Sulphate Conjugation & EMT pathways in cancer

1. Sulphonation metabolism- introduction, types of sulphate transferases, reactions, summary
2. EMT pathways explaining metastasis in cancer and molecular pathways
3. Thank you

1. Assignment On
SULFUR CONJUGATION
&
EMT PATHWAY


ASSIGNMENT SUBMITTED BY - PRIYANSHA SINGH
M.S. Pharm- PHARMACOLOGY & TOXICOLOGY (2nd SEMESTER)
SUBJECT- DRUG METABOLISM (PC-610)
SUBMITTED TO- Dr. ROSHAN BORKAR (Asst. Professor)
NIPER- GUWAHATI

2. INTRODUCTION
All creatures are continually and inadvertently exposed to xenobiotics, which include both
man-made and natural substances such as pharmaceuticals, plant alkaloids, microbe poisons,
pollution, pesticides, and other industrial chemicals. Formally, xenobiotic and endogenous
chemical biotransformation is separated into phase I and phase II processes.
On the other hand, due to the metabolic synthesis of hazardous metabolites such as reactive
electrophiles, these conjugations play an important part in the toxicity of numerous
substances. Biotransformation enzyme gene polymorphism is frequently implicated in a
variety of pathophysiological diseases. Conjugation reactions typically involve metabolite
activation by a high–energy intermediate and are divided into two types: type I (e.g.,
glucuronidation and sulfonation), in which an activated conjugating agent combines with the
substrate to yield the conjugated product, and type II (e.g., amino acid conjugation), in which
the substrate is activated and then combined with an amino acid to yield the conjugated
product.

SULPHUR CONJUGATION
Sulfoconjugation (or sulfonation) is an essential route in the metabolism of several exogenous
and endogenous chemicals. Baumann discovered the sulfonation process in 1876. Baumann
discovered phenyl sulphate in the urine of a patient who had been given phenol. The
sulfonation reactions are carried out by a supergene family of enzymes known as
sulfotransferases (SULTs). In general, these enzymes catalyse the transfer of sulfonate (SO3–)
from the universal sulfonate donor 3'–phosphoadenosine 5'–phosphosulfate (PAPS) to the
hydroxyl or amino group of an acceptor molecule.

MECHANISM OF SULPHUR CONJUGATION

Fig. 1- The Sulphonation system present in cytosol
Sulfonation of low molecular weight compounds is catalysed by members of the cytosolic
sulfotransferase multigene family (SULT) is an important determinant of the
pharmacology and toxicology of a vast array of endogenous and foreign chemicals. This
pathway involves the transfer of a sulfonate group (SO3-) from the universal donor 3’-

3. phosphoadenosine 5’-phosphosulfate (PAPS) to an appropriate substrate. Sulfonated
conjugates are often incorrectly referred to as ‘‘sulphates’’ because the transfer of an SO3-
group to a hydroxyl acceptor creates an SO4 ester. Availability of the obligatory co-
factor, PAPS, is present at relatively low concentrations and may limit the synthesis of
sulfonate conjugates catalysed by various SULTs. PAPS is formed via a single
bifunctional enzyme that contains both ATP sulfurylase and APS activities. Inorganic
sulphate and two molecules of ATP are required for each molecule of PAPS
synthesized. Sulfonate ester formation may also be reversed via the hydrolytic enzyme
arylsulfatas-c present in the endoplasmic reticulum. Sulfonate conjugates (ROSO3
-
/
ROSO3) leave and enter cells via specific transporters including mdr and organic acid
transport molecules (OAT).
Inter-individual variation in each of the above enzymatic activities imposed by genetic
and/or environmental events may limit the process of sulfonation intact cells and
organs. PAPS is a universal donor of sulfonate moiety in sulfonation reactions and has
been shown to by synthesize by almost all tissues in mammals from inorganic
sulphate. Depletion of PAPS due to lack of inorganic sulphate or due to genetic defects of
enzymes participating in PAPS synthesis may lead to reducing of sulfonation capacity
which could affect the metabolism of xenobiotics or disrupt the equilibrium between
synthesis and degradation of active endogenous compounds. The sulfonation system
resides primarily in cytosol but involves interaction with arylsulfates-c (ARSc) in the
endoplasmic reticulum and specific transport molecules localized in the plasma
membrane.
Here, through the activity of sulfo-transferases (SULT), sulphate functions as a conjugation
molecule that attaches to a substrate. At first, Sulfate is combined with ATP molecules to form
PAPS via the enzymes PAPSS1 and PAPSS2 (dual function) which consists of a sulfurylase
domain and an APS kinase domain. Then, the sulphate group is transferred from PAPS to the
real substrate by sulfotransferase that leads to the production of a sulphated substrate and a
sulphate-deficient PAP.
Sulfotransferase (SULTs) are a supergene family of enzymes that mediate sulfonation
processes. Sulfonate (SO3-) is transferred from the universal sulfonate donor 3'–
phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyl or amino group of an acceptor
molecule via these enzymes.
Here 3‘–phosphoadenosine 5‘–phosphosulfate (PAPS) acts as a universal donor of sulfonate
moiety in sulfur conjugation reactions. It has been shown to be synthesized by almost all
tissues in mammals from inorganic sulfur. The most prevalent source of sulphur in the
environment is inorganic sulphate, which is metabolised into two nucleotide precursors:
adenosine 5'-phosphosulfate (APS) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS).
Activated sulphur is donated by a variety of enzymatic processes, majorly use PAPS and
produce 3'-phosphoadenosine 5'-phosphate as a result (PAP).
To date, two large groups of SULTs have been identified- The first group includes
membrane–bound enzymes with no demonstrated xenobiotic–metabolising activity.
These enzymes are localized in the Golgi apparatus and they are involved in metabolism
of endogenous peptides, proteins, glycosaminoglycans, and lipids. Cytosolic SULTs
constitute the second group of sulfotransferases and play a major role in conjugation
of a broad spectrum of xenobiotics including environmental chemicals, natural

