Kingston University: AML Treatment with Autophagy Modulators Project

This project investigates the effect of autophagy modulators, specifically chloroquine and rapamycin, on STAG2 mutated cells in the context of Acute Myeloid Leukemia (AML). The research explores the use of these modulators as potential therapeutic approaches for AML, focusing on their impact on cell viability and DNA repair mechanisms. The study examines the effects of these drugs, along with cisplatin, on cell death and autophagy modulation in STAG2 mutated cell lines (UMUC-3) and a control cell line (ASPC-1). The project aims to determine if autophagy modulation can enhance drug sensitivity towards platinum-based chemotherapy, providing a potential targeted therapy for AML. The methods include cell culturing, drug preparation, cell viability assays (Alamar Blue), and statistical analysis. The study tests hypotheses regarding the increased cellular death in STAG2 mutated cells, the differential effects of chloroquine and rapamycin, and the enhanced drug sensitivity with autophagy modulation. The results section presents data on cell counts and cell viability, with a discussion of the findings in the context of existing literature and the implications for future research on AML treatment strategies.

Running head: MEDICAL
BIOMEDICAL SCIENCE
Name of the Student
Name of the University
Author Note

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Abstract
Aggressive leukemia (AML) has occurred with the incidence of one lakh cases and has been
observed to increase with age. The identification of genetic and molecular groups have been
increasingly studied for the treatment of AML. This disease has been characterized by
various chromosomal translocations and mutations associated with the gene differentiations
as FLT3, NPM1, CEBPA ASXL. Autophagy is the sequential process involving cell death
regulation and damaged cellular debris recycling is initiated by external and cellular cures to
maintain proper homeostasis and metabolism. This process is important for the regulation of
double-stranded DNA repair.
Table of Content

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s
Introduction................................................................................................................................4
AML (Acute Myeloid Leukaemia):.......................................................................................4
Treatment strategies:..............................................................................................................4
STAG2- Cohesin Complex:...................................................................................................5
DNA repair:............................................................................................................................5
Autophagy:.............................................................................................................................6
Chloroquine............................................................................................................................6
Rapamycin..............................................................................................................................6
Aims and Objectives..................................................................................................................7
Materials and Methods...............................................................................................................7
Reagents.................................................................................................................................7
Cell culturing and lines..........................................................................................................8
Cell subculture.......................................................................................................................8
Counting of cells....................................................................................................................8
Calculation of cell counting...................................................................................................9
Preparation of drug and addiction of drugs..........................................................................10
Alamar Blue Assay (Cell Viability).....................................................................................13
Cisplatin (Alkylating agent) - Combined drug therapy........................................................13
Statistical Analysis...............................................................................................................14
Results......................................................................................................................................14
Counting of cells:.................................................................................................................14

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Alamar Blue Assay: Cell Viability......................................................................................19
Discussion................................................................................................................................24
Conclusion................................................................................................................................29
References................................................................................................................................30

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Introduction
AML (Acute Myeloid Leukaemia):
According to various research studies, AML is a part of leukemia that occurs in
adults. The majority of the leukemia cases occurring in adults consists of AML as the root
cause (Li et al. 2019). This form of leukemia has been characterized by mutation
(translocation) in the gene differentiation and hematopoietic proliferation. This condition
farther leads to the accumulation of cells in the peripheral blood and bone marrow due to the
absence of an apoptotic mechanism. Thus, it is an important decision to choose an
appropriate method for the experiment. One of the best future methods for the conduction of
this experiment is the ((3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide) assay
(MTT) which is a colorimetric assay used for the access to cell viability and cellular toxicity
(Darwish et al. 2016). This procedure includes the use of succinate which is used to
determine the cellular viability. While MTT was added to the cells, NADH will work by
reducing it to a purple formazan that can be quantified by the use of light absorbance.
According to the statistical records, 12 people per 100,000 people over 65 years have more
chance of the disease than 1 individual per 100,000 people under 65 years (Saultz and Garzon
2016). This disease is diagnosed with a prevalence percentage of over 20% blast in peripheral
blood or bone marrow followed by the detection of myeloid progenitor cells in
myeloperoxidase test.
Treatment strategies:
Intensive induction chemotherapy with anthracycline administration for three days
and cytarabine for seven days allows a CR (complete remission) among 80% of the younger
adults. The percentage is 60% for older adults due to the reduced strength of their immune
system. Isocitrate inhibitors (IDH), FLT3 (Fms-like tyrosine kinase 3) and nuclear-exported

