oxygen available.28 Krebs cycle intermediates29 have been implicated with antioxidant activities. El Sayed suggests that due to the tendency of cancer cells to generate energy primarily by fermentation, less Krebs cycle intermediates will be available to aid in modulating ROS levels. Additionally, the Krebs cycle produces NADH, which is indirectly used to reduce oxidized glutathione. While El Sayed and his coworkers have a point that fermentation may result in diminished antioxidant capacity of Krebs intermediates, this can only be a small contributor in the elevated ROS levels. The Krebs cycle intermediates are namely weak antioxidants and require concentration levels exceeding the physiological values to have the same potency as, for instance, glutathione;29 this is due to their postulated mechanism as they likely exert their antioxidant properties by chelating reduced transition metals.29 Furthermore, lactic acid, the resulting compound from pyruvate fermentation, is known to exhibit antioxidant properties as well.30 The only strong argument in El Sayed et al’s favor is that the intermediate fumarate has recently been associated with activating the important antioxidant transcription factor Nrf2.31 On the other hand, El Sayed has not taken into account that, in addition to fermentation, cancer cells still undergo oxidative metabolism via the Krebs cycle.32 Thus, it can be concluded that the Warburg effect is unlikely the main cause for –or even a big contributor to– the elevated ROS levels.
Telomeres are the non-‐coding nucleotides at the ends of chromosomes that protect the genome from losing ‘information’ during replication of the lagging DNA template strand.33 Telomere dysfunction, which is the shortening of telomeres, may cause genomic instability and thus the initiation of cancer. Interestingly, telomere
dysfunction has recently also been linked with mitochondrial fitness.34 The shortening of telomeres may namely induce the tumor suppressor protein p53, which is involved in numerous regulatory pathways. The transcriptional coactivators PGC-‐1α and PGC-‐1β, which are important regulators for mitochondrial biogenesis and functioning –including antioxidant generation, are associated with upstream p53 signaling; telomere dysfunctioning will result in the inhibition of these proteins. As a result, the inhibition of PGC coactivators by telomere dysfunctioning will result in mitochondrial impairment, which will result in increased leakage of electrons from the ETC and thus ROS generation.34 It is however important to know that p53 has to function for this to contribute to extra ROS generation, which is not always the case.
The tumor suppressor protein p53 is a very important factor in physiological cell functioning; it is namely responsible for preventing the formation of cancer, hence the name ‘tumor suppressor’. Furthermore, p53 may also regulate the transcription of antioxidants.7 Its main functions include the activation of DNA repair mechanisms, the suspension of mitosis and, in case of irreparable damage, the induction of apoptosis.35 Many cancers are associated with acquired mutations that result in the loss of functional p53 protein.35 This loss of function can lead to a vicious cycle of genomic instability; namely, less antioxidants will be synthesized and ROS-‐induced DNA damage will not be repaired, which will lead to accumulation of damage and further dedifferentiated cells following cell division. In their turn, these cells are prone to produce even more ROS and so on.35
ROS levels beyond the threshold
Despite the importance of ROS for cancer initiation, promotion and progression, they are still inherently noxious molecules with the potential to kill cancer cells.
While the basal ROS levels are significantly elevated in cancer physiology, the concentration is still contained below the toxic threshold (Figure 1). Cancer cells manage to survive under these conditions by altering the expression of their antioxidant defenses or the proteins involved with survival and death.
As described above, the antioxidant expression of cancer cells has already been altered. Many transcription factors involved in the expression of antioxidant enzymes are known to be redox sensitive.10 High ROS levels could therefore be sensed by these transcription factors and the expression of antioxidants will subsequently be induced. Additionally, oncogenes may also induce the synthesis of various antioxidants, which could also be linked to their production of ROS molecules. Cells with high Ras activity have been determined to have high peroxiredoxin concentration,36 which is also an enzyme that is responsible for H2O2 reduction to water, while c-‐Myc activity is associated with induction of GSH synthesis.37
In addition to upregulating the synthesis of antioxidants, cancer cells may also cope with the high ROS levels by interfering with apoptosis. The redox-‐sensitive transcription factors can namely also enhance the synthesis of anti-‐apoptotic factors10 or diminish the activity of pro-‐apoptotic factors.38 However, when the ROS level somehow manages to surpass the closely guarded toxic threshold, cancer cell
cycle arrest and cell death can take place, mediated by ROS. ROS may attenuate cancer by either inducing apoptosis and senescence via pro-‐apoptotic proteins, or damaging the cancer cells to the point of irreparable damage.12, 18, 39 The various mechanisms for ROS-‐mediated cancer attenuation will be elucidated in the following section for each drug, when available.
