Metabolism of Cancer Cells

Cancer cells undergo a different
metabolism process than normal cells. The metabolism of cancer cells is
attributed to the Warburg effect. Warburg effect refers to the process of
aerobic glycolysis where glucose is converted firstly into pyruvate then into
lactate by cancerous cells despite being in the presence of sufficient oxygen (Vander Heiden, Cantley, & Thompson, 2009). This process
actually results in a lesser amount of ATP generated which is -4 mol ATP/mol
glucose than during oxidative phosphorylation at -36mol ATP/mol glucose (Vander Heiden, Cantley, & Thompson, 2009). This low amount of
ATP produced could be why cancerous cells often compete with surrounding cells
for nutrition and materials for survival. In addition to this, cancer cells
make use of glutaminolysis which is driven by Myc to support NADPH production for ATP production (Vander Heiden, Cantley, & Thompson, 2009). Other reports also
state that there was high uptake of glutamine in cancerous tissue due to the
increase in metabolic demand such as for the use in catabolism (breakdown of
substances) for the generation of ATP or for anabolism (synthesis of molecules
from smaller units) (Eagle, Oyama, Levy, Horton, & Fleischman, 1956 &
DeBerardinis, et al., 2007). Due to this factor, the uptake of glutamine shows
the importance it has in the metabolism of cancer cells.

 

In this paper, our interest lies with the
alanine, serine and cysteine transporter 2 (ASCT2) which is a neutral amino
acid transporter that mediates the uptake of neutral amino acids including
glutamine (Kanai & Hediger, 2004). Studies show the
importance of this transporter in the uptake of glutamine and how dependent
cancer cells are towards the use of glutamine as well as a higher expression of
ASCT2 in certain cancers (Geldermalsen, et al., 2015). This statement is
supported by a study done by Hassanein, M.; 2015 whereby the effects of
targeting ASCT2 resulted in apoptotic cell death in non-small cell lung cancer.
To work towards imaging ASCT2, we will be employing the use of positron
emission tomography (PET).

 

Positron Emission Tomography (PET) Imaging

Positron emission tomography (PET) imaging
is a non-invasive diagnostic technique for numerous cancer types. PET imaging
makes use of radiotracers (PET tracers) and a PET scanner to give information about
a particular cancerous mass in the body. One common PET tracer used in tumour
and cancer imaging is 18F-fludeoxyglucose (18F-FDG) (Fig.
1-1) which is an analog of glucose. Since it has a similar structure to
glucose, it will be taken up by the appropriate glucose transporter. However
since there is a slight difference in the structure, it will not be processed
the same as glucose. Instead it is retained at the site of uptake. This means
that in the area where there is glucose uptake, the radiotracer 18F-FDG
will emit a radiation signal which will be captured by the PET scanner.

 

In the case of organs which have high
glucose uptake, the use of 18F-FDG for imaging can result in high
background signal when detecting the tumour mass in that particular organ. Such
organs with high glucose uptake include the brain and the liver. The use of 18F-FDG
in imaging can also result in difficulty in differentiating between the inflammed
tissue and cancerous tumours because of the high FDG uptake in these 2 areas (Benamor, et
al., 2007).
This was further supported by Kubota et al; 1990, whereby a portion of the patients tested with FDG
resulted in false positives which were non tumour tissues showing its lack of
specificity.

 

In PET imaging, another PET tracer that
has been developed for imaging of brain cancers is 18F-fluoroethyl-L-tyrosine
(18F-FET) which has found most applied in the neurooncological
diagnosis of brain tumours. The structure 18F-FET (Fig 1-2) mimics
that of the amino acid tyrosine which follows a similar uptake process as the
amino acid however will be retained in the site of uptake and imaged. A study
stated that when 18F-FDG was compared to 18F-FET in terms
of the imaging of peripheral tumours, 18F-FET was inferior to the
other in terms of general tumour diagnosis however it displayed higher
specificity when distinguishing between tumours and inflamed tissues in
patients with squamous cell carcinoma. (Pauleit, et al., 2005)

 

 

Due to this lack of specificity in
differentiating between tumour tissue and non tumour tissues like inflamed
tissues for 18F-FDG as well as the lack of specificity in imaging
peripheral tumours for 18F-FET, a more specific PET tracer is aimed
for in this paper. Since our aim is to image ASCT2, a PET tracer mimicking the
structure of glutamine (Fig. 1-3) will
be the area of interest in this paper. This will be done through the synthesis
of molecules that mimic the structure of glutamine through a variation of cross
coupling chemical reactions with the use of a palladium catalyst.