4. compounds, drugs as well as endogenous compounds such as steroid hormones,
iodothyronines, catecholamines, eicosanoids, retinol or vitamin D. Sulfonation is
generally described as a detoxification pathway for many xenobiotics. Addition of the
sulfonate moiety to the molecule of a parent compound or most often to the molecule of its
metabolite originating in the oxidative phase of drug metabolism leads to formation of a
water–soluble compound which is then easily eliminated from the body. However, in several
cases the sulfonation reaction can lead to formation of a more active metabolite
compared to the parent compound as is the case for the hair follicle stimulant
minoxidil. Furthermore, the role of sulfotransferases in the activation of various
procarcinogens and promutagens was confirmed.

To date, four human SULT families, SULT1, SULT2, SULT4 and SULT6, have been
identified. These SULT families include at least 13 different members. The SULT1 family
comprises of 9 members divided into 4 subfamilies (1A1, 1A2, 1A3, 1A4, 1B1, 1C1, 1C2, 1C3 and
1E1). SULT2A (SULT2A1) and SULT2B (SULT2B1a and SULT2B1b) belong to SULT2 family. The
SULT4A1 and SULT6B1 are the only members of the SULT4 and SULT6 family, respectively.
Cytosolic sulfotransferase exert relatively broad tissue distribution

Fig. 2– Distribution of SULT in body tissues

SUBSTRATES OF SULTs
SULT1A1 has been shown to be one of the most important sulfotransferases participating in metabolism
of xenobiotics in humans. It has also been termed phenol sulfotransferase (P–PST) or thermostable
phenol sulfotransferase (TS PST1). In general, SULT1A1 is responsible for sulfoconjugation of phenolic
compounds such as monocyclic phenols, naphtols, benzylic alcohols, aromatic amines or
hydroxylamines. Acetaminophen, minoxidil as well as dopamine or iodothyronines undergo sulfonation
by SULT1A1. SULT1A1 also takes part in transformation of hydroxymethyl polycyclic aromatic
hydrocarbons, N–hydroxyderivatives of arylamines, allylic alcohols and heterocyclic amines to their
reactive intermediates which are able to bind to nucleophilic structures such as DNA and consequently

5. act as mutagens and carcinogens. SULT1A2 also plays an important role in the toxification of several
aromatic hydroxylamines.
SULT1A3, formerly known as thermolabile phenol SULT (TL PST) or monoaminesulfotransferase,
exhibits high affinity for catecholamines (dopamine) and contributes to the regulation of the rapidly
fluctuating levels of neurotransmitters. The human SULT1B1 was isolated and described and was shown
to be the most important sulfotransferase in thyroid hormone metabolism. SULT1E1 plays a key role in
estrogen homeostasis. This enzyme conjugates 17┚–estradiol and other estrogens in a step leading to
their inactivation. Since 17┚–estradiol and relative compounds regulate various processes occurring in
humans, inactivation of these compounds by the SULT1E1 enzyme constitute an important step in the
prevention and development of certain diseases. Down regulation or loss of SULT1E1 could be to a
certain extent responsible for growth of tumor in hormone sensitive cancers such as breast or
endometrial cancer SULT2A and SULT2B subfamilies include the hydroxysteroid sulfotransferase with
partially overlapping substrate specifities. SULT2A1 is also termed as dehydro-epi-androsterone
sulfotransferase (DHEA ST) and conjugates various hydroxysteroids such as DHEA, androgens, bile
acids and oestrone. Recently, a role of SULT2A1 in metabolism of quinolone drugs in humans was
confirmed

