Clinical and Translational Oncology

https://doi.org/10.1007/s12094-020-02325-7

REVIEW ARTICLE

Drug‑metabolizing enzymes: role in drug resistance in cancer

G. Kaur1   · S. K. Gupta1 · P. Singh1 · V. Ali1 · V. Kumar1 · M. Verma1

Received: 1 November 2019 / Accepted: 18 February 2020

© Federación de Sociedades Españolas de Oncología (FESEO) 2020

Abstract
Although continuous researches are going on for the discovery of new chemotherapeutic agents, resistance to these antican-
cer agents has made it really difficult to reach the fruitful results. There are many causes for this resistance that are being
studied by the researchers across the world, but still, success is far because there are several factors that are going along
unattended or have been studied less. Drug-metabolizing enzymes (DMEs) are one of these factors, on which less study has
been conducted. DMEs include Phase I and Phase II enzymes. Cytochrome P450s (CYPs) are major Phase I enzymes while
glutathione-S-transferases (GSTs), UDP-glucuronosyltransferases (UGTs), dihydropyrimidine dehydrogenases are the major
enzymes belonging to the Phase II enzymes. These enzymes play an important role in detoxification of the xenobiotics as well
as the metabolism of drugs, depending upon the tissue in which they are expressed. When present in tumorous tissues, they
cause resistance by metabolizing the drugs and rendering them inactive. In this review, the role of these various enzymes in
anticancer drug metabolism and the possibilities for overcoming the resistance have been discussed.

Keywords  Drug-metabolizing enzymes (DMEs) · Drug metabolism · Drug resistance · DME inhibitors · Prodrugs

Introduction to treat cancer. Drug resistance is well known to us since

At present, cancer is one of those diseases on which exten-
sive research is going on. It has become the topic of con-
cern because of its fatal nature and the increasing number
of cancer cases and deaths all over the world. Although a
huge number of resources are invested for the development
of preventive, diagnostic and therapeutic strategies for the
control of cancer, it is difficult to tackle it with the current
knowledge. The causes of cancer are very vast and so is
its prevention and cure [1]. In the 1990s, the research was
mainly focused on the development of chemotherapeu-
tic agents; those were at that time used as the first line of
therapy, until later on many heterocyclic compounds were
approved by FDA for treating cancer. After that, the research
diverted towards the development of various agents that
could overcome various resistance mechanisms (Fig. 1)
against the already existing anticancer drugs [2]. The devel-
opment of various resistances has made it more difficult
* M. Verma
malkhey.verma@cup.edu.in; malkhey@yahoo.com
1
Department of Biochemistry, School of Basic and Applied
Sciences, Central University of Punjab, Bathinda 151001,
India
very old times. It is a phenomenon when a disease becomes
resistant to pharmaceutical treatments. The concept was first
noticed in bacteria against some antibiotics after that other
diseases were also found to show this phenomenon. Cancer
is one among them. Cancerous cells can acquire resistance
against cytotoxic drugs in many ways [3]. Some of the major
mechanisms responsible for the resistance against anticancer
chemotherapeutic agents are acquired mutations (primary
and secondary), drug metabolism and inactivation, cross-
talk between kinases signaling, alteration in regulation of
cell cycle and checkpoints, blocked apoptosis, cancer stem
cells, immune system evasion, DNA damage and repair, epi-
genetic modifications like DNA methylation, histone modi-
fication, etc. [4].
Drug resistance is a major pitfall to cancer therapy as
most of the chemotherapy will lead to resistance after long
use [4]. In the last few decades, studies have shown that
cancer cells differ in their metabolism from the normal
cells. These metabolic changes are one of the main reasons
for normal cells transforming into cancerous cells and also
a cause for the resistance to various types of chemothera-
peutic drugs. The changed expression and activity of drug-
metabolizing enzymes play a considerable role in the drug
resistance due to which metabolic and signaling pathways

13
Vol.:(0123456789)

Fig. 1  Various causes of anti-
cancer drug resistance
become altered in cancerous cells [5]. These drug-metab-
olizing enzymes have been broadly divided into two cat-
egories—Phase I and Phase II drug-metabolizing enzymes.
Phase I enzymes include the oxidases, dehydrogenases,
deaminases and hydrolases. Phase II enzymes comprise
UDP-glucuronosyl transferases (UGTs), sulfotransferases
(SULTs), glutathione transferases (GSTs), N-acetyl trans-
ferases (NSTs), etc., that generate water-soluble compounds
by the conjugation of products of Phase I reactions or parent
compounds with suitable functional groups (Fig. 2). These
compounds can be easily eliminated. The Phase I enzymes
that play a major role in drug metabolism are the oxidases-
Cytochrome P450 [6]. As they take part in the metabolism of
various anticancer drugs, their elevated expression in various
Fig. 2  Phase II enzymatic reac-
tions in drug metabolism
13
Clinical and Translational Oncology
types of cancers cause rapid turnover and the elimination of
the drug before reaching to the target [7]. Some of the Phase
II drug-metabolizing enzymes that will be discussed in this
review are GSTs, UGTs and DPDs.
Cytochrome P450s
Cytochrome P450s (CYPs) are a large group of enzymes
present in the membrane of the endoplasmic reticulum.
They are terminal oxidases and belong to the heme-thiolate
multigene family. CYPs need molecular oxygen and cou-
pling to NADPH reductase. They take part in oxidative
reactions involved in the metabolism of many of the mar-
keted drugs [8]. Cytochrome P450s have been evolved to
Clinical and Translational Oncology

protect the organisms against toxic compounds [9]. How-
ever, cytochrome P450-mediated biotransformation may
result in metabolic stimulation of environmental chemical
compounds leads to the production of carcinogens which is
called ‘lethal synthesis’ [10]. CYPs play an important role in
detoxification of xenobiotics as well as various endogenous
compounds such as bile acids, steroids, prostaglandins, leu-
kotrienes and unsaturated fats (Table 1) [11].
CYP enzymes are broadly divided into two classes: Class
I and Class II. The Class I is composed of well-conserved
members CYP1A1, CYP1A2, CYP2E1, and CYP3A4; not
having important functional polymorphisms and are actively
involved in the metabolism of drugs and procarcinogens.
Class II is composed of highly polymorphic CYP2B6,
CYP2C9, CYP2C19, and CYP2D6 which actively partici-
pate in drug metabolism but not in procarcinogen metabo-
lism [4]. CYPs are divided into families and subfamilies,
drug-metabolizing enzymes being confined to subfamilies 1,
2, 3 and 4 [8]. There are about 57 genes and 58 pseudogenes
in human differing in their substrate specificity and tissue
expression profile (Table 1). There are about 18 distinct
P450s in the liver, out of which only 10 are from families 1,
2, and 3; metabolize the most of the marketed drugs. These
are CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5.
Some of these enzymes are also expressed in extrahepatic
tissues like kidneys, lungs, small intestine, brain, skin and
placenta. CYP3 group is the most abundant enzyme belong-
ing to the Phase I drug-metabolizing enzymes [8].
The enzyme CYP2C8 has been extensively studied for its
crystal structure and was resolved [13]. The enzyme exists
as a dimer and the approximate molecular weight is 54 kDa.
The structure contains a heme moiety as a prosthetic group
that can bind carbon monoxide very tightly. The name P450
has been given because the microsomes with carbon mon-
oxide exposure show a strong absorption band at 450 nm,
and P stands for pigment. The substrates of P450s range
from smaller (ethylene, MW = 28) to larger (cyclosporine,
MW = 1201) molecules [14]. P450s can be classified into
three classes according to the redox partner they need, Class
I P450s need two redox cofactors, ferredoxin (an iron–sul-
fur protein) and a FAD-containing NAD(P)H-ferredoxin
reductase; Class II P450s use NADPH-P450 reductase hav-
ing FAD/FMN and Class III does not need a separate pro-
tein for reduction [15]. The typical structure of cytochrome
P450 consists of two domains—one having a majorly
α-helix structure and is about 70% of the protein and the
other formed mainly of β-sheet. There are three conserved
residues among the P450 superfamily: (i) cysteine, present
in heme binding region, coordinates the iron present in the
heme group; (ii) glutamine and (iii) arginine, both present
in the α-helix [15].
CYPs catalyze many oxidative reactions like CYP2C8
is involved in particular hydroxylation, N-demethylations
and N-de-ethylations. CYP2C8 can catalyze substrates of
different sizes and structures, because of its structure. It is
involved in the metabolism of anti-microtubule agent pacli-
taxel, which is converted to 6α-hydroxy paclitaxel. CYP3A4
also metabolizes paclitaxel to 39-phenyl-hydroxypaclitaxel.
These metabolites are further metabolized to 6α, 3′-p-dihy-
droxy paclitaxel [16]. CYP3A4 is involved in the metabo-
lism of many of the known drugs including anticancer drugs
like taxol, tamoxifen, ifosfamide, VP-16 and Vinca alkaloids
[17]. The anticancer drugs flutamide, mitoxantrone, pacli-
taxel and docetaxel have also been proposed to be the sub-
strates of the isozyme CYP1B1 as they inhibited the activity
of this enzyme in a competitive manner [18].
Catalytic cycle of CYP enzymes
CYP enzymes have a wide range of substrate specificity and
catalyze a number of redox reactions such as hydroxylation,
heteroatom oxygenation, dealkylation, epoxidation, desatu-
ration, and several others [19, 20]. CYP mediated monoox-
ygenation reactions incorporate one oxygen atom into the
substrate, while the other oxygen atom is reduced to water
[19]. A characteristic CYP enzyme catalyzed reaction is:
RH + O2 + 2e− + 2H+ → ROH + H2 O
The amount of energy required to break the bond between
the oxygen molecules is provided by the transfer of elec-
trons from NADPH through FAD and FMN mainly by
enzyme NADPH cytochrome P450 reductase or in certain

