2. Université de Strasbourg
Faculté de pharmacie
Evaluation of cell viability and proapoptotic events in
human pancreatic cancer cell line ASPC-1, using fungi
extracts.
Strasbourg 2014
3. CONTENTS
1. Introduction__________________________________________1
1.1 What is cancer_____________________________________1
1.2 Drug Discovery____________________________________2
1.3 Cell Cycle_________________________________________4
1.3.1 Cell Cycle Information__________________________4
1.3.2 Cell Cycle Assay_______________________________5
1.3.3 Cell Viability Assay_____________________________6
1.4 Apoptosis and Cancer________________________________7
1.5 Cell Culture________________________________________8
1.6 Cytometry_________________________________________10
1.6.1 Flow Cytometry_______________________________10
1.6.2 Capillary Flow Cytometry_______________________11
1.7 Pancreatic Cancer___________________________________12
1.7.1 What is Pancreas?_____________________________12
1.7.2 Biological reasons that cause pancreatic cancer______13
1.7.3 Pancreatic Cancer Symptoms_____________________15
1.7.4 What causes Pancreatic Cancer?___________________15
1.7.5 Pancreatic Cancer Diagnosis______________________16
1.7.6 Therapy for Pancreatic Cancer____________________17
1.8 ASPC-1 Pancreatic Cancer cell line_____________________18
2. Main Part: Methods, Materials, Protocols, Results, and Comparison
with similar studies_____________________________________19
2.1 Subculture Protocol_________________________________20
2.2 Annexin V/ PI Assay________________________________20
2.3 Cell Cycle Protocol_________________________________25
2.4 APH Assay Protocol_________________________________34
2.5 Comparison of my results with other ones that have been
pubished for ASPC-1 cell line_________________________38
3. Conclusion____________________________________________39
Acknowledgments______________________________________40
References____________________________________________41
4. 1
1. Introduction
1.1 What is cancer?
Cancer can be described as an aggregation of numerous diseases. The main
cause of cancer is the unreasonable proliferation of growing cells. Cancer cells
can be found to many and different parts of the body through blood circulation
and lymph systems [1]
.
Cancer causes damage to the body because of the irrational cell division. More
specifically, cancer cells are able to create lumps and compact populace, also
known as tumors. Tumors, as time goes by, are able to develop and impede
with the digestive, nervous and circulatory systems. As a consequence, some
“inappropriate” hormones relinquish that can change the physiological
processes and mechanisms of the human body [1]
.
There are two types of tumors. The first type, which can be thought as a benign
one, shows limited development and stays in a certain place without moving
into other tissues. The second one can be considered as a malignant one and
can be seen in two occasions. The one occasion can be observed if cancer cells
move around the body via blood or lymph systems, and as a result damage
healthy tissues, in a procedure also known as invasion. The other occasion can
be seen if cells achieve to split, get bigger and create new vessels to get
themselves fed, and this can also be called as angiogenesis [1]
.
The procedure of the cancer cell growth, the spreading and the damage of the
healthy tissues can be named metastasis, which is very hard to be treated. The
main cause of that spread has to do with the weak adhesive abilities of the
cancer cells that make them move and create new tumors [1]
.
The main reasons that occur cancer are the non programmed cell death
(apoptosis-which will be described extensively later), genes-the DNA type,
carcinogens, genes- the family type and mutations [1]
.
Cancer symptoms vary a lot. They depend on the kind of cancer and the place
that cancer cells lie inside our body. The most common types of cancer can be
understood by the two of our senses which are the sight and the touch and they
have to do with our skin. Moreover, some more serious occasions can be
diagnosed if we realize that some important cognitive and physiological
functions are somehow affected. In addition, some other symptoms such as
5. 2
fever, fatigue, excessive sweating and unexplained weight loss can be observed
in some types of cancer [1]
.
Cancer is classified in 5 types:
i) Carcinoma: Cells that lie both inside and outside of the body such as lungs,
breast, colon cancer
ii) Sarcomas: Cells that are situated in bone, cartilage, fat, connective tissue,
muscle.
iii) Lymphomas: Cancers that start in the lymph nodes and immune system
tissues.
iv) Leukemia: Cancer that begins in the bone marrow and often conglomerate
in the bloodstream.
v) Adenoma: Cancer that grow in the thyroid and glandular tissues [1]
.
Nowadays, there are several ways to diagnose cancer. The most effective way
to diagnose all types of cancer is biopsy, which can be done by the extraction
of cancer cells by our body and further analysis in the microscope. Likewise,
physicians can examine the elements of our body such as sugars, proteins, fats,
and DNA at a molecular basis in order to have more accurate results. Finally,
some innovative imaging techniques such as X-rays, CT scans, MRI scans,
PET scans and ultrasound scans seem to have great utilities, and that is why we
are able to know where a tumor is situated and which other organs will
probably be in danger in the future [1]
.
1.2 Drug Discovery
Nowadays, specialists from all the medical fields are struggling to discover
innovative cancer drugs. The methods and the techniques they follow are
certain and have to do with clinical studies. There is no doubt that cancer is a
serious disease and because of that, scientists put serious efforts to discover
effective compounds from all around the globe. The challenge for them is to
invent brand new extracts, and use those extracts to make cancer drugs with the
6. 3
least possible number of side effects. This procedure is time consuming but
profitable too. The steps that scientists follow, before a drug will be on
pharmacies’ disposal, are defining, and will be described below [48]
.
Pharmacognosy seems to be the key to success for new inventors.
Pharmacognosists normally, have collected from nature a plethora of plants that
can be tested if they are able to have any effect in the types of cancer. Apart
from that, bacteria and eukaryotic organisms such as fungi are considered
really promising in cancer fields. Moreover, marine plants and organisms
appear to gain the scientists ‘interest as years go by[48]
.
In order to avoid mistakes and achieve accuracy, scientists are obliged to make
experiments that compare and contrast the behaviors of both tumor and healthy
cells. By following that route, it would be easier for them to conceive which
unpleasant function that causes cancer should be ceased with the aid of the
appropriate drug [48]
.
Researchers are required to study bibliography for certain compounds, so that
their future work will be target. Those details have to do with general
knowledge about each compound’s family, chemical structure and properties.
Furthermore, they can utilize specific PC softwares to represent the interplay of
a possible compound and its aim. This procedure enables us to make special
drugs with specific characteristics that cure cancer [48]
.
When all those theoretical studies come to an end, those compounds ought to
be practically examined. To begin with, scientists at first are obliged to make
experiments with provided cell lines. More specifically, they have to make
many kinds of assays in order to discover how effective the compounds they
examine are. The most wanted result would be to have the tumor cells killed
with the simultaneous’ least amount of compound spent [48]
.
If the results are the desired ones, then the researchers proceed to the next
phase of experiments, which are the in vivo tests. Tumor cells are transferred to
experimental animals like mice, in order to have a clear view of a simulation of
how extracts interact with an animal that suffers from cancer. The most
effective compounds that have the smallest amount of side effects, can be
further examined. Those effective drugs can be tested in humans.