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inhibitors are some novel therapies devised for the treatment for this disease. Repair of
defective DNA, stabilization of genomic instability and deregulation of transcription are also
the major processes of AML treatment. Thus it can be understood that the treatment can be
induced from the DNA level (Dombret and Gardin 2016). Thus, this research study uses
mutation of STAG2 along with abnormal replication of DNA to induce lethality after using
cisplatin and chloroquine. These two drugs have been observed to modulate autophagy to
involve the replication of DNA. AML treatment options have minimal side effects and are
also associated with higher efficacy.
STAG2- Cohesin Complex:
The gene stag2 encodes the cohesion complex component that participates in the
sister chromatid regulation of separation required during mitosis. After the activation of
stag2, protein acts as a tumour suppressor disrupting the separation of mitotic sister
chromatin. According to various research studies it can be stated that the chromosomal
deletions involving Xq25 as observed in myelodysplasia and AML along with several
mutations in STAG2 hotspots regions known as exon 9,11,12,20 were identified as acute
leukaemia causes (Nishiyama 2019). STAG2 has also been observed to participate in the
inactivation of RUNX1, NPM1, and RB1 or CDKN2A/B (tumour suppressor genes). This
process is named as leukemogenesis.
DNA repair:
The genomic DNA gets damaged due to various factors including radiation,
environmental chemicals, UV light, and other external factors. These factors result in the
generation of reactive oxygen species (ROS) which gives rise to replication errors. Induction
of DSB (double-stranded breaks) occurs by an improper activation of Chk2 and the absence
of Rad21 leading to an increased probability of chromosomal rearrangement (Zatopek et al.

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2019). One of the most significant repair techniques is BER (Base Excision repair) required
for BRCA mutant cells which cause the ovarian cell death and breast cancers associated with
improper homologous recombination. Normal cells do not get affected.
Autophagy:
The sequential process involving cell death regulation and damaged cellular debris
recycling is initiated by external and cellular cures to maintain proper homeostasis and
metabolism. These conditions protect the cell against the oncogenic development which is
deregulated in a broad range of cancers (Zhang, Shang and Zhou 2015). When a cell is
observed to experience a nutrient deprivation or metabolic stress, the cell undergoes
catabolism to recycle the cellular organelles which are damaged and extract the proteins from
damaged of long-lived organelles.
Chloroquine
Chloroquine is an antimalarial agent, which is used in the treatment of erythematous,
and rheumatoid arthritis. Chloroquine acts by the inhibition of an autophagic flux during a
later stage to prevent the fusion of autophagosomes with lysosomes. This condition causes
the degradation of autolysosome (Verbaanderd et al. 2017).
Rapamycin
Also known as RAPA which is a member of the macrocyclic class of
immunosuppressive drugs that has been used as an immunosuppressive drug. The primary
target for rapamycin (mTOR) acts as an amino acid and ATP sensor involved in the balance
of nutrient availability. The majority of cancers show hyperactivity during the switching on
of mTOR by an oncogenic signaling pathway. Rapamycin inhibits the mTORC1 activity
which can also inhibit the actions of mTORC2 (Li, Kim and Blenis, 2014).

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Aims and Objectives
The main aim of this project is to check the effect of autophagy in STAG2 mutated cells with
a defect in DNA repair by using autophagy inducer rapamycin for the inhibition of mTOR
(mammalian TOR) in order to invoke a defect in DNA repair for the destruction of mutated
cells. Another autophagy inhibitor known as chloroquine is also used here which prevents the
fusion of lysosome and phagosome. Both these functions will be studies to provide a targeted
therapy required for the treatment of AML. This experiment will also aim to provide a sub
stratum for future research studies involving a more efficient treatment strategy.
The following three hypotheses will be tested:
1. STAG2 mutated cells will have their cellular deaths increased to a higher level than the
non-mutated cell line.
2. The cells, which are incubated with chloroquine, are expected to have an increased death
as compared to the cells treated with rapamycin.
3. The modulation of autophagy is expected to increase the drug sensitivity towards platinum.
Thus, cells which are incubated with a platinum-based chemotherapy drug (Cisplatin)
and autophagy modulators are expected to have the most amount of cell death.
Materials and Methods
Reagents: DMEM (Dulbecco’s modified eagle medium) with high concentration of glucose
(4500 mg/L), FBS (Fetal bovine serum), antibiotic- Streptomycin-Penicillin, PBS
(Dulbecco’s phosphate buffer saline), Trypsin-EDTA solution, Trypan Blue, DMSO
(Dimethyl sulfoxide), RAP (Drugs- autophagy inducer Rapamycin) [3150055-EMD
Millipore), CHL (Chloroquine diphosphate salt) [C6628-25G-25G –SIGMA-autophagy