Table 1  The classification of human CYP enzymes on the basis of major substrate classes [12]
Class of substrates Human CYP enzymes

Sterols 1B1, 7A1, 7B1, 8B1, 11A1, 11B1, 11B2, 17A1, 19A1, 21A2, 27A1, 39A1, 46A1, 51A1
Xenobiotics 1A1, 1A2, 2A6, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 3A4, 3A5, 3A7
Fatty acids 2J2, 4A11, 4B1, 4F12
Eicosanoids 4F2, 4F3, 4F8, 5A1, 8A1
Vitamins 2R1, 24A1, 26A1, 26B1, 26C1, 27B1
Unknown 2A7, 2S1, 2U1, 2W1, 3A43, 4A22, 4F11, 4F22, 4V2, 4X1, 4Z1, 20A1, 27C1

13

cases, cytochrome b5 in the smooth endoplasmic reticu-
lum. Cytochrome b5 is the smaller partner of eukaryotic
cytochrome P450 enzymes which act as an allosteric
modulator for numerous CYPs involved in the regulation
of mammalian steroidogenesis. In reality, cytochrome b5
transfers the second electron to particular CYPs faster than
CYP reductase [19]. Finally, the electrons are transferred
from NADPH to CYP inside the mitochondria [9]. There
are several donors of oxygen atom for CYP enzymes such
as hydroperoxides, peracids, perborate, percarbonate, perio-
date, chlorite, iodosobenzene and N-oxides. However, after
passing through a series of reactions CYP iron–oxygen com-
plex produce intermediates such as ferric-peroxo anion spe-
cies ­(FeIIIOO−), ferric-hydroperoxo species (Fe(III)–OOH)
and Fe(III)–(H2O2) complex in CYP mediated monooxy-
genation reactions [20].
Cytochrome P450 enzymes contribute a major part to the
metabolizing enzymes in humans, catalyzing the biotrans-
formation of various exogenous compounds, foods, drugs
and also the endogenous compounds [21]. CYP1, 2 and 3
families of the CYP isozymes have also been found to cata-
lyze the biotransformation of various anticancer drugs [22].
CYPs mainly expressed in the liver (accounts for 90% of
the total body) [23] have also been shown to be conserved
in cancer cells and also been reviewed in tumors and can-
cer cell lines [24]. So, it indicates that CYPs may have a
role in detoxification and biotransformation of anticancer
drugs. Interestingly, many reports underline the coinciding
substrate specificity of CYP3A and ABCB1 (ATP-binding
cassette B1) transporters [25]. This combination of mecha-
nisms that have possibly co-evolved over billions of years
may possibly lead to drug resistance by reducing the con-
centration of active drugs in both, systemic circulation and
the target cells [26].
Various CYPs have been found to be inhibited by vari-
ous chemicals like CYP3A4 have been found to be inhib-
ited by ritonavir. As CYP3A is known to metabolize anti-
cancer drugs like paclitaxel and docetaxel, reducing their
oral availability, co-administration of ritonavir with these
drugs has been shown to enhance their bioavailability [17].
α-Naphthoflavone has also been shown to inhibit CYP1A
isoforms, in vitro. Furafylline, which is a methylxanthine
analogue also known to inhibit CYP1A2-mediated reactions,
where it is a potent inhibitor while the effect is not prominent
in case of CYP1A1 [27].
Like other anticancer drug-metabolizing enzymes, the
overexpression of CYPs in tumor cells can also be utilized
by designing prodrugs that are activated by CYPs (Table 3).
To this approach a series of chalcone derivatives have been
synthesized that showed anti-proliferative activities in
human breast cancer cell lines expressing CYP1A1 and
CYP1B1 and also showed lesser toxicity towards the normal
breast cell line having no CYP expression. The evidence for
13
Clinical and Translational Oncology
the role of CYP1 enzymes in the bioactivation of these com-
pounds was provided by the experiments carried out using
the inhibitors of CYP1, i.e, acacetin and α-naphthoflavone
[28].
Cyclophosphamide, ifosfamide and trofosfamide belong
to the oxazaphosphorine class of prodrugs. They are bio-
activated by CYP enzymes to alkylating anticancer agents.
Cyclophosphamide when introduced initially in 1960 as
an anticancer drug, it was supposed to be activated by the
enzyme phosphoramidase to bis-(chloroethyl) amine. But
later it was found to be oxidized by hepatic CYP enzymes
to 4-hydroxycyclophosphamide. So, cyclophosphamide may
be considered one of the pioneering prodrugs designed for
improving the therapeutic index of highly toxic therapeutic
agents [29]. 1,4-Bis-([2-(dimethylamino-N-oxide) ethyl]
amino) 5,8-dihydroxy anthracene-9,10-dione (AQ4N), a
bioreductive prodrug also require activation by CYP2S1 and
CYP2W1 in tumor tissues and is converted to topoisomer-
ase II inhibitor AQ4 [1,4-bis-5,8-dihydroxy-anthracene-
9,10-dione]. Other prodrugs activated by CYPs include
procarbazine, tegafur, and thiotepa. They are activated by
the enzymes in the liver [30].
Glutathione S‑transferases
Glutathione S-transferases (GSTs) belong to the family of
Phase II detoxification enzymes, which protects cellular
macromolecules from the attack of reactive electrophiles.
Precisely, GSTs catalyze the reaction in which glutathione
(GSH) is conjugated with the wide variety of exogenous
and endogenous xenobiotics. This conjugation process is
the first step of the mercapturic acid pathway, and it helps
in the elimination of toxic compounds from the cell. On the
basis of location, GSTs are divided into two super-family
members: the cytosolic and the membrane-bound micro-
somal family members. Again, the cytosolic GSTs due to
significant genetic polymorphisms in human populations are
divided into six classes, which share nearly 30% sequence
identity, and are labeled by Greek letters α, μ, ω, π, θ and ζ.
Though having few genetic similarities, the affinities of these
GSTs for the substrates differ due to genetic mutation and
have different substrate specificity (Table 2).
The GST is a dimeric enzyme and each subunit of the
enzyme has an active site with two distinctive functional
regions: hydrophilic G-Site (GSH binding site) and hydro-
phobic H-site; the binding site of structurally different elec-
trophilic substrates. The G-site is the conservative site, while
H-site has affinity for variable substrates that regulate the
specificity of GST isozymes [32]. The link between two
subunits of GST constructs the intrasubunit binding site for
ligands, which facilitates the GSH conjugate formed by one
subunit to be insulated from the adjacent subunit and hence
limit the product inhibition [33]. In the reactions catalyzed
Clinical and Translational Oncology

Table 2  Different classes of Compounds Xenobiotics GSTs

GSTs with their substrates [31]
Exogenous Polycyclic aromatic hydrocarbons π GSTs
α,β unsaturated aldehydes π GSTs
Molecule with epoxide group θ GSTs
Chemotherapeutic agents α GSTs, π GSTs
Endogenous O-Quinones of catecholamines and dopamine μ GSTs
Prostaglandins α GSTs, μ, microsomal GSTs
Lipid peroxidation products generated by reactive μ GSTs, μ, microsomal
oxygen species