Pharmacologists and specialists in Pharmacokinetics and in Pharmaceutical
Technology have to collaborate, in order those drugs to take their final shape
and form. The parameters that are studied are the dosage of the active
substance and the adjuvants that will be used in order to achieve better
bioavailability [48]
.
7. 4
1.3 Cell Cycle
1.3.1 Cell Cycle Information
Our body consists of millions of cells that are getting divided, and consequently
create a new copy of themselves. This procedure is widely known as cell cycle
[2]
.
Normally, there are four phases that constitute the cell cycle: 1) G1, 2) S, 3) G2
and 4) M. More specifically:
1) G1 Phase (Gap 1): During this phase, the cells are subjected to
complicated biochemical reactions and as a result, cells are getting ready
to enter the next phase that has to do with the composition of the
daughter DNA [2]
.
2) S Phase (DNA Synthesis): During this phase, DNA replication takes
place. This procedure is absolutely accurate and leads directly to the
creation of an identical copy of itself, which is called daughter DNA [2]
.
3) G2 Phase (Gap 2): During this phase, after the synthesis of numerous
proteins, cells are getting prepared to enter the next phase which is
called mitosis [2]
.
4) M Phase (Mitosis): The doubled DNA which is getting separated into
two identical parts, gets concentrated in order to create chromosomes, so
that each one of the daughter cells will be able to obtain a complete
duplicate of the DNA of the mother cell [2]
.
Apart from those four phases there is another one that should be
mentioned, which is called G0 (Gap 0). This phase is also known as the
resting phase, where cells exit G1 phase, in order to enter a quiescent
stage. In that stage, despite the fact that cells remain metabolically
active, they are not able to be multiplied, unless some suitable
extracellular signals force them to do so [2]
.
8. 5
Representation of the cell cycle
[3]
1.3.2 Cell Cycle Assay
Flow cytometry gives the opportunity to study and count the amount of DNA
that exists in each cell cycle phase. This fact could not be possible without the
use of certain dyes that have the ability to leash with the DNA in every phase
of the cell cycle. The most common dyes are PI, DAPI and 7-AAD. In these
cases, ethanol is considered as an obligatory element. Dyes cannot easily
invade to cells in general without any previous treatment. Consequently,
ethanol is used so that dyes gain that property. Moreover, the use f ethanol has
another benefit that has to do with our future results. The existence of ethanol
gives us results with low coefficient of variation, and that would not be
possible if we chose another substance. Samples are now able to be put in the
fridge, and be conserved for a certain period of time. Ribonuclease I in specific
concentrations, will be added in the final steps of this procedure, in order to
confirm that there will not be any amount of RNA in our samples. As soon as
we manage to include ethanol to our samples, and the leashing properties are
under control, we will be able to see more colorful cells in S phase than in G1
phase, because in G1 phase we have a bigger amount of DNA. Following this
theory, then it is more than obvious that cells in G2 phase will absorb the
double amount of the dyes, if we compare with G1 phase [54]
.
Normally, the steps that scientists follow before they proceed to the flow
cytometer, will be described below. Firstly, after a subculture of our cell line,
9. 6
we should was the cells with buffer. Ethanol should be maintained in cold
conditions, in order to be more effective. Also, the procedure of filling our
samples has to be very slow while vortexing. Afterwards, the samples should
be remained for half an hour in a certain temperature inside the fridge. Then the
cells should be rewashed with buffer. The next move is the centrifugation of
the samples, and after that we should be cautious, so that we will not eliminate
the cells that lie in the bottom. Then, the samples should be filled with a certain
quantity of ribonuclease I. Finally a specific amount of our fluorescent dye
should be pipetted to our samples. At this point of time, are ready to be
examined by our flowcytometer. The parameters and the diagrams that our PC
software will include are: forward scatter versus side scatter to determine the
cells, and pulse height versus pulse to eliminate cells doublets [54]
.
1.3.3 Cell Viability Assay
A field which seems to gain scientists’ interest is the research of the lifetime of
cancer cells, which interact with promising anti-cancer compounds and drugs.
The way this procedure is examined can be captured by a plethora of assays.
The Acid Phosphate Assay (APH assay) is meant to be one of the most
effective ones and its protocol will be described below [52]
.
APH assay is a technique that gives us the opportunity to analyze cytotoxicity
in spheroid tissue cell cultures, without using experimental animals [52]
. Its
main property is the appraisal of the cytosolic acid phosphatase potency [52]
.
The main biochemical reaction we examine at the microenvironment of living
cells, at this point of time, is the transformation of p-nitrophenyl phosphate to
p-nitrophenol [52]
. This transformation is considered as an hydrolysis, which is
connoted by the existence of intracellular acid phosphates [52]
. This procedure
requires a 96-well plate, which is the place where cells will be transferred from
the flask, with a certain amount of medium and a substance with anti-cancer
properties in different concentrations [53]
.After filling the 96-well plate, we have
to put it ti the incubator for 24 hours [53]
. When 24 hours pass away, we should
pipette a certain amount of a specific concentration of p-nitrophenyl phosphate
and Triton-X-mixed in sodium acetate buffer at pH 5,5 to all the wells and
then, the plate has to be returned back to the incubator for 2 hours [53]
.Two
hours later, we have to add NaOH to the wells, and then the plate is ready to be
put to the spectrophotometer and finally be examined [53]
.The software
measurements have to be adjusted into 405 nanometers. The results that will be
shown on the computer screen depict the absorbance [53]
. The percentage of
10. 7
absorbance is expressed by this arithmetic operation: (absorbance treatment-
absorbance media) / (absorbance control – absorbance media) [53]
.
1.4 Apoptosis and Cancer
Apoptosis is the programmed cell death. Every day millions of cells that
seem to be useless, die, in order to avoid some unpleasant diseases like
cancer. This is the main reason why biologists and scientists in general
are keen on studying about apoptosis and apoptotic cells. The reasons
why some cells do not follow the apoptotic path may vary. Nevertheless,
some of them will be explained shortly. [4]
In the case of cancer, the failure of apoptosis is associated with genetic
defects, which are located in the tumor protein, p53, the protein which
activates the mechanism of apoptosis, when the DNA of the cell has
been severely damaged, which cannot be repaired. Other proteins
involved in the mechanism of apoptosis are the Bcl-2 proteins, mainly in
lymphocytes, wherein cell death is blocked by the excessive production
of this protein. The contribution of the Bcl-2 gene in cancer is in contrast
with the action of other cells such as melanocytes, which produce
melanin, which darkens the skin and protects other epidermal cells by
ultraviolet radiation. So if the melanocytes were easily getting damaged,
then all the other cells would be put in danger too. For this reason the
melanocytes produce large amounts of Bcl-2 protein, which prevents
their death. It is also worth mentioning, that some other molecules like
TNFR1 and Fas play a profound role in apoptosis. More specifically,
those two molecules are death receptors that transfer the signal of the
programmed cell death. Many irregularities in the death signaling
pathways that may drive to prevarication of the outer route of
apoptosis have been examined. Those irregularities that have to do with
the structure of those molecules, include the damage of those receptors
from external factors, and consequently apoptosis is inhibited.
