Screening methods of anticancer
Drugs
Introduction:
Cancer terminology
Cell cycle
Apoptosis
ROS etc.
Normally, when cells become old or damaged,
they undergo programmed cell death, i.e.
apoptosis and new cells replace them to fulfill
the need of the body. Whereas in cancer case,
this orderly process is disrupted and as the
cells become old or damaged, instead of
dying, they survive. These cells can divide
into less specialized cells (tumor) and are
able to ignore the signals which stop division
or by which apoptosis is started in normal
cells
Types
Depending on the potential clinical behavior, a tumor
can be divided into two categories:
Benign and malignant.
Benign tumor, termed by attaching the suffix “-oma” to
the type of cells in which the tumor arises, for
example, fibroma, adenoma, and papilloma, will
remain localized and the patient generally survives to
local surgical procedures.
Malignant tumors of solid mesenchymal tissues are
called sarcomas; for example, cancer of fibrous tissue
is known as fibrosarcoma while those ascending from
the mesenchymal cells of the blood are leukemias or
lymphomas.
However, malignant tumors of epithelial cells are
termed as carcinomas irrespective of the origin of
tissue (as the epithelial cells are originated from
three germ cell layers).
Therefore, malignant tumors arising in the renal
tubular epithelium (mesoderm), skin (ectoderm),
and lining epithelium of the gut (endoderm) are all
carcinomas.
Carcinomas are subdivided further.
Carcinomas with glandular pattern, squamous
cells, and undifferentiated cells are called
adenocarcinomas, squamous cell carcinomas, and
undifferentiated carcinoma, respectively.
cancer was primarily considered a disease of
uncontrolled cell division, by measuring the
regression in tumor size, identification of a
cytotoxic or an antiproliferative compound was
considered as the main objective endpoint of
efficacy of a compound in preclinical and clinical
anticancer drug development for decades.
For rapid screening of new anticancer
compounds, murine models of rapidly growing
cancer were developed, for example, sarcoma
180, carcinoma 755, and L1210 mouse leukemia
model which were later replaced by the P388
murine leukemia model.
Several clinically important anticancer agents
such as methotrexate, actinomycin D, 6-
marcaptopurine, 5-fluorouracil were identified
using these murine models;
however, successes were achieved mainly in
the cases of rapidly growing cancers, e.g.
lymphomas, childhood leukemia, and germline
tumors while relatively limited successes were
seen in the treatment of the slow-growing
common solid tumors of the adults, e.g. lung,
breast, and colorectal cancers.
Content:
Introduction of Cancer.
Screening methods:
A. In-vivo models:
1. EAC model.
2. N-Methyl-N-Nitrosourea induced mammary tumour.
3. Solid tumor model using DLA cell ( Daltons Lymphoma Ascitic)
4. DPPH assay
5. Hemolytic assay.
B. In-vitro models:
1. Brine Shrimp Lethality Assay.
2. Trypan blue exclusion assay.
3. MTT assay
4. XTT assay
5. SRB assay.
In vitro Screening Methods
Large-scale screening using animal systems as
practiced in the past is highly unethical and in certain
countries such as Europe and India is strictly regulated.
The Committee for the Purpose of Control and
Supervision of Experiments on Animals in India
regulates the screening using animals and has
inculcated the credo of 4Rs: Replacement, reduction,
refinement, and rehabilitation of animals used in
experimentation.
Therefore, in vivo evaluation of anticancer drugs
usually preceded by either cellular or target-based
high-throughput assays.
Cell Line Prescreens
Cellular screens in cancer research mainly consist of
permanent human tumor cell lines; most suitable test
system, in terms of management, because of their
immortal nature and reproducible growth behavior.
A panel of sixty different human tumor cell lines from
nine different types of cancer (leukemia, nonsmall cell
lung cancer, colon cancer, brain cancer, melanoma,
ovarian cancer, renal cancer, prostate cancer, and
breast cancer) constitute the NCI in vitro primary
screen, against which compounds are tested over a
defined range of concentrations to determine the
relative growth inhibition or cytotoxicity against each
of the cell line.
