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By Abul Qasim Asadi
By Abul Qasim Asadi
Abstract: The level of multi drug resistance caused by the expression of the MDR-1 gene and
consequently P-glycoprotein efflux pumps in Hepg2 cancer cells can be determined.
At the mRNA level, the MDR-1 gene codes for an efflux pump called P-glycoprotein
which causes the chemotherapeutic resistance in the Hepg2 cells sine this channel
protein pumps out foreign chemicals. When Quinidine and Doxorubicin are added to
the Hepg2 cells, the expression of MDR-1 gene and P-glycoprotein changes. The
concentrations of the drugs to be used on Hepg2 cells were optimised by an MTT
assay. The protein and RNA of the treated cells was extracted. These were used to
carry out gel electrophoreses and Western Blots to determine the change in MDR-1
expression and P-glycoprotein respectively. The change in MDR-1 expression was
more observable whereas for the P-glycoprotein, the change was more subtle. The
change in MDR-1 expression was further quantified by using a real time quantitative
PCR. There was a link established with relative evidence showing that MDR-1 gene
expression plays a key role in Hepg2 cells with the interaction of chemotherapeutic
drugs.
MDR-1 in HepG2 cells and its role in the interaction of
chemotherapeutic drugs.
Introduction:
MDR-1(Multi-drug resistance) is a gene which codes for a protein known as P-
glycoprotein. This is found in all animal cells and protects the brain cells from toxins
as well as its involvement in excretory tissues (kidney, liver, large intestine). This
channel protein acts as an efflux for foreign chemicals/substances (Dr. Christopher
Mee: 2015). In some cancer cells such as Hepg2 (cell line originating from a 15 year
old male- (Hepg2.com, (2015) Hepg2 (Liver Hepatocellular Carcinoma)) the MDR-1 gene is
upregulated leading to an over expression in the P-glycoprotein. Therefore
chemotherapy is less effective on these cancer cells due to their mutated MDR-1 gene.
Figure 1 P-glycoprotein- is a channel protein found on cell membranes and is an ABC-transporter.
(Google.co.uk, (2015) P-Glycoprotein)
Doxorubicin is a pre-existing anti-cancer drug which is used for many types of cancer
namely: liver, lung, lymphomas, mesothelioma, multiple myeloma and many more. It
can be used with a combination of another drug to enhance its chemotherapeutic
effect on the cancer cells. This is called combination therapy.
Quinidine which was originally used and which is still a widely used anti-malarial
drug, can be used with Doxorubicin to enhance
Chemotherapeutic effects on cancer cells such as Hepg2 cells. This is because
Quinidine acts as an inhibitor to the efflux pump. Since it is a substrate for the P-
glycoprotein which blocks it hence reducing the chemotherapeutic resistance of the
cancer cells.
P-glycoprotein- is a channel protein
found on cell membranes and is an
ABC-transporter.
These ATP-binding cassette (ABC)
transporters are involved in MDR. P-
Glycoprotein/ABCB1 is a member
of the ABC transporter family, and
facilitates the efflux of various
anticancer drugs
Previous studies for e.g. The modulation of ABC-transporter mediated multidrug resistance in
cancer ( Department of Pharmaceutical Sciences, College of Pharmacy and Health
sciences, St. John’s University, (2014) ) have shown there to be a link between the use
of these drugs and their effect on cancer cells. In this study, this link is being verified
and tested by using different concentrations of the drugs.
Aims:
Main Aim: To see how MDR-1 gene affects the chemotherapeutic
resistance of Hepg2 cells.
1. To find suitable concentrations of Doxorubicin and Quinidine to be
used on Hepg2 cells.
2. Identify the change in MDR-1 gene expression and protein expression
after treatment of Hepg2 cells with Doxorubicin and Quinidine in
isolation.
3. To observe the change in MDR-1 gene expression and protein
expression with combination therapy of Doxorubicin and
Quinidine.
4. To compare these treatments between naïve Hepg2 cells and MDR-
1 over expressing Hepg2 cells.
There were alternative aims such as experimenting on kidney and
intestinal cancer cells. However these cancer cell lines were harder to
obtain for our research project and were more sensitive to drug treatment
for the drugs that were being used. Hepg2 cancer cells were readily
available and had a good expression of MDR-1 that could easily be
identified by PCR and Gel electrophoresis. This is why it was chosen to
focus on Hepg2 cancer cells and its engineered family member which
was MDR-1 over expressing Hepg2 cell line.
Project Strategy:
Spend first 2-3 days familiarising with lab equipment and
procedures such as composition of reagents.
Sub-culture Hepg2 flasks
Determine suitable drug concentrations to use on Hepg2 flasks by
MTT assay.
Extract RNA & Protein from treated Hepg2 flasks.
Image RNA & Protein by Gel Electrophoresis and Western Blot.
Determine MDR-1 gene expression in both Hepg2 cells & MDR-1
cells – treated with drugs by using PCR
Compare results
What was required to complete the project:
Assistance of supervisor and ambassadors
Pipettes and pipette tips
T25 & T75 flasks
Media, PBS, T.A.E, T.B.E & various reagents mentioned in
methodology.
Machines such as; orbital shaker, vortex, centrifuge etc.
IMS (Industrial Methylated spirit) for sterilising equipment and
experiment material.
Hepg2 cells and MDR-1 over expressing (engineered) cells.
Laminar – flow cupboards
Safety & Precautions:
Whilst in the lab some strict safety precautions needed to take place
otherwise immense damage to project work could happen and possible
risk of harm to people working in the lab. Every time the laminar – flow
cupboards were used they had to be sterilised down with 70% IMS along
with any equipment or reagents that would go inside it.