Furthermore, there are some proteins named IAPs (Inhibitor of
Apoptosis Proteins). ΙΑPs are a conglomeration of identical proteins that
are able to control apoptosis and transfer the appropriate signals that
have to do with the cell death, and also won a baculovirus IAP repeat
11. 8
(BIR) protein domain. To conclude with, IAPs are inhibitors of caspases
and as a result, they have the ability to cease caspase operation by
tethering their stored BIR domains to the effective sites of caspases, by
impelling relegation of active caspases or by dissociating the caspases
from their substrates. [4]
1.5 Cell Culture
For more than five decades, scientists own a very important tool for the
study of cancer cells. This tool is called cell culture. Cell culture is a
procedure which enables us to treat cancer cells with various ways, in
order to keep them alive and make experiment with them. This process
also facilitates us to study the biological and physiological properties of
each cancer cell line we examine [49]
.
More specifically, culture can only be named the first treatment of the
human cancer cell line. The treatments that will follow are known as
subcultures, and each time the content of the subculture will be
transferred to a brand new substrate. The majority of cell lines have cells
that seem to adhere to those substrates, but there is also a number of cell
lines without adhesive properties, where cells are able to waft[49]
.
A scientist, in order to subculture cells, is obliged to have certain
products on his disposal and certain conditions too. One of the most
important ingredients is media. Media is a liquid that consists of
carbohydrates, salts, aminoacids and vitamins. Its main ability is to
nourish cells and keep them alive. Nowadays, there are plenty of media
in the market, and scientists are able to choose the one that fits better
with their cell lines’ needs. Another important tool is a liquid buffer in
pH 7,4 named PBS, and its main property is to wash the cells. Also,
another liquid that plays an important role is serum. Serum is necessary
because it has the ability to deputize some main cell components like
growth factors and proteins. Its absence would cause harm, because
those components fade away as time goes by. Moreover, serum can be
added directly to the bottle that contains the media. The amount that will
be put does not have to be more than the one fifth of the quantity of the
media. The exact amount and the kind of the serum we will utilize,
depends on the cell line we examine too. If our cell lines consist of
adherent cells, trypsin/EDTA is the only solution to have our cells
detached. The place where we will keep the ingredients of the subculture
12. 9
, also plays a profound role. Cell development can be achieved if we
store the cells in certain containers. Those containers have to own a flat
bottom and have to be made by specific materials, such as plastic or
glass. The most frequent ones are made of polysterene and plycarbonate.
The most common name of those containers is flask. The conditions
where the flasks should be kept, are very certain and severe. More
specifically, the place that we leave the cells to grow is called incubator.
The temperature of the incubator has to be the human’s body one, and
that means that it should be around 36,7o
C. Lower or higher
temperatures can cause harm to the cell lines. Last but not least, the
percentage of the CO2 in the incubator has to be exactly 5%[49]
.
The subculture procedure begins by checking the confluence of cells in
the substrate we have stored them in. If the percentage of confluence,
does not seem to be enough, we are not able to proceed to subculture.
The key to a successful subculture is to avoid contamination. That is
why scientists are obliged to use the laboratory robe, gloves and sterile
equipment in general. The first step is to put out the whole amount of
medium from the flask. After that, we should shortly wash our cells with
PBS. The amount of PBS we will put has to be around the half quantity
of the medium that was previously in the flask. If the cells are adherent,
we are obliged to put a small amount of trypsin/EDTA, in order to cease
this situation. Since the interaction of trypsin and serum causes
malfunction to trypsin’s protein, we have to make sure there is no
vestige of serum in the flask. Of course, trypsin/EDTA is absolutely
unnecessary to non-adherent cell lines. Afterwards, the flask should be
put back to the incubator, so that trypsin will be effective t cells. The
duration may vary according to each cell line, but in general lines, 10
minutes are more than enough. Further microscope analysis has to be
done, so that we will make sure our cells are not attached to the surface
of the flask anymore. A new amount of medium should be put to this
flask, to cause interaction with detached cells. Then, the counting
procedure begins, where firstly, a small volume of cells in medium and
an exact same volume of a certain dye should be mixed very well. This
mixture should be transferred in a slide that will be examined by us in
the microscope. Since the appropriate calculations will be made, a
specific volume of the cells in medium and a specific volume of new
medium should be put together in a new flask, in order to have the
desired concentration of cells in that. Finally, the new flask is ready to
13. 10
be put back to the incubator continuously, before the time for a new
subculture will come [49]
.
1.6 Cytometry
1.6.1 Flow Cytometry
Flow cytometry (Flow Cytometry, FCM) is an automated cell analysis
technique that gives us the possibility to measure individual particles
like cells, nuclei etc. Those particles pass through a laminar flow from a
standard checkpoint, in which a laser beam incidents. In flow cytometry,
the molecules we examine ought to be in suspension (blood , bone
marrow or any other suspension from cells that has been taken from a
tissue). Those molecules are treated in each occasion differently, with
specific monoclonal antibodies, which are wedded with fluorescent dyes
or fluorescent substances, each time depending on the chemical
parameter that is need to be found. The measurement of fluorescenece
takes place after the appropriate preusage of each specimen that will be
examined afterwards, with special fluorescent substances, which are
chosen depending 1)on the origin of the cell aggregation, 2)on the
disease we are looking into and 3)on the quantity of the surface or of the
intracellular antigen we examine. Nowadays, researchers seem to have
used around twenty one fluorescent substances. [5]
A flow cytometer is composed of three main
subsystems: 1)the hydrodynamic flow system, 2)the visual system and
3) the online data analysis system. [5]
14. 11
Representation of a typical flow cytometer
[5]
Flow cytometers are specialized to study apopoptosis in cancer cells.
There are two specific substances that help scientists examine apoptotic
properties. Those are Annexin V and Propidium Iodide (PI) [50]
.
Phosphatidylserine (PS) is a protein, where in healthy cells lies in the
cytoplasmic surface of the cell membrane. In cancer cells, this protein
lies outside this membrane and it can be found to the outer cellular
location [1]
. Annexin V combined with green fluorescent dye is capable
to find out the amount of cells in apoptosis. PI combined with red
fluorescence is able to find out the amount of cells in necrosis [51]
.
After a proper manipulation of cells in a plate full of wells, including of
course the addition of Annexin V and PI, the software of flow cytometer
indicates the number of apoptotic cells in green color, the number of
dead cells in red and green color, and living cells without any specific
color [51]
.