by GSTs, GSH does the nucleophilic attack on electrophilic
substrates after bringing the substrate into nearby vicinity
with GSH at the active site of the enzyme. The distribution
of GSTs among organ is also diverse and depends on age,
sex and species. GSTA1, GSTA2 and GSTM1 are highly
expressed during entire life, while GSTP1 are expressed in
embryonic and fetal organs that are diminished at the end of
prenatal period [34]. The allocation of GSTs also depends on
the organ, like GSTA1 is expressed in the liver, kidney and
testis; GSTP1 in lung and brain; GSTM2 is highly expressed
in the brain while GSTM1 in expressed at the highest level
in the liver [35]. It is found that the expression of GSTs
also varies from tissue to tissue within the same organ, e.g.,
GSTA1 and GSTA2 is found in the duodenum and small
intestine, while GSTP1 is found in the stomach [36]. The
distribution of GSTs also depends on the cell division stage,
like the erythroleukemia cells (K562) have GSTP1 in its
proliferating stage and during differentiation, this GST is
replaced by GSTA1 [37]. The expression of GSTs in the
human body is also regulated by chemicals and xenobiotics.
The expression of GSTs in the cancer cell lines makes them
resistant to drugs, had led the idea to use GSTs as the marker
of tumor progression.
Among all GSTs, GSTP1 expression is highly coupled
with the development of drug resistance in tumor cell lines.
The GST families include many isozymes with wide sub-
strate specificities, but the polymorphisms within these
isozymes not only contribute to inter-individual differ-
ences in response to the xenobiotics but also to anti-cancer
drugs [31]. In the tumor cells drug resistance develops after
induction and activation of the efflux transporter proteins,
the mutation in the topoisomerase II, enhancement in DNA
repair, alteration in genes which are responsible for apopto-
sis and at last by expressing the detoxifying enzymes. The
alkylating agents which act as the anticancer agents are the
substrates of the GST. The evidence showed that the over-
expression of GSTs and GSH in tumors is associated with
the development of drug resistance.
The level of GST increases by the action of the nuclear
factor-erythroid 2 p45-related factor 2 (Nrf2). Under normal
conditions, the Nrf2 is seized in the cytoplasm due to its
association with the Kelch-like ECH-associated-protein 1
(Keap1) and cullin3, where Nrf2 is degraded by ubiquitina-
tion. But, oxidative stress interrupts the cysteine residue in
the Keap1 and so disruption of (Keap1) and cullin3 ubiq-
uitination system occur. This further leads to the translo-
cation of Nrf2 from the cytoplasm to nucleus, and in the
nucleus Nrf2 carry out the transcription of antioxidative
genes. The nucleophilic attack by GSTs on the sulfur atom
of GSH on the electrophilic group of several xenobiotics
could reduce the activity of the drugs and favor their elimi-
nation by making them water soluble. Hence, the catalytic
activity of GSTs could play a key role in the detoxification
of anticancer drugs such as cyclophosphamide, chlorambu-
cil, carmustine, melphalan, cisplatin, ifosfamide, busulfan,
thiotepa and mitoxantrone [38].
Besides its role as the detoxifying agent, GSTs also regu-
late apoptosis by acting as the inhibitor of c-Jun N-termi-
nal kinase 1 (JNK1), a kinase involved in stress response,
apoptosis, and cellular proliferation. Firstly, the GST was
predicted to be ligand because of its binding capability with
the substrates, which are not linked to the enzymatic mecha-
nisms and hence entitled as ‘ligandin’ [31]. Recently, the
regulatory role of the π and μ classes of GSTs have been
identified in the mitogen-activated protein (MAP) kinase
pathway, one of the signaling pathways in the cell. In nor-
mal cellular condition, low JNK activity was observed due
to the formation of GSTπ–JNK complex, but under oxida-
tive stresses, the complex is degraded, and further GSTπ is
oligomerized and induction of apoptosis occurs [39]. The
JNK is also activated either by immunodepletion or inhibi-
tion of GSTπ. So, for the treatment of cancer, the researchers
are focusing on the GSTs inhibitors, which might break the
GST–JNK complexes and then JNK will be free to carry out
the apoptosis in the cancer cells.
The efflux pumps were found to be over-expressed in sev-
eral cancers and they mediate the extrusion of drugs from
the cells. Similar associations have been found between
GSTs and efflux pumps which lead to a reduction in drug
accumulation and further triggers drug resistance. The com-
monly found efflux transporters are the ABC transporters
Pgp, ABCG2 and mitoxantrone-resistance proteins (MRPs)

13

[40]. Out of these transporters, the MRPs are involved in
the transport of GSH, glucuronate or sulphate conjugates
of xenobiotics that result from after the detoxification reac-
tions carried out by Phase II conjugating enzymes such as
GSTs, sulfotransferases and UDP-glucuronosyltransferases
(UGT). GSTs coexpress with MRP result in drug resistance
in the melanoma cells against vincristine [41], and also
against other anticancer drugs, i.e., chlorambucil, vincris-
tine, ethacrynic acid, and etoposide [42]. The cis-acting
regulatory elements ARE, the promoter site for the antioxi-
dant enzymes, also play a central role in the coexpression of
GSTs and MRPs. Nrf2 also regulates the expression of the
efflux pumps, MRP1 and MRP2 efflux pumps [40].
To cope with the drug resistance, the researchers devel-
oped inhibitors of GSTs and the prodrugs (Table 3). One
of the most specific GSTs inhibitors is TER 199/TLK199
(γ-Glutamyl-S-(benzyl)-cysteinyl-R(-)-phenylglycine
diethyl ester). TLK199 has a phenylglycine moiety which
interacts precisely with a hydrophobic pocket close to the
GSH-binding site of GST and enhances the effect of anti-
tumor drugs, for example, melphalan and chlorambucil
[43]. Another GST inhibitor, the 7-nitro-2,1,3-benzoxadi-
azole derivative NBDHEX binds to the cytosolic GST and
induce the dissociation of JNK–GST complex that leads to
the activation of the JNK-mediated pathway [44]. Prodrugs
are the class of cytotoxic agents that comprise molecules
which exploit the catalytic capability of GSTs, and these
drugs have different affinities for the various isoenzymes
of GST. Findlay and his colleagues synthesized a prodrug
called ­[O2-{2,4-dinitro-5-[4-(N-methylamino) benzoyloxy]
phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate]
(PABA/NO) which had the capability to release cytotoxic
levels of nitric oxide [45]. PABA/NO is an anticancer pro-
drug which transfers its aryl groups to nucleophiles (i.e.,
GSH) along with cogeneration of ions which instinctively
release NO more efficiently at an appropriate pH after
metabolization by GSTs [46]. Later, in vitro transport studies
revealed that PABA/NO are effluxed by MRP1, and hence
the case of drug resistance was observed [45]. Additionally,
PABA/NO are found to be not very much soluble and are
suddenly hydrolyzed. After this, Cui and colleagues syn-
thesized pH-controlled NO donors which are 4-Aryl-1,3,2-
oxathiazolylium-5-olate (OZO) derivatives [47]. The OZO
derivatives react with GSH only in the presence of GST
and further the reactions affect the catalytic properties of
GSTs and activation of JNK to carry out the apoptosis in
the cancer cells. Few other prodrugs include GST-activated
alkylating generators, for example, TER286 [48], ethacra-
platin [49], 2-crotonyloxymethyl-2-cyclohexenone (COMC)
[50], and brostallicin [51, 52].
The catalytic activity and binding properties of some
members of the GST superfamily have been exploited to
generate anticancer agents efficient at overcoming drug
13
Clinical and Translational Oncology
resistance. GST-activated prodrugs use the catalytic power
of these enzymes, particularly GSTP1-1, to activate them-
selves into cytotoxic agents. Some of these molecules yield
promising results and are currently under clinical trials for
the treatment of specific forms of advanced cancers. While
great strides have been made in overcoming drug resistance,
further investigation of both existing and novel strategies
may be the approach necessary to surmount this phenom-
enon [38, 53]. In recent years there has been particular
interest in the link between the GSTP1-1 isoenzyme and
the MAPK pathway that mediates both stress and apoptotic
cellular responses. In fact, several findings indicate that a
promising new strategy for tumor treatment may be the acti-
vation of this pathway through molecules that selectively
target GSTP1-1. After first attempts with the analogues of
GSH, the search for new proapoptotic agents that do not
interact with the multidrug transporters is yielding encour-
aging results and are currently underway at the preclinical
level [38].
In the past few years, along with the GSH-conjugating
catalytic activity, GSTs are additionally found to have the
non-enzymatic activity. It regulates several signaling path-
ways related to cell division, differentiation and cell death
[54]. Among different GST isozymes, GSTP1-1 found
to be highly expressed in cancer cells where it provides
drug resistance to those cells [55]. So, the GST inhibitors,
especially for GSTP1-1, may provide the chemotherapy
for cancer and other diseases linked with abnormal cell
proliferation.
Uridine diphospho‑glucuronosyltransferases
Like GSTs, uridine diphospho-glucuronosyltransferases
(UGTs) also belong to Phase II enzyme classification and it
catalyzes the reactions related to glucuronidation. In glucu-
ronidation, UGTs transfer glucuronosyl group from uridine
5′-diphospho-glucuronic acid (UDPGA) to the compounds
having oxygen, nitrogen, sulfur, or carboxyl functional
group. UGTs are the undoubtedly one of the important con-
jugating enzymes of the liver, which ease the elimination of
several endobiotics (thyroid and steroid hormones, bilirubin,
bile acids and retinoids), as well as xenobiotics (environmen-
tal chemicals, pollutants and drugs). Due to glucuronidation,
these compounds become highly soluble in blood and are
easily excreted from the body via kidney. The process of
glucuronidation is carried out by UGTs in the liver, intesti-
nal mucosa and kidney; and the resultant glucuronides are
secreted in bile and urine. These are highly expressed in the
liver but are also widespread in the gastrointestinal tract,
kidney, lungs, brain, and reproductive tissues [56]. Similar
to GSTs, human UGT superfamily is divided into three sub-
families: UGT1A, 2A, 2B, which after alternative splicing
Clinical and Translational Oncology
and translation further categorized into different functional
proteins. The UGT1A is located on chromosome 2q37 and
codes for nine functional proteins (UGT1A1, UGT1A3,
UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8,
UGT1A9, and UGT1A10), while the UGT2 family is located
on chromosome 4q13 and encodes for eight functional pro-
teins (UGT2A1, UGT2B4, UGT2B7, UGT2B10, UGT2B11,
UGT2B15, UGT2B17, and UGT2B28). This mechanism of
alternative splicing is conserved in human, mouse, and rat
UGTs [57].
UGTs are type-I transmembrane glycoproteins mainly
located within the smooth endoplasmic reticulum (ER),
although some isoforms can be found in the nuclear enve-
lope [58]. The detoxification enzymes cytochromes P450,
CYP (Phase I enzyme) and UGTs (Phase II enzyme) are
found to be related, as CYP add the functional groups to
the molecules that have to be metabolized by UGTs [59].
Apart from the process of detoxification, few times the glu-
curonidation can activate metabolites, like in case of mor-
phine which is converted into morphine 3- and 6-glucuron-
ides and proved to be more potent analgesic than morphine
[60]. Rarely, it happens that glucuronidation metabolizes
substrates into their toxic forms, such as steroid D-ring
glucuronides, which could be the mediator for intrahepatic
cholestasis of pregnancy [61].
The UGT1A1 gene codes for an enzyme which conjugates
bilirubin to facilitate its removal from the human body. The
UGT1A1 gene comprises a TA repeat promoter polymor-
phism, located 41 nucleotides upstream of the translational
start site adjacent to a presumed TATA box, is responsible
for the decreased enzymatic activity of UGT1A1. In the
normal state, the serum bilirubin has the antioxidant and
cytoprotective properties, but in the individuals with seven
thymine-adenine (TA) repeats the condition of hyperbiliru-
binemia occurs. This elevation in bilirubin level may further
cause brain damage in neonates and could also modulate
susceptibility to oxidative damage and cancer. The polymor-
phism in UGT1A1 gene causes the production of UGT1A1
variants, and these variants may cause jaundice. Presently,
around 135 genetic variants of UGT1A1 have been reported
in which the most common variant allele is UGT1A1*28
[62]. This variant has seven TA repeats within the TATA
box promoter region in comparison to 6 TA repeats of the
wild-type allele [63].
UGT1A1*28 is also associated with drug toxicity,
which leads to neutropenia following intravenous and iri-
notecan therapy. Irinotecan (brand name Camptosar), is
an anti-cancer drug for colorectal cancer and the role of
UGT1A1 enzyme in its metabolization is highly studied.
This drug is a topoisomerase I inhibitor and is converted
into its active form (SN-38) in the human body. SN-38 is
further inactivated and detoxified by enzyme encoded by the
UGT1A1 gene [64]. But the presence of modified structure
of UGT1A1 gene, i.e., UGT1A1*28 results in reduced excre-
tion of irinotecan metabolites, and due to the accumulation
of these metabolites in blood, the development of neutrope-
nia occurs [65].
The role of UGTs in breast cancer, the most common can-
cer, is also detected. For the growth of breast, the estrogen is
required, but excessive estrogen level in cell signaling cause
breast cancer. The relation between the activity of UGT1A1
alleles and breast cancer is mixed type because of results
from different studies. Four studies reported link between
low activity UGT1A1 alleles (i.e., *28, *34) and increased
breast cancer threat [66], while this increased risk was not
identified by few other studies [67]. A case study of Nigerian
women confirmed a negative association between low activ-
ity UGT1A1*28 allele and breast cancer [68].
A case study showed the potential involvement of the
UGT1A1 promoter TATA box polymorphism and endome-
trial cancer risk [69]. The results from this study revealed
that women having homozygous (*28/*28) or heterozy-
gous (*1/*28) UGT1A1*28 alleles had a reduced risk of
endometrial cancer in comparison to women homozy-
gous for UGT1A1*1 allele (*1/*1). Normally, UGT1A*1
allele of UGT1A1 in the uterus show high activity toward
2-hydroestradiol, an antiproliferative metabolite of estradiol,
while UGT1A1*28 allele diminishes the capability of the
body to glucuronidate 2-hydroestradiol in the endometrium
and hence reduces the risk of developing endometrial cancer
[69].
Since last 15 years, numerous case–control studies have
evaluated that the polymorphism in UGT genes (primarily
UGT1A and UGT2B genes), increase the risk of cancers
(bladder, breast, colorectal, endometrial, esophageal, head
and neck, liver, lungs, prostate, and thyroid). Few reports
proved that polymorphism in a single UGT allele might give
rise to multiple cancers. In the coming future, the assured
association of several cancers with UGTs polymorphism
requires detailed studies with a large number of sample size
in different situations to end the conflicts between the results
of different studies.
Dihydropyrimidine dehydrogenases (DPDs)
DPD is also known as dihydrouracil dehydrogenase, dihy-
drothymine dehydrogenase and uracil reductase. DPD plays
an important role in catabolizing various nitrogen-contain-
ing molecules like pyrimidines: uracil and thymine. Along
with these endogenous nitrogenous bases, DPD also play
an important role in 5-FU (5-fluorouracil, an anticancer
drug) metabolism (Table 3). As it is the major enzyme in
5-FU catabolism, metabolizing about 85% of the adminis-
tered amount, into a compound 5-fluoro-5,6-dihydrouracil
(5-FUH2). This inactivation of 5-FU reduces its anabolism
and thus its overall metabolism. So, the increased level of
13
 Clinical and Translational Oncology

