Letter to the Editor

To the Biochemical Pharmacology Editor

Barcelona, July 12th 2015.

Dear Editor,

Please find in the attachment a manuscript entitled "New specific G6PD inhibitors reduces
viability, induce cell cycle arrest and promote apoptosis in cancer cells”. This manuscript was
written by Surabhi Mishra, Roldán Cortés, Jaime Rubio, and Marta Cascante. This manuscript
describes a the biological effects of a set of four previously designed and synthesized small
molecules (named 1, 2, 3 and 4) over cell viability, G6PD activity, cell cycle phase distribution
and apoptotic state of three different cancer cell lines: A549 (lung), SKOV-3 (ovary) and SW-
1990 (pancreas). Our study evidenced that these compounds, which were designed to inhibit
G6PDH following in silico studies, are able to inhibit the enzyme’s activity in all three cell lines,
which is concomitant with the inhibition of cell viability through arrest of the cell cycle and,
eventually, the induction of apoptosis. G6PD is the main regulatory enzyme of the pentose
phosphate pathway, and our results help to better understand the relationship between this
pathway and the accelerated proliferation rates characteristic of cancer cells. Furthermore, they
reinforce the utility of exploiting vulnerabilities associated to cancer metabolic reprogramming
as a promising strategy to impair tumor proliferation, and open new avenues to improve the
design and synthesis of a new family of metabolic inhibitors as putative therapeutic agents in
cancer treatment.

For all these reasons, we would like to have this manuscript considered for publication in
Biochemical Pharmacology and we look forward to learning your decision.

Sincerely yours,

Dr. Marta Cascante, Full Professor.

martacascante@ub.edu
 New specific G6PD inhibitors reduces

viability, induce cell cycle arrest and

promote apoptosis in cancer cells

Surabhi Mishra1, Roldán Cortés1, Jaime Rubio3, and Marta Cascante1,2

1
Department of Biochemistry and Molecular Biology, Faculty of Biology, and IBUB, Universitat de

Barcelona, Unit Associated with CSIC Diagonal 645, E-08028-Barcelona, Spain. E-mail:

martacascante@ub.edu

2
Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

3
Universitat de Barcelona, Facultat de Quimica, Departament de Química Física, Martí i Franqués

1, 08028 Barcelona, Spain

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the oxidative branch of the

pentose phosphate pathway, which is essential for cell proliferation and therefore commonly up-

regulated in cancer cells. Here we present a study of the biological activity of four previously

synthesized small molecules (compounds 1, 2, 3 and 4)which were designed to inhibit the

formation of the active G6PD dimer and, therefore, inhibit the enzyme’s activity. The activity of

the compounds was tested over three different tumor cell lines: A549 (lung cancer), SKOV-3

(ovarian cancer) and SW-1990 (pancreatic cancer). Obtained results evidenced that this family of

compounds is able to inhibit the enzyme’s activity in all three cell lines, which is concomitant

with the observed inhibition of cell viability through the arrest of the cell cycle and, eventually,

the induction of apoptosis. Results help to better understand the relationship between the activity

of G6PD and the accelerated proliferation rates which are characteristic of cancer cells.

Furthermore, they reinforce the utility of exploiting vulnerabilities associated to cancer metabolic

reprogramming as a promising strategy to impair tumor proliferation, and open new avenues to

improve the design and synthesis of a new family of metabolic inhibitors as putative therapeutic

agents in cancer treatment.

Keywords: metabolic reprogramming, Glucose-6-phosphate dehydrogenase, pentose

phosphate pathway, cell cycle, apoptosis, cancer.

Abbreviations
BCA- Bicinchoninic acid assay
DMEM- Dulbecco's modified eagle medium
DMSO- Dimethyl sulfoxide
ELISA- Enzyme-linked immunosorbent assay

