Increasing oxidative stress contributes to the mechanism of action of anti-leukaemia chemotherapeutics
This section will discuss how some front-line anti-leukaemia chemotherapeutics alter the cellular redox balance, which could be important for their pharmacologic effect. A better understanding of this question would help to improve their therapeutic effect and overcome drug resistance.
AML still presents a great therapeutic challenge, of which the initial treatment cycle consists of arabinocytosine (a.k.a. cytarabine), a purine analogue that interferes with DNA replication, during 7 days, followed by anthracyclines antibiotics for 3 days. This is followed by additional chemotherapy or transplantation [
137]. An increase in intracellular ROS has been found to be associated with the mechanism of action of cytarabine [
138]. In this study the source of ROS was identified as coming from mitochondria. However, as reviewed elsewhere [
139], anthracyclines could induce ROS formation via other mechanisms: (i) interacting with free iron and (ii) as by-products of their own metabolism by enzymes such as cytochrome P450 reductase, NADH dehydrogenase or XO and thioredoxin reductase (TrxR) [
138]. NADPH oxidases are also implicated in anthracyclines-induced increase of ROS [
140‐
142]. A recent study related the efficacy and toxicity of anthracyclines to NADPH oxidase polymorphisms [
143]. The importance of ROS in the mechanism of action of anthracyclines was also highlighted by the fact that administration of antioxidants reduced their cytotoxicity [
144]. In addition, it has been suggested that the enhancement of cellular antioxidant systems, such as SOD2 [
145] or glutathione [
146], could drive resistance to anthracyclines.
There is a significant number of novel compounds for AML treatment, whose feasibility is currently being tested in clinical trials [
137]. Interestingly, the mechanism of action of some of these agents might be related to the alteration of redox homeostasis. Enzymes that control DNA epigenetic modifications, such as histone deacetylases (HDACs), are among their targets. HDACs inhibitors (HDACi), such as vorinostat, show anti-tumour capacity and increase ROS production. The use of antioxidants consistently reduces HDACi cytotoxicity [
147], thus suggesting the importance of increased ROS production in the mechanism of action of HDACi. In addition, there is a study showing that AML cells resistant to HDACi exhibit higher antioxidant defences [
148]. The aforementioned increase in ROS may be derived from mitochondria [
147] and NADPH oxidases [
148]. The mechanism of action of other epigenetic enzyme inhibitors currently being tested for AML treatment, such as Lysine-specific demethylase 1 (LSD1) [
149] and Bromodomain and Extra-Terminal proteins (BET) inhibitors [
150], could also be related to increases in ROS.
The cell cycle is another interesting molecular target in AML and cyclin-dependent kinases (CDKs) inhibitors are currently being studied in this respect [
137]. Some work supports the fact that the antiproliferative effect of this type of inhibitors is linked to the induction of oxidative stress [
151].
Leukaemic cells have a high concentration of ubiquitinated proteins [
152]. Therefore, a number of proteasome inhibitors have been tested for anti-leukaemic activity. The first proteasome inhibitor approved by the FDA, bortezomib, has shown effectiveness against multiple myeloma (MM) [
153], and it could also be used for AML [
154] or CLL [
155] treatment; induced oxidative stress is important for bortezomib cytotoxicity [
156].