1.6.2 Capillary Flow Cytometry
Capillary flow cytometry can be also referred as a cytometric analysis
technique. The main difference from the usual techniques is that the
molecules that we analyze are being automatically absorbed by the
specimen and lead to the analysis area without the use of sheath fluid
15. 12
and hydrodynamic focusing. There are many benefits of using capillary
flow based instruments. First of all, those who use that method do not
have to worry about the amount of waste that is produced, if we
compare it with the traditional ones. Furthermore, capillary flow
cytometry can directly measure the quantity of cells we examine and,
also simplified and uncomplicated fluidics may be used. Three basic
systems contribute to achieve those results. The first one helps the cells
to collimate, the second one enlightens the cells with the aid of the laser
light and third one owns light detectors which transform light signals to
electric signals [7]
.
The procedure begins with the gathering of cells in suspension from the
small vial, and then this quantity is being transferred to the flow cell.
One difference is that with capillary flow cytometry, cells are moving
through the laser beam and disperse the laser light. Some parts of these
machines such as the optical lenses, the beam splitters and filters drive
both forms of light to photomultipliers. Photomultipliers are able to
transform the light into an electric signal, which will be afterwards
examined by our PC [8]
.
Representation of a Capillary Flow Cytometer
[9]
1.7 Pancreatic Cancer
1.7.1 What is pancreas?
16. 13
Pancreas is one of the most important and basic organs that lie inside our
body. Its length is around 15 cm and is located between the backbone
and the stomach surrounded by the duodenum. Pancreas consists of
three parts. The most expanded part is the head, the part that lies in the
middle is called the body and the slimmest one is called the tail [24]
.
Pancreas has the ability to create pancreatic juices. Their typical
function is to disrupt the food we consume. Those juices circulate
through conduits, and we can see them ending up in the duodenum,
which is the initial part of the small intestine. Moreover, pancreas
achieves to create a variety of hormones such as insulin. Those
hormones are getting transferred via blood circulation to our body, and
as a result, our body is capable to utilize or supply energy from food [24]
.
Representation of pancreas
[24]
1.7.2 Biological reasons that cause pancreatic cancer
Pancreatic cancer is considered as one of the most aggressive types of
cancer. The most significant biological reasons that justify that can be
described below:
i) A malfunction of the CDKs may cause pancreatic cancer. To be
more specific, CDKs normally phosphorylate Rbs and help E2F
genes proceed to S-phase [10], [11]
. A gene product named
p16INK4A coalesces to CDK4 and CDK6 kinases, impeding the
configuration of drastic complexes such as D-CDK4/CDK6 [12],
[13],[14]
.
ii) Consequently, the phosphorylation of the Rbs stops, and that can
be considered as an obstacle for genes to enter the S-phase [12],
17. 14
[13],[14]
. The grand majority of pancreatic cancers have to do with
poor functionality of the p16ΙΝΚ4Α[12], [13],[14]
.
iii) High amounts of cyclin D and further development of CDK4s
and CDK6s phosphorylate the Rb, giving the opportunity to the
cells to be transcripted. Normally, p16INK4 ceases the
multiplication of the cells, and as a result the Rb is brought into
action [15], [16]
. Meanwhile, a gene sheathing with p16, which is
named p19ARF achieves the identical result, through the startup
of p53 by giving an end to its proteolysis [17]
. Consequently,
scientists have concluded that, the failure of transcription of ARF
and INK4 simultaneously lead to pancreatic cancer [17]
. Mutations
can happen in p16 gene but not necessarily in ARF, and that
connotes that only the absence of INK4 plays a profound role in
pancreatic cancer [17]
.
iv) The gene that is responsible for a plethora of occasions that have
to do with pancreatic cancer is the TP53 [12]
. HDM2 is a human
oncoprotein, that lets p53 stay in poor percentages in our body
[10]
. Under certain circumstances of cellular stress, p53 and
HDM2 lose contact and as a result the amount of p53 is getting
augmented [10]
. This augmentation causes trouble to the normal
procedure of apoptosis and causes cancer in pancreas [10]
.
v) Another important gene that causes pancreatic tumors is Smad4.
A shift in chromosome 18, and more specifically in MADH4
spot, can wrongfully codify Smad4 [18], [19]
. It is considered as a
genetic alteration as long as heterozygosity is lost [18], [19]
. In
addition, in the majority of pancreatic cancers, Smad4 has totally
missed its right properties [18], [19]
. The absence of Smad4 may
cause serious reactions on the tumors that lie in our body, and
enchantment of the onrush [20], [21], [22]
.
vi) Smad4 belongs to the big Smad family,(whose function is to give
the opportunity to our genes to transcript), which is responsible
for one important process. This process refers to conveyance of
the signal of the TGF-β cytokines. The lack of Smad4 is able to
nullify TGF-β’s properties that have to do with the non-
expression of tumor operations. As a result, people who suffer
from pancreatic cancer own high amounts of TGF-β lignands and
TGF-β receptors in their blood, and both lignands and receptors
can be considered as indicators for pancreatic cancer [23]
.
18. 15
1.7.3 Pancreatic Cancer Symptoms
In early ages of pancreatic cancer, people usually are not able to confirm
any symptoms, but as the tumor augments, some those symptoms are
conceivable [24]
:
1) Soreness in the upper place of the abdomen.
2) Unstoppable ache in the back.
3) Wan excrement, obscure urine, and general symptoms of jaundice.
4) Qualm and throwing up.
5) Excrement that waft in the toilet.
6) Lack of energy and fatigue.
7) Lack of the feeling of hunger.
8) Unreasonable weight loss [24]
.
1.7.4 What causes Pancreatic Cancer?
There are numerous factors that may cause pancreatic cancer. To begin
with, chronic tobacco smokers might suffer from pancreatic ductal
adenocarcinoma [25]
. Apart from that, age plays an important role. More
specifically, aged people are more likely to create pancreatic tumors
than the younger ones [25]
. Furthermore, people who suffer from diabetes
have stronger possibilities to be patients from this disease [26]
. Other
maladies that have to do with pancreas like chronic pancreatitis or
hereditary pancreatitis are able to cause pancreatic cancer [27], [28], [29]
. Of
course, patients with hereditary pancreatitis have to deal with cancer
more seldom, if we compare them with those who have chronic
pancreatitis for a period of two or three decades [25]
. The kinds of food
we consume also effect the predisposition of creating pancreatic tumors
[25]
. More specifically, people who are obese and consume big amounts
of red meat , and do not normally consume food with nourishing factors
like folate or methionine are more likely to appear a tumor [30], [31],[32]
.
Moreover, recent studies have confirmed that pancreatic cancer can be
considered as a family illness. Despite the fact that the outbreaks seem
to be really infrequent , the familial pancreatic cancer has to do with
BRCA2 germline mutations, because approximately over the one fifth of
the relatives who suffer from familial pancreatic cancer, appear to have
that kind of mutation[33]
.
19. 16
1.7.5 Pancreatic Cancer Diagnosis
People who may have the kinds of symptoms we mentioned previousy,
should visit a doctor in order to identify if it is pancreatic cancer or not.
There are many tests that have been invented through the years, and the
most common ones are described below [24]
:
1) The first checkpoint of diagnosis has to be a simple physical exam.