The dual action of ROS may seem paradoxical, however recently, research has found evidence that O2–• and H2O2 may have differential activity in cancer cells.40 In their review article Pervaiz and Clement present evidence that high O2–• levels are linked with apoptosis resistance in cancer cells, as SOD isoforms seem to be downregulated whereas the anti-‐apoptotic Bcl-‐2 is up-‐regulated. On the other hand, high H2O2 levels are associated with apoptosis. This may potentially be due to hydrogen peroxide’s ability to diffuse away from its source and interact with cellular structures, such as the mitochondrial membrane or caspases. Interaction with the membrane could result in an alteration of its permeability, resulting in the release of cytochrome C;
interaction with caspase proteases could result in its activation. Apoptosis will be induced in both these scenarios. In any case, the exact mechanisms are still unclear, however evidence suggests that high H2O2 levels may act as pro-‐apoptotic factors.
Reactive oxygen species as a potential therapeutic
Equipped with the knowledge that cancer cells have significantly higher ROS levels than healthy cells, viable targeted treatments could thus be aimed at turning an important carcinogenic to our own advantage.
Indeed, researchers have found three distinct ways to fight cancer cells through ROS-‐
related mechanisms. Namely, cancer cells can be killed by generation of additional ROS; or ROS levels can be further raised by diminishing their antioxidant capacity;
and finally, the high ROS levels could be employed to activate prodrugs specifically in cancer cells.
This section of the essay will elaborate on the mechanisms of action by which the three ‘classes’ of ROS-‐related anti-‐cancer drugs work. The three classes will be divided in three separate paragraphs and drugs in each class will include its proof of concept in the form of experimental in vitro or in vivo data, if available.
Cancer cells rigorously maintain their ROS levels below the toxic threshold. As ROS levels are constitutively elevated significantly in cancer cells and antioxidant systems have already been enhanced in order to survive, additional generation of ROS could thus be able to tip the balance towards toxicity and cancer-‐killing. Cancer selectivity in this case is based on the ability of normal cells to easily deal with the additional ROS.
Motexafin gadolinium, commercially known as Xcyclin, is a synthetic porphyrin, which is heterocyclic macrocycle. 41 After showing high potential in preclinical studies for the treatment of lung cancer brain metastases, the drug was ultimately still disapproved by the FDA (reason not specified);
researchers are now investigating Xcyclin for non-‐
small cell lung cancers.42
Xcyclin can be listed under both the ROS Generation or Antioxidant modulation paragraphs as the compound was found to deplete the reducing agent NADPH and the antioxidants ascorbate (vitamin C) and GSH, as well as induce ROS generation through redox cycling.41 The resulting high levels of ROS are
suspected to interact with the mitochondrial membranes, causing release of cytochrome c and subsequent induction of apoptosis.41
In both in vitro and in vivo studies, researchers found that the compound is selectively taken up by cancer cells.43 This selective localization was expected, as natural porphyrins are known to be taken up by cancer cells, although the reason for
N Gd HO
O O O
Structure 1 Motexafin gadolinium
this is still unclear.44 The subsequent clinical studies have determined that Xcyclin could synergistically enhance the activity of other cancer treatments. In the case of brain metastases, the drug improved the neurological functioning of the patients over exclusively brain radiation; however, no improvement in anti-‐cancer activity was found.45
Hispidin is a natural compound obtained from a medicinal mushroom used for centuries in traditional Asian medicine.46 Included in its indications was the treatment for cancer. Researchers have recently discovered that Hispidin induces apoptosis in colon cancer cells through ROS level manipulation.47
As the discovery of Hispidin’s activity is recent, only fundamental proof of concept experiments were thus far conducted in vitro. Firstly, colon cancer cells were dose-‐
dependently killed by Hispidin as determined by Annexin V staining.47 Pre-‐incubation of the cells with a ROS scavenger showed that the activity of Hispidin was diminished, thus proving ROS the likely executioner for Hispidin. Subsequently, during gene expression studies, the researchers found that Hispidin, through ROS, induced the expression of p53 protein and its downstream pro-‐apoptotic factors, whereas anti-‐apoptotic factors were downregulated. Even more interestingly, the researchers discovered that in p53-‐null cells, extrinsic apoptotic factors were induced, thus effectively bypassing the necessity of p53 mediation.47 Figure 6 shows the protein expression over the Hispidin concentrations.
