Goo

Pharmacological and toxicol

dm
ogical screening

orn
Presented by: RUJU VASHI
M.PHARM (SEM-I) Pharmacology

in g !
18MPH210
GUIDED BY: DR. SNEHAL PATEL
SCREENING OF ANTI-CAN
CER DRUGS
 Sustaining proliferative signa
lling ICON Resisting cell death

ICON ICON

Evading growth suppresso

rs HALLMARK Inducing angiogenesis
S OF
ICON CANCER ICON

Activating invasion and metas Enabling replicative immorta

tasis lity
ICON

3
Add an image
• CANCER: Uncontrolled proliferation of genetically
altered cells derived from the repeated divisions of a
mutant cell.
• Cancer-promoting mutations may be acquired
1 through errors in DNA replication.
• Activate the cancer promoting oncogenes and/or
inactivate the tumor suppressor genes.
•They are mostly cytotoxic in nature and act by a very
limited number of molecular mechanisms.
•Thus, the need for novel drugs to treat malignant
disease requiring systemic therapy is still pressing.
INTRODUCTION • A preselection, called the screening process, is
therefore required.
•The aim of screening efforts is to identify products
that will produce antitumor effects matching the
activity criteria used to define which compounds can
progress to the next stage in the preclinical
development program.
ANTI-TUMOUR MODELS

Add an image

IN-VITRO
&
IN-VIVO

5
 1 MTT Assay

2 SRB(Sulphorhodamine B assay)

In-vitro 3 3-H-Thymidine uptake

assays
4 Membrane integrity assay(dyes)

5 Clonogenic test

6 LDH assay

6
 Microculture Tetrazolium Test(MTT Ass
ay)
• MTT assay is a quantitative colorimetric assay for measuring cellul
ar growth, cell survival and cell Proliferation based on the ability of l
iving cells.
• Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro
mide) a tetrazolium salt is reduced to purple formazan by mitochon
drial dehydrogenase of living cells in which tetrazolium ring gets clea
ved in mitochondria .

7
 Method

• It is performed to determine the Enzymatic properties.

• Cells from particular cell lines in log phase of growth are trypsinised,
• It is counted in a hemocytometer and adjusted multiwell plates (96 well plates)
• The cells are treated with a various concentration of drug for specified duration
• After MTT dye is added in each well and plates are incubated at 37° C for 4 hrs in a
CO2 incubator.
• The plates are taken out from the incubator and darkblue colored formazan crystal
are thoroughly dissolved in DMSO(dimethyl sulphoxide) in room temperature.
• The plates are then read on a ELISA reader at 570nm
• To calculate the percent cell viability with respect to control is calculated .
8
The Power of PowerPoint | thepopp.com 9
 Evaluation

The most common routine method for cell counting which is e

fficient and accurate is with the use of a haemocytometer.
• % cell viability =(OD of treated cells/ OD of control cells) × 100

10
XTT
(2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has be
en processed to repulse into other tetrazolium salt assay, to yield more sensitive
and greater effective range. The advantage of this assay is, obtained formazan p
roduct is water-soluble, which will avoid final addition of solubilizing solvent.

MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium), 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.
11
The Power of PowerPoint | thepopp.com 12
 Wsts (Water-soluble Tetrazolium salts)

8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfop
henyl)-2H-tetrazolium), this assay has been developed to obtain
different absorption spectra of the formed purple colored for
mazan. The assay has an advantage over other salt based assay
that they are reduced outside the cells, combined with alread
y 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.
13
 SRB assay (sulforhodamide-b assay)

SRB is an anionic dye and it is aminoxanthene, which can react with basic amino ac
id residues of protein to forms an electrostatic complex under moderately acid co
nditions, which contribute to a susceptible and linear response.

METHOD
• The Sulphorhodamine B assay measures whole-culture protein content, which should be pro
portional to the cell number.
• Cell culture are stained with a protein staining dye, Sulphorhodamine B.
• SRB is a bright pink anionic dye that binds to basic amino acid of cell.
• Unbound dye is then removed by washing with acetic acid.
• During the procedure, the dead cells either lyse or are lost, the amount of SRB binding is propo
rtional to the number of live cells left in a culture after drug exposure.