In the year 1995, to weed out inactive agents and to
find out potential agents from the pool of compounds,
NCI adopted an in vitro prescreen which consists of
MCF-7 breast, H460 lung, and SF268 brain cancer cell
lines. This prescreen is used to check the presence of
toxicity at a drug concentration of 10−4 M and could
eradicate a large fraction of the inactive agents but
retain active agents for multidose 60-cell-line testing.
The efficiency of 60-cell-line screen was increased with
limited loss of information as approximately 50% of the
agents could be removed without a significant drop in
the capability to identify active anticancer agents.
Cell Growth Determination
Cell growth can be determined by various
accepted methods that utilize the exclusion of
certain dyes by live cell membranes. Selection
of a particular method depends on factors
such as minimum number of cells required,
sensitivity, speed, and ease of handling. The
various preferable methods for cytotoxicity
studies are 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium (MTT) assay,
sulforhodamine B (SRB) assay, propidium
iodide (PI) assay, and luciferase assay.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Assay
The tetrazolium salt is an electron acceptor which is
reduced to a colored formazan by accepting electrons
from NADH, NADPH, and other oxidized substrates or
appropriate coenzymes. The reduction of MTT occurs
at multiple cellular sites including mitochondria.
MTT assay is simple, rapid, and convenient, but the
endpoint of assay is influenced by various factors such
as concentration of D-glucose in the culture medium at
the time of spectrophotometric assessment.
Furthermore, the kinetics of MTT formazan production
vary in a cell line-specific manner and quantitation of
drug cytotoxicity is influenced by the length of
exposure to MTT
In-vitro assay:
1. MTT assay method:
Principle of assay:
1. This is colorimetric assay that measures the reduction
of 3-(4,5-Dimethylthiazol-2 yl)-2,5 diphenyl tetrazolium
bromide by mitochondrial succinatedehydrogenase.
2. MTT enters & passes into mito. where it is reduced to
an insoluble coloured (Dark purple) formazan pdt.
3. The cells are then solubilised with formazen reagent &
meseure spectrophotometrically.
Requirements:
Procedure:
1. The cell count is adjusted to 3 lac cells/ml using medium
2. Pre-incubate cells- 6.5% CO2 incubator at 370C for 3 hr.
3. Then cells will be seeded at conc. of 5X104 cells/well in 100µl
medium (incubation=370C, 3hr.)
4. Then add diff. Conc. of Test Comp, Std. to respective wells
5. After 72 hr. (inspecting cells granularity, shrinkage, swelling)
6. Then add 10µl MTT dye in each well(Shake & incubate for 4 hr. at
370C. 5% CO2 incubator)
7. Supernatant removed & 100µl isopropanol added in each plate to
solubilize formazen
8. Absorbance measure on Microplate reader at 590 nm with
reference filter 620 nm.
Evaluation:
Measure cytotoxicity:
(Control - Blank) - (Test - Blank)
% cytotoxicity=------------------------------------------------X100
(Control - Blank)
2. Trypan Blue Exclussion assay:
Trypan blue is an AZO dye derived from Toluidine.
It is extensively used for assessment of viability of
cells
It works on principle of cell permeability
dependent dye exclusion.
Normal cells= Intact cell membrane
Dead cells/ Late phase Apoptosis= Permeable to dye
Time sensitivity is more
Requirements:
Precedure:
Place 50µl of cell Susp. In vial
Add equal parts of 0.4% Trypan blue to cell Susp. to
obtain 1,2 dilution. (Mix well)
Incubate mix. In 7% CO2 incubator for 3hr. At 370C
Load the cell Susp. On Haemocytometer on stage of
light microscope focus on cells
Count 16 squares of Haemocytometer.