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inhibitor). AlamarBlue cell viability reagent (Thermofisher Scientific, UK) and 100mM
Cisplatin (Sigma).
Cell culturing and lines: ASPC-1 and UMUC-3 were frozen and thawed inside a 37-
degree centigrade water bath. UMUC3 (cells obtained from the cell line of the human urinary
bladder associated with a STAG2 mutation). APSC1 (adenocarcinoma pancreas cell line
without the absence of STAG2 mutation will be used as a control). The thawed samples were
then transferred into falcon tubes and then washed with a 10mL media. This apparatus was
then centrifuged at 1000 rpm for 5 minutes. Following this centrifugation, the tubes were
taken out and the supernatant was discarded. Then the pellet was farther re-suspended into a
10mL media which was prepared by the addition of 50 mL of 10% FBS. Penicillin-
Streptomycin (5 mL) 1% was added to 500 mL DMEM and mixed with the previous solution.
The complete mixture was then transferred to a flask and incubated with 5% CO2 required
for cell growth and 37-degree centigrade. The cells were isolated from the mixture and then
sub-cultured to use in the project. Tissue culture hood was the chamber used to perform all
the cell culturing and subculturing after using 70% alcohol spray to sterilize the chamber.
Cell subculture: The cells were allowed to grow up to a 75% confluency level after every
two days. The T75 flasks were removed from the incubator and the media from one flask is
then transferred equally to two falcon tubes. Then 5 mL PBS was added to the mixture and
incubated for 5 minutes. These were then transferred to both the falcon tubes and incubated
for 5 minutes with 5% CO2 at 37-degree centigrade. Then the supernatant was discarded
followed by the pellet with cells suspended in DMEM 20 mL media. The final solution is
then transferred to 2 separate T75 flasks and then placed into the incubator again with 5%
CO2 and 37-degree centigrade.

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Counting of cells: The assay used in this experiment to count the viable cells is named as
Trypan blue exclusion assay. The viable cells are differentiated from the dead cells in terms
of their cell membranes. The cell membrane of viable cells is intact which prevents the entry
of Trypan blue into the cells and thus does not have a blue colour. The dead cells have a blue
colour and the viable cells have a clear cytoplasm. 50 microlitre of cells were then suspended
in the media which is then placed in an Eppendorf, followed by an addition of 50 micro liter
of Trypan blue. The whole apparatus was then incubated at 25-degree centigrade (room
temperature) for three minutes. The cells were then counted using a FastRead-102 10-
chamber grid for counting cells (haemocytometer). Twenty micro liter of the sample was
added into the chamber present at the sample application area and then a 4X4 grid was
counted under a light microscope fixed at a magnification of 10x.
Calculation of cell counting: The chambers of the haemocytometer has 10, 4X4 grids (Fig
1),
Counts/mL= total counts
number of complete 4 X 4 grids counted X 104 X dilution of the sample
As an example, the figure below shows that five chambers having 67 live cells is counted if
the dilution of the sample was 3 mL. According to the formula stated,
Cell count= 67
5 x 104 x 3 = 402, 000

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Fig 1: Haemocytometer (10 chambers were counted for live cells)
Source:
Preparation of drug and addiction of drugs: Determination of the effect of rapamycin
and chloroquine on the cell viability. An approximate value of 500,000 ASPC-1 and UMUC3
cells in the 2.5 mL medial was then divided into eight wells having several concentrations of
drugs being added to each of the wells. None of the drugs were added to the control wells
(A1) (Fig 2).
Fig 2: Plate representation

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Source:
Preparation of Chloroquine: Four of the chloroquine stock concentrations were prepared
(10 mM, 10 micromoles, 1 micromole, and 0.1 micromole) as stated below-
Chloroquine (Molecular weight) = 515.86 g/mol
Final volume (Desired) = 10 ml = 0.01 L
Final concentration (Stock) [Desired] = 10 mM = 0.01 mol/L
Using: Mass (g) = Concentration(mol/ L)×Volume (L)× Formula Weight ( g/mol)
= 0.01 mol /L ×0.01 L× 515.86 g / mol
Mass = 0.051586g
Thus, it can be said that chloroquine of 0.0516 g was weighted off and then added to
PBS of 10 mL to prepare the primary stock solution with a concentration of 10 mM [Tube 1].
The secondary stock concentration was 10 micromolar, prepared by transferring 2.5
microlitres from tube 1 to 2.5 mL PBS. This solution was then diluted by 1000 factors to
make the desired concentration to 10 micromoles for tube 2. Ten microlitres were taken from
tube 2 and then transferred to PBS (900 ml) to prepare a final concentration of 1 micromole.
This is for tube 3. Ten microliters from the sample were taken from tube 3 followed by an
addition to 990 ml PBS in order to prepare a 0.1 micromole final concentration for tube 4.
Then, 2.5 microlitres will be taken from each of the chloroquine tubes and then added to B1,
B2, B3, B4 wells to prepare 10 μM, 0.01 μM, 0.001 μM, 0.0001 μM as the final
concentration.
In well B1,
Using the following equation, C1V1= C2V2