FACS- Fluorescence activated cell sorter

FITC- Fluorescein isothiocyanate
G1- First growth phase
G2- Second growth phase
G6PD- Glucose-6-phosphate dehydrogenase
M- Mitotic phase
MTT- 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide
NADH- Nicotinamide adenine dinucleotide
NADPH- Nicotinamide adenine dinucleotide phosphate
NSCLC - Non-small cell lung cancer
PPP- Pentose phosphate pathway
PS- Phosphotidylserine
ROS- Reactive oxygen species
RPMI- Roswell park memorial institute medium
S- Synthesis phase
TBS- Tris buffered saline
1. Introduction
Cancer is a generic term that includes a large group of diseases in which abnormal cells divide

without control and are able to invade other tissues. In 2012, more than14.6 million new cases of

cancer were diagnosed and around 8.2 million cancer deaths were registered worldwide[1]. The

development and progression of cancer requires that malignant cells acquire certain capabilities

along the carcinogenic process. These required capabilities, which are known as the hallmarks of

cancer, include classical requirements such as sustaining proliferative signaling, evading of

growth suppressors, invasivity, and metastasis. Over the last few years, cancer research has

promoted the appearance of an emergent new hallmark of cancer: the reprogramming of cancer

metabolism. In order to maintain an accelerated proliferation rate, cancer cells must reorganize

their metabolic network to serve increased demands of energy and macromolecules[2].

Pentose Phosphate Pathway (PPP) is one of the metabolic pathways with higher impact in cell

proliferation. The pathway comprises two different phases, named the oxidative phase(non

reversible) and the non-oxidative phase (reversible). Both phases of the PPP produce of 5 carbon

sugars(pentoses), and therefore the pathway is essential pathway for nucleotide (and therefore

DNA) production. Besides, the oxidative phase leads to the reduction of NADP into NADPH

[3], whose reductive power is essential not only in the protection against ROS, but also in the

synthesis of biomolecules such as fatty acids[4]. Because of this essential role in cell

proliferation through the synthesis of macromolecules, PPP is found overactivated in many

cancer types. The pathway is connected to the regulation of many aspects of tumor biology such

as proliferation, survival, tumor invasion, angiogenesis, response to chemotherapy and

radiotherapy, and it has been reported that enhanced activation of the entire PPP can be regarded

as one of the hallmarks of cell transformation [5]. G6PD is considered to be the main controlling
enzyme of metabolic flux through the oxidative phase of PPP [6]. The enzyme works at a low

basal rate in non transformed cells but it exerts a strong proliferative role when it becomes

deregulated [7, 8]. The increase in its activity inhibits apoptosis [5], and its inhibition slows

tumor growth in vivo[9].

Lung cancer is the most common cancer in the world, with 1.8 million new cases diagnosed in

2012 alone [1]. 85% of lung tumors are non-small cell lung cancer (NSCLC), with

adenocarcinoma as the most common type of cancer both in smoker and non-smoker groups. It is

one of the most aggressive cancer types, with 50% of mortality one year after diagnosis, and a 5-

year survival rate below 15%. Platinum-based chemotherapy, which remains the main option for

more than 80% of patients diagnosed with advanced NSCLC, is characterized by great

heterogeneity both in terms of efficacy and toxicity, and its long-term effectiveness is generally

very limited, with platinum-treated patients exhibiting a median progression-free survival of

three to seven months, and a median overall survival of less than one year[10]. Pancreatic cancer

remains one of the challenging disease because of its late diagnosis and a short survival rate of

the patient [11] . It is one of the 4 deadliest cancer types, with a survival rate close to zero[12].

The 3 year survival rate for these patients is 3% while 5 year survival rate has increased to a still

low 20%, the cancer spreads very quickly in the body and its recurrence is likely to happen[13].

Gemcitabine and Marimastat constitute the first-line therapy for patients with unrespectable

pancreatic cancer[14][15][16]. Their effectiveness is very limited, since they usually increase the

life expectancy by one year maximum. Epithelial ovarian cancer is the most common cause of

gynecological cancer and has high incidence of death among women[17]. Its frequency varies

among different ethnic groups, but the risk of having an ovarian cancer increases with age [18].

Cisplatin/Paclitaxel and Carboplatin/Paclitaxel combined therapies are used as first-line

treatment of ovarian cancer [7], but they has many side effects like kidney damage, loss of

fertility, and loss of taste and appetite. The 5 year survival rate of a ovarian cancer patient is

40%, and therefore both optimization of efficacy and tolerability remain important issues to be

solved.

All these problems highlight the need to find new therapeutical targets that can be exploited in

novel treatments against these cancer types. Since G6PD activity in tumors has been shown to be

significantly different from that of normal cells, and considering how essential PPP metabolic

pathway is for cell proliferation, the enzyme can be an interesting target whose inhibition could

be exploited to impair proliferation rates in these cancer tissues. In order to validate this

hypothesis, in this study we have evaluated the effect of putative G6PD inhibitors over the

phenotype of three cancerous cell lines:A549 (lung), SW1990 (pancreas), and SKOV3 (ovary).