If disposable equipment was being used (single use) I would make sure
that it was discarded after the single use each time despite the fact that it
could be reused. I did this to prevent any chance of contamination or
mixture of reagents which would invalidate results.
Methodology:
Day 1:
Buffers were made for the different processes to be carried out over the course
of the placement.
MTT (3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium Bromide) assay
was prepared.
Protocols for the following buffers was given: PBS (Phosphate buffered
saline), T.B.E, T.A.E. Equipment required to compose these were: 4x500ml
glass bottles, weighing scales (+/- 0.0005g) and weighing boat-of varying
capacities, spatula, auto clave tape, measuring cylinder, stirrer & magnetic flea
and pH adjuster.
PBS recipe:
4g of NaCl
0.1g of KCl
1.445g of Na2HPO4.12H20
0.1g of KH2PO4
500ml of R/O water.
T.B.E recipe:
10.8g of TrisBase
5.5g of Boric Acid
0.95g of EDTA-Na2.2H20
Adjust pH to pH 8 using the pH adjuster*
Add 500ml of distilled water(i.e. R/O water)
*pH Adjuster: This is a device which with the aid of known pH solutions can
assist in the adjustment of a desired solution (i.e. T.B.E for this instance). The
pH meter must be calibrated first with pH 7.0 by swirling the pH probe in
distilled water. Then the probe was swirled in a pH 8.0 solution so that pH
meter knows what pH 8.0 is. Then probe re-swirled in pH7.0-distilled water
(prevents contamination). After that, an alkaline solution- NaOH(Sodium
Hydroxide) was slowly added -drop-wise to T.B.E solution with probe
swirling, until the meter read 8.0( +/- 0.1 pH). This meant the T.B.E was
successfully calibrated.
Sub Culturing:
The remaining cells in the T75 flasks were to be sub-cultured. This was done to
maintain a stock of Hepg2 cells from the same original sample for future treatments in
the project.
1. Remove old media from flasks
2. Wash down with 5ml of PBS then discard PBS
3. Add trypsin enzyme 3ml – place in incubator for 3 min
4. Check for movement of cells under microscope in suspension – otherwise
manually dislodge
5. Add 9ml of media to original flask so now there is a total of 12ml
6. Pipette out 6ml, from each of the two T75 flasks being used, and pipette into 2
new T25 flasks.
7. Take 12ml of fresh Media into new pippeted and add 6ml to each new T75
flask so that they both have a total of 12ml of solution within them.
MTT preparation:
HepG2 cells cultured in T75 flasks were acquired at the start of the study. Some of the
cells from these flasks were to be used to be treated with drugs on a 96/well plate.
96/well plate preparation:
Flask preparation:
Firstly, 75 flasks were gathered from the 37 degrees C/ 5% CO2 incubator. The
laminar flow cabinet and all materials were completely sterilised in 70% IMS before
use.
Then the flasks were washed down with 8ml of PBS to remove any dead cells and any
leftover media that would inhibit trypsinisation. The PBS was discarded after wash.
To complete this destruction 2ml of Trypsin (an enzyme which breaks down proteins)
was added to the flask and then it was placed in the 37°C-optimal temperature-
incubator for 3 minutes to optimize the action of the Trypsin and re-suspend them in
solution. After 3 min 8ml of fresh media was added to the flask to stabilize the cells
since the media contains essential substances for the survival of the cells such as
glucose, FBS (Foetal Bovine solution).Also the media inhibits further trypsinisation.
Cell counting:
To ensure full re-suspension of the Hepg2 cells, the flasks were given a firm tap on
the sides to dislodge any remaining bound cells otherwise the cell count can be
inaccurate. The flask was put under microscope for a quick check as to their
movement showing that they are suspended. Then a 20µl sample each of the T75
Hepg2 flask was obtained and carefully pippeted into the cavity of a haemocytometer
for cell counting.
Figure 2 Haemocytometer (Ruf.rice.edu, (2015) Microscope Counting Chamber).
On Haemocytometer there was a grid on which the cells in the sample could be
counted when placed under a microscope. The sample was x10^4 smaller than the
actual flask therefore the mean of the cells was multiplied by this factor. Therefore the
actual mean was 68.75x10^4 so 6.88x10^5 cells in standard form. These cells were to
be added to a 96/well plate evenly. The optimal number of HepG2 cells per well is
4x103 and 100µl capacity. Therefore the following calculation was used
(4000/6.88x10^5) * 100µl = 5.8µl of Hepg2 solution per well.
However for convenience the Hepg2 solution was diluted 10 fold so 1ml (hepg2):9ml.
Once the wells were filled the plate was left to incubate in 37 dC/5 % CO2 incubator
overnight so that the cells could proliferate and form a monolayer.
Preparation of 2 new Hepg2 flasks:
Firstly, fresh media, PBS and trypsin had to be gathered from the fridge and
heated in 37dC water bath to bring the solutions to the appropriate temperature
(i.e. 37dC) for the Hepg2 cells.
1. Remove current media from the T75 flask and wash down with 5ml of
PBS to initiate the breaking down of the protein monolayer formed by
the Hepg2 cells by breaking the calcium bonds between the cells, then
discard PBS.
2. Add 3ml of Trypsin to further breakdown the protein monolayer. Then
incubate the flask for 3 min at 37dC in incubator.
3. Observe cells under microscope for movement if not manually
dislodge.