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Where C1= Stock concentration = 10mM = 10000 μM
V1 = volume taken from the stock (tube 1) and added to each well = 2.5 μL
C2= final concentration in each well =?
V2= final volume in each well. = 2502. 5 μL
C2 = C1V1/V2= (10000*2.5)/2502.5=10 μM.
Preparation of Rapamycin: 100 UG was present in the pack of Rapamycin and then DMSO
(400 microlitre) was added to it.
The molecular weight of rapamycin is 914.172 g/mol.
According to the formula,
Mass (g) = Concentration(mol/ L)×Volume (L)× Formula Weight (g/mol)
= 0.0001/ (0.0004*914.172)
= 0.0001/0.3656688
= 0.0002734715 mol/L = 273.5 μM = 273500 nM.
From the stock solution, 25 microliters were extracted and added to the A2 well. Then
2.5 microlitre was extracted from the stock and transferred to A3 well. 0.25 microlitre from
the stock solution was transferred to the A4 stock in order to prepare a final concentration of
2710 nM, 273 nM and 27.3.
As an example, inside well A2, 25 micro liter was then extracted from the stock
solution and then added to the well. This well contained 500,000 cells in the 2.5 mL media.
According to the formula,
C1V1= C2V2

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Where C1 = Stock concentration = 273,500 nM
V1 = volume taken from the stock and added to each well = 25 μL
C2= final concentration in each well =?
V2= final volume in each well. = 2525 μL
C2 = C1V1/V2= (273500*25)/2525=2710 nM.
This experiment was then performed three times for each of the cell lines. The total
number of live cells were counted on the 5th day and 10th day. Then the average was
performed at the end.
Alamar Blue Assay (Cell Viability)
This method has been observed to provide a suitable method required to monitor the
cell viability and health required to detect the changes in the key cell indicators such as the
changes of the plasma membrane and the reducing cellular environment. The healthy cells are
maintained properly in a reducing environment inside the cytosol. Thus, when Alamar blue
cell viability reagent has been added, a non-toxic and active resazurin (non-fluorescent) and
after its entry into the cell, gets reduced to resorufin (red) which is fluorescent and thus
increasing the complete fluorescence inside the viable cells detected by an absorbance based
plate reader. 90 micro liter of media with cells were then transferred to a 96 well plate. Then
10 microlitre of Alamar blue cell viability reagent was added and protected from the effect of
direct sunlight followed by incubation for 2 hours at 37-degree centigrade. The plate was read

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by using the M200 pro plate reader at 560 nm excitation and a 590 nm emission wavelength
required to measure the viability of the cell.
Cisplatin (Alkylating agent) - Combined drug therapy
The cells were prepared and set up with various concentrations of CHL and RAP.
Cisplatin (100 mM) of 2.5 microlitre was transferred to each of the wells except the controls
in order to check the synergistic effects of drugs on ASPC-1 and UMUC-3 cells. The plates
were then incubated and counted after five days of incubation. This assay was then performed
for another two to three times to produce an extra reliable outcome. The test for cell viability
was also carried out by alamarBlue assay.
Statistical Analysis
A two-tailed t-test was performed to compare the cell viability analysis and determine
the statistical significance. The P values were then considered to be significant at <0.5.
Results
Counting of cells:
Table 1 (Day 5 UMUC3-Chloroquine, ASPC1-Chloroquine, ASPC1-
Chloroquine+Cisplatin)-
Concentration
(micromole)
Controls Cell viability
for UMUC3-
Chloroquine
Cell viability
for ASPC1-
Chloroquine
Cell viability for
APSC1-
Chloroquine+Cisplatin
10,000 100 109 74 29
10 100 125 85 27
1 100 143 77 23

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0.1 191 95 25
10000 10 1 0.1
0
50
100
150
200
250
UMUC3-Chloroquine ASPC1-Chloroquine APSC1-Chloroquine+Cisplatin
Fig 3: The graph shows the cell viability in UMUC3 and ASPC-1 after the completion of 5
days treatment with various concentrations of chloroquine as the monotherapy and in a
combination of 100 nM cisplatin. Then the cells were counted by using a trypan blue
exclusion hemocytometer placed at 10x magnification. The results shown here are the
average of the three repeat experiments which are expressed as a percentage of untreated cells
(control) where the control is stated as 100%.
Table 2 (Day 5 UMUC3-Rapamycin, APSC1-Rapamycin)-
Concentration
(micromole)
Cell viability for UMUC3-
Rapamycin
Cell viability for APSC1-
Rapamycin
0.1 98 95
0.001 125 74
0.0001 102 77
Concentration (micro Mole)
Cell viability (percentage of controls)