DPD in tumors results in the nitrogen heterocycle based anti-
cancer drug resistance [2]. The enzyme in its cytosolic form
is present in a many of the tissues with major activity being
found in liver, catalyzes the reduction of the pyrimidines,
uracil and thymine in an NADPH dependent manner, leading
to the 5-FUH2 formation (Fig. 3) [70]. Researchers are able
to measure 5-FU metabolites with the innovation of new
techniques like high-pressure liquid chromatography [71].
On the basis of these findings it is confirmed that DPD is the
first rate-limiting enzyme involved in the catabolic process
of 5-FU, but it did not show the clinical importance of these
results [71]. Shortly after that in clinical literature, they iden-
tified the key role of DPD activity in catabolism of fluoropy-
rimidine and the consequences of DPD deficit for fluoropy-
rimidine related severe adverse events (AEs). The first case
related to this catabolic enzyme reported that patient had
oral ulceration, neurotoxicity and severe myelosuppression,
and further analysis of case confirmed that patient had famil-
ial pyrimidinemia and pyrimidinuria as a result of uracil
and thymine in blood and urine [72]. The removal of 5-FU
in cancer patients takes place via DPD dependent metabo-
lism. Based on this study it was found that the removal of
more than 80% of systemic 5-FU, along with 95% of the
ultimate metabolite via urine is solely responsible for the
catabolic pathway (Fig. 3) [73]. Diasio and his colleagues
issued a case report after the confirmation of DPD as the
vital metabolic enzyme causative for 5-FU elimination, the
case report was about a female patient with rigorous fluoro-
pyrimidine-induced neurotoxicity, a familial DPD deficiency
[74]. Various case reports identified the relation between
DPD deficiency and fluoropyrimidine toxicities [75]. For
the identification of the molecular background of this heredi-
tary defect, cloning of DPYD stage was set [76]. The most
common familial DPD deficiency was recognized which is
related to a fault in the processing of the DPD precursor
mRNA, specifically an exon-skipping abnormality that cause
the decline of 165 nucleotides from the fully spliced mRNA
[77]. One year later, two different researchers published the
first DNA sequence level recognition of this DPYD varia-
tion; they represented similar results within two different
families. Both identified a single nucleotide polymorphism
(SNP) inside DPYD that depicts a new splice site, which
results into exon 14 skipping. The resulting DPD protein has
loss of its function completely [78]. Now this DPYD variant
is generally called as DPYD*2A (or called as rs3918290 or
c.1905 + 1G > A). Around 2% of Caucasians of European
descent possess this DPYD*2A allele. 50% reduction of
DPD activity is exhibited in heterozygous carriers allele,
besides that is very rare (~ 1:1000), homozygous DPYD*2A
patients determine complete DPD deficiency [79].
Knowing the DPD activity is essential for understanding
the link between DPD deficiency and severe fluoropyrimi-
dine-related AEs. DPD activity follows a circadian rhythm
and its activity was recorded at peak in the midnight with the

Table 3  Drug-metabolizing enzymes overexpressed in various tumors and their substrates and prodrugs

Sr. no. Enzymes Tumor overexpression Resistance against drugs Prodrugs

1. CYPs Breast cancer, colon cancer, stomach Paclitaxel, taxol, tamoxifen, VP-16, Chalcone derivatives, cyclophosphamide,
cancer flutamide, mitoxantrone docetaxel ifosfamide, trofosfamide
2. GSTs Colorectal tumor, prostate cancer, ovar- Cis-platin, chlorambucil adriamicin, Acrylic acid, sulphonamide derivative of
ian cancer, mantle cell lymphomas, melphalan doxorubicin, ANS-etoposide, ANS-
breast cancer cells DOX
3. UGTs Human colorectal tumor, human Irinotecan sorafenib, PR-104A, –
esophageal tumor, lung cancer cells GS-1101 methotrexate
breast cancer cells, chronic lympho-
cytic leukemia
4. DPDs Lung cancer, colorectal cancer, breast 5-Fluorouracil 1-Ethoxymethyl 5-fluorouracil, tegafur
cancer

Fig. 3  Inactivation and elimination of 5-FU by the action of an onate (FUPA) and the third enzyme beta-ureidopropionase (UPB1)
enzyme dihydropyrimidine dehydrogenase (DPD) that converted into activity finally terminates in urinary elimination of fluoro-beta-ala-
5-dihydrofluorouracil (5-DHFU). The second enzyme dihydropy- nine (FBAL)
rimidinase (DPYS) causes the formation of fluoro-beta-ureidopropi-

