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Anti-Cancer Agents in Medicinal Chemistry
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Anti-Cancer Agents in Medicinal Chemistry, 2022, 22, 215-222
215
REVIEW ARTICLE
Reactive Oxygen Species (ROS): Key Components in Cancer Therapies
Biswa Mohan Sahoo1,*, Bimal Krishna Banik2, Preetismita Borah3 and Adya Jain4
1Roland Institute of Pharmaceutical Sciences (Biju Patnaik University of Technology Nodal Centre of Research), Berhampur-760010,
Odisha, India; 2Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin
Fahd University, Al Khobar, Kingdom of Saudi Arabia; 3CSIR-Central Scientific Instruments Organization, Chandigarh, India;
4Department of Chemistry, MRK Educational Institutions, IGU Rewari, Haryana, India
A R T I C L E H I S T O R Y
Received: November 12, 2020
Revised: March 31, 2021
Accepted: April 05, 2021
DOI:
10.2174/1871520621666210608095512
Abstract: Reactive Oxygen Species (ROS) refers to the highly reactive substances which contain oxygen radicals.
Hypochlorous acid, peroxides, superoxide, singlet oxygen, alpha-oxygen, and hydroxyl radicals are the major exam-
ples of ROS. Generally, the reduction of oxygen (O2) in molecular form produces superoxide (•O2
−) anion. ROS are
produced during a variety of biochemical reactions within the cell organelles, such as endoplasmic reticulum, mito-
chondria, and peroxisome. Naturally, ROS are also formed as a byproduct of the normal metabolism of oxygen. The
production of ROS can be induced by various factors such as heavy metals, tobacco, smoke, drugs, xenobiotics, pollut-
ants, and radiation. From various experimental studies, it is reported that ROS acts as either a tumor-suppressing or a
tumor-promoting agent. The elevated level of ROS can arrest the growth of tumors through the persistent increase in
cell cycle inhibition. The increased level of ROS can induce apoptosis by both intrinsic and extrinsic pathways. ROS is
considered to be a tumor-suppressing agent as the production of ROS is due to the use of most of the chemotherapeutic
agents in order to activate cell death. The cytotoxic effect of ROS provides impetus towards apoptosis, but in higher
levels, ROS can cause initiation of malignancy that leads to uncontrolled cell death in cancer cells. In contrast, some
species of ROS can influence various activities at the cellular level, including cell proliferation. This review highlights
the genesis of ROS within cells by various routes and their role in cancer therapies.
Keywords: Reactive oxygen species, generation, cancer, pathway, therapy, free radical.
1. INTRODUCTION
Reactive Oxygen Species (ROS) are defined as reactive, unsta-
ble, and reduced forms of oxygen derivatives that are produced as a
by-product of normal metabolic processes [1]. ROS mainly com-
prise superoxide anion (•O2-), singlet oxygen (1O2), hydrogen per-
oxide (H2O2), hypochlorous acid (HOCl), and hydroxyl radical
( •OH). The reactive nature of ROS is due to the presence of extra-
unpaired electrons at outer shells and their half-life. The half-life
ROS is measured in milliseconds [2]. The hydroxyl radical is the
primary damaging species because it cannot be easily scavenged in
the human body. ROS acts as a secondary messenger in cell signal-
ing pathways and plays a major role in various biological processes
in the case of normal and cancer cells [3]. From various studies, it is
reported that ROS acts as a tumor-suppressing or promoting agent.
ROS is considered a tumor-suppressing agent when the production
of ROS is a mechanism shared by most of the chemotherapeutic
agents due to their involvement in producing cell death [4]. How-
ever, some species of ROS can affect various functions at the cellu-
lar level, including cell proliferation. In the case of cancerous cells,
ROS are usually observed as oncogenic because these are associ-
ated with the initiation, progression, and metastasis of tumors [5].
However, this hypothesis is not clear as ROS may also be crucial
for clearance of tumor. The effect of ROS in the development of
tumors involves damage of DNA directly during the transformation
of carcinogenesis [6].