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0.1 0.001 0.0001
0
20
40
60
80
100
120
140
Cell viability for UMUC3-Rapamycin Cell viability for APSC1-Rapamycin
Fig 4: The graph shows the cell viability in ASPC-1 and UMUC3 after the completion of 5
treatment days with various rapamycin concentrations as a monotherapy. The cells were then
counted using trypan blue exclusion and at a 10x magnification using a hemocytometer. The
results here are calculated by an average of 3 repeat experiments which are expressed as a
percentage of untreated cells (control) and the control is 100%.
Table 3 (Day 10 UMUC3-Chloroquine, ASPC1-Chloroquine)-
Concentration (micro mole) Cell viability for UMUC3-
Chloroquine
Cell viability for ASPC1-
Chloroquine
10,000 58 52
10 63 79
1 108 114
0.1 72 100

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10000 10 1 0.1
0
20
40
60
80
100
120
Cell viability for UMUC3-Chloroquine Cell viability for ASPC1-Chloroquine
Concentration (micro mole)
Fig 5: This graph shows the viability of cells in UMUC3 and ASPC-1 after the treatment is
performed for 10 days with several chloroquine concentrations. These concentrations are
adjusted for chloroquine as a monotherapy. The cells were then counted using trypan blue
exclusion at a 10x magnification of hemocytometer. The outcomes shown are the average of
three repeats which are expressed as a control percentage where there is a control of 100%.
Table 4 (Day 10 UMUC3-Rapamycin, APSC1-Rapamycin)
Concentration
(micromole)
Cell viability for UMUC3-
Rapamycin
Cell viability for APSC1-
Rapamycin
0.1 83 71
0.001 70 67
0.0001 57 72

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0.1 0.001 0.0001
0
10
20
30
40
50
60
70
80
90
Cell viability for UMUC3-Rapamycin Cell viability for APSC1-Rapamycin
Fig 6: The graph shows the viability of cells in UMUC3 and ASPC-1 after a ten-day
treatment with various concentrations of rapamycin as a monotherapy. The cells were then
counted by using trypan blue exclusion and at a 10x magnification of hemocytometer. The
outcomes shown here are of three repeat experiments expressed as a percentage of control
where the control is set at 100%.
For the investigation of the modulating potential for autophagy in STAG2 mutated
cells which acts as a specific and effective treatment for AML patients which have their
STAG2 mutated. The ASPC-1 without the mutation in STAG2 was used as a control and
UMUC3 line with a mutation in STAG2 was further treated with rapamycin to induce
autophagy and chloroquine to inhibit autophagy. The approximate value of 100,000 cells on
an average was treated with varying concentrations of rapamycin and chloroquine as a
nanotherapeutic treatment or in a combination along with 100 nM cisplatin. The cells were
then counted on the 5th day (Fig 3, Fig 4) and the 10th day after treatment (Fig 5, Fig 6).

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An overall increase in the viability of cells in UMUC3 cells that are treated with
chloroquine has been observed after treatment was done for five days where there is an
increase in the cellular viability with a decrease in the concentration. APSC-1 cells, on the
other hand, show a decrease in the viability of cells with a decrease starting from 10 μM (Fig
3). When UMUC3 cells are treated with rapamycin, an increase in cell viability is observed
after a treatment of 5 days. However, the viability shows a drastic fall from 0.001 micromole
concentration. ASPC-1 has been observed to show a decrease in cell viability (Fig 4). Both
UMUC3 and ASPC-1 when treated with the combined therapy including rapamycin with
cisplatin, the most significant decrease in cell viability has been observed on day 5 (Fig 3).
The highest percentage of viability (190%) was seen in the cells which were treated with the
lowest concentration of chloroquine (0.1 micromoles) (Fig 3).
After a chloroquine treatment was performed on day 10, cell viability fluctuation was
observed. UMUC3 cells showed an increase in viability from 10,000 micromoles and
suddenly dropping from the peak at 1 micromole (Fig 5). On the other hand, ASPC-1 has
shown a slow increase in cell viability from 10,000 micromoles to 10 micromoles. Thus it
rises to a peak point at 1 micromole in an exponential manner and then falls from there to 0.1
micromole (Fig 5). However, it has been observed that the cells, which are treated with a
combination of chloroquine or rapamycin with Cisplatin, showed the highest decrease in cell
viability.
Alamar Blue Assay: Cell Viability
Table 5 (Day 5 UMUC3-Chloroquine, ASPC-Chloroquine)
Concentration in microM Absorbance at 590 nm for
UMUC3-Chloroquine
Absorbance at 590 nm for
ASPC-Chloroquine
10,000 2494 17763