4. Add 7ml of fresh media to the T75 flask so that there is now a total of
10ml solution – 3ml Trypsin + 7ml media.
5. Pipette 5ml each into 2 new T25 flasks. Then add 5ml of fresh media
to each new T25 so that each has 10ml volume of solution.
6. Flasks labelled (Hepg2 AQA 24/7/15) and put into incubator (37dC,
5%CO2).
T75 flask from 22/7/15 used to extract 20µl sample for 96/well plate. Same procedure
as above was used up till step 4 inclusive.
Then transfer 10ml solution into a sterilin.
Centrifuge sterilin for 5 minutes at 1500rpm, this will cause all the cells to
converge to the bottom of the sterilin creating a pellet (white deposit).
Then using automated pippeted, re-dissolve cells- this should leave any dead
cells at the bottom so only the living cells are now in the solution.
From this solution pippeted 20µl of solution onto a haemocytometer.
Place haemocytometer under a microscope and count number of cells on grids
(4 sections) and obtain a mean value.
Therefore mean = 24.25x10^4 cells i.e. 2.43x10^5/ml cells.
1. For T25 flask – 5x10^5 cells required
Therefore (5x10^5)/ (2.43x10^5) = 2.06 ml
2.06 x 1000 = 2060 µl required in flask which is ~ 2ml.
2. For 96/well plate 4x10^3 cells required per well.
Therefore (4x10^3)/ (2.43x10^5) = 0.0165ml
0.0165 ml x 1000 = 16.5 ~ 17µl/well in 96/well plate.
For 96/well plate it was found to be very time consuming to use a single pipetted to
add 17µl of hepg2 cell solution into each well. Therefore a 4 fold dilution was taken,
so 1ml of hepg2 cell solution: 3ml of media mixed together. Now 68-70µl was added
to the wells using a multipippete. 70µl of extra media added to each well to sustain
the cells over the weekend since some media will evaporate off.
The remaining cells were sub cultured.
30x10^4 31x10^4
18x10^4 18x10^4
Preparation of Drugs:
Two drugs were to be used: Doxorubicin and Quinidine. The whole MTT assay is
solely to identify which concentrations of these drugs are most suitable to be used on
the cells specifically later on for further procedures.
Calculations:
Doxorubicin was acquired 1mM.
This was made into a 5µM solution by a 1:200 dilution since 5µM is 200 times more
diluted than a 1mM solution. Therefore 10µl Dox was added to 1990µl of media
which gave 5µM solution.
A serial dilution method was used so the previous step’s concentration was diluted to
make a new more diluted concentration.
1. 10µl Dox + 1990µl Media = 5µM
2. 600µl Dox (from step 1) + 400µl Media = 3µM
3. 333µl Dox (from step 2) + 767µl Media = 1µM
4. 333µl Dox (from step 3) + 767µl Media= 0.3µM
5. 333µl Dox (from step 4) + 767µl Media= 0.1µM
Two 5µM solutions were made so that if there was ever a lack of the drug for a
particular concentration the stock version could be used and this proved to be useful.
Quinidine originally came as a 60mg/ml solution.
This was essentially a 60µg/µl concentration when both units are divided by 1000.
1. This concentration was diluted 60 fold by 1µl Quinidine (60µg/µl) + 59µl
Media which made it into a 1µg/µl concentration.
2. 1µl Quin (from step 1) + 999µl Media = 1µg/ml
3. 2µl Quin (from step 2) + 998µl Media = 2µg/ml
4. 3µl Quin (from step 2) + 997µl Media = 3µg/ml
5. 5µl Quin (from step 2) + 995µl Media = 5µg/ml
6. 7.5µl Quin (from step 2) + 992.5µl Media = 7.5µg/ml
The drugs were stored at 20°C.
Drug addition:
The diagram below shows where the drugs concentrations were added on the 96/well
plate. Two of these plates were used for the two different cell lines- Hepg2 & MDR-1.
Doxoru
bic
in
(µM
)
Control 0.1 0.3 1 3 5 Control
Quin
idin
e
(ng/m
l)
Control 1 2 3 4 5 Control
Original copy
Figure 3 96/well plate MTT assay
The outer layer of wells was left empty to optimize the reading of the plate in the
absorbance reader.
Once the drugs were added to the 96/well plate, the plate was placed in the 37dC/5%
CO2 incubator for 24 hours.
MTT Addition:
After the 24-hour incubation the media from the 96/well plate was removed and 50µl
of MTT solution*1 was added to each well along with 50µl of fresh media. The plate
was left to incubate overnight (37dC 5% CO2).After the overnight incubation, the
media&MTT solution was removed from the wells and 50µl of DMSO( Dimethyl
Oxide) was added to each well. DMSO is a substances which lyses cells and is
miscible in a wide range of solvents); therefore it was used so that the level of
formazan produced, representing the number of living cells*2, could be released into
the wells. Now the plate had varying shades of formazan (typically purple could be
observed) corresponding to the varying concentrations of drugs used.
1*MTT protocol:
5mg/ml vial of MTT in PBS
*2 MTT assay explanation:
The MTT solution is metabolized by the Hepg2 cells over the 4 hour period
and a purple substrate called formazan is produced within the cell. The
mechanism by which this happens is when NADPH-dependent cellular
oxidoreductase enzymes reduce the tetrazolium dye-MTT to its insoluble
formazan. The level of this formazan found by absorbance reading, reflects the
number of viable cells within the wells. Therefore the MTT assay is
technically a proliferation assay, however the negative of that can be used to
observe the chemotherapeutic effects on the Hepg2 cells. DMSO is required so
that the cells lyse and the purple formazan is released into solution. After this,
the 96/well plate is placed in a microplate reader machine which reads the
absorbance of the purple formazan and gives arbitrary values relative to a
known sample for the formazan.