Judging from the in vitro data, Hispidin seems to be a compound with great potential. The question however remains whether the same promising results are expressed in vivo. Furthermore, the source of the ROS molecules is still unclear, however judging by the structure, a quinone-‐related redox cycling might be its mechanism. On the other hand, cancer selectivity still has to be proven as the researchers have not reported the effect of hispidin in healthy cells.
Structure 2 Hispidin
Figure 5 Gene expression study carried out on rat colon cancer cells (CMT-‐93). Bax is responsible for mitochondrial pore opening and cytochrome C release. Bcl-‐2 is an inhibitor of apoptosis. As can be seen in (A), increase of Hispidin concentration results in the decrease of Bcl-‐2 and increase of Bax and p53. In (B), Hispidin induces the activation of both caspases 1 and 8, as well as the death receptor 3.
Furthermore, the cleavage of Parp signals the start of apoptosis. These proteins are associated with the extrinsic apoptosis pathway.
Hirsutanol A (HirA) is a natural product obtained from fungi that has recently been identified to show promising anti-‐cancer activity; apoptosis can be induced in breast cancer cells, colon cancer cells and hepatocellular carcinomas.48, 49 By probing the breast-‐
and colon cancer cells that were exposed to HirA with
Annexin V, researchers were able to determine that the compound induced apoptosis in a dose-‐dependent manner. In addition, experiments with colon cancer xenografts in mice show that HirA is also able to attenuate tumor growth by half in vivo (Figure 6).49 The researchers did not report on any observed side effects.
HirA induces apoptosis by generating ROS that interacts with the mitochondrial membrane resulting in the release of cytochrome c to the cytosol; cytochrome c is then able to activate the caspases, the apoptotic executioners. Inhibition of Hirsutanol A activity by incubating the cell lines with ROS scavengers supports the ROS-‐based apoptosis idea. The source for the extra ROS generated by HirA is still unknown, however by testing HirA on ETC-‐impaired cell lines, researchers discovered that apoptosis is still induced in the same manner. Thus, the conclusion can be made that HirA induced ROS stem from somewhere else than the mitochondria.
Furthermore, the group also suggested, by using fluorescent oxidative stress indicators, that H2O2 was the main molecule to be generated, as the CM-‐H2DCF-‐DA probe showed significantly more fluorescence than the DHE probe in a dose dependent fashion.49 The DHE probe is specific for O2–•, whereas CM-‐H2DCF-‐DA is unspecific. The researchers suggested that since DHE showed minor fluorescence, it is most likely H2O2 that is causing CM-‐H2DCF-‐DA to fluoresce, as O2–• and H2O2 are the most frequently occurring ROS species.
As result of the HirA induced extra ROS, cells were found to induce the anti-‐
apoptotic JNK signaling pathway to diminish the effect of HirA; thus, treatment with HirA paired with a selective JNK signaling inhibitor should further improve results. As
Figure 6 The effect of Hirsutanol A on in vivo colon cancer xenografts in mice plotted with normal saline (NS), the topoisomerase 1 inhibitor HCPT and DMSO. The tumor volume is approximately cut by half with Hirsutanol A. The experiment was terminated following measurements on day 21.
Structure 3 Hirsutanol A
is with Hispidin, the researchers did not report on the cancer selectivity of HirA and they have not reported the effects of HirA on healthy cells.