Note down viable , Non-viable cells
Evaluation:
Calculate the % of viable cells-
NO. of viable cells
% Cytotoxicity---------------------------X 100
Total No. of cells
Sulforhodamine B Assay
SRB assay is a rapid, sensitive, and inexpensive
method, which utilizes a bright pink anionic dye, that
binds electrostatically to the basic amino acids of
trichloroacetic acid fixed cells.[18]
The protein-bound dye is extracted with Tris base
(tris (hydroxymethyl) aminomethane), after washing
off the unbound dye, and thus, protein content can be
quantified indirectly spectrophotometrically.
This method is suitable for an ordinary laboratory
as well as for a very large-scale antitumor
screening.[20]
The endpoint of SRB assay is nondestructive, not
time critical (stable) and comparable with other
fluorescence assays.[21]
Although this labor intensive method (several
washing steps) offers practical advantage of high
flux screening of anticancer drugs, results obtained
with SRB assay are not significantly different from
the results obtained with the MTT assay
In vivo Screening Methods
The cell line screens, although provide faster results
in a cost and time effective manner yet only cytotoxic
compound can be identified by these screens. Many
new anticancer agents (molecule targeted cytostatic
drugs) would be considered inactive by these
screens.
Furthermore, in vitro cytotoxicity is only one of the
many factors which play critical role in clinical
efficacy of a given compound. Factors such as
physiochemical properties, pharmacokinetics, and
toxicological assessments are equally important
along with the outright effectiveness of the
anticancer agent.
Regardless of an agent's affinity to its
targets, poor aqueous solubility, inadequate
bioavailability, and metabolic instability can
lead to the failure of the compound at clinical
trial.
Moreover, in vitro cell line screens are
inadequate in the evaluation of the off-target
effects, which may contribute to the potency
or toxicity, of a novel agent.
In vivo tumor models used in preclinical drug
development express the targets for the new
generation anticancer agents and are both disease
focused and target based.
In vivo tumor models include either human tumor
explants/xenografts or specifically bred transgenic
mice
In vivo tumor models are proven for predicting
clinical effects when compared with in vitro assays
because multiple parameters such as
pharmacokinetics, efficacy, and therapeutic index
of drug candidates are assessed simultaneously.
Screening methods:
In- vivo method:
EAC Model:
Purpose & Rationale:
The cells are maintained in vivo in Swiss albino mice
by I.P. transplantation.
EAC cells aspirated from the peritonial cavity of
mice, washed with saline & given I.P. to develop
ascetic tumour.
Requirements:
Experimental design:
15 Days Model:
Sr. Groups Treatment
No.
1. Group I Normal with Sodium CMC Susp. (0.1%)
2. Group II Induced EAC cell (2X106) with Sodi.
CMC Susp.
3. Group III EAC Cell + Test comp.
4. Group IV EAC Cell + Test comp.
5. Group V EAC cell Susp. + Cyclophosphamide
Procedure:
Result & Evaluation:
Evaluation of Hematological parameters
Cell viability count
Packed cell volume count
2. N-methyl-n-nitrosourea induced mammary
tumour.
Purpose & Rationale:
1. To determine chemopreventive property of test
compound
2. Herbal plant screening purpose.
3. Antioxidants mainly evaluated
Requirements:
Experimental design:
N=10 animals, 15 days model
Procedure:
Select animals randomly divided as per groupings.
Animals of normal group receive no any treatment
for 21 weeks.
Test group receive investigating sample+ NMU
Accordingly other groups receive treatments .
At last day, Parameters will be measured
Evaluation:
Parameters to be measured:
1. Body wt. twice a week:
2. Tumorological Parameters:
Tumour parameters: Tumour incidence: Tumour yield:
Tumour volume: Tumour burden: Tumour size:
Tumour mass
3. Hematological parameters:
RBC, WBC, Hb, DLC etc.
Tumor Xenograft Model
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