13
Clinical and Translational Oncology
trough in the early afternoon [80]. An interesting discovery
was related to the difference in the systemic 5-FU disclosure
through persisting uninterrupted infusion, with a change of
2.3-fold from peak to trough in the level of systemic 5-FU
during the 5-day course [81]. Despite that there is little
agreement on the rhythm’s physiological consequence [82].
Generally, it was comprehended that hepatic DPD activity
is causative for the bulk of 5-FU clearance [74], but at the
population level it follows an ordinary division [83]. The
DPD activity from the frozen liver samples was measured
by radiolabeled biochemical assay. They showed a power-
ful connection among level of DPD protein expression and
enzyme activity [83]. Alternate work has been done to esti-
mate the DPD activity required to correlate mRNA expres-
sion with DPD activity. But, later the conclusion of the study
was demolished as it was understood that elevated expres-
sion of mRNA might not recompense for inactive enzyme.
Thus, DPD mRNA levels might not provide satisfactory con-
sideration of 5-FU elimination. Throughout the examination
of peripheral blood mononuclear cells (PBMCs), researchers
have desired to understand population difference in DPD
activity, however significant correlation among PBMC DPD
activity and hepatocyte DPD activity R2 < 0.6 [84], that cat-
egorize the significance of PBMC DPD activity studies fur-
ther difficult. Besides that, the relationship among PBMC
DPD activity and systemic 5-FU clearance established weak
associations than PBMC DPD activity and hepatic DPD
activity [84, 85]. Finally, PBMC DPD activity established
larger difference than established in examination of hepatic
DPD activity, wherein PBMC DPD confirmed difference of
activity among 8- to 21-fold based on the study [85]. Thus,
the gain of PBMC DPD activity in illustrating the popula-
tion difference of endogenous DPD activity remains tough
to understand. Beside that DPD activity is acknowledged to
Fig. 4  Deactivation of 5-FU by
DPD
differ among healthy and malignant tissues of the identical
organ [86].
DPD enzyme has been isolated and purified from various
sources like pig, rat, bovine and human. It is homodimer
of each monomer being 111 kDa. It has a large number of
redox cofactors. These are one FAD, one FMN, 16 acid-
labile sulfur and 16 non-heme-iron atoms, present on each
(1025 amino acid residue long) subunit. According to the
amino acid sequence analysis, the enzyme most likely has
four [4Fe–4S] cluster in each subunit, two being in an N-ter-
minal Cys-rich stretch, and the other two in a 50 amino acid
long stretch at the C-terminus. According to two-site ping-
pong kinetic mechanism, there are two separate binding sites
for NADPH/NADP+ and pyrimidine/5,6-dihydropyrimidine,
respectively (Fig. 4) [70].
DPD is an important regulatory enzyme in the metabo-
lism of the anticancer drug 5-FU [2]. 5-FU is counted in the
most widely used drugs and has been in use for a long time.
In 1994, for the first time, DPD activity was shown to be
significantly related to the sensitivity of 5-FU. An experi-
ment was performed to check the co-relation between DPD
activity and 5-FU sensitivity, in which 19 human tumor cell
lines, from digestive tract, breast, head and neck cancer cells
were taken into the investigation. Marked differences were
shown between the cell lines. DPD activity was shown in
all cell lines except four. The cell lines showed the inverse
relation between DPD activity and 5-FU sensitivity [87].
Further several studies show that DPD can be a major fac-
tor for variation in effectiveness of anti-tumor agent 5-FU
among various tumors as well as people. Few studies on
DPD have shown that there is variable expression of DPD
in various tumors. These varying levels of DPD in tumors
are in accordance with the variance in the responses of the
tumors towards 5-FU [2]. In another study, head and neck
13