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10 2439 15768
1 1846 15334
0.1 1664 15146
10000 10 1 0.1
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
Absorbance at 590 nm for UMUC3-Chloroquine Absorbance at 590 nm for ASPC-Chloroquine
Fig 7: The graphical representations show the cell viability in ASPC-1 and UMUC3 after a
treatment of 5 days with various concentrations of chloroquine as the monotherapy. Alamar
blue viability- Cell viability was determined by using ELISA based assay. The absorbance
was measured by using an Infinite M200 Pro plate by using a 560nm excitation followed by a
590 emission wavelength after a two to four hours incubation with the help of Alamar blue
reagent. The results show the average from three replica plate sets.
Table 6 (Day 5 UMUC3-Rapamycin, ASPC-Rapamycin)
Concentration in microM Absorbance at 590 nm for
UMUC3-Rapamycin
Absorbance at 590 nm for
ASPC-Rapamycin
0.1 1656 8626

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0.001 2152 10127
0.0001 1711 9347
0.1 0.001 0.0001
0
2000
4000
6000
8000
10000
12000
Absorbance at 590 nm for UMUC3-Rapamycin Absorbance at 590 nm for ASPC-Rapamycin
Fig 8: This graph shows the cell viability in ASPC-1 and UMUC3 after a 5 days treatment
with various rapamycin concentrations in a monotherapy. The ELISA based assay was used
to determine the viability of cells by Alamar blue viability. The absorbance measurement was
done by using an Infinite M200 Pro plate reader by using a 560 nm excitation and 590 nm
emission wavelength after a time period of two to four hours for incubation with Alamar blue
reagent.
Table 7 (Day 10 UMUC3-Chloroquine, ASPC1-Chloroquin)
Concentration in mM Absorbance at 590 nm for
UMUC3-Chloroquine
Absorbance at 590 nm for
ASPC-Chloroquine
10,000 2258 5459
10 2372 8450

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1 2256 6625
0.1 2087 8318
10000 10 1 0.1
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Absorbance at 590 nm for UMUC3-Chloroquine Absorbance at 590 nm for ASPC1-Chloroquine
Fig 9: This graph shows cellular viability in ASPC-1 and UMUC3 after a time length of 10
days for treatment with various concentrations of chloroquine used as a monotherapy. The
cell viability was determined by using an Infinite M200 Pro plate reader by using the ELISA
based assay. The reader was used at an excitation wavelength of 560 nm and an emission
wavelength of 590 nm after the time length of two to four hours. The incubation was done
with an Alamar blue reagent. The results are shown as an average from three replica plates.
Table 8 (Day 10 UMUC3-Rapamycin, ASPC-Rapamycin)
Concentration in mM Absorbance at 590 nm for
UMUC3-Rapamycin
Absorbance at 590 nm for
ASPC-Rapamycin
0.1 2196 5891
0.001 2185 2515

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0.0001 1711 6281
0.1 0.001 0.0001
0
1000
2000
3000
4000
5000
6000
7000
Absorbance at 590 nm for UMUC3-Rapamycin Absorbance at 590 nm for ASPC1-Rapamycin
Fig 10: The graph is showing the cell viability in UMUC3 and ASPC-1 after the time period
of 10 days of treatment with several concentrations of rapamycin as a monotherapy. The cell
viability was determined by using an Infinite M200 Pro plate reader by using the ELISA
based assay. The reader was used at an excitation wavelength of 560 nm and an emission
wavelength of 590 nm after the time length of two to four hours. The incubation was done
with an Alamar blue reagent. The results are shown as an average from three replica plates.
The Alamar blue cell viability assay was prepared for both ASPC-1 and UMUC3
which were treated with either rapamycin or chloroquine as a monotherapy. For five days
after the treatment with chloroquine was performed, a small difference in UMUC3 cell
viability was observed. Increased cell viability was shown by ASPC-1. On day 10, there was
a little change in the UMUC3 cell viability pattern. However, a drastic change was observed
by an increase in proliferation in ASPC-1 with chloroquine. An abrupt decrease in cell
viability was observed when cells were treated with rapamycin for both UMUC3 and ASPC-
1.