The function of ROS is observed as conflicting in cancer che-
motherapy. In some cases, the excess levels of ROS exhibit
*Address correspondence to this author at the Roland Institute of Pharmaceutical
Sciences (Biju Patnaik University of Technology Nodal Centre of Research), Berham-
pur-760010, Odisha, India; Tel: +91 -9040442719;
E-mail: drbiswamohansahoo@gm ail.com
anti-tumorigenic activities by inducing the cell cycle arrest and cell
death. In addition to this, it is reported that tumor cells elevate their
survival through augmentation of manipulation mechanisms of
reactive oxygen species that include elevated antioxidant levels or
increased ROS production to maintain the optimum balance of ROS
level that promotes proliferation and survival [7]. In the case of
cancer cells, the oxidative stress induced by drugs results in ROS
mediated cytotoxic effect that preferentially destroys tumor cells
and reduces their proliferation.
Similarly, the cancer cells comprise elevated levels of both
intracellular and extracellular reactive oxygen species. The en-
hanced level of intracellular ROS is due to inadequate production of
ROS or detoxification methods that can transform the normal cells
into m alignant cells. Therefore, attempts are made for the accurate
measurement of ROS in order to characterize their nature, localiza-
tion, and time-course of generation and spread at the cellular level.
This review is mainly focused on the role of ROS involved in dif-
ferent pathways of killing cancer cells during the development of
anti-cancer drugs for achieving good therapeutic efficacy and
minimal side effects. Therefore, there should be a better under-
standing of the molecular mechanisms of the signaling pathway of
ROS in cancer cells and the identification of specific ROS targets
that can provide novel therapeutic avenues for the treatment of
cancer [8].
2. SOURCES AND PRODUCTION OF ROS
ROS is grouped into two categories such as radical ROS and
non-radical ROS. Superoxide anion (•O2
-), hydroxyl radical ( •OH),
nitric oxide (NO•), alkoxyl radicals (RO•), thiyl radicals (RS•),
sulphonyl radicals (ROS•), and lipid peroxyl radical (•LOO-) are
the major examples of radical ROS whereas hydrogen peroxide
(H2O2), singlet oxygen (1O2), dinitrogen dioxide (N2O2), ozone (O3)
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216 Anti-Cancer Agents in Medicinal Chemistry, 2021, Vol. 22, No. 2 Saho o et al.
and hypochlorous acid (HOCl) are considered as non-radical ROS
(Table 1). ROS is commonly generated by different biochemical
and physiological oxidative processes inside the cellular system.
The generation of ROS is generally involved via two routes, includ-
ing (a) in the cell membranes by utilizing NADPH oxidases (b) in
the mitochondria by using the enzyme-like NADH oxidoreductase
as depicted in Fig. (1) [9-11].
Table 1. List of radical and non-radical reactive oxygen species.
Sl. No. Free radicals Non-radicals
1 Superoxide anion (•O2-) Hydrogen peroxide (H2O2)
2 Hydroxyl radical ( •OH) Hypochlorous acid (HOCl)
3 Lipid peroxyl (•LOO-) Singlet oxygen (1O2)
4 Alkoxyl radicals (RO•) Dinitrogen dioxide (N2O2)
5 Sulphonyl radicals (ROS•) Ozone/trioxygen (O3)
Mitochondria and the enzyme systems are considered as the
main sources of generating the reactive oxygen species. The univa-
lent oxygen undergoes a reduction in the mitochondria to produce
superoxide anion (•O2
-), which can be further decreased by the
action of mitochondrial superoxide dismutase (SOD) into non-
radical species like hydrogen peroxide (H2O2) and singlet oxygen
(1O2). This reaction involves the Electron Transport Chain (ETC) of
mitochondria without any implication of enzymatic activity by the
action of oxido-reductive reaction intermediates (Fig. 2) [12, 13].