In order to determine the chemotherapeutic effects of the drugs on the Hepg2
cells, the wells in which the drugs were added are compared to the control set
of wells in which 0% drugs was present. Therefore the control wells have
100% cell viability and 100% formazan production relative to the drugged
wells. So the wells with subsequent lower absorbance readings have lower cell
viability which is what reflects on the potency of the drugs.
RNA& Protein extraction from naïve Hepg2 cells:
In order to determine the effects of Doxorubicin and Quinidine on the cells other than
general cell viability, the protein and RNA was to be extracted to observe what
changes have occurred. This would show changes on the mRNA level (for MDR-1
gene) and subsequent protein expression for the P-glycoprotein.
Reagents required:
Chloroform
Isopropyl
75% ethanol(in DEPC-treated water)
DEPC treated water(BIO-38030)
Methods: (Bioline.com, (2015) Bioline)
Phase Separation:
1. Remove media from the treated T25 flasks
2. Wash the flasks down with 2ml of PBS- discard PBS
3. Add 1ml of TRIsure (lyses cells without damaging any of the organelles and
components such as the RNA and protein which is to be extracted), to the
flasks.
4. Incubate at room temperature on Orbital shaker for 5 minutes
5. Add each 1ml of TRIsure Hepg2 solution from the T25 flasks into 1ml
Eppendorf’s.
6. Add 200µl of Chloroform per 1ml of TRIsure added- so 1ml.
7. Close eppendorfs and shake vigorously for 15s
8. Open cap and incubate for 3 minutes at room temperature however within the
sterile compounds of the laminar-flow cupboard which keeps the air sterile (to
prevent any pathogenic or human protein contamination)
9. Centrifuge samples at 12,000xg for 15 minutes at 4dC.
This is the general method of obtaining separating the protein& DNA from the
RNA. This is because at this stage, within the Eppendorf there are 3 distinct
layers observable. Organic phase below-containing Protein &lipids. White
interphase containing DNA. Aqueous phase above containing RNA.
Figure 4 Cell phases after lysis with TRIsure (Google.co.uk, (2015) Protein&RNA Layers
on Extraction).
Organic phase: Protein &
lipids
In order to obtain just the aqueous phase for the RNA. A combination of P-200
and P10 pipettes were used in order to extract all of the aqueous phase from the
Eppendorf but not take up any of the lower phases. For maximum RNA yield.
Once the aqueous phase containing RNA is extracted, it is placed into a separate
Eppendorf and 3 further stages are required.
RNA Precipitation:
Precipitate the RNA by mixing with cold isopropyl alcohol. 500µl of isopropyl used.
Then incubate samples for 10 minutes at room temperature. After this centrifuge at
12,000xg for 10 minutes at 4dC.
RNA Wash:
Remove the supernatant (aqueous content) leaving the pellet at the bottom of
Eppendorf. Wash the pellet once with 75% ethanol, adding at least 1ml of ethanol.
Then Vortex samples and centrifuge at 7500xg for 5 minutes at 4dC.
Re-dissolving the RNA:
Air-dry the pellet and dissolve in DEPC-treated water by pipetting the solution up
and down. Incubate for 10 minutes at 60dC. Store RNA at -70 ̊ C.
From the original Eppendorfs with the phases separations, there should only be the
DNA interphase and Protein &Lipid organic phase remaining. From there the
following steps are taken:
Protein Precipitation:
To the retained supernatant from step 2(Phase separation) add 1.5ml of isopropyl
alcohol. Mix samples for 10 mins at room temperature. Then centrifuge at 12,000xg
for 10 minutes at 4 ̊ C.
Protein Wash:
Pellet should have formed in Eppendorf from previous step. Remove the supernatant
and wash the protein pellet twice. To wash the protein add 2ml of 0.3M guanidine
hydrochloride in 95% ethanol. Mix for 20 minutes at room temperature then
centrifuge at 7500xg for 5 minutes at 4 ̊ C.
Following these steps, add 2ml of ethanol and vortex. Mix for 20 minutes at room
temperature then centrifuge again at 7500xg for 5 minutes.
Re-dissolving the Protein:
Vacuum dry the protein pellet for -10 minutes. Dissolve in 1% SDS by pipetting up
and down. For difficult samples, incubate at 50 ̊ C. Remove any insoluble material by
centrifugation at 10000xg for 10 minutes and then transfer the supernatant to another
tube. The protein sample was then added to sample buffer so that it could be loaded
into the western blot gel.
Nano Drop-spectrophotometer:
This was a device which was used to determine the volume of protein/RNA extracted
per µl/ml of sample. The device first must be blanked with milliq water (i.e. purified)
to prevent any contamination which could lead to undesired peaks in the results. Then
2µl of the protein/RNA extract sample was pipetted onto the small reading spot on the
device. The device then emitted radiation that was absorbed by the sample and the
relative remittance was converted into meaningful peaks which would show the level
of protein/RNA in the sample.
Figure 5 Nano Drop spectrophotometer device (Google.co.uk, (2015) Nano drop
Spectrophotometer)
Gel Electrophoresis:
This a process by which the levels of RNA in a sample of cells can be seen. The gel is
of a converging porous nature with pores sizable to RNA, DNA and protein.
Therefore the smallest macromolecules will travel furthest and larger molecules will
stop progress closer to the start of the gel; this is reflected in the UV image of the
bands of molecules shown.