squamous cell carcinoma (HNSCC) were used to check the
relation between DPD expression and cytotoxic activity of
5-FU. DPD cDNA was constitutively expressed and 5-FU
cytotoxicity was examined. The cytotoxicity was shown to
be decreased with the overexpression of DPD [88].
Knowing the role of DPD in the varying sensitivity to
5-FU and also their negative correlation, it became a point
of interest for researchers to consider inhibiting DPD in
order to eliminate the varying responses to 5-FU. Few years
ago, attempts have been made to synthesize the inhibitors
of DPD. New fluoropyrimidine drugs have been introduced
using the DPD inhibition agents called as dihydropyrimidine
dehydrogenase inhibitory fluoropyrimidines (DIFs).
There are four DIF drugs: UFT, ethynyluracil, S-1, and
BOF-A2. These drugs differ in both type and degree of DPD
inhibition produced. UFT was the first DIF drug to be syn-
thesized. It is a combination of uracil, the naturally occur-
ring pyrimidine, and the 5-FU prodrug-tegafur. Actually, it
is competitive inhibition of DPD. In the presence of excess
uracil, there is a competition at DPD level, so the 5-FU will
not be degraded rapidly and its presence will be prolonged.
This effect is reversible, so it is better than the earlier inhibi-
tors [89]. Ethynyluracil (eniluracil) is a pyrimidine structur-
ally similar to uracil and 5-FU. It is an irreversible inhibitor
of DPD. In its presence, the oral bioavailability of 5-FU
is increased to 100%, which was earlier hampered due to
the variable activity of DPD enzyme. Phase I and Phase II
trials have been done, which shows the increased half-life
and decreased clearance of 5-FU. So, eniluracil is a promis-
ing approach that allows reliable and safer oral administra-
tion of 5-FU and overcomes the resistance caused by the
overexpression of DPD [90]. S-1 is a combination of three
drugs—tegafur (prodrug); 5-chloro-2,4-dihydroxypyridine
[CDHP] (a DPD inhibitor); and potassium oxalate in a molar
ratio of 1:0.4:1. This combination along with the previous
inhibitor provides the relief from GI-tract toxicity. Potassium
oxalate selectively inhibits 5-FU phosphorylation caused by
the enzyme, orotate phosphoribosyltransferase, in the gas-
trointestinal tract and not in tumor cells [89, 91]. BOF-A2
is still another attempt towards improved fluoropyrimidine.
In this drug combination, 1-ethoxymethyl 5-fluorouracil
(EM-FU)-the prodrug is combined with the DPD inhibitor
3-cyano-2,6-dehydropyrimidine (CNDP) in a molar ratio of
1:1. EM-FU is resistant to degradation and is converted to
5-FU by liver microsomes [89]. TAS-114 also inhibits DPD
enzyme, it targets the metabolism of 5-FU to enhance its
anticancer activity and also modulates the catabolic path-
way to enhance the systemic availability of 5-FU. It affects
the DPD enzyme moderately and in irreversible manner. It
enhanced the bioavailability of 5-FU on co-administration
with capecitabine in mice and improved the therapeutic
index of capecitabine [92].
13
Clinical and Translational Oncology
There are several other anticancer drugs involved in the
cure of cancer through different mechanisms. Angiostatin
K13 is a potent inhibitor of endothelial cell growth and
angiogenesis and acts as an anticancer agent that targets
VEGF and prevents angiogenesis [93]. Staurosporine is a
powerful inhibitor that stops angiogenesis by suppressing
up-regulated VEGF expression in tumor cells and prevents
their angiogenesis [94]. A well-documented inhibitor of
microsomal UGT1 A4 is 1-naphthol UGTI-3. The mode of
action of 1-naphthol includes its higher glucuronidation via
UGT1 A6, which results in the decreased level of UDP-glu-
curonic acid and a higher level of UDP and plays an impor-
tant role in competitive inhibition of UDP-glucuronic acid
[95]. Recently, kinase inhibitors against UGTs such as lapat-
inib UGTI-8, pazopanib UGTI-9, regorafenib UGTI-10, and
sorafenib UGTI-11 are reported to inhibit the UGTs enzyme
[96]. Pilocarpine and sulfaphenazole both inhibit CYP2C9,
the ketoconazole inhibits CYP3A4, and quinidine inhib-
its CYP2D6 vigorously [97]. Cimetidine is a well-known
inhibitor of P450-linked reaction. It has been shown that
cimetidine interacts with human hepatic CYP1A, 2C, 2D,
2E, and 3A forms and inhibits P-450-linked reactions [98].
Gemcitabine (2′,2′-difluoro-2′-deoxycytidine, dFdC) is intra-
cellularly metabolized to 5′diphosphate (dFdCDP). It inhib-
its ribonucleotide reductase and an encouraging anticancer
drug [99]. HDAC inhibitors such as vorinostat and romidep-
sin have been approved by the Food and Drug Administra-
tion, USA, as anticancer drugs [100]. The metal-binding
compounds such as clioquinol (a zinc ionophore) is another
anticancer drug that targets cyclin D1 gene at the levels both
transcriptional and post-transcriptional regulation in cancer
cells. It encourages mRNA degeneration of the cyclin D1
gene controlled by miR-302C. It means the metal-binding
compounds have an impact on gene expression at different
regulatory levels. In this case, the post-transcriptional gene
regulation can be a probable target for chemotherapy [101].
Conclusion
Drug-metabolizing enzymes are one of the important fac-
tors causing resistance against various anticancer drugs. In
the last two decades, people have worked on DMEs, but
yet their role in the anticancer drug resistance has not been
fully understood. In this review, significant data have been
collected to show the role DMEs play in drug resistance.
So, while designing new drugs, the role of these enzymes
should be taken into consideration. Various inhibitors of
these enzymes have been discussed that can be used as a
combination with the anticancer drugs to overcome the
resistance. The overexpression of these enzymes in cancers
can be turned into an advantage by the use of prodrugs that
are being activated by these enzymes. In that way, they will
Clinical and Translational Oncology
help in making the anticancer drugs more specific to the
cancer cells. As these enzymes are overexpressed in cancer
cells, the prodrug will remain inactive in the normal cells
decreasing the cytotoxicity of the drugs on normal cells.
Hence, there is still a need for research to be done in this
area of anticancer drug resistance and should be taken as a
potential factor of consideration while designing anticancer
drugs.
Acknowledgements  The authors acknowledge the support received
from the Central University of Punjab, Bhatinda, India, in writing this
manuscript.
Authors’ contribution  MV conceived and designed the research work.
GK and SKG both have first authorship. PS and VA further discussed
and improved the review. All authors read and approved the final
manuscript.
Compliance with ethical standard  together in restricting the oral bioavailability of paclitaxel. Int J
Conflict of interest  The authors declare no conflicting interests.
Ethical approval  This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent  For this type of study, formal consent is not required.
References
1. Kumar S, Ahmad MK, Waseem M, Pandey AK. Drug targets for
cancer treatment: an overview. Med Chem. 2015;5:115–23.
2. Pathania S, Bhatia R, Baldi A, Singh R, Rawal RK. Drug metabo-
lizing enzymes and their inhibitors’ role in cancer resistance.
Biomed Pharmacother. 2018;105:53–65.
3. Housman G, Byler S, Heerboth S, Lapinska K, Longacre M, Sny-
der N, Sarkar S. Drug resistance in cancer: an overview. Cancers.
2014;6(3):1769–92.
4. Leary M, Heerboth S, Lapinska K, Sarkar S. Sensitization of
drug resistant cancer cells: a matter of combination therapy. Can-
cers. 2018;10(12):483.
5. Rochat B. Importance of influx and efflux systems and xenobiotic
metabolizing enzymes in intratumoral disposition of anticancer
agents. Curr Cancer Drug Targets. 2009;9(5):652–74.
6. Jain AK, Jain S, Rana AC. Metabolic enzyme considerations in
cancer therapy. Malays J Med Sci. 2007;14(1):10.
7. Doehmer J, Goeptar AR, Vermeulen NP. Cytochromes P450 and
drug resistance. Cytotechnology. 1993;12(1–3):357–66.
8. Penner N, Woodward C, Prakash C. Drug metabolizing enzymes
and biotransformation reactions. In: Zhang D, Surapaneni S, edi-
tors. ADME enabling technologies in drug design and develop-
ment. Hoboken: Wiley; 2012. p. 545–65.
9. Guengerich FP, Waterman MR, Egli M. Recent structural
insights into cytochrome P450 function. Trends Pharmacol Sci.
2016;37(8):625–40.
10. Wahlang B, Falkner KC, Cave MC, Prough RA. Role of
cytochrome P450 monooxygenase in carcinogen and chemo-
therapeutic drug metabolism. Advances in Pharmacology. Cam-
bridge: Academic Press; 2015. p. 1–33.
11. Rendic S, Guengerich FP. Survey of human oxidoreductases
and cytochrome P450 enzymes involved in the metabolism
of xenobiotic and natural chemicals. Chem Res Toxicol.
2014;28(1):38–42.
12. Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM,
Nebert DW. Comparison of cytochrome P450 (CYP) genes from
the mouse and human genomes, including nomenclature recom-
mendations for genes, pseudogenes and alternative-splice vari-
ants. Pharmacogenetics Genomics. 2004;14(1):1–8.
13. Schoch GA, Yano JK, Wester MR, Griffin KJ, Stout CD, John-
son EF. Structure of human microsomal cytochrome P450 2C8
Evidence for a peripheral fatty acid binding site. J Biol Chem.
2004;279(10):9497–503.
14. Testa B, Krämer SD. The biochemistry of drug metabolism–an
introduction: part 2. Redox reactions and their enzymes. Chem
Biodiversity. 2007;4(3):257–405.
15. Graham-Lorence S, Peterson JA. P450s: structural similarities
and functional differences. FASEB J. 1996;10(2):206–14.
16. Backman JT, Filppula AM, Niemi M, Neuvonen PJ. Role of
cytochrome P450 2C8 in drug metabolism and interactions.
Pharmacol Rev. 2016;68(1):168–241.
17. Hendrikx JJ, Lagas JS, Rosing H, Schellens JH, Beijnen JH,
Schinkel AH. P-glycoprotein and cytochrome P450 3A act
Cancer. 2013;132(10):2439–47.
18. Rochat B, Morsman JM, Murray GI, Figg WD, McLeod HL.
Human CYP1B1 and anticancer agent metabolism: mechanism
for tumor-specific drug inactivation? J Pharmacol Exp Ther.
2001;296(2):537–41.
19. Isin EM, Guengerich FP. Complex reactions catalyzed by
cytochrome P450 enzymes. Biochimica et Biophysica Acta
(BBA)-General Subjects. 2007;1770(3):314–29.
20. Hrycay EG, Bandiera SM. Monooxygenase, peroxidase and per-
oxygenase properties and reaction mechanisms of cytochrome
P450 enzymes. Monooxygenase, peroxidase and peroxyge-
nase properties and mechanisms of cytochrome P450. Cham:
Springer; 2015. p. 1–61.
21. Ingelman-Sundberg M, Daly AK, Oscarson M, Nebert DW.
Human cytochrome P450 (CYP) genes: recommendations
for the nomenclature of alleles. Pharmacogenet Genomics.
2000;10(1):91–3.
22. Iyer L, Ratain MJ. Pharmacogenetics and cancer chemotherapy.
Eur J Cancer. 1998;34(10):1493–9.
23. Krishna DR, Klotz U. Extrahepatic metabolism of drugs in
humans. Clin Pharmacokinet. 1994;26(2):144–60.
24. Doherty MM, Michael M. Tumoral drug metabolism: per-
spectives and therapeutic implications. Curr Drug Metab.
2003;4(2):131–49.
25. Huang L, Wring SA, Woolley JL, Brouwer KR, Serabjit-Singh
C, Polli JW. Induction of P-glycoprotein and cytochrome
P450 3A by HIV protease inhibitors. Drug Metab Disposition.
2001;29(5):754–60.
26. Cummings J, Zelcer N, Allen JD, Yao D, Boyd G, Maliepaard
M, Friedberg TH, Smyth JF, Jodrell DI. Glucuronidation as a
mechanism of intrinsic drug resistance in colon cancer cells:
contribution of drug transport proteins. Biochem Pharmacol.
2004;67(1):31–9.
27. Pelkonen O, Mäeenpäeä J, Taavitsainen P, Rautio A, Raunio
H. Inhibition and induction of human cytochrome P450 (CYP)
enzymes. Xenobiotica. 1998;28(12):1203–53.
28. Ruparelia KC, Zeka K, Ijaz T, Ankrett DN, Wilsher NE, Butler
PC, Tan HL, Lodhi S, Bhambra AS, Potter GA, Arroo RR. The
synthesis of chalcones as anticancer prodrugs and their bioac-
tivation in CYP1 expressing breast cancer cells. Med Chem.
2018;14(4):322–32.
29. Stella V, Borchardt R, Hageman M, Oliyai R, Maag H, Tilley J,
editors. Prodrugs: challenges and rewards. Springer Science &
Business Media; 2007.
13