With this method, the doctor has o touch patient’s belly in order to
understand if there are any alterations to some organs that are close
to the pancreas like the spleen and the liver. Specialists also have to
investigate if they can feel an unusual accumulation of liquids in the
belly. Apart from that, patient’s eyes and dermis should be examined
to check if they suffer from jaundice which is considered to be a
symptom for those who suffer from pancreatic cancer [24]
.
2) The method of CT scan is also very widespread. To begin with, if we
follow this test, X-rays are able to show us some images from the
pancreas, the spleen, the liver and the gallbladder, so that doctors
make a right diagnosis. Sometimes, patients are obliged to i)
consume a certain amount of water before the test and to ii) have an
injection of certain pharmaceutical products in order to have a more
accurate result [24]
.
3) A very common test is the ultrasound. Specialists put the ultrasound
device in the belly, moving it around the place that is examined. This
device creates sounds that are not able to be heard by people’s ears.
So, those sounds give us the opportunity to appear some images of
patients’ organs and thereafter to conclude if they suffer from
pancreatic cancer [24]
.
4) One of the most arduous tests is the endoscopisis, which is also
known as EUS. Doctors put very carefully to the patient a long,
flexible, but not heavy pipe that starts from the esophagus and ends
up in the small intestine. Ultrasound wave lengths are transferred to
the examined abdominal area, and as soon as this procedure is
finished, and the pipe is put out from our body, echoes create certain
images of the pancreatic area that seem to be really effective. Some
details that can be found with EUS are the size of the tumor, or the
depth of the cancer that lies inside our vessels [24]
.
5) Another efficient technique is the PET scan. Patients undergo an
injection with radioactive sugars. Normally, cancer cells absorb
20. 17
quicker the sugars than the normal cells do. Conclusively, the images
that will be printed, can show us a possible tumor in pancreas if we
can distinguish some luminous spots in those. Moreover, this test can
also examine the possibility of metastasis [24]
.
6) Biopsy can also be a helpful method. Scientists use a needle in order
to take specimen from the area they examine. Further analysis in the
microscope can be done, so that tumor cells will be tested properly.
[24]
1.7.6 Therapy for Pancreatic Cancer
Nowadays, it is widely known that pancreatic cancer is considered the
most difficult kind of cancer to be treated. Despite that fact, there is a
certain number of possible treatments. There are also some factors that
lead us to choose a specific treatment. [24]
Those factors are:
i) The place that pancreatic tumor lies inside our body
ii) The purpose of metastasis of pancreatic cancer
iii) The medical history and the age of the patient
The most effective therapeutical techniques will be described below:
1) Surgery: Patients who undergo this procedure, appear to be in the
premature levels of the development of pancreatic cancer. Doctors, in
the majority of cases have to abstract a certain part of the organ, but in
some purposes they are obliged to take away the whole pancreas and
sometimes some parts of the nearby organs. The manipulation of this
surgery is very hard and patients have to stay at least two weeks in the
hospital, because some complications like haemorrhage and
contamination may happen. Patients have to take the appropriate
medications for a period of time after the operation. To conclude with,
patients should also take the appropriate advice from the doctors for
their diet in order to avoid unpleasant conditions. [24]
2) Chemotherapy: Chemotherapy is the most popular treatment for those
who have pancreatic tumors. It can also be combined with radiation
therapy or targeted therapy. In premature levels of cancer, patients
undergo this procedure after the operation. Furthermore, patients who
suffer from pancreatic cancer take their drugs with IV Bolus injection
form in order to accelerate bioavailability in the blood circulation.
21. 18
Unfortunately, chemotherapy causes some [24]
unpleasant side effects
like hair loss, fatigue, weakness and nausea.
3) Radiation Therapy: Radiation therapy is a way of treatment for those
who have pancreatic tumors. It can also be combined with chemotherapy
in order to be more effective. A specific machine sends radiation to the
area of the belly, so that cancer cells get extinguished. Patients are
obliged to visit the hospital frequently for a short period of time. Despite
the fact that this procedure is anodyne, there are some side effects like
flatulence and gastrointestinal issues in general. [24]
4) Targeted Therapy: Patients who are not able to proceed in to the
surgery take per os medication, in order to avoid metastasis, and the
augmentation of the tumor. Targeted therapy can also be combined with
chemotherapy. Of course some similar unpleasant side effects can easily
appear. [24]
1.8 ASPC-1 Pancreatic Cancer Cell Line
ASPC-1 is the pancreatic cancer cell line I made experiments with.
Those cells is suspension have been taken by a 62 years old Caucasian
woman, and have been conserved for a period of time in liquid nitrogen.
The most important properties of this cell line will be described below:
1) Adhesion: Each cell line has its own adhesive capacity. Adhesion
has to do with the ability of certain extracellular matrix testimonials
to merge with cells. By studying this property, we are able to
understand, how much the tumor will be developed in the future and
if the patient should worry about further metastasis. The most
examined extracellular matrix testimonials are: collagen I, collagen
IV, and fibronectin. ASPC-1 cells show strong adhesive properties in
collagen I, and collagen IV and rather poor adhesive properties in
fibronectin [34]
.
2) Cell Migration/invasion: Cell migration is an unpleasant, but usual
phenomenon where cancer cells enter blood [35], [36]
. The reason we
study this factor is because scientists would like to investigate the
internal cellular processes that preceded metastasis [35], [36]
. Cell
invasion has been also studied a lot in pancreatic cancer, because
pancreatic tumors have been considered as malingnant ones [35], [36]
.
More specifically, the reason why pancreatic cancer cannot be
punctually diagnosed is because pancreatic cancer cells are able to
22. 19
expand very quickly to nearby organs, and this is a fact that enhances
metastasis [35], [36]
. The difference between cell migration test and
invasion tests, is that in cell migration tests we examine the mobility
of cells without the presence of a characteristic extracellular matrix,
which is named Matrigel that consists of Collagen IV, heparin sulfate
laminin and entactin [37]
. Recent researches show that, fortunately
ASPC-1 cell line is one of the least invasive pancreatic cell lines.
[38,39]
3) Angiogenesis: Metastasis and tumor developments can be also
connoted by angiogenesis, which is the procedure where tumor cells
give the opportunity to endothelial cells to multiply, and as a result,
new blood vessels appear [40]
. The consistency of those vessels can
be referred as an indicator of the evolution of the tumor and
metastasis in general [41]
. Recent studies have shown that this
indicator is not very reliable in the case of pancreatic cancer [42], [43],
[44]
. Despite that, those researches that were focused on ASPC-1
cells, have proved that this cell line appears to own very few pro-
angiogenic factors [42], [43], [44]
.
4) Tumorigenicity: Tumorigenicity is a technique that enables us to
examine some tumor factors such as the predisposition of the tumor
size, the tumor bulk, and its further development, with the aid of
experimental animals, like mice [45], [46]
. Mice receive an injection of
pancreatic cancer cells in suspension, and the results of those
parameters are on scientists’ disposal [45], [46]
.Those parameters show
evidence of how human body may react to pancreatic cancer [45], [46]
.