The reactive oxygen species are generated at the intracellular
part by stimulation of nicotinamide adenine dinucleotide phosphate
(NADPH), oxidases, and peroxidases. NADPH-oxidase (NOX) is
present in different isomeric forms such as Nox1, Nox3, Nox4,
Nox5, Duox1, and Duox2. These isoforms are responsible for gen-
erating the different types of ROS in various normal or pathological
cells. The elevated level of ROS by NOX4 regulates the growth
factor (EGFR) involved in the survival of cells. NOX is one of the
major factors necessary for the immunosuppressant activity of
Myeloid-Derived Suppressor Cells (MDSCs). MDSCs are hetero-
geneous groups of cells that suppress the responses of T-cell and
increase during inflammation and cancer. MDSCs also induce the
progression of cancer via ROS. MDSCs are found to enhance angi-
ogenesis, cell division, and metastasis of tumors [14].
ROS is also generated as an alternate product by other enzyme
systems such as cytochrome-P450, Xanthine Oxidase (XO), and
nitric oxide synthase. The production pathway of ROS also in-
volves the conversion of the hypoxanthine into xanthine in the
presence of XO and xanthine into uric acid that yields superoxide
radicals. Similarly, the pathological conditions involving ischemia,
reperfusion, inflammation, and infection also play a significant role
in regulating the generation of ROS. It has been noted that there is
an increase in the production of ROS in endothelial cells induced by
tumor necrosis factor (TNF) [15].
Fig. (1). Sources of reactive oxygen species.
H2O2
Hydrogen
peroxide
OH•
Hydroxy
radical
Cl−
HOCl
Hypochlorous acid
NO2
NO2
•
Nitrogen dioxide
e− + 2H+
O22−
Peroxide
ion
NO
ONOO•
Peroxy nitrate
HO2
•
Perhydroxyl radical
H+
e−
O2
• −
Superoxide
radical ion
e−
3O2
Triplet
Oxygen
UV
O• −
Oxygen
radical
O31O2
Ozone S inglet Oxygen
O2Irradiation/
Photosensitizer
Fig. (2). Production of Reactive Oxygen Species (ROS).
Role of Reactive Oxygen Species in Cancer Therap ies Anti-Cancer Agents in Medicinal Chemistry, 2022, Vol. 22, No. 2 217
The smoke of cigarette is primarily composed of carcinogenic sub-
stances such as polycyclic aromatic hydrocarbons (PAHs) and nitrosa-
mines that causes accumulation of 8-hydroxydeoxyguanosine (8-
OHdG) in lungs. Due to this effect, cigarette smoking persons exhibit 2
to 3 folds higher level of 8-OHdG that leads to mutations, some of
which might be stimulated by oxygen free radicals, which results in
inflammatory responses, fibrosis, and development of tumor. ROS is
also produced from lymphocytes via 5-lipoxygenase (5-LO) pathway.
In contrast, the cyclooxygenase (COX) generates ROS after stimulation
of TNFα, interleukin-1, bacterial lipopolysaccharide, and tumor pro-
moter-4-O-Tetradecanoyl-Phorbol-13-Acetate (TPA). The neurotrans-
mitter dopamine undergoes oxidation to produce reactive oxygen spe-
cies [16].
Furthermore, most of the chemotherapeutic agents produce
ROS in cancer cells. Anthracyclines such as Doxorubicin, Daun-
orubicin, and Epirubicin cause an increase in the cellular levels of
ROS. Alkylating agents (Busulfan, Cyclophosphamide, Chloram-
bucil, Cisplatin, carmustin, thiotepa) and topoisomerase inhibitors
(Topoisomerase-I inhibitors: camptothecin, topotecan, irinotecan
and Topoisomerase-II inhibitors: doxorubicin, etoposide, Daunoru-
bicin, Epirubicin, mitoxantrone and epipodophyllotoxin) induce a
high level of cellular ROS. In contrast, vinca alkaloids, taxanes,
nucleotide analogues, and anti-metabolites, including anti-folates,
create lower levels of ROS. New chemotherapeutic agents are also
bio-engineered to target the specific compartments at the cellular
level for the generation of ROS and continue for a definite period of
time [17].
