Cancer- It is the leading cause of mortality. Cancer is the disease which is categorized by abnormal cell growth with the dormant to spread to other parts of body.
In vitro methods of screening of anticancer agents
1. IN-VITRO METHODS OF
SCREENING OF
ANTICANCER AGENTS
Presented by
Nikita Savita
32/MPH/2019
Pharmacognosy and
phytochemistry
Presented to
Dr. Mahaveer Dhobi
2. CANCER
Introduction
Cancer is the leading cause of mortality.
Cancer is the disease which is characterized by
the abnormal cell growth with the dormant to
spread to other parts of body.
3. Normal cell
Grow, divide and
die in orderly
fashion
Cancer cells
They don’t die,
they continue to
grow and divide in
disorderly fashion
4. They form a set of tumor or neoplasm.
A neoplasm or tumor is a group of cells, which is
also called malignancy, have undergone unregulated
growth of cell and often form a mass and lump.
Various types of treatments are available for cancer
patients like surgery, chemotherapy, radiations, etc.
But there are lots of side effects related to these
treatments.
5. Hence now a days scientist are focusing towards
development of new anticancer drugs.
A pre-selection, called the screening process is
required to identify products that will able to perform
anti-tumor effects.
The biological evaluation of these newly synthesize
compounds include various in-vitro or in-vivo
techniques.
6. Direct screening by in-vivo technique requires lot of
expenses and approval from animal ethics committee
hence it is always better to screen the synthesized
drugs by various in-vitro techniques which are
cheaper.
After screening by in-vitro technique, the promising
compounds can be screened further by in-vivo
technique.
7. Methods for in-vitro screening
1. MTT Assay: (Colorimetric Assay
Based Upon Tetrazolium Salt)
2. SRB assay (sulforhodamide-b assay)
3. Clonogenic Assay
9. 8. DNA fragmentation assay
9. Trypan blue exclusion assay
10. Potato disc tumor induction assay
10. The amount of colour produce by a specific reagent in
the assay can be measure by calculating amount of light
absorbed by the compound or newly form product at
particular wavelength, this is called colorimetric base
assay.
It is a type of method which determines the
concentration of a chemical element or chemical
compound in a solution with the aid of a colour reagent.
MTT Assay
11. Micro culture tetrazolium salt based assays (MTAs) are
colorimetrically based assay
they based on the reduction of a tetrazolium salt by a
mitochondrial enzyme
which lead to the production of a coloured compound
which is called formazan
Quantified by spectrophotometry.
12. Types of Tetrazolium Salts
1. MTT - A yellow coloured tetrazole, which was
converted into purple formazan in the cell.
The formazan is insoluble in the medium which could
be solublize by the use of solution, which make the
medium clear.
The quantification is done by taking absorbance of this
solution at specific wavelength, normally it is taken at
500 and 600 nm by the use of spectrophotometer.
14. 3. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2Htetrazolium)
by the addition of phenazine methosulfate (PMS)
obtained formazan product has an maximum
absorbance at 490-500 nm with the use of phosphate-
buffered saline.
The assay has usually known as a 'single-step' MTT
assay, which will offers us for straight addition of cell
culture without the other additional steps, which is
essential in the MTT assay. However this convenience
makes the MTS assay sensitive to colorimetric
interference.
15. 4. Wsts (Water-soluble Tetrazolium salts) 8(2-(2-
methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium)
this assay has been developed to obtained different
absorption spectra of the formed purple coloured
formazan.
The assay has an advantageous over other salt based
assay is that they are reduced outside cells, combined
with already added PMS solution which behave as a
electron mediator, and produce a water-soluble
formazan. Water-soluble tetrazolium salts are more
recent alternatives to MTT.
16. MTT assay is a type of colorimetric assay, which had
been extensively used for the estimation of the cell
metabolic activity.
Under certain specific conditions NADPH dependent
cellular oxido-reductase enzymes may, equal to the
number of viable cells present in the medium.
They are able to reduced tetrazolium dye into its purple
coloured formazan product.
MTT assays can also be used to quantitate loss of viable
cell(cytotoxicity) and shifting of proliferation to
quiescence (cytostatic) by the use of potential medicinal
agents and toxic materials.
17. The alteration of MTT by cells in culture is time
dependent. The accumulation of colour can be happen if
incubation time will increase and sensitivity is also
affected.
Because of the toxic nature of the reagents, the
incubation time should be limited otherwise they will
the incubation time is limited because of the toxic
nature of the detection reagents which promote to the
energy from the cell to generate a signal (reducing
equivalents such as NADH).
18. These salt based assays are usually done in the dark
since the salt is sensitive to light.
The cell get lose there ability to produce MTT they die,
and color formation serves as a essential and convenient
marker for the viable cells.
19. Example- Justicia tranquibariensis
• Justicia tranquibariensis a native of India and many
other tropical regions is a common garden plant.
• The hydro-alcoholic extract of the roots of the plant
have been tested for anticancer activity. The extract
was prepared by cold maceration method,
hydroalcohol was used as the solvent.
20. • The in-vitro anticancer studies were performed
against human cervical adenocarcinoma cell line
(HeLa) and MTT assay was used to analyze the cell
growth inhibition.