These inhibitors (compounds 1, 2, 3, and 4)have been developed by Dr. Jaime Rubio’s team by

using structural based virtual screening, which focused on mimicking the protein protein

interaction of the homodimer of the G6PDH.The evaluation of the effect of the compounds over

cell viability, G6PD activity, cell cycle phase distribution and apoptosis induction has been

conducted in the present study. Results show that the inhibition of G6PD is a promising strategy

in the impairment of cancer cell proliferation.

2. Materials and Methods

2.1. Cell Culture

Human lung carcinoma epithelial cell line A549(ATCC® CCL-185™)(ATCC® CRL-2172™),

human ovarian adenocarcinoma epithelial cell line SKOV3 (ATCC® HTB-77™), and human

pancreatic adenocarcinoma epithelial cell line SW1990 (ATCC® CRL-2172™)were obtained

from American Type Culture Collection (Manassas, VA, U.S.A). A549 and SKOV 3 cell lines
were cultured in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum and 0.5%

penicillin/streptomycin. SW1990 cell lines were cultured in RPMI (Sigma-Aldrich)

supplemented with 10% fetal bovine serum and 0.5% penicillin/streptomycin. All cell lines were

cultured under standard culture conditions (humidified air with 5% CO2 at 37˚C).

2.2. Cell Viability Assay

4 compounds, referred as 1, 2, 3 and 4, were used in the cell viability tests Stock solutions of

those 4 molecules were prepared in DMSO, at concentrations of 11 mM (for compounds 1 and 3)

or 33 mM (compounds 2 and 4), depending on their solubility. To obtain the final assay

concentrations, compounds were diluted in DMEM (for assays over A549 and SKOV3 cell lines)

or RPMI (for assays over SW1990 cell line) immediately before the start of the incubation

period. Final concentration of DMSO was identical for all conditions, and was always kept lower

than 1%. The assay was performed by a variation of the MTT assay described by Mosmann[19]

as specified by Matito and coworkers [20], which is based on the ability of live cells to cleave

the tetrazolium ring of the MTT thus producing formazan, which absorbs at 550 nm. In brief, a

fixed number of cells (2500 cells/well for A549, 4000 cells/well for SKOV3, and 4500 cells/well

for SW1990) were seeded in 96 well plates 24hr before the addition of the different compounds,

in triplicate. Once the compounds were added, cells were cultured for an additional 72h,

supernatant was aspired and 100µL of filtered MTT (0.5 mg/ml) were added to each well.

Following 1 h of incubation with the MTT, the supernatant was removed and the precipitated

formazan was dissolved in 100µL DMSO. Relative cell viability, compared to the viability of

untreated cells, was measured by absorbance at 550 nm on an ELISA plate reader (Tecan Sunrise

MR20-301, TECAN, Salzburg, Austria). Concentrations that inhibited cell growth by 50% (IC 50)

after 72 h of treatment were subsequently calculated.

2.3. Enzymatic Assay (G6PD Activity)

Activities of PPP enzyme glucose-6-phosphate dehydrogenase (G6PD) were determined from

cell culture extracts in two independent experiments performed in triplicate. Cell extracts were

prepared and enzyme activities were measured on the Roche Cobas Mira Plus chemistry analyzer

from the rate of production of NADPH from NADP. Each enzyme activity was normalized by

determining the protein concentration using BCA method (Thermo Fisher Scientific, IL, U. S.

A.). Enzyme activities are expressed as milliunits per milligram of protein (mU/mg prot).