3. REGULATION OF ROS
Homeostasis of ROS is needed for the survival of normal cells
and suitable cell signaling. The decrease in the level of ROS can
stimulate signaling pathways to control the metabolic process, dif-
ferentiation and cellular proliferation in a controlled way. In com-
parison with normal cells, cancer cells cause increase in production
of localized ROS that hyperactivates the cell signaling pathways
essential for cellular transformation and carcinogenesis. The accu-
mulation of ROS can arise as a result of activation of the oncogene,
loss of tumor suppressor gene, increased metabolic activity, defi-
ciency of nutrient (low glucose), or low level of oxygen (hypoxia).
The excess levels of ROS can induce oxidative damage and cell
death. So, the cancer cells regulate the accumulation of ROS by
increasing their antioxidant capacity [18].
Cancer cells activate the transcription factor NRF2 (nuclear
factor erythroid-2 related factor-2) to enhance the expression of
antioxidants such as peroxiredoxins (PRXs), superoxide dismutases
(SODs), catalase (CAT), and glutathione peroxidases (GPXs). Can-
cer cells also trigger the genes involved in NADPH production,
glutathione (GSH) synthesis and enzymes detoxification. This type
of balance between production and elimination of ROS maintains
an optimum level of ROS for pro-tumorigenic signaling (Fig. 3)
[19].
4. METHODS FOR MEASUREMENT OF ROS
There is a significant challenge in bio-analytical techniques for
the estimation of reactive oxygen species in both cells and living
organisms. So, it is difficult to identify ROS directly in complex
biological systems because most of the ROS are highly reactive,
short-lived, and do not accumulate at elevated levels. Recently, the
advancement of analytical methods, especially in electron par-
amagnetic resonance and Mass Spectroscopy (MS), afford quantita-
tive and precise measurement of ROS at the cellular level. The
assay methods involved in the measurement o f ROS are susceptible
to numerous artifacts that result from the analytical techniques and
redox sensor (Table 2). These methods are inadequate in th eir func-
tion to distinguish among different types of ROS. The real-time
imaging of oxidative changes by applying redox-sensitive green
fluorescent proteins is recently utilized to provide information
about the location of ROS at the subcellular level. The elevated
levels of ROS are identified in cancer cells due to high metabolic
activity, cellular signaling, peroxisomal activity, mitochondrial
dysfunction, activation of oncogene, and increased enzymatic activ-
ity of oxidases, cyclooxygenases, lipoxygenases, and thymidine
phosphorylases [20, 21].
5. ROLE OF ROS IN CANCER THERAPIES
Cancer is recognized as one of the major heterogeneous dis-
eases with high morbidity and mortality with poor prognosis. It was
estimated that there are 18.1 million new cases of cancer and 9.6
Fig. (3). Balance of reactive oxygen species in cancer cells. (A higher resolution / colour version of this figure is available in the electronic copy of the arti-
cle).
Table 2. Detection methods for measurement of ROS.
Sl. No. Types of ROS Detection Methods
1 Superoxide anion (•O2-) Cyto chrome-c reduction Chemiluminescence reactions Electron paramagnetic resonance
2 Hydroxyl radical ( •OH) Horseradish peroxidase assay Dichlorofluorescein fluorescen ce Dihydrorhodamine-123 fluorescence
3 Sin glet oxygen (1O2) Scanning-laser method I nfrared phosphorescence spectroscopy Electro n paramagnetic resonance spec-
troscopy
218 Anti-Cancer Agents in Medicinal Chemistry, 2021, Vol. 22, No. 2 Saho o et al.
million cancer deaths globally during 2018. Current developments
in different research also reveal that cancer is one of the leading
causes of death and will be a major barrier to increasing life expec-
tancy worldwide. Further, more than 90% of cancer deaths are
caused due to the metastasis of tumor cells. Metastasis is the condi-
tion in which the primary tumor cells spread to distant organs of the
body, and this process is considered the primary cause of morbidity
and mortality of cancer [22].