14
EVALUATION
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.
These all evaluation is depend on the uptake or incorporation of the pink amino x
anthine (negatively charged) dye by amino acids (basic) in the cells. The greater a
mount of dye is taken up by the cell if adequate amount of cell is present. Aft
er washing and fixing, the released dye will give a more acute color and greater ab
sorbance when get lysed. It is rapid, susceptible, sensitive, and inexpensive metho
d for measuring the cellular protein content of the cell.

15
 3H-Thymidine
THYMIDINE INCORPORATION ASSAY

• This assay is the most common assay which utilizes a strategy by the
use of radioactive nucleoside, 3H-thymidine. The new stands of DNA
form during the mitotic division, in which it is consolidated. The limit
of cell division that had occurred by the effect of a test agent is reflect
ed to radioactivity of DNA obtained from assay which can be measur
e by the used of commercially available scintillation kit.
• The advantage of these incorporation assays is related to the direct
measures of proliferation.

16
METHOD
• The overall performance of the assay started from addition of lymphocyt
es for few days with the provocative chemical compound which is related
to growth factor (3H-Thymidine).
• The 3H-Thymidine get incorporated into the newly formed DNA which is
to be synthesized, when the cell get proliferate.
• TCA-precipitation may be used historically for the separation of cell comp
onent. This separation help us in washing of incorporated 3H-Thymidin
e.
• then proceed towards the reaction(solubilization) with 1M NAOH and m
ake it neutral with the addition of HCL(neutralization).
• Then remaining sample was shifted towards scintillation vial for quanti
fication.
• Other cell component and specifically newly synthesis DNA which is to be
process get incorporated into the harvesting step , which get conquer int
o the filtermat, in which buffer will be using as a washing solvent.
• Before reaching to the harvesting step the cell which get adhere will requi
re a trypsin to slacken them.
• Before counting in the microbeta the, solution get dried and scintillation
cocktail is adapted.
17
MEMBRANE INTEGRITY ASSAY
• Possibly the simplest assay for cell death is measurement of plasma membrane in
tegrity. This can be assessed in two ways: The ability of a cell to prevent a fluoresc
ent dye from entering it and the ability of a cell to retain a fluorescent dye within i
t.
• As a cell dies it's plasma membrane becomes permeable allowing fluorescent dyes
present outside the cell to enter it and fluoresce.
• The most common dyes used for this purpose are dyes that label nucleic acids.

18
 DYE EXCLUSION TEST
• This assay is based on the structural integrity of the cells.
• Live cells possess intact cell membranes that exclude certain dyes, such as trypha
n blue, Eosin, or propidium, whereas dead cells would have lost membrane integr
ity.
• Hence they would take up the dyes while the live cells exclude it.
METHOD
• Cell lines are counted, cultured and inoculated in 96 well plates.
• Cells were incubated with different concentrations of test compounds
for 4days.
• Number of cultured cells in different wells were counted using hemo
cytometer after staining with suitable dyes.

19
EVALUATION

20
The Power of PowerPoint | thepopp.com 21
 CLONOGENIC ASSAY

The clonogenic assay determines cell proliferation, it is an in-vitro type o

f cell survival assay. The ability of a cell to proliferate indefinitely is s
aid to be clonogenic, with the ability to form a reproductive and lar
ge colony or a clone. The principle need of the assay is the ability of a c
ell to raise into a colony. The set of at least 50 cell is defined as colony. T
he ability of a single cell to grow into a large colony that can be visualize
d with the naked eye is proof that it has retained its capacity to reprodu
ce. The assay will determine the effectiveness of cytotoxic agents, it c
an also evaluate the cell reproductive death after programming with ion
izing radiation.
22
The Power of PowerPoint | thepopp.com 23
LDH ASSAY
The assay is based on consideration that tumor cells possess high concentration of intracellular
LDH and the cleavage of a tetrazolium salt when LDH is present in the culture supernatant.

