screening methods for anticancer activity

1. PRESENTED BY,
SHAFNA HUSSAIN
FIRST YEAR MPHARM
JAMIA SALAFIYA PHARMACY COLLEGE

3.  Cancer is uncontrolled division of cells.
 It has the ability to invade other tissues either by
direct growth into adjacent tissue , through
invasion or by implantation by metastasis.
 Metastasis is the stage in which cancer cells are
transported through the bloodstream to sites far
removed from the original site.
 Treated with a combination of surgery ,chemo
therapy & radio therapy.
 Many non toxic herbs are used now a days thus
reducing toxic effects of chemo & radio therapies.

4.  Madagascar periwinkle ( Catharanthus roseus
or Vinca roseus )
 Taxus baccata :Arrest multiplication of
cancerous cells by cross linking.
 Curcumin :Inhibit the growth of cancer by
preventing pdn of harmful eicosanoids.
 Vinca alkaloids & podophyllin : Act by
breaking the cytoskeletal organelle.

5. TWO MODELS ARE THERE :
 INVITRO METHOD
 INVIVO METHOD

6.  Can be screened about 85000compounds in a
short term assay.
 Provide information about the fun of specific
organs &how a toxic agent can adversely affect
them.
 More specific
 Accurate
 Less time consuming

7. THREE CATEGORIES
1.To indicate drug has altered cell viability.
a) Trypan blue dye uptake
b) Lactate dehydrogenase (LDH) leakage.
c) Neutral red dye retension.
d) Propidium iodide or Flourescent marker attachment to double-stranded
nucleic acids.
2.To assess if the drug has an effect on cell growth & cell
proliferation.
a) [³H]thymidine uptake assay.
b) Total DNA synthesis measured by [1251] Udr uptake.
c) Cell cycle analysis with propidium iodide.
3. To assess whether the toxicant has affected cellular
metabolic processes.
a) MTT dye conversion by mitochondria.
b) Alamar blue reduction by respiring cells.
c) Fluorescent dye loss.

8. 1.Trypan blue dye uptake:
 Simplest & cheapest method
 Qualitative & indicates only if a cell is active
PRINCIPLE:
 Based on the ability of a cell with an intact plasma
membrane, to exclude trypan blue dye which is
impermeable to healthy cells but can permeate through
compromised membrane of dying cells.
 Can be counted manually using a haemocytometer.
 Detects loss of memberane integrity during later
stages of cell death.

9.  PROCEDURE:
1. 20µl cells +20µl trypan blue dye in an Eppendroff tube.
2. Mixture is stand for 5 min.
3. Surface of heamocytometer is cleaned with 70% alcohol &
cover slip is placed.
4. Each chamber is allowed to fill by capillary action.
5. Total no: of stained cells & total no: of cells in the four 1 mm
squares are counted.
6. Cells which touches the top & left middle line are included.
7. The cells in the 4 corner squares in both chambers are
counted
Cells/ml =avg cell count per sq × dil. Fac × 10
% Cell viability =[total viable cells (unstained cells) ÷ total
cell]×100
Dil fac=[vol of sample+vol of diluent]÷vol of sample

10. [³H] Thymidine uptake to cell growth:
 Relatively complex
 Sensitive procedure
 Requires use of radio-labelled chemicals.
 Used as an indication of cytotoxicity because toxicants
can decrease the rate of cell proliferation.
 Used as an indicator of cell proliferation as
[³H]thymidine incorporates to the nucleic acids of
proliferating cells.

11. PROCEDURE:
1. 1×10 cells are placed wells of a flat bottomed 24well micro titer plate.
2. Allowed to settle for 24 hrs.
3. Medium from the wells are replaced with 1ml serum free chemically
defined medium ,24-48 hrs before treating cells with test cpds.
4. 50µl drug/vehicle control is added.
5. 50µl/well thymidine soln is added for last 2hrs of incubation with the
test substance & incubated @ 37˚c.
6. Reaction is stopped by placing on ice bath & cells are washed 3 times
with 1ml ice cold serum free medium
7. Then carefully removed from the medium by vaccum aspiration.
8. 1ml of ice cold trichloro acetic acid is added to each well.
9. The plates are left on ice for 10 min to allow precipitation of DNA &
protein.
10. 1ml 10%TCA is added & left ice for again 5min.
11. Washed again with TCA.
12. Then TCA is removed.
13. Precipitated protein is solubilised with 0.5ml of 0.3N sod.hydroxide by
incubating for 30-60 min @ room temp.
14. 250µl is transferred to a mini- scintillation vial, with 5ml scintillation fluid
and count.

12. MTT dye conversion by mitochondria:
[3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide]
 A std colorimetric assay for measuring cellular proliferation.
 Quantitative & very sensitive test.
 Growth &death rates can be measured easily.
PRINCIPLE:
Based on the ability of a mitochondrial dehydrogenase enzyme
present in viable cells to cleave the tetrazolium rings of the
pale yellow MTT & form dark blue formazan crystals which
are largely impermeable to cell memb thus resulting in its
accumulation with in healthy cells.

13. PROCEDURE:
o 500-10000 cells plated in 200µl media per well in a 96 well
plate.
o 8 wells are kept as blank.
o Incubated (37˚C,5% carbon dioxide) overnight.
o 2µl drug + DMSO is added to each well.
o Placed on a shaking table @150rpm for 5min.
o 20µl MTT soln is added to each well.
o Incubated 2hr @ 37˚C.
o 100µl of lyses buffer is added to each well.
o Incubate overnight @ 37˚C.
o Quantification done on a micro plate reader @ wavelength
600nm.
% Cell viability= (absorbance of treated grp ÷ absorbance of
control grp) × 100.

14.  Common models are rats.
 Models are ;
A. Models of spontaneous & chemically induced
colorectal cancer (CRC).
B. Models of chemically induced breast cancer.
C. Hollow fibre assay.
D. Xenograft tumour models.
E. Orthotropic & metastasis tumour models.
F. Autochthonous models.
G. Genetically engineered cancer models.

15.  Frequently used carcinogens are dimethylhydrazine
(DMH), its metabolite azoxy methane
(AOM),MNNG,MNU.
 71 Sprague-Dawley/Wistar rats (20mg/kg/s.c.)- 20
wks injected with DMH .
 Barium enema is performed to see the size of tumour.
 Animals divided to 3 grps & subjected to 5-FU, 1(2-
tetra hydro furyl)-5 FU(FT) & mixture of UFT & FT.
 After 5 wks barium enema was repeated.
 Response was assessed on basis of tumour doubling
time.
 Response rate 5-FU, FT, UFT were 25%, 33%,36%.

16.  1-methyl-1-nitrosourea (MNU) is the carcinogen used to
induce adenocarcinomas in the rat΄s mammary glands.
 It induced in female Sprague-Dawley rats by the
i.p.administration of MNU (50mgMNU/kg bodywt) @21
days of age.
 Divided into 4 grps.
• Ad libitum fed.
• Restriction of calorie intake 90%, 80%, 60% of ad libitum
intake.
• Energy restriction (ER) reduces the ductal extension of the
mammary gland into fat pad.
• Animal΄s breast density is predictive to its carcinogenic
response.
• ER inhibits cell proliferation & induce apoptosis in
premalignant &malignant mammary gland lesions.

17. THANK YOU