All thr experiments that have been made indicate that ASPC-1 cell
line that all the mice grew pancreatic tumors [45], [46]
.
5) Mutations: ASPC-1 is the pancreatic cell line with the biggest
number of mutations that may happen in its genes. The most
common ASPC-1 cell line mutations can be seen below[47]
:
Tumor source Mutuant gene Zygosity Geen Sequence Protein sequence
2. Main Part: Methods, Materials, Protocols,
Results and Comparison with similar studies.
23. 20
2.1 Subculture Protocol
The liquids that are used in this procedure are: medium (RPMI Medium
1640 (1x) + GlutamaxTM
– I with 5,7 mL of Penicillin/ Streptomycin and 57
mL of FBS), buffer (DPBS (1x) Dulbecco’s Phosphate Buffered Saline/
gibcoTM
by life technologies) and trypsine. All the liquids are warmed first at
36o
C. To begin with, I have to put out of the incubator the previously
subcultured fask. After that I have to take out of the flask alla the liquid from
the inside. Then I put in the flask 7-8 mL of buffer and then I mix. After mixing
I should get rid of the liquid that is inside the flask. Next, I have to add to the
flask 1-1,5 mL of trypsin. After that, the flask must stay to the incubator 5-10
minutes so that the cells are detached. The next step is to add 10 mL of medium
to the flask and mix that quantity. As soon as I do this, I have to put the whole
amount of liquid that is inside the flask to a tubeand mix well. Then, I have to
procced to the counting procedure. First, I have to mix in a small ependorf tube
50 μL of the liquid that exists in the tube, and 50 μL of a blue dye (trypan blue
solution 0,4%, SIGMA CELL CULTURE). We put a small amount of the
mixed liquid in a small surface that is called slide, which is covered by a small
piece of a flat glass. After that we transfer that to the microscope to analyse it,
and count the cells. In the surface of the slide you can see two big crosses, that
have 16 squares which are next to each side of the cross’s area. So, in total we
count the number of cells that exist in those 128 squares. Afterwards, we have
to add all the numbers we found in each square, and then the total should be
divided by 8 first, and then we should multiply that number by 20.000 in order
to identify the number of cells we have in the bottle per mL. Finally, in a brand
new flask I add a specific number of mL of cells and a specific number of mL
of medium in order to have an appropriate concentration of cells in my new
subcultured flask. In my ASPC-1 cell line for example, I use 5 mL of cells and
10 mL of buffer.
2.2 Annexin V/ PI Assay
Guava Technologies Inc. has developed systems for automated cell counting
and flow cytometry. We used Guava EasyCyte plus in order to study the
effectiveness of certain compounds, and in order to study the cell cycle.
In order to study the efficiency of our compounds we have to use this
machine and follow a certain protocol. A 96-well plate is used firstly in order to
24. 21
fill the wells with different concentrations of different compounds. Some wells
are filled with a certain amount of celastrol, some with a certain amount of
Dimethyl sulfoxide, A.C.S. spectophotometric grade (DMSO)/ SIGMA-
ALDRICH, and some others are only filled with buffer. Afterwards, I have to
put the same quantity of my cells in suspension in each well. The total volume
of each well will not be more than 200μL. After this preparation, this 96-well
plate should be maintained to the incubator for 24 hours. After 24 hours I have
to follow certain steps so that my plate can be analysed with Guava machine.
Firstly, I have to bring a brand new 96-well plate. I put out 190 μL of each well
from the old plate with a multiple pipette, and then I put each amount to the
exact same well like previously. Then, I have to put in the old plate 50 μL
buffer, so that the cells will be washed, and then I throw back all that quantity
soon. Afterwards, I have to fill each well with 40 μL of tryspine to the old
plate, and as soon as I do that, I should put m plate to the incubator for 10
minutes so that my cells will be detached. For further analysis I can watch my
plate to the microscope. The amount of liquid that lies inside the new flask (190
μL), should be put back to the old plate. The mixing plays a profound role at
this point of time. The last step is to fill each well with 3μL of Annexin V FITC
(500μΙ/ Immuno Tools), and 3 μL of Propidium Iodide (MACS, Miltenyi
Biotec). Some wells that contain only buffer will be filled with Annexin and PI
in order to create the zero control, and some others like them will not be filled
with Annexin and PI in order to create negative control. Then, the plate is ready
to be analyzed in Guava machine.
Half maximal effective concentration, also known as EC50 indicates the
concentration where the 50% of its highest effect can be observed. According
to our data, EC50 can be translated as an indicator of the concentration that
induces apoptosis in the exact half number of our available cells. The
compounds I used were kindly given by Dr. Paivi Paulina Jarvinen. Dr Jarvinen
has collected and elaborated those extracts herself in Finland. The only
information I have about those extracts is that they are taken from fungi. The
names of those extracts that were given to me were HKI B1, HKI B2, HKI C1,
HKI D2, HKI F2, HKI G1, HKI H2.
After a number of experiments, our results were combined and the diagrams of
EC50 curves can be seen below. The x axis represents the concentration of each
compound in μΜ, and the y axis represents the percentage of dead and
apoptotic cells. Apart from that, we can also observe the percentage of dead
and aoptotic cells that appears in the in the highest concentration of oure
extracts (300μM), and conclude if those compounds have any potential for
further studies in pancreatic cancer cell lines.
25. 22
First of all, the results of the experiments with the compounds that were tested
once will be presented below:
C1
F2
G1
The following curves come from combined experimental results that have been
repeated twice or thrice:
B1
27. 24
Apart from the compounds, some experiments have been made with celastrol,
in order to have a positive control, and as it is obvious, a percentage that
approaches 100% can be observed in our highest concentration (100μM):
CELASTROL
In each experiment, we also examined how much DMSO affects apoptois,
since all our extracts were diluted in DMSO. The concentration we used in
each experiment was 50 μΜ and the average percentage that caused apoptosis
was 1,9%. That means that DMSO had no serious effect in our extracts and did
not influence or caused harm to our experiment.
To sum up, after all those diagrams, it is clear that those compounds I used
induce apoptosis. The percentages of dead and apoptotic cells in our highest
concentration may vary, but the grand majority of them are more than 90%, and
some of them approach 100% (C1, D2, F2, G1, and H2). The results of
celastrol can be considered as normal ones, since celastrol completely induces
apoptosis, and DMSO does not seem to have any serious effect. So, according
28. 25
to this assay, and according to the results I own, all those extracts I tested can
be further examined, so that new anti-cancer properties will be found.