2.4. Cell Cycle Analysis

Cell cycle was analyzed by using a fluorescence activated cell sorter (FACS). A fixed number of

cells (45000 cells/well for A549, 59000 cells/well for SKOV3, and 167000 cells/well for

SW1990) were seeded in 6 well plates with 2ml of medium, in duplicate. After 24h, compound 1

was added at their IC50 value (2.8 µM for A549, 1.40 µM for SKOV3, and 4.65 µM for

SW1990) and twice their IC50 value (5.6 µM, 2.8 µM, and 9.3 µM). After 72h of incubation,

cells were harvested by trypsinization followed by centrifugation. The resulting pellet was

resuspended in 50% Tris buffered saline (TBS) and 50% Vindelov Buffer (which contained 50

µg/ml Propidium Iodide, 10 µg/ml DNAse-free RNase and 0.1% Igepal CA-630). The cells were

then incubated for 30 min at 4˚C, and then FACS analysis was carried out at 488nm in an Epics

XL flow cytometer (Coulter Corporation, Hialeah, FL). Data were analyzed using the Multicycle

Program (Phoneix Flow Systems, San Diego,CA).

2.5. Apoptosis Assay

Apoptosis was evaluated by the binding of annexin-V to phosphotidylserine (PS), which is

exposed to the outside surface of the cells during the first stages of the apoptotic process. A fixed
number of cells (45000 cells/well for A549, 59000 cells/well for SKOV3, and 167000 cells/well

for SW1990) were seeded in 6 well plates with 2ml of medium, in duplicate. After 24h,

compound1 was added at their IC50 value (2.8 µM for A549, 1.40 µM for SKOV3, and 4.65 µM

for SW1990) and twice their IC50 value (5.6 µM, 2.8 µM, and 9.3 µM). After 72h of incubation,

cells were harvested by trypsinization followed by centrifugation. The resulting pellet was

resuspended in 95µL of binding buffer (10mM HEPES/NaOH, 140mM NaCl, and 2.5mM CaCl 2,

pH 7.4) 3µL of Annexin-V FITC conjugate (1 µg/ml) were then added and the suspension was

kept in dark for and incubated for 30 minutes at 4˚C. Just before analysis, 500 µL of binding

buffer and 10 µL of PI (1mg/ml) were added to each sample. The sample was then analyzed via

FACS.

3. Results

3.1. Antiproliferative activity

Antiproliferative activity of the compounds 1,2,3 and 4 was evaluated invitro by performing

viability assays against human lung, ovarian and pancreatic cancer cell lines (A549, SKOV3, and

SW1990 respectively). The effect of the compounds on the growth rate of the selected cell lines

was evaluated after 72h (see Figure 1), and IC50 values (which indicate the needed concentration

to inhibit 50% of the viability of the control condition) were obtained (see Table 1). All four

compounds are able to inhibit cell viability in all the lines tested, with IC50 values in the lower

micromolar range. However, compound 1 exhibited a greater antiproliferative activity when

compared to compounds 2,3 and 4:it has considerably lower IC50 values in all the three cell lines.

IC50 values for compound 1 are 6 times lower inA549 cells, 17 times lower in SKOV3 cells, and

5 times lower in SW1990 cells when compared to compound 3, which is the second most potent

compound in all three cell lines.

3.2. Enzyme activity

The principal intracellular reductant is NADPH, which is mainly produced by the pentose

phosphate pathway through the actions of glucose-6-phosphate dehydrogenase (G6PD), the rate-

limiting enzyme of the pentose phosphate pathway. G6PD activity plays a critical role in cell

growth by providing NADPH for redox regulation and macromolecule synthesis and, through the

PPP, by providing pentoses to synthesize nucleid acids. Since all tested compounds were initially

designed to inhibit G6PD, we assessed whether the effect over cell viability was related to a

reduction in the enzyme’s activity. Compound 1, which was the compound with a higher

biological activity over cell viability in all cell lines tested, was selected for this evaluation.

Compound 1’s effect over G6PD activity was tested in all three cell lines. Enzyme activities

were normalized by total protein concentration, which was determined using BCA method.

Normalized enzyme activities are represented in Figure 2. Results show how G6PD enzyme

activity decreased after the cells incubation with compound 1 at its IC50(2.8µM) and 2xIC50

(5.6µM) concentrations. It was observed in SKOV3 and SW1990 that the decrease of 20% in

enzyme activity can be achieved at their IC50. This effect was even higher and was around 30%

in the case of A549 cell line.