The treatment of acute promyelocytic leukaemia (APL) with the pro-differentiation agent
all-trans retinoic acid (ATRA) is an effective strategy that would be interesting to treat other types of leukaemia [
157]. In cases of relapsed APL, arsenic trioxide (As
2O
3, Trisenox) is a good drug choice, as it has been approved by the FDA against this disease in combination with ATRA and anthracyclines chemotherapy. Currently, As
2O
3 efficacy is being tested in newly diagnosed APL patients, with complete remission in 83–86% of patients and 3-year overall survival. As
2O
3 acts through different mechanisms such as stimulation of differentiation, induction of apoptosis, and NF-κB inhibition [
158]. However, it seems that its main effect would be the induction of ROS accumulation. As
2O
3 can alter the cellular redox homeostasis at different levels: (i) by inducing electron leakage from the mitochondrial respiratory chain [
159] and (ii) through the expression of the genes that code for the Nox2 complex-forming proteins [
160]. As suggested by the irreversible inhibition of TrxR [
161] and the depletion of peroxiredoxin III (Prx III) [
162], As
2O
3 may also diminish the cellular antioxidant capacity, which would contribute to raise ROS levels. It is noteworthy that As
2O
3 cytotoxicity inversely correlates with the level of glutathione [
163]. This is of key relevance, since the measurement of antioxidant defences in patient samples could help to predict their responsiveness to As
2O
3 or other pro-oxidant treatments, as previously reported for paclitaxel, a chemotherapeutic that also increases ROS [
164]. This information could also help to improve the effect of As
2O
3, by simultaneously targeting various antioxidant cellular systems. In this regard, the reduction of glutathione has shown to increase the effectiveness of As
2O
3 [
165]. Jeanne et al. have shown the importance of ROS production in the molecular mechanism of action of As
2O
3. The oxidative stress induced by As
2O
3 allows for PML/RARα dimerization through the formation of a disulfide bridge. Direct binding of As
2O
3 to PML/RARα dimers induces the formation of multimers and their association with the nuclear matrix in the so-called nuclear bodies (NBs), where eventually sumoylation and degradation of PML/RARα occurs [
166].
A striking observation is that, despite differences between the mechanisms of action of all the previously described chemotherapeutics, they share oxidative stress as a mediator of their cytotoxic activity. This implies that in the past leukaemia was unintentionally targeted through redox mechanisms. The awareness of this fact may have important practical consequences, since it could help to improve therapeutic results, overcome resistances and reduce toxic side effects. Regarding the latter issue, the use of the tubulin inhibitor vincristine to treat ALL is associated with neurotoxicity [
167]. The experimental evidence suggests that the cytotoxicity of vincristine and other tubulin inhibitors, such as paclitaxel, is related to the induction of oxidative stress [
168]. Importantly, the concomitant use of antioxidant molecules reduced the toxic side effects of paclitaxel and other chemotherapeutics without compromising, or even improving, therapeutic results [
169].
Given the complexity of leukaemia and the multiple factors contributing to its development, single-agent treatments are sometimes not satisfactorily effective; thus, combinatorial treatments are very common. The combination of two of the previously mentioned chemotherapeutics would ensure that two different biological processes relevant to leukaemic cells were targeted. Additionally, the combined treatment would increase oxidative stress (Table
1). Preclinical studies in leukaemic cell lines have shown a synergistic effect when combining HDACi with proteasome inhibitors, which is dependent on ROS production [
170]. This result has led to two recently conducted phase II clinical trials in which the combination of bortezomib with vorinostat was tested in AML (NCT00818649) and ALL (NCT01312818). The combination of As
2O
3, bortezomib and L-ascorbate was recently evaluated in a clinical trial against relapsed or refractory MM with encouraging results [
171]. Recent clinical trials have tested the combination of As
2O
3 with other compounds that also can increase ROS levels in AML patients. Examples are decitabine (NCT00671697, Phase I; NCT03381781, Phase II); citarabine and idarubicin (NCT00093483, Phase I); combination of altezumab with As
2O
3 and other chemotherapeutics (NCT00454480, Phase II/III).
The ongoing search for novel chemotherapeutics that increase ROS levels
Given the oxidative stress “addiction” displayed by cancer cells, the promotion of their death through a ROS “over-dose” is becoming a popular idea. The realisation that many chemotherapeutics increase the oxidative stress has paved the way for the active search for novel compounds that can increase ROS levels, as recently reviewed [
135]. Although some of these compounds may not be very effective as single agents, some reports suggest that they could potentiate the effect of traditional therapy and help to overcome resistance [
172].
In the search for agents that alter cellular redox homeostasis, the mitochondrion should be one of the first targets considered. As reviewed elsewhere, the use of mitochondrial inhibitors is a suitable strategy for inducing oxidative stress with a therapeutic purpose in leukaemic cells [
94]. Moreover, mitochondrion-targeting drugs might also activate the intrinsic apoptotic pathway. Interestingly, metformin, an antidiabetic drug, has been shown to inhibit mitochondrial ATP production and increase ROS [
173]. This drug is currently under study in clinical trials for CLL (NCT01750567, phase II; NCT02948283, phase I), relapsed childhood ALL (NCT01324180, phase I), and relapsed/refractory AML (NCT01849276, phase I). Adaphostine, initially described as a TKi, increases ROS levels by inhibiting mitochondrial respiration [
174], and can overcome resistance against imatinib in primary CML cells [
175]. The safety of tigecycline, an antibiotic that inhibits mitochondrial biogenesis, has already been tested in AML patients (NCT01332786, Phase I) [
176]. An in vitro study on the anti-leukaemic effect of tigecycline against cells from CML patients (NCT02883036) is also scheduled.