The set up consists of: a tank, a gel, gel box, electrode buffer, electrodes and an
electric supply.
The RNA travels from negative electrode to the positive electrode because it has a
phosphate head which makes it negatively charged. Therefore the RNA molecules are
attracted to the positive electrode and repel- therefore move away from the starting
position which is close to the negative electrode.
The gel was run at 72V, 60mA so that the RNA could slowly disperse across the gel.
Figure 6 Gel Electrophoresis tank (Google.co.uk, (2015) Gel Electrophoresis)
Making of the gel:
Mix 40ml of T.A.E with 0.6g of Agarose in a 100ml glass bottle.
Microwave this for 1 minute with the cap lose to prevent overheating and possible
explosion. Then let solution cool down to 50-60 ̊ C.
Pour gel solution into gel box and let the gel set.
Once the gel is set remove sides of gel box and place it in the main tank.
Then pour 50ml of electrode buffer into cavities of the main tank- this maintains the
pH whilst the current is passing through the tank.
RNA sample calculation for Gel Electrophoresis:
In the Eppendorf containing the RNA sample there was found to be 886.5 ng/µl.
1500µl of sample was required for loading into the wells of the gel.
Therefore the calculation was 1500/886.5 = 1.7µl of RNA sample required.
Western Blot:
The Western Blot is a similar process to Gel Electrophoresis. The main difference is
that Western blot shows specific protein bands rather than RNA or DNA bands.
Making of the gel:
There are two types of gel required for the western blot process.
Resolving gel:
This was the main part of the whole gel; this is where the protein bands separate and
show. This is an 8% gel.
It is composed of the following reagents:
4.7ml of milliq water
2.7ml of Acrylamide
2.5ml of gel buffer
0.1ml of 10% SDS
100µl of 10% APS & 10µl of TEMED to initiate polymerisation
This gel solution was poured into the glass slide cassettes for the western blot
chamber. Once this resolving has polymerised the next gel can be made.
Stacking gel:
This is the top part of the whole gel where the samples are loaded into the wells.
This is a 4% gel.
It is composed of the following reagents:
6.1ml of milliq water
1.3ml of Acrylamide
2.5ml of gel buffer
0.1ml of 10% SDS
100µl of 10% APS & 10µl of TEMED to initiate polymerisation
The stacking gel is poured into the same glass cassettes. A plastic comb was carefully
placed into the stacking gel; this comb indents the loading wells for the protein
samples. Once this stacking gel has polymerised the comb is taken out without
disrupting the shape of the wells.
Figure 7 Western Blot tank with loaded wells
Preparing the protein samples for Western Blotting:
The protein that had been extracted previously from the drug treated Hepg2 cells was
to be used for western blotting. The protein had to be denatured into its
primary/secondary form so that it could be easily transferred through the gel.
The protein extracts were defrosted from the -20̊C freezer.
Then the following steps were taken:
1. Mix protein extract with 25µl of sample buffer
2. Add 19.5µl of 1% SDS
3. Sonicate for 10-15 seconds to completely denature proteins.
4. Heat 20µl sample to 95̊C for 5 minutes; cool on ice.
5. Micro centrifuge at10000xg for 5 minutes.
Now the protein samples were ready to be loaded into the wells of the western blot
gel. The samples were added in the following order:
Control, Doxorubicin (treated cell protein), Quinidine (treated cell protein), Mix of
drugs (treated cell protein) and a ladder sample (containing all the proteins within
cells so the other samples could be compared to in order to see which proteins are
present within them).
Wells containing protein samples.
Blotting onto membrane:
Once the gel had fully run through; this meant that the bands from the ladder had
reached the end of the tank. The gel was carefully taken out of the cassette and placed
on filter papers.
Diagram below shows how the blotting chamber was set up.
Figure 8 Blotting chamber configuration (Google.co.uk, (2015) Western Blotting).
There were 7 sheets of filter paper either side of the gel. The diagram above shows
how 2 gels can be placed together in a larger chamber. However it is the same
configuration.
The blotting chamber was placed in the blotting machine for 20 minutes (optimized
time since 10-15 minutes did not give clear results). The membrane was then placed
in a 10ml sterilin with 7ml of 5% milk in the fridge for 24 hours.
The milk was added to block the membrane completely so that there would be no
unspecific binding from undesired protein antigens for the next step.
After 24 hours the membrane was washed with 10ml of TBST solution 5 times for 5
minutes each time whilst placed on an Orbital shaker device.
Then the primary antibody-P-glycoprotein monoclonal antibody (C219) was added to
the blotted membrane for 1 hour. During this time this specific antibody bound to its
specific antigens of the P-glycoprotein.
After the 1 hour, the primary antibody was taken out and 5x 5 min TBST washes were
carried out again.
Then the secondary antibody was added- this was a fluorescent antibody that would
fluoresce as a green colour once bound to the primary antibody. This anti-body acts a
marker and means that the location of the antibodies where the target protein is can be
seen. This 2̊ antibody is left on for 1 hour as well.
Then 5x5 minute TBST washes are carried out once more to ensure that any unbound
antibodies are removed to prevent inaccurate results.
Now the membrane is almost ready for imaging. Then 500µl of an enzyme called
HRP (Horse radish peroxidase) reduces the secondary anti body so that it releases the
green fluoresce onto the membrane so that it is visible for imaging.
PCR (Polymerase chain reaction):
The extracted RNA from previous procedures must be converted into CDNA for
PCR. PCR essentially isolates the target DNA section and amplifies it- so that it can
be seen clearly and quantified in terms of its level within the sample used.