24
 Scratch assay (wound healing assay)

A wound healing assay is one of the simple and common assays used to monitor c
ancer cell migration . In a normal wound healing assay, attached cancer cells are e
xposed to tested compound (s) and incubated for 1–2 hours. A scratch is then ma
nually made using a sterile pipette tip or needle and the width of the scratch is rec
orded at different time interval. The effects of a testing drug on invasiveness of cel
ls can also be monitored using this assay. Becker et al. (2016) showed that rocagla
mide can inhibit migration of prostate (PC‐3) and triple negative breast (MDA‐MB ‐
231) cancer cells using the wound healing assay. Although this assay is easy to car
ry out, it is time consuming and requires a large number of cells. Sometimes, dead
cells can be trapped near the edges of scratches, which can interfere with measur
ements of the width of the scratch. However, this assay cannot be carried out with
non‐adherent cells. 25
 Matrigel Assay
Angiogenesis, the process of new blood vessel growth, is fundamental to human health, as it
plays key roles in wound healing and tissue growth. More recently, the Matrigel plug assay h
as become the method of choice of many studies involving in vivo testing for angiogenesis.

Method:
Matrigel containing experimental cells or growth factors is injected subcutaneously, where it solidifies to form a plug. The plug is
recovered after six days in vivo and examined for new blood vessels. Assessment of angiogenesis in the Matrigel plug is achieved
either by scoring selected regions of histological sections for vascular density or by measuring hemoglobin content. These
analytical indices serve merely as an estimate and are not accurate enough to follow the angiogenic progression in vivo.

26
Evaluation:
Angiogenesis was evaluated six days post-implantation by fluorescence-activated cell sorting (F
ACS) analysis of the enzymatically liberated endothelial cells, via labeling with an established en
dothelial-specific marker, CD31. The precise quantification of endothelial cells represents a signi
ficant advancement since it better reflects the efficiency of the angiogenesis process, as well as
provides a specific means to distinguish the effects of varying therapeutic treatments on angiog
enesis.

27
28
FIGURE- Schematic representation of assays and techniques available for the detection of cancer cell viability/proliferation based on cellular enzyme activity, pro
tein levels, DNA synthesis/DNA content, ATP levels and membrane integrity
29
The Power of PowerPoint | thepopp.com 30
31
 1 Carcinogen induced models

2 Viral infection models

In-vivo 3 Transplantation models

Genetically engineered mouse mode

assays
4 ls

5 In vivo hollow fibre assay

32
 CHEMICAL CARCINOGEN MODELS

DMBA induced mouse skin papillomas

• Two stage experimental carcinogenesis
› Initiator – DMBA (dimethylbenz[a]anthracene),
› Promotor – TPA (12-O-tetradecanoyl-phorbol-13-acetate)
• Mice : Single dose – 2.5 μg of DMBA , 5 to 10 μg of TPA in 0.2 ml of acetone twic
e weekly.
• Papilloma begins to appear after 8 to 10 weeks – Tumor incidence & multiplicity
of treatment group is compared with DMBA control group

33
 METHOD

• Mice are topically applied a single dose of 2.5 μg DMBA in acetone, followed by
5-10 μg of TPA in 0.2 ml acetone twice weekly on the same site starting one wee
k after DMBA application.
• Percent tumor incidence and multiplicity of treatment groups is compared with
DMBA control group.
• Drug under test can be administered either topically or oral route.
• The tumor incidence in this model is usually about 100% DMBA controls.
• In repeated topical application of DMBA alone has also been shown to induced
carcinogenesis.
• Drug efficacy is measured as percent reduction in carcinoma incidence, compar
ed with that of carcinogen control.
34
 GENETICALLY ENGINEERED MICE
• Transgenic mice
• Knockout mice

Transgenic mice
The transgenic mouse is the resultant progeny of the pronucleus of a fertilized eg
g that is injected with a foreign gene. This progeny then carries and expresses t
his exogenous gene and passes it on to its descendants. Genes can be transferred
to the pronucleus by microinjection, retroviral infection, or embryonal stem cell (E
SC) transfer. Transgenic animals are excellent models for studying the oncogenic
phenotype that results from the dysregulation of a known gene.
Examples in transgenic mice, which provided invaluable information regarding the
characteristics of oncogenes, include the NF1 gene in the case of neurofibromatos
is, c-fos, N-myc, erb B2, and others.
35
Knockout mice
A knockout is an animal model that is generated by omitting both alleles of a specific gene.
The Nkx 3.1 knockout mice: Nkx 3.1 is a prostate specific tumor suppressor gene. It is essential
for prostate differentiation and function. Loss of function of this gene results in histopathologic
al defects that resemble prostate cancer in humans. This model provides a model for studying
mechanism of prostate cancer initiation as well as to explore the tissue specific features of the
disease.
Homozygous p53 knockout mice: Mutation of p53 tumor suppressor gene is the most frequent
ly observed genetic lesion in human cancer. Over 50% of all human tumors have identifiable p5
3 gene point mutation or deletions. These mice are highly susceptible to spontaneous tumorige
nesis particularly lymphomas.
Brca1 conditional knockout model: Brca1 deletion is induced using Cre Ioxp system by expres
sing Cre under the control of MMTV-LTR or WAP. Animal develop mammary tumor by the age of
10 to 13 months .
36
 VIRAL INFECTION MODELS