24Running head: MEDICAL
Discussion
A heterogeneous disease, which is caused by the accumulation of undifferentiated
immature cells present in the peripheral blood and bone marrow, is termed as AML.
However, significant treatment procedures are still absent from this disease since progress
has only be made in the diagnosis of genetic and molecular backgrounds responsible for the
cause of the disease. Thus, the need for a specific and efficient treatment procedure has
become primarily important for the present human community. The treatment procedure will
be devised based on the genetic and molecular backgrounds responsible for the cause of the
disease. This function will help in the innovation of an efficient therapeutic target for disease
treatment. The biochemical pathways responsible for the occurrence of the disease (AML)
can be identified. A mutation spotted in the STAF2 gene has been observed as a part of the
cohesion complex which is functional in the double-stranded DNA repair. This function is
also responsible for the regulation of transcription and an autophagy mutation in genes as
ATG7 and ATG5 (Gomez‐Puerto et al. 2016).
An evolutionarily conserved process aiding the removal of damaged proteins and organelles
is defined as Autophagy. This process is of significant importance in the homologous DNA
repair, which functions as a tumor suppressor acting to reduce oxidative stress, genome
instability and inflammation (Zhong, Sanchez-Lopez and Karin 2016). These processes are
very much essential for the survival of cells. Other mechanisms of survival controlled by
autophagy include starvation, stress, hypoxia, growth factor deprivation, radiation, and
chemotherapy in order to recover the cytoplasmic sub-components to provide the cell with
the essential nutrients required for the survival of cancer cells.
The malfunctioning of autophagy is stated to be associated with the different types of
human diseases which leads to the initiation and an aggressive progression of the disease.
Autophagy has also been found to be associated with the cellular resistance occurring due to

25Running head: MEDICAL
chemotherapy (Levy et al. 2017). These studies suggest that autophagy inhibition also
triggers apoptosis occurring in the leukemic cells which makes autophagy an interesting
target for the newly developed therapy. However, there are various clinical trials that have
been performed to treat AML, which is associated with Autophagy.
This paper worker by developing a treatment strategy involving a synthetic lethality
occurring in STAG2 mutated cells that have a defective DNA repair mechanism process.
Autophagy modulation occurs by using rapamycin and chloroquine, which acts as a single
treatment process (monotherapy) and in a combination with cisplatin on the cell bladder
(Duffy et al. 2015). There are two main modulators of autophagy, rapamycin that induces
autophagy and chloroquine which inhibits autophagy. A naturally occurring mTOR inhibitor
is Rapamycin, which deregulates autophagy in various cancers and has been considered as an
anticancer therapy.
There have been several conflicting reports which talk about rapamycin application in
the therapy of cancer. Anti-proliferative effect on ASPC-1 and Panc-1 cells which are treated
with rapamycin monotherapy or with the other drugs combined. Anti-angiogenesis therapy
has been observed to strongly inhibit metastatic and primary tumor growth (Mirzaei et al.
2017). This condition is also supported by the use of the UMUC3 cell line which was found
to induce an arrest of G0-G1 phase cells. Another author reported that there was no effect of
rapamycin on cellular viability.
The cellular death, which was observed in cellular treatment with rapamycin between
day 5 and day 10, which results from cellular counting. There are several studies, which
support rapamycin and suggest the induction of autophagy, which uses rapamycin in turn,
suggests it to be a good treatment strategy. The results, which are obtained from Alamar blue
assay, have been found to show very little change in the cellular viability in UMUC3. There

26Running head: MEDICAL
is a reduction in the ASPC-1 cellular line occurring between the 5th day and 10th day, which
acts in a reduction in the control which does not have drugs.
The death of cells has also been observed because of the method required to separate
the cells from the plate. These separated cells are counted and then Alamar blue assay was
performed after the detachment of cells from the plate. While the experiment was performed,
the scrapper was used in the detachment of cells from the plate and this method also damages
the cells by increasing its permeability to trypan blue. Cellular proliferation has been
observed to occur by the inability of rapamycin for the inhibition of mTOR2 and permanently
stopping the feedback loop which is associated with S6K1 to PI3K-AKT pathways that
produce a protein kinase B AKT activation (Lindblad et al. 2016). This function regulates
cellular proliferation that results in the growth, differentiation, and survival of cells. Thus, the
need for an inhibitor becomes prominent which inhibits the activities of mTOR1 and
mTOR2.
On the other hand, chloroquine is a drug that acts against the malaria parasite having
the lysosomotropic ability (Hounjet et al. 2019). This ability has been observed to give a new
function as an autophagy inhibitor at a later stage which inhibits the lysosome protease thus
preventing the fusion of lysosome and phagosome. This condition inhibits autophagy. The
antitumor effect of chloroquine present in cells occurs by targeting autophagy and cellular
proliferation (Chen et al. 2017). The effect of lethality with STAG2 mutated cells have not
been reported in many studies. The highest concentration used for this project was 10,000
micromole. The lowest concentration used for this experiment was 0.1 μM. Thus no
significant death of cells was found for the cells which were treated with chloroquine. The
lines were almost horizontal which shows that there is a low significant death of cells after
the addition of chloroquine.