3.3. Cell cycle analysis

Cell cycle is typically divided into mitosis and interphase, which is further divided into quiescent

and gap 1(G0/G1 phase), synthesis(S phase) and gap 2 (G2 phase). One of the main hallmarks of

cancer is cell cycle dysregulation. To study the effect of compound 1 on the distribution of tumor

cells in each phase of the cell cycle, A549, SKOV3 and SW1990 cells were treated with the

compound (at a concentration equal to its IC50 and at twice its IC50 values) for 72h and then

analyzed by FACS.
As seen in Figure 3, treatment with compound 1 provokes a redistribution of the percentage of

cells in each phase of cell cycle. A significant increase of cells in G0/G1 phase was seen in A549

cells treated with IC50(2.8µM) and 2xIC50(5.6 µM) concentrations of compound 1. Changes in

cell distribution were even more apparent in SKOV3 cells, but in this case the cells were arrested

mostly in the G2/M phase.

3.4. Apoptosis analysis

When the cells get arrested in a particular phase of the cell cycle and they are not able to cross

the cell cycle checkpoints, they get accumulated in the phase before that checkpoint. This arrest

of cell cycle can sometime lead to apoptosis, so after assessing compound 1’s clear effect over

cell cycle distribution, apoptotic assays were performed to see whether apoptosis was involved in

the antiproliferative effect of compound 1. When the apoptotic process begins, cell membrane

loses its symmetry, which entails a translocation of phosphatidylserine from the inner membrane

to the outer membrane. Once phosphatidylserine is exposed to the external cell environment, it

can bind to the annexin V-FITC conjugate, for which it has high affinity. Once the cells reaches

the final apoptitic stages, cell membranes completely lose their integrity, which gives propidium

iodide (a nucleotide binding dye which intercalates between the DNA bases) access to the

nucleus. Flourescence activated cell sorting(FACS) was used together with annexin V-FITC and

Propidum Iodide staining in order to differentiate non apoptotic cells (annexin V- and PI-) from

early apoptotic cells (annexin V+ and PI-) and necrotic or late apoptotic cells (PI+). After

incubating the cells with compound 1, we could see a decrease in the number of healthy cells and

an increase in the number of early apoptotic and late apoptotic/necrotic cells (see Figure 4). This

effect was hardly evident at 24h, but became more and more apparent with longer incubation

times, which indicates that the mechanism of action of compound 1 may not be directly related
to apoptosis, but rather that arises as a consequence of the compound’s effect over cell cycle

arrest (accumulation of cells in one of the cell cycle phases eventually leads to the induction of

apoptosis and their controlled death).

4. Discussion
The current study focuses on validating the biological effects of four compounds (1, 2, 3 and 4)

which were previously designed to prevent the G6PD dimer formation by inhibiting its protein-

protein interaction. The activity of these four compounds, whose structure is tailored to inhibit

the hydrophobic contact points as well as the van der Waals contact points while also targeting

hydrogen bond donor and acceptor in G410, was tested on three cancer in vitro models: lung

cancer cell line (A549), ovarian cancer cell line (SKOV3) and pancreatic cancer cell line

(SW1990). Since G6PD is the enzyme controlling the oxidative phase of PPP (one of the most

important metabolic pathways for tumor proliferation), our goal was to assess whether

incubating cancer cells with these inhibitors was able to inhibit their proliferation rate. Therefore,

first we decided to assess the antiproliferative potential of the four molecules, which were

selected previously amongst other candidates after molecular dynamic stimulation and structural

based virtual screening.

It was found that all four compounds exhibit antiproliferative activity. After 72h of incubation,

drastic reductions in cell viability were observed in the three cancer cell lines. The difference in

the observed IC50 values could be associated with the different proliferative behavior of these cell

lines, but in any case those were not very important and IC50 values were found to be in the low

micromolar range in all cases. 1 showed to be the most potent of the four tested compounds, as it

had the lowest IC50 value in all three cell lines(2.8µM in A549, 1.4µM in SKOV3 and 4.65 µM

in SW1990).Structure-activity relationship (SAR) is the relationship between the 3D structure of

a molecule and its biological activity, and SAR analysis can help in determining which chemical

groups can evoke a particular biological activity. Since we know that the compound 1 is the most

potent, its structure can be used as a starting point for the development of a new family of more

potent compounds with enhanced biological activities.

Once the existence of an antiproliferative effect was established, and considering the compounds

were designed to inhibit the formation of the active G6PD dimer, we decided to assess the effect

of the most potent compound (compound 1) over G6PD activity in all three cancer cell lines.