30. Nishida CR, Lee M, De Montellano PR. Efficient hypoxic activa-
tion of the anticancer agent AQ4N by CYP2S1 and CYP2W1.
Mol Pharmacol. 2010;78(3):497–502.
31. Townsend DM, Tew KD. The role of glutathione-S-transferase
in anti-cancer drug resistance. Oncogene. 2003;22(47):7369.
32. Deponte M. Glutathione catalysis and the reaction mechanisms of
glutathione-dependent enzymes. Biochimica et Biophysica Acta
(BBA) Gen Subj. 2013;1830(5):3217–66.
33. Wu B, Dong D. Human cytosolic glutathione transferases:
structure, function, and drug discovery. Trends Pharmacol Sci.
2012;33(12):656–68.
34. Raijmakers MT, Steegers EA, Peters WH. Glutathione S-trans-
ferases and thiol concentrations in embryonic and early fetal tis-
sues. Hum Reprod. 2001;16(11):2445–50.
35. Sherratt PJ, Hayes JD. Glutathione S-transferases. Enzyme Sys-
tems Metab Drugs Other Xenobiotics. 2001;2002:319–52.
36. Coles BF, Chen G, Kadlubar FF, Radominska-Pandya A. Inter-
individual variation and organ-specific patterns of glutathione
S-transferase alpha, mu, and pi expression in gastrointestinal
tract mucosa of normal individuals. Arch Biochem Biophys.
2002;403(2):270–6.
37. Schnekenburger M, Morceau F, Henry E, Blasius R, Dicato M,
Trentesaux C, Diederich M. Transcriptional and post-transcrip-
tional regulation of glutathione S-transferase P1 expression dur-
ing butyric acid-induced differentiation of K562 cells. Leukemia
Res. 2006;30(5):561–8.
38. Sau A, Tregno FP, Valentino F, Federici G, Caccuri AM.
Glutathione transferases and development of new princi-
ples to overcome drug resistance. Arch Biochem Biophys.
2010;500(2):116–22.
39. Adler V, Yin Z, Fuchs SY, Benezra M, Rosario L, Tew KD, Pin-
cus MR, Sardana M, Henderson CJ, Wolf CR, Davis RJ. Regula-
tion of JNK signaling by GSTp. EMBO J. 1999;18(5):1321–34.
40. Meijerman I, Beijnen JH, Schellens JH. Combined action and
regulation of phase II enzymes and multidrug resistance pro-
teins in multidrug resistance in cancer. Cancer Treat Rev.
2008;34(6):505–20.
41. Depeille P, Cuq P, Mary S, Passagne I, Evrard A, Cupissol D,
Vian L. Glutathione S-transferase M1 and multidrug resistance
protein 1 act in synergy to protect melanoma cells from vincris-
tine effects. Mol Pharmacol. 2004;65(4):897–905.
42. Depeille P, Cuq P, Passagne I, Evrard A, Vian L. Combined
effects of GSTP1 and MRP1 in melanoma drug resistance. Br J
Cancer. 2005;93(2):216.
43. Morgan AS, Ciaccio PJ, Tew KD, Kauvar LM, Ciaccio FJ.
Isozyme-specific glutathione S-transferase inhibitors potenti-
ate drug sensitivity in cultured human tumor cell lines. Cancer
Chemother Pharmacol. 1996;37(4):363–70.
44. Federici L, Sterzo CL, Pezzola S, Di Matteo A, Scaloni F, Fed-
erici G, Caccuri AM. Structural basis for the binding of the
anticancer compound 6-(7-nitro-2, 1, 3-benzoxadiazol-4-ylthio)
hexanol to human glutathione S-transferases. Cancer Res.
2009;69(20):8025–34.
45. Findlay VJ, Townsend DM, Saavedra JE, Buzard GS, Citro ML,
Keefer LK, Ji X, Tew KD. Tumor cell responses to a novel glu-
tathione S-transferase–activated nitric oxide-releasing prodrug.
Mol Pharmacol. 2004;65(5):1070–9.
46. Saavedra JE, Srinivasan A, Bonifant CL, Chu J, Shanklin AP,
Flippen-Anderson JL, Rice WG, Turpin JA, Davies KM, Keefer
LK. The secondary amine/nitric oxide complex ion R2N [N (O)
NO]-as nucleophile and leaving group in SNAr reactions. J Org
Chem. 2001;66(9):3090–8.
47. Cui H, Shen J, Lu D, Zhang T, Zhang W, Sun D, Wang PG.
4-Aryl-1, 3, 2-oxathiazolylium-5-olate: a novel GST inhibitor
to release JNK and activate c-Jun for cancer therapy. Cancer
Chemother Pharmacol. 2008;62(3):509–15.
13
Clinical and Translational Oncology
48. Vergote I, Finkler N, Del Campo J, Lohr A, Hunter J, Matei
D, Kavanagh J, Vermorken JB, Meng L, Jones M, Brown G.
Phase 3 randomised study of canfosfamide (­ Telcyta®, TLK286)
versus pegylated liposomal doxorubicin or topotecan as third-
line therapy in patients with platinum-refractory or-resistant
ovarian cancer. Eur J Cancer. 2009;45(13):2324–32.
49. Ang WH, Khalaila I, Allardyce CS, Juillerat-Jeanneret L,
Dyson PJ. Rational design of platinum (IV) compounds to
overcome glutathione-S-transferase mediated drug resistance.
J Am Chem Soc. 2005;127(5):1382–3.
50. Hamilton DS, Zhang X, Ding Z, Hubatsch I, Mannervik B,
Houk KN, Ganem B, Creighton DJ. Mechanism of the glu-
tathione transferase-catalyzed conversion of antitumor 2-crot-
onyloxymethyl-2-cycloalkenones to GSH adducts. J Am Chem
Soc. 2003;125(49):15049–58.
51. Lorusso D, Mainenti S, Pietragalla A, Ferrandina G, Foco G,
Masciullo V, Scambia G. Brostallicin (PNU 166196), a new
minor groove DNA binder: preclinical and clinical activity.
Expert Opin Investig Drugs. 2009;18(12):1939–46.
52. Pezzola S, Antonini G, Geroni C, Beria I, Colombo M, Brog-
gini M, Mongelli N, Leboffe L, MacArthur R, Mozzi AF, Fed-
erici G. Role of glutathione transferases in the mechanism of
brostallicin activation. Biochemistry. 2009;49(1):226–35.
53. Ruzza P, Calderan A. Glutathione transferase (GST)-activated
prodrugs. Pharmaceutics. 2013;5(2):220–31.
54. Cho SG, Lee YH, Park HS, Ryoo K, Kang KW, Park J,
Eom SJ, Kim MJ, Chang TS, Choi SY, Shim J. Glutathione
S-transferase mu modulates the stress-activated signals by sup-
pressing apoptosis signal-regulating kinase 1. J Biol Chem.
2001;276(16):12749–55.
55. Tew KD, Monks A, Barone L, Rosser D, Akerman G, Montali
JA, Wheatley JB, Schmidt DE. Glutathione-associated enzymes
in the human cell lines of the National Cancer Institute Drug
Screening Program. Mol Pharmacol. 1996;50(1):149–59.
56. Riches Z, Collier AC. Posttranscriptional regulation of uridine
diphosphate glucuronosyltransferases. Exp Opin Drug Metabol
Toxicol. 2015;11(6):949–65.
57. Caspersen CS, Reznik B, Weldy PL, Abildskov KM, Stark RI,
Garland M. Molecular cloning of the baboon UDP-glucurono-
syltransferase 1A gene family: evolution of the primate UGT1
locus and relevance for models of human drug metabolism.
Pharmacogenetics Genomics. 2007;17(1):11–24.
58. Fry DJ, Wishart GJ. Apparent induction by phenobarbital of
uridine diphosphate glucuronyltransferase activity in nuclear
envelopes of embryonic-chick liver. Biochem Soc Trans.
1976;4:265–6.
59. Fremont JJ, Wang RW, King CD. Coimmunoprecipitation of
UDP-glucuronosyltransferase isoforms and cytochrome P450
3A4. Mol Pharmacol. 2005;67(1):260–2.
60. Abbott FV, Palmour RM. Morphine-6-glucuronide: anal-
gesic effects and receptor binding profile in rats. Life Sci.
1988;43(21):1685–95.
61. Vore M, Slikker W. Steroid D-ring glucuronides: a new class
of cholestatic agents. Trends Pharmacol Sci. 1985;1(6):256–9.
62. Strassburg CP. Pharmacogenetics of Gilbert’s syndrome. Phar-
macogenomics. 2008;9(6):703–15.
63. Hall D, Ybazeta G, Destro-Bisol G, Petzl-Erler ML, Di AR.
Variability at the uridine diphosphate glucuronosyltransferase
1A1 promoter in human populations and primates. Pharmaco-
genetics. 1999;9(5):591–9.
64. Dean L. Irinotecan therapy and UGT1A1 genotype. Medical
Genetics Summaries 2018;. National Center for Biotechnology
Information (US).
65. Liu X, Cheng D, Kuang Q, Liu G, Xu W. Association of
UGT1A1* 28 polymorphisms with irinotecan induced
Clinical and Translational Oncology
toxicities in colorectal cancer: a meta-analysis in Caucasians.
Pharmacogenomics J. 2014;14(2):120.
66. Justenhoven C, Winter S, Dünnebier T, Hamann U, Baisch C,
Rabstein S, Spickenheuer A, Harth V, Pesch B, Brüning T, Ko
YD. Combined UGT1A1 and UGT1A6 genotypes together with
a stressful life event increase breast cancer risk. Breast Cancer
Res Treatment. 2010;124(1):289–92.
67. Clendenen T, Zeleniuch-Jacquotte A, Wirgin I, Koenig KL, Afa-
nasyeva Y, Lundin E, Arslan AA, Axelsson T, Försti A, Hallmans
G, Hemminki K. Genetic variants in hormone-related genes and
risk of breast cancer. PLoS ONE. 2013;8(7):e69367.
68. Huo D, Kim HJ, Adebamowo CA, Ogundiran TO, Akang EE,
Campbell O, Adenipekun A, Niu Q, Sveen L, Fackenthal JD,
Fackenthal DL. Genetic polymorphisms in uridine diphospho-
glucuronosyltransferase 1A1 and breast cancer risk in Africans.
Breast Cancer Res Treatment. 2008;110(2):367–76.
69. Duguay Y, McGrath M, Lépine J, Gagné JF, Hankinson
SE, Colditz GA, Hunter DJ, Plante M, Têtu B, Bélanger
A, Guillemette C. The functional UGT1A1 promoter poly-
morphism decreases endometrial cancer risk. Cancer Res.
2004;64(3):1202–7.
70. Dobritzsch D, Schneider G, Schnackerz KD, Lindqvist Y. Crystal
structure of dihydropyrimidine dehydrogenase, a major determi-
nant of the pharmacokinetics of the anti-cancer drug 5-fluoroura-
cil. EMBO J. 2001;20(4):650–60.
71. Sommadossi JP, Gewirtz DA, Diasio RB, Aubert C, Cano JP,
Goldman ID. Rapid catabolism of 5-fluorouracil in freshly iso-
lated rat hepatocytes as analyzed by high performance liquid
chromatography. J Biol Chem. 1982;257(14):8171–6.
72. Tuchman M, Stoeckeler JS, Kiang DT, O’Dea RF, Ramnaraine
ML, Mirkin BL. Familial pyrimidinemia and pyrimidinuria
associated with severe fluorouracil toxicity. N Engl J Med.
1985;313(4):245–9.
73. Heggie GD, Sommadossi JP, Cross DS, Huster WJ, Diasio RB.
Clinical pharmacokinetics of 5-fluorouracil and its metabolites
in plasma, urine, and bile. Cancer Res. 1987;47(8):2203–6.
74. Diasio RB, Harris BE. Clinical pharmacology of 5-fluorouracil.
Clin Pharmacokinet. 1989;16(4):215–37.
75. Beuzeboc P, Pierga JY, Stoppa-Lyonnet D, Etienne MC, Milano
G, Fouillait P. Severe 5-fluorouracil toxicity possibly second-
ary to dihydropyrimidine dehydrogenase deficiency in a breast
cancer patient with osteogenesis imperfecta. Eur J Cancer.
1996;32(2):369–70.
76. Yokota H, Fernandez-Salguero P, Furuya H, Lin K, McBride OW,
Podschun B, Schnackerz KD, Gonzalez FJ. cDNA cloning and
chromosome mapping of human dihydropyrimidine dehydroge-
nase, an enzyme associated with 5-fluorouracil toxicity and con-
genital thymine uraciluria. J Biol Chem. 1994;269(37):23192–6.
77. Meinsma R, Fernandez-Salguero P, Van Kuilenburg AB, Van
Gennip AH, Gonzalez FJ. Human polymorphism in drug metab-
olism: mutation in the dihydropyrimidine dehydrogenase gene
results in exon skipping and thymine uracilurea. DNA Cell Biol.
1995;14(1):1–6.
78. Vreken PA, Van Kuilenburg AB, Meinsma R, Smit GP, Bakker
HD, De Abreu RA, Van Gennip AH. A point mutation in an
invariant splice donor site leads to exon skipping in two unre-
lated Dutch patients with dihydropyrimidine dehydrogenase
deficiency. J Inherit Metab Dis. 1996;19(5):645–54.
79. Amstutz U, Henricks LM, Offer SM, Barbarino J, Schellens JH,
Swen JJ, Klein TE, McLeod HL, Caudle KE, Diasio RB, Schwab
M. Clinical Pharmacogenetics Implementation Consortium
(CPIC) guideline for dihydropyrimidine dehydrogenase geno-
type and fluoropyrimidine dosing: 2017 update. Clin Pharmacol
Ther. 2018;103(2):210–6.
80. Harris BE, Song R, Soong SJ, Diasio RB. Relationship
between dihydropyrimidine dehydrogenase activity and plasma
5-fluorouracil levels with evidence for circadian variation of
enzyme activity and plasma drug levels in cancer patients
receiving 5-fluorouracil by protracted continuous infusion.
Cancer Res. 1990;50(1):197–201.
81. Petit E, Milano G, Lévi F, Thyss A, Bailleul F, Schneider M.
Circadian rhythm-varying plasma concentration of 5-fluoroura-
cil during a five-day continuous venous infusion at a constant
rate in cancer patients. Cancer Res. 1988;48(6):1676–9.
82. Jacobs BA, Deenen MJ, Pluim D, van Hasselt JC, Krähenbühl
MD, van Geel RM, de Vries N, Rosing H, Meulendijks D,
Burylo AM, Cats A. Pronounced between-subject and circa-
dian variability in thymidylate synthase and dihydropyrimidine
dehydrogenase enzyme activity in human volunteers. Br J Clin
Pharmacol. 2016;82(3):706–16.
83. Lu Z, Zhang R, Diasio RB. Population characteristics of
hepatic dihydropyrimidine dehydrogenase activity, a key meta-
bolic enzyme in 5-fluorouracil chemotherapy. Clin Pharmacol
Ther. 1995;58(5):512–22.
84. Fleming RA, Milano G, Thyss A, Etienne MC, Renée N, Sch-
neider M, Demard F. Correlation between dihydropyrimidine
dehydrogenase activity in peripheral mononuclear cells and
systemic clearance of fluorouracil in cancer patients. Cancer
Res. 1992;52(10):2899–902.
85. Etienne MC, Lagrange JL, Dassonville O, Fleming R, Thyss
A, Renee N, Schneider M, Demard F, Milano G. Population
study of dihydropyrimidine dehydrogenase in cancer patients.
J Clin Oncol. 1994;12(11):2248–53.
86. Guimbaud R, Guichard S, Dusseau C, Bertrand V, Aparicio
T, Lochon I, Chatelut E, Couturier D, Bugat R, Chaussade S,
Canal P. Dihydropyrimidine dehydrogenase activity in normal,
inflammatory and tumour tissues of colon and liver in humans.
Cancer Chemother Pharmacol. 2000;45(6):477–82.
87. Beck A, Etienne MC, Cheradame S, Fischel JL, Formento P,
Renee N, Milano G. A role for dihydropyrimidine dehydroge-
nase and thymidylate synthase in tumour sensitivity to fluoro-
uracil. Eur J Cancer. 1994;30(10):1517–22.
88. Yasumatsu R, Nakashima T, Uryu H, Masuda M, Hirakawa N,
Shiratsuchi H, Tomita K, Fukushima M, Komune S. The role
of dihydropyrimidine dehydrogenase expression in resistance
to 5-fluorouracil in head and neck squamous cell carcinoma
cells. Oral Oncol. 2009;45(2):141–7.
89. Robert B, Diasio M. Clinical implications of dihydropyrimi-
dine dehydrogenase inhibition. Phys Pract. 1997;7(3):13–21.
90. Schilsky RL, Kindler HL. Eniluracil: an irreversible inhibitor
of dihydropyrimidine dehydrogenase. Expert Opin Investig
Drugs. 2000;9(7):1635–49.
91. Shigeishi H, Biddle A, Gammon L, Rodini CO, Yamasaki M,
Seino S, Sugiyama M, Takechi M, Mackenzie IC. Elevation
in 5-FU-induced apoptosis in Head and Neck Cancer Stem
Cells by a combination of CDHP and GSK 3β inhibitors. J Oral
Pathol Med. 2015;44(3):201–7.
92. Yano W, Yokogawa T, Wakasa T, Yamamura K, Fujioka A,
Yoshisue K, Matsushima E, Miyahara S, Miyakoshi H, Tagu-
chi J, Chong KT. TAS-114, a first-in-class dual dUTPase/DPD
inhibitor, demonstrates potential to improve therapeutic effi-
cacy of fluoropyrimidine-based chemotherapy. Mol Cancer
Ther. 2018;17(8):1683–93.
93. Wahl ML, Kenan DJ, Gonzalez-Gronow M, Pizzo SV. Angio-
statin’s molecular mechanism: aspects of specificity and regu-
lation elucidated. J Cell Biochem. 2005;96(2):242–61.
94. Stepczynska A, Lauber K, Engels IH, Janssen O, Kabelitz
D, Wesselborg S, Schulze-Osthoff K. Staurosporine and con-
ventional anticancer drugs induce overlapping, yet distinct
pathways of apoptosis and caspase activation. Oncogene.
2001;20(10):1193.
13