27Running head: MEDICAL
The results obtained for Alamar blue assay when the cells were treated with
chloroquine, a drastic decrease in cellular viability was observed between the 5th and 10th day
for ASPC-1 (Fig 7 and Fig 9). However, there was no difference in the viability of cells
occurring in UMUC3 between the 5th and 10th days. However, these results were different
from those obtained in the cell counting assay and thus suggested the presence of error in the
counting of cells. This condition can arise due to various disadvantages of cell counting due
to the use of trypan blue in Alamar blue assay. The trypan blue has been observed to exert a
toxic effect on the cells due to the sudden exposure and giving rise to false-negative results
(Pan et al. 2017). These results occur from the irreversible changes made in the cell
membrane. This condition occurred due to the sudden initiation of apoptotic pathways having
intact membranes. This experiment is completely based on cell counting by human and thus
human errors, loss of cells during the dispersion, poor dispersion of cells, the presence of air
bubbles and improper filling of chambers can give rise to false-negative results.
As the general precautions, care must be taken during the placing of cells inside the
chambers while ensuring the absence of air bubbles. The use of pipette must be gentle in
order to prevent cellular death. This condition can also arise due to Alamar blue assay which
is easy to use and nontoxic along with cost-effective in nature. Resazurin is also responsible
for the cause of oxidative stress that leads to cell death and autophagy, which allows the
production of ROS (Reactive Oxygen Species) (Chen et al. 2017).
An accurate viable cell count along with the cellular growth determination has been
an important part of the project, which requires the monitoring effect of the drugs on the
cells. Thus, it is an important decision to choose an appropriate method for the experiment.
One of the best future methods for the conduction of this experiment is the ((3-(4,5-
dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide) assay (MTT) which is a colorimetric
assay used for the access to cell viability and cellular toxicity (Darwish et al. 2016). This

28Running head: MEDICAL
procedure includes the use of succinate which is used to determine the cellular viability.
While MTT was added to the cells, NADH will work by reducing it to a purple formazan that
can be quantified by the use of light absorbance. This method has been observed to be more
effective in the production of reproducibility, easy and cost-effective the however the false
negativity and false positivity have been observed to occur because of the background
interference that leads to the overestimation of the cellular viability.
The second method which can be used in the future is the ATP (Adenosine
triphosphate) assay or the Lactate dehydrogenase (LDH) leakage assay (Salvestrini et al.
2017). While cell death is occurring, the concentration of ATP reduces and LDH has been
observed to be released inside the media. This condition can be visualized by the
quantification tests performed by fluorometric, colorimetric and luminometric assays. An
essay is also present which is sensitive and reproducible during the determination of cell
viability as compared to the conventional method. This assay is named as CellTiter-Glo
Assay (Drenberg et al. 2016). This method has also been able to monitor cellular proliferation
which distinguishes the cells which are undergoing apoptosis and necrosis which becomes
different when the cells are tagged with fluorescent dyes. However, this experiment becomes
very expensive.
This experiment successfully showed that the number of cells was higher for ASPC-1 as
compared to UMUC3 which was expected as the ASPC-1 cells do not have STAG2 mutation
(Pelt et al. 2018). The use of rapamycin or chloroquine monotherapy in the modulation of
autophagy occurring in cancer therapy has been conflicted by many scientists. Apoptosis has
been shown to enhance the effectiveness of the therapeutic procedure. The combination of
rapamycin and cytotoxic chemotherapy has been observed to enhance the apoptotic
procedure in vitro to enhance the anti-tumor efficacy in vivo.

29Running head: MEDICAL
Cytarabine is one of the treatment processes used for AML which induces
cytoprotective autophagy in AML cells (mTOR dependent). This treatment procedure is very
much effective for targeted treatment of leukemic cells which has been reported to increase
the cellular sensitivity which is treated with rapamycin and cisplatin (Sujobert et al. 2015).
The alkylating agent (cisplatin) is used in the therapy for cancer. Combined therapy using
chloroquine and the existing anti-cancer treatment has been studied in various preclinical
research (both in vitro and in vivo). In this experiment, chloroquine and rapamycin have been
used in a combination with cisplatin to arrest the progression of cancer cells (Nalbandian et
al. 2015).
Conclusion
The use of autophagy has been used as a therapeutic approach for various cancer and
has become a major interest. More deaths of cells have been found in the study in the cell
population of controls as compared to the cells which are STAG2 mutated. The results from
this study also showed that there is higher in cellular growth when chloroquine is used than
rapamycin. Thus, the second hypothesis is not supported by this study. A higher number of
cellular viability has been observed in the case of cells treated with chloroquine than
rapamycin. Thus, a higher concentration of rapamycin could have been used to get a result
that could have supported the hypothesis. However, this experiment confirmed the fact that
modulation in autophagy increases the cellular sensitivity towards the anti-cancer drug
cisplatin. This compound has only been used once in this study during the combination of
chloroquine and cisplatin. A more effective treatment is the hope of provided the anti-cancer
therapy is modulated by adjusting the concentrations of chloroquine and rapamycin to their
accurate concentration levels.

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