CDNA = 8µl of master mix solution + 12µl of milliq water and according RNA
sample.
Master Mix contains nucleotides and other DNA forming material such as reverse
transcriptase enzyme that converts the mRNA into CDNA.
The volume of the RNA sample required is determined by the following calculation.
What is required/what there is = xµl of RNA sample.
Therefore for Doxorubicin treated cell’s RNA, 1500/525.5= 2.9µl of RNA sample. So
12-2.9= 9.1µl of milliq water added.
Control = 1500/1438.9 = 1.04µl of RNA sample therefore 11µl of milliq water.
Quinidine = 1500/1310.1= 1.14µl RNA sample therefore 10.9 µl of milliq water.
Mix = 1500/330.9 = 4.5µl RNA sample therefore 7.5µl of milliq water.
CDNA put on heating block- 50 ̊ C then placed in -20 ̊ C freezer to denature.
The PCR that was to be carried out was to test for 4 sections of the cDNA or also
known as genes which code for specific proteins such as MDR-1(main gene of
interest) codes for P-glycoprotein.
Below is a table representing the 96/well PCR plate used.
B-actin
Hepg2
Control
B-actin
Hepg2
Control
B-actin
Hepg2
Control
MDR1
Hepg2
Control
MDR1
Hepg2
Control
MDR1
Hepg2
Control
VGF
Hepg2
Control
VGF
Hepg2
Control
VGF
Hepg2
Control
CYP1B1
Hepg2
Control
CYP1B1
Hepg2
Control
CYP1B1
Hepg2
Control
B-actin
Hepg2
Quin
B-actin
Hepg2
Quin
B-actin
Hepg2
Quin
MDR1
Hepg2
Quin
MDR1
Hepg2
Quin
MDR1
Hepg2
Quin
VGF
Hepg2
Quin
VGF
Hepg2
Quin
VGF
Hepg2
Quin
CYP1B1
Hepg2
Quin
CYP1B1
Hepg2
Quin
CYP1B1
Hepg2
Quin
B-actin
Hepg2
Dox
B-actin
Hepg2
Dox
B-actin
Hepg2
Dox
MDR1
Hepg2
Dox
MDR1
Hepg2
Dox
MDR1
Hepg2
Dox
VGF
Hepg2
Dox
VGF
Hepg2
Dox
VGF
Hepg2
Dox
CYP1B1
Hepg2
Dox
CYP1B1
Hepg2
Dox
CYP1B1
Hepg2
Dox
B-actin
Hepg2
Mix
B-actin
Hepg2
Mix
B-actin
Hepg2
Mix
MDR1
Hepg2
Mix
MDR1
Hepg2
Mix
MDR1
Hepg2
Mix
VGF
Hepg2
Mix
VGF
Hepg2
Mix
VGF
Hepg2
Mix
CYP1B1
Hepg2
Mix
CYP1B1
Hepg2
Mix
CYP1B1
Hepg2
Mix
B-actin
MDR1
Control
B-actin
MDR1
Control
B-actin
MDR1
Control
MDR1
MDR1
Control
MDR1
MDR1
Control
MDR1
MDR1
Control
VGF
MDR1
Control
VGF
MDR1
Control
VGF
MDR1
Control
CYP1B1
MDR1
Control
CYP1B1
MDR1
Control
CYP1B1
MDR1
Control
B-actin
MDR1
Quin
B-actin
MDR1
Quin
B-actin
MDR1
Quin
MDR1
MDR1
Quin
MDR1
MDR1
Quin
MDR1
MDR1
Quin
VGF
MDR1
Quin
VGF
MDR1
Quin
VGF
MDR1
Quin
CYP1B1
MDR1
Quin
CYP1B1
MDR1
Quin
CYP1B1
MDR1
B-actin
MDR1
Dox
B-actin
MDR1
Dox
B-actin
MDR1
Dox
MDR1
MDR1
Dox
MDR1
MDR1
Dox
MDR1
MDR1
Dox
VGF
MDR1
Dox
VGF
MDR1
Dox
VGF
MDR1
Dox
CYP1B1
MDR1
Dox
CYP1B1
MDR1
Dox
CYP1B1
MDR1
Dox
B-actin
MDR1
Mix
B-actin
MDR1
Mix
B-actin
MDR1
Mix
MDR1
MDR1
Mix
MDR1
MDR1
Mix
MDR1
MDR1
Mix
VGF
MDR1
Mix
VGF
MDR1
Mix
VGF
MDR1
Mix
CYP1B1
MDR1
Mix
CYP1B1
MDR1
Mix
CYP1B1
MDR1
Mix
Results:
MTT assay:
Figure 9 MTT assay results for varying concentrations of Quinidine.
Figure 10 MTT assay results for varying concentrations of Doxorubicin on Hepg2 cells
0102030405060708090
100110120130140150
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
Re
lait
ive
Pe
rce
nta
ge A
bso
rban
ce (
%)
Quinidine Concentration (μg/ml)
Effects of varying concentrations of Quinidine on HepG2 cells after 24hrs.
0102030405060708090
100110120130140150160
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5
Re
lati
ve P
erc
en
tage
Ab
sorb
ance
(%
)
Doxorubicin Concentration (μM)
Effects of varying concentration of Doxorubicin on HepG2 cells after 24 hours
From the MTT assay the following results were observed.
Figure 9 MTT assay results for Hepg2 cells
This graph shows the relative cell viability which has been normalised to the control
set of wells. The control have been given 100% cell viability, therefore any
percentage less than 100 means cell death and above 100 means cell proliferation has
taken place.