95. Landmann H, Proia DA, He S, Ogawa LS, Kramer F, Beißbarth
T, Grade M, Gaedcke J, Ghadimi M, Moll U, Dobbelstein M.
UDP glucuronosyltransferase 1A expression levels determine the
response of colorectal cancer cells to the heat shock protein 90
inhibitor ganetespib. Cell death & disease. 2014;5(9):e1411.
96. Miners JO, Chau N, Rowland A, Burns K, McKinnon RA, Mac-
kenzie PI, Tucker GT, Knights KM, Kichenadasse G. Inhibition
of human UDP-glucuronosyltransferase enzymes by lapatinib,
pazopanib, regorafenib and sorafenib: implications for hyper-
bilirubinemia. Biochem Pharmacol. 2017;1(129):85–95.
97. Bourrié M, Meunier V, Berger Y, Fabre G. Cytochrome P450
isoform inhibitors as a tool for the investigation of metabolic
reactions catalyzed by human liver microsomes. J Pharmacol
Exp Ther. 1996;277(1):321–32.
98. Knodell RG, Browne DG, Gwozdz GP, Brian WR, Guengerich
FP. Differential inhibition of individual human liver cytochromes
P-450 by cimetidine. Gastroenterology. 1991;101(6):1680–91.
13
Clinical and Translational Oncology
99. Pereira S, Fernandes PA, Ramos MJ. Mechanism for ribonucleo-
tide reductase inactivation by the anticancer drug gemcitabine. J
Comput Chem. 2004;25(10):1286–94.
100. Campas-Moya C. Romidepsin for the treatment of cutane-
ous T-cell lymphoma. Drugs Today (Barcelona, Spain: 1998).
2009;45(11):787–95.
101. Zheng J, Benbrook DM, Yu H, Ding WQ. Clioquinol suppresses
cyclin D1 gene expression through transcriptional and post-tran-
scriptional mechanisms. Anticancer Res. 2011;31:2739–47.
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.