• The results showed that the hydroalcoholic extract of
roots of Justicia tranquibariensis possessed a
moderate amount of anticancer activity and the IC50
value was greater than 200 μg/ml
21. IN-VITRO EVALUATION OF ANTICANCER
ACTIVITY BY MTT ASSAY
Cell Culture
The HeLa cell lines (human cervical adenocarcinoma
cell line) was provided by National Centre for Cell
Science, Pune and was grown in Eagles Minimum
Essential Medium (EMEM) which contained 10% fetal
bovine serum (FBS).
All cells were maintained at 37°C, 100% relative
humidity, 5% CO2, 95% air and the culture medium was
changed twice a week.
22. Cell Treatment
1. The monolayer cells were detached and single cell
suspensions were made using trypsin-ethylene diamine
tetra acetic acid (EDTA).
2. A hemocytometer was used to count the viable cells
and the cell suspension was diluted with a medium
containing 5% FBS, to obtain final density of
1x105cells/ml.
23. 3. 96-well plates at plating density of 10,000 cells/well
were seeded with one hundred microliters per well of
cell suspension and incubated for cell attachment at
37°C, 5% CO2, 95% air and 100% relative humidity was
maintained.
4. The cells were treated with different concentrations
of the test samples after 24 hours prepared by serial
dilution method.
5. Cells were initially dissolved in dimethylsulfoxide
(DMSO), further diluted with serum free medium to
achieve twice the desired final maximum test
concentration.
24. 6. The required final drug concentrations of 25, 50, 75,
100, 200, 300μg/ml were obtained by adding aliquots
of 100 μl of the different drug dilutions to the
appropriate wells already containing 100 μl of medium.
7. After addition of the drug the plates were incubated
for an additional 48 hours at 37°C.
8. The medium without samples served as control and
triplicate was maintained for all concentrations.
25. MTT Assay
After 48hours of incubation, to each well added 15μl of
MTT (5mg/ml) in phosphate buffered saline (PBS) and
incubated at 37°C for 4 hours. The medium with MTT
was removed and the formed formazan crystals were
solubilized in 100μl of DMSO solution. Using a micro
plate reader the absorbance was measured at 570 nm.
The percentage cell inhibition was determined using the
formula.
Percentage Cell Inhibition = [100- Abs (sample)/Abs
(control)] x100.
26. S.No. Concentration of extract
(μg/ml)
Absorbance Percentage
inhibition of
cell growth
1. 25 0.60698±0.01184 -3.26077
2. 50 0.49257±0.021085 1.03429
3. 75 0.36369±0.005142 5.54823
4. 100 0.30834±0.004967 24.65825
5. 200 0.239645±0.00532 36.65734
6. CONTROL 0.5976±0.00579 0
TABLE : Percentage cell growth inhibition of hydro-alcoholic extract
of Justicia tranquibariensis on HeLa cell lines by MTT assay
27. RESULTS AND DISCUSSION
In Vitro Anticancer Activity
The results for cell growth inhibition by the extract
against HeLa cell lines for various concentrations shows
in table that as the concentration increases there is an
increase in the cell growth inhibition but is found to be
very less with only 36.65734% growth inhibition at 200
μg. The regression value was difficult to analyze. The
results obtained showed that hydro-alcoholic extract of
Justicia tranquibariensis had a very moderate
anticancer activity.
28. CONCLUSION
The results obtained from the in-vitro cancer studies
performed using the HeLa cell lines reveals that
the hydro-alcoholic root extract of Justicia
tranquibariensis has a moderate anticancer activity.
Even though there was increase in the cell growth
inhibition when concentration of sample was increased,
the IC50 value was more than 100 μg/ml for the cell line
studies as shown by the MTT assay method.
Hence the level of cytotoxicity of the hydroalcoholic
extract of Justicia tranquibariensis roots can be
concluded to be less effective.
29. The basic principle of MTT or XTT reagents is relies
on the formation of color compound, that depend on
the activity of the mitochondria.
So variable results can be obtained if the variations is
done by the cellular levels of glucose or other cellular
content or functions of these reagents is inhibited and
if the cells were not alive or not proliferating then
similar result has been shown.
SRB Assay
[Sulphorhodamine- b assay]
30. These types of limitations can be overcome by the
second major technique for testing cell viability or
cytotoxicity is the sulphorhodamine b (SRB) assay,
which is more preferred.
SRB is an anionic dye and it is amino xanthene,
which can react with basic amino acid residues of
protein to forms an electrostatic complex under
moderately acid conditions, which contribute to a
susceptible and linear response.
The cells get prepare by thoroughly washed it, fixed
and stained it with the dye.
31. The reagents which were incorporated get liberated
from the cells with a tris base solution.
The formation of color is quick and stable and the
absorbance can be readily measured at wavelength
between 560 and 580nm.
The concomitant change in the amount of dye which
is incorporated in the culture contribute to the
increase or decrease in the total number of cell.
These changes will show the degree of cytotoxicity or
cell viability caused by the test compound.
32. These all evaluation is depend on the uptake or
incorporation of the pink amino xanthine (negatively
charged) dye by amino acids (basic) in the cells.
The greater amount of dye is taken up by the cell if
adequate amount of cell will present and after washing
and fixing, the released dye will give a more acute
color and greater absorbance when get lysed.
It is rapid, susceptible, sensitive, and inexpensive
method for measuring the cellular protein content of
the cell.