There are no suitable therapeutic approaches available to sup-
press the metastasis of cancer cells. However, the resistance devel-
oped by conventional chemotherapy and disease relapse remains a
persistent challenge clinically. These observations suggest that
there is no clear information regarding the mechanism of action at
the cellular level and biotic heterogeneity in the cancer cells. Clini-
cally, it is also observed that cancer may be a genetic disorder that
results from both internal factors (e.g., inherited mutations, immune
conditions, hormones) and external factors (e.g., environment, diet,
tobacco, diet, infection, radiation). These factors can also affect
essential genes, including proto-oncogenes, tumor suppressor
genes, and deoxyribonucleic acid repair genes via cellular interme-
diates like reactive oxygen species [23].
In the case of normal cells, ROS are produced in a highly con-
trolled manner as these are associated with the regulation of signal-
ing processes of cell division, immune regulation, autophagy, in-
flammation, and stress-related responses. However, the uncon-
trolled production of ROS can lead to oxidative stress and cytotox-
icity, which impart loss of cellular functions and development of
heterogeneous disease states like cancer. In the early stages of can-
cer, the intracellular ROS promote the initiation of cancer via in-
duction of oxidation and base-pair substitution mutations in pro-
oncogenes such as Ras and tumor suppressor genes such as p53.
Oxidative stress mainly contributes to aging and many other dis-
eases such as cancer, diabetes, and obesity. Oxidative stress arises
due to the excess accumulation of ROS in the cell. This is again due
to an imbalance of oxido-reductive activities that results in damage
to the cellular system, as presented in Fig. (4) [24].
Generally, it is reported that ROS may promote either cell pro-
liferation or cell death depending on the intensity or location of the
oxidative burst and th e activity of the antioxidant system. The abil-
ity of ROS to stimulate cell growth or cell death mainly d epends on
the intensity or duration of redox signals and the defense mecha-
nisms of antioxidants. The existing anti-cancer drugs exert harmful
effects on normal cells, which are partially activated by ROS. These
species exert reverse cellular effects by promoting either cell prolif-
eration and tumor progression or cell death. ROS act as “double-
edged sword” by acting not only as disease inducers or sustainers
but also therapeutic weapons in cancer cells. The increased level of
ROS in mitochondria is found to induce cell proliferation, cell sur-
vival, cell migration, and epithelial-mesenchymal transition through
mitogen-activated protein kinase (MAPK) and R as-ERK activation,
as presented in Fig. (5) and Table 3 [25, 26].
Edderkaoui et al. demonstrated that ROS produced by an ex-
tracellular matrix increases the survival of pancreatic cancer cells
through 5-lipoxygenase (LOX) and NOX [27]. For pancreatic can-
cer, some of the treatment strategies have proved to be effective
that include combination therapy of gemcitabine with trichostatin-
A, epigallocate-3-gallate (EGCG), capsaicin, and benzyl-
isothiocyanate (BITC) [28]. All of these drugs produce their action
based on the same mechanism via elevation of intracellular ROS
levels to trigger apoptosis (Fig. 6). Currently, FDA-approved non-
steroidal anti-inflammatory drugs (NSAIDs) like Sulindac are
tested for their potential in cancer therapy. Sulindac enhances the
intracellular level of ROS that renders colon and lung cancer cells
more sensitive to H2O2 promoted apoptosis [29, 30].
Similarly, the anticancer drug like Aminoflavone promotes cell
death in MCF-7 and MDA-MB-468 breast cancer cells. Chemi-
cally, it is 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-
methylchromen-4-one. The treatment of cancer cells with
Fig. (4). Role of Reactive Oxygen Species (ROS) in carcinogenesis.