Figure 10 MTT assay results for MDR-1 cells
Gel Electrophoresis results:
Figure 11 First Gel Electrophoresis- preliminary attempt for optimisation
Figure 12 Gel Electrophoresis with both cell lines. L=Ladder, C=Control, D=Doxorubicin, Q=Quinidine,
M=mix.
MDR1 Hepg2
L C D Q M C D Q M L
Nano Drop spectrophotometer results:
Figure 13. Hepg2 first RNA extraction
Figure 14 Hepg2 first protein extraction
Main results from RNA extraction:
Figure 15 Hepg2 control
Figure 16 Hepg2 mix
Figure 17 Hepg2 doxorubicin
Figure 18 Hepg2 Quinidine
Western Blot Results:
Figure 19 Good image 20 min blotting
Figure 20 20 min blot ladder _ High molecular weight
Figure 21 Blot over exposed image
Figure 22 Ladder at lower exposure
PCR results:
Google.co.uk, (2015)
Protein Bands
Figure 23 CYP1B1 results
Figure 24 MDR-1 results
Figure 25 Melt curve of whole PCR
Discussion of results:
MTT assays:
The first set of MTT assay results show how varying the concentrations
of Doxorubicin and Quinidine separately affect the number of viable
Hepg2 cells. The controls were set at 0% of the drug therefore were given
100% cell viability and the corresponding percentages for the increasing
drug concentrations were made relative to this- normalised.
For Quinidine the cell viability did not change substantially overall, it
generally stayed near the 100% cell viability mark. This trend was
expected of Quinidine since it is not specifically an anti-cancer drug.
Quinidine on its own only inhibits the P-glycoprotein efflux pumps since
it is a substrate for the P-glycoprotein. From the results it was decided
that 5µg/ml was the most suitable concentration to treat the Hepg2 flask
with. This concentration gives ~ a 12% proliferation in the hepg2 cells,
this is appropriate because the quinidine does not aim to terminate the
hepg2 cells. A higher concentration was not used because it would be too
potent and could terminate all the cells and this is not ideal because the
RNA and protein could not be extracted then from the hepg2 cells. Lower
concentrations were either resulting in cells termination slightly or would
not have strong inhibition for the efflux pump therefore 5µg/ml was used.
For Doxorubicin the general trend was that as the concentration was
increased the cell viability relative to the control decreased. This trend
was also expected because Doxorubicin is a specific anti-cancer drug
therefore will terminate more cells with increased potency. For treatment
on Hepg2 flasks, a concentration of Doxorubicin was required that would
terminate a significant level of cells but also leave a significant level of
cells not terminated so the stages of the gene expression and protein
expression could be observed. At 2µM there was almost 100% cell
viability which is too high because some cells need to be terminated so
the drug is not potent enough at this concentration. At 3µM there was 75-
80 % cell viability this means that ~25% of cells have been terminated –
this was slightly too high. Therefore the concentration 2.5µM was used
since it gave 85% cell viability which was more suitable.
The second MTT assay, shows how the drug concentrations identified
from the first MTT assay, affected the Hepg2 cells and the MDR-1 over
expressing Hepg2 cells. The Quinidine (5µg/ml) as expected, did not
cause any cell termination instead it cause cell proliferation by 10%.
Doxorubicin caused a 65% termination of cells – this is significantly high
however this is on the Hepg2 cells which do not have many efflux pumps.
When the drugs were mixed there was an 80% cell termination. This high
mortality was also expected because the combination of the drugs
enhances the overall chemotherapeutic effect.
For the MDR-1 cells, Quinidine’s individual effect was not much
different- only causing 5% more cell proliferation. However for
Doxorubicin there was ~ a 25% decrease in cell termination from Hepg2
cells (which was 65% cell termination) therefore 25% higher cell
viability. This was because the MDR-1 cells have more efflux pumps to
pump out the Doxorubicin therefore are more have more resistance.
When the drugs were combined there was a 24% increase in cell viability
from the effect of the mixed drugs on Hepg2 cells.
Gel Electrophoresis:
From the Gel electrophoresis it can be seen that there are bright bands
towards the middle. This shows the quality of the RNA. 2 bright bands
can be seen in the RNA lane. These are called 28s and 18s RNAs. The
upper band (28s should be brighter than the lower band). Sharp bands
with no small band at the bottom of the gel show no degradation of RNA
and a pure sample. In the first run there was clearly not a clear sample.
In the second gel with 8 samples loaded – 4 for Hepg2 and 4 for MDR-1
cells, it is still not an entirely pure sample. However it can be seen that
for the MDR-1 the Quinidine has the brightest bands. This may be
because when Quinidine inhibits the efflux pump, to compensate the
MDR-1 cell begins to upregulate the expression of the MDR-1 gene.
Nano Drop:
This is quantified the level of RNA and protein extracted. In the first
extraction of RNA there is 886.5ng/µl of RNA- this is a good yield
because the yield should be close to 1000ng/µl. For the protein extraction
the result is even better because 1.79mg/ml was extracted whereas only
1mg/ml was required. For the rest of the RNA extractions there were
varying quantities. This was not something that could be controlled.
However this was not a big problem because they were mostly above the
desired value and for the ones which were not, the higher value extracts
were diluted down.