This would also help us establish if there is any relationship between the viability of the cells and

the levels of G6PD activity. It was found that the levels of G6PD activity decreased significantly

in all cell lines after their incubation with compound 1. The decrease in enzyme activity after

cells incubation with compound 1 at its IC50 concentration was of 20% for SKOV3 and SW1990

cell lines, and 30% in the case of A549 cell line. Although other factors (such as unspecific

inhibitions of other enzymes) might be involved in the antiproliferative effect of the compounds,

this result points out to the existence of a direct relationship between G6PD activity and cell

viability in the three cell lines, especially considering that the molecules were specifically

designed to inhibit G6PD dimer formation. Interestingly, decreases of around 20% in G6PD

activity were related to a decrease of around 50% in cell viability, highlighting the key play

G6PD protein plays in cancer proliferation. The effect of compound 1 was over G6PD activity

was not evaluated over a dilution of the pure G6PD enzyme for two reasons. First, human G6PD

is not available commercially. Second, these compounds are designed to inhibit the formation of

dimer, so when the cells are incubated with the compound the formation of the dimer throughout

the incubation time is hindered. The compound would not be able to cause the same effect to a
dilution of commercially obtained pure enzyme, since the dimer would be already formed and

the G6PD would therefore be already active.

Considering G6PD’s relationship with cell cycle regulation [21], and since one of the important

hallmarks of cancer is the dysregulation of cell cycle [22], we then checked whether compound 1

could lead to a redistribution of the cells in the different phase of the cell cycle. In the case of

A549 cells, flow cytometery analysis showed a significant (p<0.05) increase in G0/G1

population after treatment with compound 1 when compared to control conditions. This result

suggests that compound 1 induces cell cycle arrest in G0/G1 phase, which would explain the

decrease in cell proliferation observed in the viability assays. The arrest in G0/G1 is concordant

with the inhibition of the enzyme G6PD, since PPP is essential to produce the nucleotides that

are needed in DNA synthesis. Without those nucleotides, S phase of the cell cycle can’t take

place, and over time cells accumulate over time in the previous phase of the cell cycle (G1). In

the case of SKOV3, however, the cell cycle arrest after incubation with compound 1 was seen in

G2/M phase, and not in G0/G1 phase. A possible explanation could be that the metabolism of

ovarian cancer cells lines is more prepared to use the non-oxidative branch of pentose phosphate

pathway, and therefore they can synthesize nucleotides (and, through them, DNA) even if G6PD

is inhibited. This would allow the cells to go through G1 and S phases, but since G6PD is also

required for NADPH production, a lack of NADPH would still prevent those cells to support the

production of macromolecules such as fatty acids, needed to generate cell membrane just before

mitotic division. In this case, even if SKOV3 cells are able to avoid G1 arrest, they could not go

through the mitotic phase, and therefore would accumulate over time in the G2 phase of the cell

cycle, which agrees with our observed results. The fact that ovarian cancer cells overexpress

TKT (a key enzyme in the non-oxidative branch of the PPP) while lung cancer cells overexpress
G6PD support this hypothesis about their divergent capacities to avoid G1 phase arrest after

G6PD inhibition [23].

G6PD has also been previously connected to apoptosis inhibition [24], so we decided to check

whether compound 1 was able to induce apoptosis in the tested cancer cell lines. When. So it was

checked if the compound was only cytostatic in nature or it was cytotoxic too. Results show that

when cells were incubated with the compound 1 for 24 hours, there was no significant changes in

the apoptotic state of the cells, which suggests that the compound does not exercise its

antiproliferative effect through induction of apoptosis. However, after 48h of incubation the

number of early apoptotic and late apoptotic/necrotic cells was increased, an effect that was even

more noticeable after 72h of incubation. This suggests that compound 1 is not directly cytotoxic,

but rather that the arrest and accumulation of the cells in one of the phases of the cell cycle due

to compound 1’s effect, which is already evident after 24h, eventually entails their cell death

through apoptosis.

In any case, our results prove that the previously synthesized compounds are able to inhibit

G6PD activity, and that this is related with a decreased of the viability of cancer cells through

cell cycle arrest, which eventually leads to apoptosis induction. This reinforces the utility of

exploiting vulnerabilities associated to cancer metabolic reprogramming as a promising strategy

to impair tumor proliferation, and opens new avenues to improve the design and synthesis of a

new family of metabolic inhibitors as putative therapeutic agents in cancer treatment.