Fig. 4(a) – Sulphur conjugation of Minoxidil by SULT and PAPS

Fig. 4(b) - Sulphur conjugation of Acetaminophen

6. Summary of Xenobiotics/Endogenous chemicals metabolised by sulphur conjugation

7. REFERENCES:-

1. Jančová, P., & Šiller, M. (2012). Phase II Drug Metabolism. In (Ed.), Topics on
Drug Metabolism. IntechOpen. https://doi.org/10.5772/29996

2. Kauffman F. C. (2004). Sulfonation in pharmacology and toxicology. Drug
metabolism reviews, 36(3-4), 823–843. https://doi.org/10.1081/dmr-200033496

8. EMT PATHWAY

An overview of EMT—causes and consequences. EMT is regulated by multiple signaling pathways, such as
TGF-β, HIF-1α, and Notch. These signaling pathways often regulate EMT via a core regulatory circuit
consisting of two mutually inhibitory feedback loops—miR-34/SNAIL and miR-200/ZEB. The miR-
34/SNAIL/miR-200/ZEB functions as a three-way decision-making circuit regulating the transitions among
epithelial, mesenchymal and hybrid E/M phenotypes. EMT is closely connected with tumor metastasis,
acquisition of stem-like properties and evasion of immune attack.
Introduction- Breast cancer is the most common cancer in women, and approximately 90% of breast
cancer deaths are caused by local invasion and distant metastasis of tumor cells. Epithelial
mesenchymal transition (EMT) is a vital process for large scale cell movement during morphogenesis at
the time of embryonic development. Tumor cells usurp this developmental program to execute the
multistep process of tumorigenesis and metastasis. Several transcription factors and signals are involved
in these events. In this review, we summarize recent advances in breast cancer researches that have
provided new insights in the molecular mechanisms underlying EMT regulation during breast cancer
progression and metastasis.
An epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell,
which normally interacts with basement membrane via its basal surface, to undergo multiple
biochemical changes that enable it to assume a mesenchymal cell phenotype, which includes enhanced
migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of
ECM components. The completion of an EMT is signalled by the degradation of underlying basement
membrane and the formation of a mesenchymal cell that can migrate away from the epithelial layer in
which it originated.
Here in this pathway, the epithelial cells lose the adherent and tight junctions that keep them in
contact with their neighbours and gain mesenchymal properties, including fibroblastoid morphology,
characteristic gene expression changes, and increased potential for motility, enabling them to break
through the basal membrane and migrate over a long distance. The concept of EMT was developed in
the field of embryology but has recently been extended to tumor progression and metastasis
A number of distinct molecular processes are engaged in order to initiate an EMT and enable it to reach
completion. These include activation of transcription factors, expression of specific cell-surface
proteins, reorganization and expression of cytoskeletal proteins, production of ECM-degrading

9. enzymes, and changes in the expression of specific microRNAs. In many cases, the involved factors are
also used as biomarkers to demonstrate the passage of a cell through an EMT

EMT is classified into three types based on the biological context under which it occurs:-
1. Type 1 EMT describes the transition events that allow epithelial cells to become motile
mesenchymal cells during implantation, embryo formation, gastrulation, and neural crest
migration. These primary mesenchymal cells act as progenitors and generate secondary
epithelia in mesodermal and endodermal organs via mesenchymal­ epithelial transition (MET).

2. Type 2 EMT is associated with wound healing, tissue regeneration, and organ fibrosis. In this
process, tissue fibroblasts are generated from epithelial or endothelial cells during injury and
chronic inflammation.