2.3 Cell Cycle Protocol
Cell cycle can be also examined in Guava Machine. The first step is to put
200 μL of our cells in medium in some wells of a 96-well plate and put it to the
incubator for one day. The other day, three wells have to be filled with a certain
amount of a compound, three others with a certain amount of this compound
with the presence of a certain amount of DMSO, and some others have to be
stayed the way they were, so that we create a zero control. As soon as this
treatment will be finished we mark the exact time we finish. Those plates can
be examined after 6 or 24 hours. As soon as 6 or 24 hours pass away, we have
to collect the medium from each 3 wells that have the same consistence, and
put this quantity in small eppendorf tubes. Afterwards, we have to wash the
wells we used with 50 μL of buffer, and then we should throw it away. Then,
we should put 40 μL of trypsin to the same wells, and after that the plate should
be put to the incubator for 10 minutes so that the cells will be detached. Then,
we fill the same wells with 60 μL of buffer and we mix that quantity.
Afterwards, we transfer the content of those wells to the same eppendorf tubes.
Then, the eppendorf tubes should be centrifuged for 5 minutes in 1500 rpm.
The supernatant must be thrown to waste, and then we put 500 μL of PBS and
we mix that quantity. After that, we centrifuge again for 5 minutes in 1500 rpm,
and the supernatant is thrown to waste. Then, we fill the eppendorf tubes with
300 μL of PBS and we mix. After that, the eppendorf tubes should be filled
with 1200 μL of ice cold ethanol. Now, the eppendorf tubes have to be placed
in the freezer over night. To prepare the samples for measurement in Guava, I
have to centrifuge the tubes for 5 minutes in 2500 rpm and remove the
supernatant. Then, the tubes should be filled with 500 μL of PBS and
centrifuged again in 2500 rpm for 5 minutes. Later, I remove the supernatant
and I add 300 μL of a mixture of 150 μL of PBS and of 150 μL of PI/RNase
(PI/RNase Staining Solution/ molecular probes by life technologies). Finally, I
put the samples in a 96-well plate and I place it for 30 minutes in a dark place
where the temperature should be around 25o
C. When 30 minutes pass away, the
plate is ready to be analyzed by Guava.
The normal cell cycle procedure was described in the introduction. Cell cycle
assay helps us discover if the extracts we used force the cells to go through
apoptosis. Cells that undergo the apoptotic process normally appear to have
less quantity of DNA in comparison with G0/G1 phase, because of DNA
29. 26
fragmentation. Extracts’ presence as time goes by (from 6 to 24 hours), will
normally cause an increase of the percentages in Sub G0/G1 phase. In my
experiments we used B1 and D2 extracts, DMSO in the same concentration as
the extracts, and zero controls for 6 and 24 hours. The extracts’ concentration
that used for those experiments is taken from IC-50 curves. More specifically,
the concentrations we used were the same ones that cause 70% of cell death for
each extract. The x axis of the diagram depicts the cellular DNA content and
the y axis the count.
B1 6H
B1 24H
32. 29
ZERO CONTROL 6H
ZERO CONTROL 24H
The following chart depicts all the previous results gathered:
33. 30
After the presentation of the results, we can deduce that D2 is more effective
than B1 in both 6 and 24 hours, but still we cannot claim that those results are
sufficiently representative ones if we compare these results with those from
Annexin V/PI assay. To be more specific, the percentages in Sub G0/G1 phase
should have been higher in order to support that our extracts induce apopotosis
for sure. With the data I own, I can undoubtedly say that there is a slight effect
in the cell cycle, but we cannot certainly name this effect apoptosis. DMSO and
zero control results are reasonable and they do not seem to influence cell cycle
in a remarkable way.
In cell cycle assay we also examined celastrol’ s influence. We counted IC 30
and IC 70 values from celastrol’s combined results and we adjusted these
concentrations for our experiments for both celastrol and DMSO for 6 and 24
hours. Zero control was examined too. The results are the following ones:
IC 30 6H
0
20
40
60
80
100
Percentage(%)
G2/M
S
G0/G1
SubG0/G1
37. 34
The following chart depicts all the previous results gathered:
As we expected, celastrol induces apoptosis in both concentrations and time-
points because the percentage s in sub G0/G1 phase are high enough. DMSO
and zero control have approximately the same percentages and that means that
DMSO does not have any serious influence in this case too.
2.4 APH Assay Protocol
The first 96-well plates we filled were only with cells and PBS in different
concentrations. More specifically, we filled 5 wells with 10 different
concentrations, so 50 wells in total. We took a certain number of cells and that
was around 4.000.000. Then, we centrifuged that quantity for 5 minutes in
1500 rpm, and we threw away the supernatant. Afterwards, we suspended cells
in PBS, so that we have a concentration of 1.000.000 cells/mL. Then we used
three effervescent tablets of PNPP (PNPP Tablets/ Thermo Scientific), and we
dissolved them with 7,5 mL of PBS. Then, we warmed that liquid for 10
minutes, and we kept that liquid away from the light because it is
photosensitizing. Then, each well was filled with 100 μL of that solution. The
10 different quantities we used (μL of cells/well), were: 100, 90, 80, 70, 60, 50,
40, 30, 20, 10. Then we analyzed our results in spectrophotometer every 30
minutes for 2 hours, and after those 2 hours we had to add 10 μL of NaOH in
each well and have a measurement as soon as all the wells are filled. Finally
we made EC-50 curves.
0
20
40
60
80
100
Percentage(%)
G2/M
S
G0/G1
SubG0/G1
38. 35
The most recent 96-well plates we filled had a totally different consistence. The
main difference is the use of celastrol, the DMSO, and the zero control. The
preparations before the analysis in the spectrophotometer have been done in
two days. The first day we filled each well with 180 μL of cells and 20 μL of
our sample. We used 10 different concentrations of celastrol and we filled 3
wells for each one, so 30 in total. The concentrations of celastrol (μM) were the
following ones: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.39063,
0.19532. We also filled 3 wells with 180 μL of cells and 20 μL of DMSO,
which came from a dilution of 16 μL of DMSO in 144 μL of buffer. We also
used 6 wells for zero control which contained 180 μL of cells and 20 μL of
PBS. The concentration of the cells in suspension was 200.000 cells/mL. After
filling all those wells, we put this plate in the incubator for 24 hours. And as
soon as 24 hours pass away, we had to throw away 160 μL of cells of all te
wells, and after that we had to put 160 μL of PBS inside those wells. Then, we
had to throw away again 100 μL out and finally we had to put in each well 100
μL of PNPP assay buffer. Now, the plate should be measured for 2 hours every
30 minutes, and after those 2 hours we had to add 10 μL of NaOH in each well
and have a measurement as soon as all the wells are filled. At last, we analyzed
our result in the computer and we made EC-50 curves.
APH assay’s diagrams depict how much the absorbance (nm) is affected by the
concentration at each point of time (0h, 0,5h, 1h, 1,5h, 2h, 2h + NaOH). The
assays with dilution in PBS contain diagrams with linear curves where the
absorbance augments, while the concentration (cells/well) augments too. On
the other hand, APH assays with celastrol do not show linear curves in their
diagrams, and the absorbance decreases as the concentration of the celastrol
increases. This fact is reasonable because celastrol causes death to the cells,
and as a result absorbance decreases more and more in higher celastrol
concentrations.