A recent report has shown the importance of limiting ROS production by cellular antioxidant defences for cancer initiation [
177]. This work revealed that glutathione and thioredoxin are required for tumour initiation, and that inhibition of these antioxidant systems hinders tumour growth in a synergistic manner. This evidence strongly suggests that targeting the cellular antioxidant defences is an interesting strategy for fighting against tumour cells. In line with this, there is a growing list of preclinical studies testing the anti-tumour capacity of inhibitors for different antioxidant cellular systems, some of which will be discussed below.
The glutathione/glutathione peroxidase (GSH/GPx) system is a major regulator of redox homeostasis; therefore, its impairment may induce a severe oxidative stress in the cells. NOV-002, a complex of oxidised glutathione (GSSG) with cisplatin in a ratio of 1000:1, has shown effectiveness against non-small cell lung cancer (NSCLC) (NCT00347412
, Phase III). Imexon acts by depleting glutathione levels [
178]. Recent clinical trials have tested imexon in MM (NCT00327249, phase I/II) and aggressive lymphomas (NCT01314014, phase II). Similarly, glutathione depletion by buthionine sulfoximine (BSO) activates apoptosis in ALL cells [
179] and increases As
2O
3 activity [
180], suggesting that the use of this type of compounds to treat leukaemia could be an interesting option.
Different SOD inhibitors, such as ATN-224 [
172] or 2-methoxyestradiol (2-ME, panzem) [
181], have shown anti-leukaemic capacity in preclinical studies. Interestingly, a recent report has identified 2-ME as capable of targeting T-ALL pre-leukaemic stem cells without affecting normal HSCs in a high-throughput screening [
182]. Recent clinical trials have tested the combination of ATN-224 and bortezomib in MM patients (NCT00352742, phase I/II), and 2-ME for targeting relapse or plateau phase myeloma (NCT00592579, phase II).
A conceptually opposing strategy could be proposed on the basis of some reports showing that SOD inhibits cancer cell growth. [
183]. With this respect, the use of SOD mimetics in combination of other chemotherapeutics that increase ROS against cancer is being assessed [
184]. These compounds may have antioxidant activity by decreasing superoxide levels, despite the fact that they can also increase the cellular level of H
2O
2. There is strong evidence supporting the idea that these mimetics would preferentially target highly dividing tumour cells, thus potentiating the effectiveness of other chemotherapeutics and reducing their toxic side effects as shown by a recent clinical trial (NCT00727922) [
185].
Another body of research suggests that the thioredoxin redox system is an interesting drug target in cancer [
186]. Several molecules interfere with this system, and some of them, such as PX-12 [
187], gliotoxin [
188], and chaetocin [
189], have shown anti-leukaemic effects in preclinical studies. Recent clinical trials have tested PX-12 against solid tumours (NCT00736372, NCT00417287).
The heme oxygenase enzyme-1 (HO-1) catalyses the degradation of heme group to ferrous iron, biliverdin and carbon monoxide (CO). Under oxidative stress, HO-1 expression is induced by the NRF2 transcription factor as part of the cellular antioxidant defence response. HO-1 is upregulated in some leukaemic cells [
190], which could be a compensatory mechanism to cope with oxidative stress [
95]. It is noteworthy that HO-1 overexpression has been related to drug resistance [
191]. There is experimental evidence showing that targeting HO-1 may be an interesting strategy to i) fight haematological malignancies, and ii) overcome the resistance to pro-oxidant drugs [
190].
As previously mentioned, the activity of many transcription factors is inhibited by oxidation, where APE/Ref-1 catalyses the reduction of several of them [
19]. APE/Ref-1 inhibition induces sensitivity to anti-tumour drugs [
192]. This finding, as well as the development of several APE/Ref-1 inhibitors [
193], suggests that this protein could be a promising therapeutic target in the treatment of leukaemia [
192].