Mouse Mammary Tumor Virus (MMTV) was the first mouse virus, isolated at Jacks
on labs as the “nonchromosomal factor” that caused mammary tumors in the C3H
strain of mice.
• Some viruses cause cancer via random integration in certain cells
• Some viruses carry cellular oncogenes
› Abelson murine leukemia virus – Abl
› Moloneymurine sarcoma virus – Raf
• Engineered viruses now used routinely in the laboratory to induce cancer.

37
 TRANSPLANTATION MODELS

Tumor cells or tissues (mouse or human) transplanted into a host mouse.

• Ectopic – Implanted into a different organ than the original (typically subcutaneo
us or kidney capsule)
• Orthotopic – Implanted into the analogous organ of the original tumor.
Advantages :
› Typically cheap, fast & easy to use.
› Not covered by patents

38
 Human Tumour Xenografts
Xenograft models of human cancer play an important role in the screeni
ng and evaluation of candidates for new anticancer agents. The models,
which are derived from human tumor cell lines and are classified accordi
ng to the transplant site, such as ectopic xenograft and orthotopic xenog
raft, are still utilized to evaluate therapeutic efficacy and toxicity. The me
tastasis model is modified for the evaluation and prediction of cancer pr
ogression. Recently, animal models are made from patient-derived tumo
r tissue. The patient-derived tumor xenograft models with physiological
characters similar to those of patients have been established for person
alized medicine. In the discovery of anticancer drugs, standard animal m
odels save time and money and provide evidence to support clinical trial
s.
NUDE MICE: hairless, lacks normal thymus gland, has defective immune
system. 39
In Vivo Hollow Fibre Assay

• In vivo screening tool implemented in 1995 by NCI

• 12 human tumor cell lines (lung, breast, colon, melanoma, ovary,
and glioma
• Cells suspended into hollow polyvinylidene fluoride fibers implan
ted IP or SC in lab mice
• After in vivo drug treatment, fibers are removed and analyzed in
vitro
• Antitumor (growth inhibitory) activity assessed

40
The Power of PowerPoint | thepopp.com 41
 References

• Jung J. Human Tumor Xenograft Models for Preclinical Assessment of Anticancer Drug Development. Toxicological Research. 2014;
30(1):1-5. doi:10.5487/TR.2014.30.1.001.
• Ashish A., Sonia SY, Mark AH, and Minas TC., Pressure Related apoptosis in Neuronal Cel Lines., Journal of Neuroscience Research 2
000 60: 495-503
• In vitro assays and techniques utilized in anticancer drug discovery Meran Keshawa Ediriweera Institute of Biochemistry,
Molecular Biology and Biotechnology, University of Colombo, Colombo 03, Sri Lanka
• Jung, Joohee. “Human Tumor Xenograft Models for Preclinical Assessment of Anticancer Drug Development.” Toxicological Researc
h 30.1 (2014): 1–5. PMC. Web. 19 Sept. 2018.
• ANIMAL MODELS OF CANCER: A REVIEW by Archana Chavan in International Journal of Pharmaceutical Sciences and Research 4
(1):19-28 · January 2013
• J Immunol Methods. 2009 Mar 15;342(1-2):78-81. doi: 10.1016/j.jim.2008.11.016. Epub 2008 Dec 25. Matrigel cytometry: a novel
method for quantifying angiogenesis in vivo.

The Power of PowerPoint | thepopp.com 42

Thank You!