3. Type 3 EMT, which occurs in epithelial cancer cells, is the process through which cancer cells
at the invasive front of primary tumours undergo a phenotypic conversion to invade and
metastasize through the circulation and generate a metastatic lesion at distant tissues or organs
by MET.
The role of EMT in breast cancer has been demonstrated via numerous studies in vitro normal and
malignant mammary epithelial cells and via studies using mouse models of breast cancers. Breast
tumors undergo EMT and show a basal like phenotype, suggesting that EMT occurs within a specific
genetic context in breast tumors. Because breast cancer is a heterogeneous disease in terms of tumor
histology, clinical presentation, and response to therapy

11. The epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells
lose their cell identity and acquire the characteristics of mesenchymal cell. EMT is commonly
seen during the development of organisms, wound healing, and tissue fibrosis.
Here, a polarised epithelial cell, which normally interacts with the basement membrane via its
basal surface, undergoes a series of biochemical changes that allow it to adopt a mesenchymal
cell phenotype, which includes increased migratory capacity, invasiveness, resistance to
apoptosis etc. The disintegration of the underlying basement membrane and the creation of a
mesenchymal cell that can move away from the epithelial layer in which it originated signify
the completion of an EMT.
A hallmark of EMT is losing expression of E­cadherin, a key cell­cell adhesion
molecule. As a caretaker of the epithelial phenotype, E­cadherin helps to assemble
epithelial cell sheets and maintain the quiescence of the cells within these sheets. The
vast majority of known signaling pathways have been implicated in the regulation of EMT.
Several transcription factors, including the Snail/Slug family, Twist, EF1/ZEB1,
SIP1/ZEB2, and E12/E47, respond to these signals and function as master molecular
switches of the EMT pathway.

 MOLECULAR PATHWAYS OF EMT TRANSCRIPTION: -
A series of molecular processes are involved in initiating the EMT pathway and enable it to
reach completion. These include activation of several transcription factors, expression of cell-
surface proteins, cytoskeletal protein rearrangement and expression, synthesis of certain
degrading enzymes, and alterations in the expression of specific microRNAs. Factors or
components involved in this process are often used as biomarkers to demonstrate the passage
of a cell through an EMT.
Throughout this process, epithelial cells lose their junctions and apical–basal polarity,
restructure their cytoskeleton, modify the signalling programmes that determine cell shape,
and re-programme gene expression. This allows for the development of an invasive phenotype
as well as increased cell motility. The underlying processes of EMT are significant in wound
healing, fibrosis and cancer progression.

12.  PROTEINS INVOLVED IN EMT:
Multiple signalling pathways are involved in the initiation and progression of EMT process,
like- epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor
(FGF), transforming growth factor β (TGFβ), β-catenin–dependent canonical and β-catenin–
independent non-canonical WNT signaling, bone morphogenetic protein (BMP) etc and they
often activate SNAIL expression.
Repression of epithelial phenotype and activation of mesenchymal phenotype involve master
regulators, including SNAIL, TWIST and zinc-finger E-box-binding (ZEB) transcription
factors. Their expression is activated early in EMT.

13. Homo-dimeric and hetero-dimeric basic helix–loop–helix (bHLH) transcription factors
function as master regulators of differentiation and lineage specification. For example-E12 and
E47, TWIST1 and TWIST2, and inhibitor of differentiation (ID) proteins have key roles in EMT
progression. There are few recently identified EMT transcription factors, their roles in EMT
and functional relationships with SNAIL, TWIST or ZEB transcription factors are not
established yet.
So, it is explicit that diverse transcription factors are involved in coordinating gene expression
reprogramming during EMT pathway, also some of these are master regulators and others
may have more restricted functions depending on the tissue context.

14. REFERENCES:-
1. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/mesenchymal-epithelial-transition
2. Epithelial-mesenchymal transition in breast cancer progression and metastasis- Wang
Y, Zhou B
3. Kalluri, R., & Weinberg, R. A. (2009). The basics of epithelial-mesenchymal transition.
The Journal of clinical investigation, 119(6), 1420–1428. https://doi.org/10.1172/JCI39104

4. Lamouille, S., Xu, J., & Derynck, R. (2014). Molecular mechanisms of epithelial-
mesenchymal transition. Nature reviews. Molecular cell biology, 15(3), 178–196.
https://doi.org/10.1038/nrm3758

5. Georgakopoulos-Soares, Ilias; Chartoumpekis, Dionysios V.; Kyriazopoulou,
Venetsana; Zaravinos, Apostolos (2020). EMT Factors and Metabolic Pathways in
Cancer. Frontiers in Oncology, 10(), 499-. doi:10.3389/fonc.2020.00499