 

 

 

The palladium catalyst in question makes
use of the ligand TIP Cy*Phine and precatalyst PdCl2(Cy*Phine)2
which was developed in 2014 as reported in a paper by Yong Y. et al., 2014. The
state of art palladium catalyst was used in a variety of cross coupling
reactions including Mizoroki-Heck (Tay et al., 2015), N-arylation reactions
(Yong F. F. et al., 2017) and copper-free Sonogashira cross coupling reactions
(Yong Y. et al., 2014) all of which displayed high cross coupling efficacy.

 

Heck Cross Coupling Mechanism

 

 

Figure 1-4. Catalytic Cycle for Heck Cross Coupling (Jutand, 2009)

 

Mizoroki-Heck or Heck cross coupling
reaction is where an aromatic halide reacts with an alkene in the presence of a
base and a palladium catalyst. This reaction is explained in the catalytic
cycle (Fig. 1-4), where the palladium precursor is reduced from a state of (2+)
to (0) prior to the reaction. Following this, oxidative addition (a) occurs
whereby the palladium catalyst inserts itself in between the aryl halide R-X.
The palladium then forms a ? complex with the alkene molecule which in this
paper is the allylglycine. Step (b) which is alkene coordination is where the
alkene molecule inserts itself in between the palladium-carbon bond. The
?-hydride elimination (c) is where the hydrogen atom from the beta-position of
the ligand is transferred to the metal center. After this step, reductive
elimination (d) occurs resulting in the dissociation of the arylated alkene from
the palladium and the regeneration of the Pd(0) complex. The cycle repeats. (Jutand, 2009)

 

Usually, the conditions for Heck cross
coupling makes use of polar aprotic solvents such as dimethyl formamide (DMF) (Jagtap, 2017). In this paper, however,
we made use of polar protic solvents which are water and acetonitrile for our
reaction because the amino acids used are only soluble in water and
acetonitrile is miscible in water. Heck cross coupling reactions also may
generate acid so in order to sequester this acid, compounds such as
tetrabutylammoniumbromide (TBAB) is added (Jagtap, 2017). In this paper, TBAB has another
function whereby it serves as a phase-transfer catalyst. Since we are making
use of an amino acid (only soluble in water) as well as our catalyst (soluble
in organic solvent), TBAB is crucial to allow the catalyst to react with the
amino acid in the water.

 

Sonogashira Reaction

Another common cross coupling reaction is
the Sonogashira Reaction. This cross coupling reaction is the formation of a
C-C bond between an aryl or vinyl halide and a terminal alkyne in the presence
of 2 catalysts, a palladium catalyst and a copper (I) co-catalyst. In the
catalytic cycle for Sonogashira there are 2 cycles happening simultaneously (Fig
1-5); the palladium cycle and the copper cycle. In recent times, the
advancement of Sonogashira optimization has resulted in the Sonogashira cross
coupling not needing a copper co-catalyst. Sometimes the use of the copper
catalyst had undesirable effects such as the formation of homocoupling side
products (Glaser coupling) which renders the use of copper-free Sonogashira
cross coupling. The copper-free Sonogashira reaction makes use of no copper
co-catalyst which decreases the production of side products.

 

Reductive elimination

Oxidative addition

(d)

(e)

(c)

(b)

(a)

Copper Cycle

Palladium Cycle

Transmetallation
 

Figure 1-5. Catalytic Cycle for Sonogashira Cross
Coupling (Shroder, n.d.)

 

Palladium cycle

Prior to the catalytic cycle (Fig. 1-5),
the palladium catalyst is reduced from a state of (2+) to (0). The palladium
(0) catalyst will then react with the aryl halide which in this paper is
5-bromo-2-fluoropyridine through a process called oxidative addition, to form
complex (a). Complex A will then undergo transmetallation with the copper
acetylide (e) derived from the copper cycle. This complex will then undergo cis-trans
isomerization since it is in cis configuration after which, the synthesized
alkyne is produced and the former palladium catalyst is regenerated.

 

Copper Cycle

Usually for Sonogashira, bases typically
used for the reaction are amides which due to its low basicity has to have the
formation of a ?-alkyne-copper complex in order to increase the acidity of the
alkene. This ensures that the alkyne is able to be deprotonated. Following this
step, the copper acetylide will be formed which will react with complex (a) (Fig
1-5).

 

The palladium catalyst PdCl2(Cy*Phine)2
was found to be highly efficient in the copper-free Sonogashira cross coupling
reaction with over 70% yield for its substrate scope and demonstrating
excellent conversion rates.

 

In this paper, the Sonogashira cross
coupling will make use of our palladium catalyst PdCl2(Cy*Phine)2.
The aryl halide used is 5-bromo-2-fluoropyridine in the presence of a base
(NaHCO3) and CuI in 1:1 acetonitrile:water as the amino acid used
(propargylglycine) is only soluble in water. A copper co-catalyst (CuI) will
also be used to test the efficacy of the precatalyst in this reaction.