Another line of work in the search for pro-oxidant chemicals is testing the anti-tumour activity of natural derivatives, as some, such as parthenolide, triptolide and avocatin B, have shown effectiveness against AML cells [
194]. Phenethyl isothiocyanate (PEITC), a compound present in cruciferous vegetables, has made a stellar apparition in the field of cancer therapeutics. There is a vast number of preclinical studies revealing the anti-tumour activity of PEITC, which is related to the induction of ROS [
195]. Experimental evidence suggests that PEITC may increase sensitivity to chemotherapy in B-cell prolymphocytic leukaemia (B-PLL) patients [
196], and overcome resistance in CLL [
197] and CML [
198]. Recent (NCT00005883, Phase I) and ongoing (NCT03034603) clinical trials will elucidate the potential benefit of using PEITC as a nutritional supplement against cancer.
Natural flavonoids can work either as ROS scavengers or pro-oxidants. Several preclinical reports showed that some natural flavonoids and their derivatives have pro-apoptotic and cytotoxic effects against different types of haematological malignancies. Interestingly, many of the anti-leukaemic effects described in the literature for these compounds are linked to an increase in oxidative stress [
199].
Reducing ROS levels as a chemotherapeutic strategy
It can be suggested that reducing ROS levels may restrain the development of these diseases, given the high degree of oxidative stress typical of haematological malignancies. In spite of this, and as discussed above, during the last decade most efforts have been made to kill tumour cells by a ROS overload. In this regard a common assumption is that an antioxidant-rich diet might reduce the incidence of leukaemia. However, the benefit of the antioxidants used during the oncologic treatment is a matter of debate. Some reports show that antioxidants can reduce the cytotoxicity of many chemotherapeutics. Taking this into account, it could be suggested that the use of antioxidants during an oncologic treatment would be unadvisable. However, there is also evidence showing that antioxidants could reduce the toxic side effects caused by pro-oxidant drugs [
200]. Therefore, the use of antioxidants as adjuvants of oncologic treatments indeed requires further evaluation to discover the way in which therapeutic benefits can be attained [
201].
An alternative to ROS-scavengers could involve the inhibition of the source(s) of ROS production, whose identification and further inhibition would be more effective than the use of antioxidants. As discussed above, NADPH oxidases could be one of the sources of ROS in leukaemic cells. In addition, the over-expression of some genes related to chemotherapeutic resistance, such as HO-1, is under NADPH oxidases control [
202]. Considering this, NADPH oxidases appear to be suitable therapeutic targets in leukaemia. Preclinical data show that the inhibition of NADPH oxidases is an effective strategy to block the signalling cascades initiated by the BCR-ABL and FLT3-ITD oncokinases in CML and AML cells, respectively. This evidence supports the hypothesis that the aforementioned proteins induce NADPH oxidase-driven ROS production to maintain the signalling cascade fully active. Thus, the use of TKis and NADPH oxidase inhibitors presents a strong synergistic effect [
81]. As discussed above, several oncogenes increase ROS production through NADPH oxidases, which turns these enzymes into desirable targets against leukaemia. However, the development of novel and more specific inhibitors against NADPH oxidases is still a challenge [
203]. The safety and efficacy of two novel NADPH oxidases inhibitors (GKT136901 and GKT137831) have been tested in diabetic patients (NCT02010242). Once the suitability of these agents is demonstrated, their use could be extended to the treatment of certain types of leukaemia.
The inhibition of ROS production by XOD is another possibility for targeting leukaemic cells. Allopurinol, a XOD inhibitor currently prescribed to gout patients, has been considered since almost 50 years ago for the treatment of haematological malignancies [
204]. In addition to modifying ROS levels, XOD is involved in the metabolism of several drugs. Recent reports suggest the utility of allopurinol to reduce chemotherapy toxicity in leukaemia patients [
205]. An ongoing clinical trial is testing the feasibility of allopurinol to improve 6-mercaptopurine regimen in paediatric ALL treatment (NCT03022747, phase II).
IDHs mutations in AML patients induce an increase in ROS [
206], and the use of IDH inhibitors can be seen as a promising strategy against AML [
207]. Thus, some interesting questions to be addressed in future studies are whether these inhibitors have the ability to reduce the oxidative stress in leukaemic cells, and whether their anti-tumour activity may be increased by the use of other ROS-modifying agents.