Methyl 3-‐(4-‐nitrophenyl) propiolate
Methyl 3-‐(4-‐nitrophenyl) propiolate (NPP) is a synthetic ROS-‐
inducing compound that was identified in high-‐throughput screening; NPP showed potential as researchers determined that it could induce apoptosis selectively in leukemia, breast-‐, liver-‐, lung-‐, prostate-‐, colon-‐, skin-‐ and cervical cancer over healthy cells in vitro (Figure 7).7
Similar to HirA, NPP was also determined to induce
apoptosis in a dose-‐dependent fashion (by Annexin V) with the mode of action being the release of cytochrome C to the cytosol.7 The researchers observed that ROS was generated dose-‐dependently in many different cell lines, including non-‐transformed cells.7 Furthermore, pre-‐incubation with antioxidants could attenuate cell death;
thus proving that apoptosis is indeed induced by the generated ROS. Monitoring the ROS levels showed that the molecules were induced within 10 minutes, peaked at 30 minutes and started diminishing within the hour.7
To determine the source of the ROS molecules, researchers first examined NPP on ETC impaired cells and determined that no significant difference in effect was detectable. Subsequently, the possibility of ROS generation by CYP-‐mediation was investigated as the researchers determined that the reduced propargyl ester of NPP might behave similarly to quinones (Figure 9). By inhibiting the CYP enzymes, researchers were able to attenuate the ROS formation dose-‐dependently;
specifically the CYP3A4 inhibitors showed this effect, as shown in Figure 8. The importance of the propagyl ester was found when carboxylesterases in hepatocyte cell lines could help the cell withstand NPP exposure.7
Figure 8 ROS levels are dose-‐dependently decreased with CYP3A4 inhibitors TAO (oleandomycin triacetate) and CHL (chloramphenicol). The CYP1A inhibitors ANF (α-‐
naphthoflavone) lacked this effect.
Structure 4 Methyl 3-‐(4-‐
nitrophenyl) propiolate (NPP). The propagyl ester is colored red.
Figure 7 The percentage of viable cells is plotted against the concentration of NPP exposure. As can be seen, the non-‐cancerous Wi-‐38 (lung) and LO2 (liver) are significantly less susceptible to NPP exposure compared to the breast carcinoma (Hs578T) and breast adenocarcinoma (MDA-‐MB-‐468)
In terms of selectivity, the researchers found that NPP induces apoptosis preferentially in cells with high ROS levels and low antioxidant defenses; leukemia was given as an example. In line with this discovery, researchers found that p53-‐
dysfunctional cells are more susceptible to NPP. This is a profound discovery as loss of functional p53 is found in the majority of all cancers and enables tumors to form and proliferate in the first place, as well as evade apoptosis. In this case, no p53 will be able to upregulate the synthesis of antioxidants following NPP-‐induced ROS generation.7
Finally, the researchers demonstrated that due to the high reactivity of ROS species, the location of formation might be more important than the quantity of ROS. NPP is believed to induce ROS near membranes, as CYP enyzmes are membrane associated.
While the plasma membrane is not one of the main locations for CYP enzymes, it is known to have CYP enzymes.50 The researchers postulated that the enzymes on the plasma membrane might play an important role in NPP activity. Near the plasma membrane, the researchers namely found that ROS molecules could inhibit the JAK/STAT signaling pathway, possibly by oxidizing the cysteine residues of the JAK tyrosine kinase, as determined by Western blotting; as a result, the signaling pathway is impaired and the STAT-‐promoted expression of anti-‐apoptotic factors is attenuated. A summary of the postulated action of NPP can be found in Figure 9.
While the researchers were able to demonstrate the significant effects of NPP in vitro, the question remains whether its potential stays intact in vivo. The main concern is if sufficient CYP enzymes are functional in cancer cells, especially in highly dedifferentiated forms, and if they are at the correct location to generate the ROS in order for NPP to exert its effect.
ROS generation by secondary mechanisms
Many current chemotherapeutics are known to generate ROS, either as a secondary mode of action or without making use of the intrinsically elevated ROS levels in
N+ O -O
O O CYP450
Figure 9 The proposed mechanism of action of NPP. NPP can undergo redox cycling by first being reduced to an allenic structure. This allenic structure then passes its electron to oxygen and thus reducing it to superoxide anion. O2–• can further be reduced to other ROS molecules and these may cause apoptosis by interacting with mitochondria and inhibiting the JAK/STAT anti-‐apoptotic pathway.