5. Acknowledgements

This work was supported by European Commission Seventh Framework Programme

FP7 COSMOS 7CAiNF-312941; METAFLUX (Marie Curie FP7-PEOPLE-2010-ITN-264780);

Spanish Government and European Union FEDER funds (SAF2011-25726, SAF2014-56059-

R); ICREA Academia prize (MC); and Generalitat de Catalunya (2014SGR1017).

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pathway in the human melanoma xenograft mouse model. BMC Cancer, 2013. 13: p. 251.
Legend/Caption for Figures

Table 1: IC50 values

IC50 values for compounds 1, 2, 3 and 4 over A549 lung, SKOV3 ovarian and SW1990

pancreatic cancer cell lines. Data are represented as the mean values for a minimum of three

independent experiments performed in triplicate. Compound 1 showed to be most potent with

least IC50 value.

Figure 1: Cell viability assay

Cell viability assay for compounds 1, 2, 3 and 4 over A549 lung cancer cell line, SKOV3

ovarian cancer cell line and SW1990 pancreatic cancer cell line. The graphs were made by using

Graphpad Prism 6. (A) represents the effect of the compounds (1, 2, 3 and 4) on A549 lung

cancer cell line, (B) represents the effect of the compounds (1, 2, 3 and 4) SKOV3 ovarian

cancer cell line and (C)represents the effect of the compounds (1, 2, 3 and 4) SW1990 pancreatic

cancer cell line. Compound 1 showed to be most potent with least IC50 value in micromolar

range.

Figure 2: Compound 1’s effect over G6PD activity for A549, SKOV3 and SW1990 cell

lines.

All the represented graphs were made by using the data collected after G6PD assay which were

normalized by doing a BCA assay. Figure(A) represents the change in the levels of G6PD when

compounds are administered to A549 cells at a concentration of 2.8µM (IC50 value)and 5.6

µM(2x IC50 ) Figure(B) represents the change in the levels of G6PD when compounds are
administered to SKOV-3 cells at a concentration of 1.4 (IC50 value)and 2.8 µM(2x IC50 ).

Figure(C) represents the change in the levels of G6PD when compounds are administered to

SW-1990 cells at a concentration of 2.8µM (IC50 value)and 5.6 µM(2x IC50 ). (Mean ± SD). p <

0.05 (*); p < 0.01 (**); p < 0.001 (***) were considered to indicate statistic significant

differences between treated cells and control cells.

Figure 3. Cytometeric plots of A549, SKOV-3 and SW-1990 for cell cycle assay

(A) Representative flow cytometric plots showing the distribution of SKOV3 cells in the

different phases of the cell cycle after 24h incubation with compound 1. (B)Bar graphs depicting

the % of cells in each phase of the cell cycle for A549, SKOV3 and SW1990 cells

untreated(control) and treated with compound 1 at its IC50 and 2xIC50concentrations for 24h, 48h

and 72h. (Mean ± SD). p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) were considered to indicate

statistic significant differences between treated cells and control cells.

Figure 4: Cytometeric plots of A549, SKOV-3 and SW-1990 for apoptosis assay

Figure(A)Representative flow cytometricplots showing the distribution of SKOV3 cells in the

different phases of the cell cycle after 72h incubation with compound 1. Quadrants 1 and 2

contain late apoptotic /necrotic cells, Quadrant 3 contains early apoptotic cells and quadrant 4

contains unaffected/healthy cells. Figure(B) Bar graphs depicting % of healthy, early apoptotic

and late apoptotic/necrotic A549, SKOV3 and SW1990 cellsuntreated (control) and treated with

compound 1 at its IC50 and 2xIC50 concentrations for 24 h, 48 h and 72 h. (Mean ± SD). p <

0.05 (*); p < 0.01 (**); p < 0.001 (***) were considered to indicate statistic significant

differences between treated cells and control cells.

Table 1:

CellLines
Compounds A549 SKOV3 SW1990

1 2.84±1.1µM 1.40±1.1 µM 4.65±1.2 µM

2 26.5±1.2 µM 32.3±1.2 µM 34.5±1.1 µM
3 7.30±1.5 µM 24.2±1.6 µM 23.7±1.4 µM
4 13.2±1.1 µM 60.2±1.3 µM 63.6±1.3 µM
Figure 1:

Figure 2:
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