Western Blot:
This shows which proteins are present in the samples and highlights one
specific protein. The P-glycoprotein was the target protein here. This
protein has a molecular weight of 140 kD. Next to figure 20 in the results
above there is a sample ladder next to the image of the real ladder. The
sample ladder helps makes the bands clearer. However it can be seen that
just under 150kD looking across from the ladders there is a dark coloured
band on the actual treated samples of MDR-1 cells. This means that there
was P-glycoprotein present on these cells. However it is not easy to
distinguish between the intensity of the lanes for the different samples
used. This may be because the cells were only left in the drugs for 24
hours, so although changes at the mRNA level can be observed, protein
expression may not change until 72hours or more.
PCR:
This shows the level of MDR-1 gene in the different samples. Greatest
relative level of MDR-1 was in the Hepg2 Quinidine sample- slightly
above 1.0 - after the controls. Lowest level of MDR-1 is in Hepg2 mix
near 0.4 relative to control (i.e. 1.0). MDR-1 mix has more MDR-1 than
Hepg2 mix by ~ 0.35 arbitrary units this is because the MDR-1 cells are
engineered to express more MDR-1. The PCR results complement the
results from the MTT assay earlier by showing that when the drugs are
combined there is a reduction in MDR-1 – since MDR-1 Hepg2 for just
Doxorubicin was ~ 0.6 AU whereas in the Hepg2 mix the MDR-1 level
was only ~ 0.4AU, therefore the mix would have less efflux pumps hence
the cell viability with the combination of drugs is lower. The MDR-1 cell
samples follow the same trend however there is more MDR-1 overall
across the samples- this is because this cell line was engineered to have
more MDR-1 expression.
CYP1B1 is an enzyme which metabolizes drugs and allows them to take
their effect. Therefore where there are greater levels of CYP1B1 there
should be less MDR-1 meaning lower cell viability. From the results
above this hypothesis is proven to be true because for the Hepg2 mix
which had the lowest MDR-1 expression hence lowest cell viability had
the highest level of CYP1B1 which was 10AU relative to control (1).
For Quinidine which had the highest MDR-1 expression in both Hepg2
and MDR-1 cells, both had less than 1.0AU of CYP1B1 which explains
why they also had the highest cell viability and MDR-1 gene expression.
Conclusion:
From this project it can be concluded that Doxorubicin and Quinidine do
alter the MDR-1 gene expression of both Hepg2 cells and MDR-1over
expressing cells. It was found that the MDR-1 gene was almost always
more abundant in the MDR-1 cells than the Hepg2 cells. The P-
glycoprotein expression could not be easily concluded since the results
were limited to a time frame of 24 hours of incubation with the drugs;
therefore, although the mRNA may change during this time period, the
protein expression may not. It may take 72+hours for the protein
expression to adjust. The MDR-1 expression was clearly shown to
change (PCR & MTT assay) across the samples of drugs when incubated
for 24 hours. It was seen that when the drugs are combined the MDR-1
expression lowers and the cancer cells become less resistant;
subsequently a decrease in cell viability is observed. Therefore it can be
concluded that MDR-1 plays an essential role in the interaction between
Hepg2 cells (including MDR-1 over expressing version) and
chemotherapeutic drugs because it determines the resistance of the cancer
cells. To a certain extent it can be said that the greater the level of MDR-
1gene expression, the greater the resistance of the Hepg2 cells,
subsequently the greater the cell viability.
Critical evaluation:
However this conclusion cannot be generalised to all types of cancer cells
since only the Hepg2 family of cancer cells were used and tested in this
project.
If this project was to be continued a wider range of cell lines could be
experimented on for e.g. intestinal and kidney cancer cells.
Therefore the aims could be:
1. To identify whether intestinal and kidney epithelia express P-
glycoprotein and whether they respond to cancer treatments by
modulating P-glycoprotein expression.
2. To identify changes in P-glycoprotein distribution and trafficking
under anti-cancer treatments in liver, gut and kidney cells.
3. To further elucidate the possible mechanism of action of the drug
synergy observed between our compounds of interest.
Other than this the project could have been more optimised. During the
first and second week there was a lot of learning to be done about lab
procedures and methods such as: composing reagents, pipetting, loading
samples into wells, and optimising methods by varying different factors
for e.g. blotting time for Western Blot and so on.
This meant that essentially there was 1-2 less weeks of actual project time
in which meaningful results could be obtained. However results from 2
cell lines were still obtained therefore the project was relatively
successful as some conclusions could be made especially regarding
MDR-1 gene expression.
Contaminations were prevalent throughout the project. Contaminations
were mainly bacteria that would easily proliferate in flasks and 96/ well
plates. This proved to be a very sensitive matter because the slightest of
unsterile practice in the lab could nullify a whole week’s worth of
experimentation. Therefore in future, sterile practice will be emphasised
to prevent any contamination.
What I have learnt from this project is that MDR-1 gene expression is a
big factor for the resilience and resistance of cancer cells to
chemotherapeutic drugs. Cancer cells are very prone to contamination
especially when put under artificial body conditions. Science and
experimentation involves a lot trial, testing and mistakes. However it is
important to understand and know exactly where they were made and
how they could be prevented in the future- not just for myself but for
other people who could benefit from the knowledge. Sharing knowledge
and findings is key because it helps increase reliability.
Bibliography:
References
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Acknowledgements:
Supervisor: Dr.Christopher Mee (much appreciated assistance and
providing us with knowledge).
Ambassadors: Eliot Barson ( much appreciated assistance throughout this
project) & Ben Knowles (much appreciated help) & Sarah Siverns (for
keeping up us up to date).
Nuffield staff: Farzana Aslam & Steve Joiner.
Lab Partner: Kashfia Akhtar (much appreciated company and assistance
throughout the project).