In order to confirm that our results are reliable, a statistical parameter was
examined at each point of time which is named Z value. Z value depict the
trustworthiness of the assay signal dynamic range and the data variation
associated with the signal measurement. The equation of Z value is the
following one:
Z = 1-
Sample is considered each result from 0,5h, 1h, 1,5h, 2h, 2h+ NaOH, and
background is considered each result from 0h. Z value can be less than 0 but
39. 36
those results are nt trustworthy. Results from 0 to 0,5 are quite reliable and the
best ones are those from 0,5 up to 1.
The assays with dilutions in PBS were combined, so that we own more credible
and gathering results. The total results of Z values are the following ones:
As we can see, the most reliable values come from the time-point “ 2h+
NaOH” and that means that the more time the 96-well plate stays to the
incubator, the better results we have. NaOH also plays a profound role in this
procedure too. So, the diagrams that have to be presented are the following
ones:
10000cells/well20000cells/well30000cells/well40000cells/well50000cells/well60000cells/well70000cells/well80000cells/well90000cells/well100000cells/well
0,5h -116,364 -0,48375 -105,225 -0,30656 -13,436 -37,6606 -2,97564 -1,85817 0,46132 -0,21435
1h -1,91242 -0,16043 -3,73793 0,234691 -2,15184 -2,12957 -5,5491 -2,07726 0,499137 0,487024
1,5h -3,41435 -0,52836 -2,46241 0,519126 -0,7412 -0,67794 -0,84583 -0,14942 0,57374 0,60752
2h -3,17111 -0,4196 -1,95781 0,348977 -0,53661 -0,30033 -0,34173 -0,05824 0,752588 0,610757
2h+NaOH -0,67216 0,420527 0,364915 0,466929 0,401932 0,483418 0,389507 0,619297 0,704419 0,633222
y = 3E-07x + 0,1278
R² = 0,5123
0
0,05
0,1
0,15
0,2
0,25
0 20000 40000 60000 80000 100000 120000
Absorbance(nm)
Cells/well
0h
y = 9E-06x + 0,0798
R² = 0,9923
0
0,5
1
1,5
0 20000 40000 60000 80000 100000 120000
Absorbance(nm)
Cells/well
2h+ NAOH
40. 37
The results for the plates that were filled with celastrol were also combined and
the Z values were counted this way at each point of time:
Z = 1-
And
Z = 1-
The combined Z values for DMSO are the following ones:
Time Z AVERAGE
0,5H 0,18976737
1H -4,29994819
1,5H 0,21436299
2H 0,303233
2H+NaOH 0,51940775
The combined Z values for zero control are the following ones:
Time Z AVERAGE
0,5H -0,3279757
1H -20,132658
1,5H -0,3494147
2H -0,1014631
2H+NaOH 0,21639876
Again, the most reliable results come from the time-point “2h+ NaOH” for the
same reasons like previously. So, the diagrams that have to be presented are the
following ones:
41. 38
DMSO does not show any serious effect because the values of absorbance in
the wells filled with DMSO seem to be really low in comparison with those
with celastrol, and really similar with the ones of zero control.
Now, if we have to compare the pros and cons of Annexin V/PI assay with
APH assay, first we have to mention that Annexin V/PI assay is a more time-
consuming method of studying apoptosis, comparing to APH assay. On the
other hand, a usual phenomenon which is observed is the deficient dissolution
of NPP tablet. This fact causes harm to our values, and as a result we can say
that APH assay is considered to be a more sensitive method. To conclude with,
Annexin V/PI assay provides more detailed results that have to do with the
number of cells that follow the path of apoptosis, while APH assay gives us a
more general view of cell viability.
2.5 Comparison of my results with other ones that have
been published for ASPC-1 cell line
0
0,2
0,4
0,6
0,8
0,1 1 10 100
Absorbance(nm)
Concentration (μΜ)
0h
0
0,2
0,4
0,6
0,8
0,1 1 10 100
Absorbance(nm)
Concentration (μΜ)
2h + NaOH
42. 39
Numerous articles have been published from scientists that have made
experiments with ASPC-1 cell line. Dr Jarvinen’s compounds were taken from
fungi. Apart from fungi, there are plenty of natural extracts that have been
examined. Of course the methods, the protocols, and the assays they followed
are not identical with mine, but still we have the ability to compare and contrast
both sides’ results.
A natural product named sansalvamide A which is taken from marine fungi
appears to have useful results. A 10 μM analogue was used for this occasion,
and cell viability assay showed that this compound was not cytotoxic and
provokes G0/G1 arrest in cell cycle assay [55]
. Sanguinarine is an alkaloid with
anti-cancer properties. The experiments that have been made with ASPC-1 cell
line showed that sanguinarine extracts urge apoptosis, suspend cell viability
and cell growth, and provoke G0/G1 arrest in cell cycle. The main difference is
that the concentrations they used for those experiments were from 0,1 μΜ up to
10 μΜ [56]
. Another interesting article refers to a flavonoid whose name is
apigenin and shows important anti-cancer properties. Annexin V/ PI assay
proved that in a concentration of 50 μΜ approximately 25% of cells go through
apoptosis. This percentage is quite similar to some of Dr. Jarvinen’s extracts in
the same concentrations and that demonstrates their effectiveness [57]
.
3. Conclusion
Dr. Jarvinen’s fungi extracts were tested in two assays (Annexin V/PI assay
and cell cycle assay) with pancreatic cancer cell line ASPC-1. Annexin V/PI
assay proved that B1, B2, C1, D2, F2, G1 and H2 extracts cause apoptosis in
high percentages. Cell cycle assay demonstrated that B1 and D2 affect cell
cycle procedure too. In APH assay no extracts were used, but despite that fact,
the method has been validated and proved that celastrol caused apoptosis in
high levels.
To conclude with, all those results made clear that Dr. Jarvinen’s fungi extracts
should be further analyzed and examined because according to my
experimental data, they appear to have serious anti-cancer properties.
43. 40
Acknowledgements
The experiments have been made in the University of Strasbourg and more
specifically in the laboratory of Dr. Christian D. Muller in the faculty of
pharmacy from February until July 2014. I am so thankful to Dr. Muller who
let me worked with his professional team and gave me the opportunity to
discover the world of cytometry. I have to thank Dr. Paivi Jarvinen, and the
department of pharmacy in the University of Helsinki who gave me the chance
to make experiments with their own extracts. Apart from that, I have to thank
her for teaching me cytometry from the very beginning, for the moral support
and for the guidelines she gave me. I feel also obliged to say thank you to Dr.
Serge Dumont for his presence in the laboratory and for his willingness to help
me whenever I had a need. Of course, I ought to thank Maria Karvouni for the
great collaboration and the nice memories we shared in the laboratory. Finally,
I have to say special thanks to: Daniel Karas, Dominik Nabergoj and Sanja
Vrbek.
44. 41
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