SCREENING METHODS OF CANCERBy

Arijit ChakrabortyM.Pharm

(Pharmacology)

What is Cancer?

Cancer is a disease which characterized by uncontrolled proliferation of cell that have transformed from the normal cells of the body.

CAUSE: • External Factors – chemicals, radiation,

viruses, and lifestyle• Internal Factors – hormones, immune

conditions, and inherited mutations• Theories

– Cellular change/mutation theories– Carcinogens– Oncogenes/ protooncogenes

Factors Believed to Contribute to Global Causes of Cancer

Methods

In Vitro: 1. Tetrazolium salt assay. 2. Sulphorhodamine B assay. 3. 3H-Thymidine uptake. 4. Dry exclusion test. 5. Clonogenic test. 6. Cell counting assay. In Vivo: 1. DMBA-induced Mouse Skin Papillomas. 2. DMBA-induced Rat Mammary Gland

Carcinogenic. 3. MNU-induced Tracheal Squamous cell

Carcinoma in Hamster. 4. Azoxymethane-induced Aberrant Crypt

Foci in Rat.

Tetrazolium salt assay (IN VITRO)

It is performed to determine the Enzymatic properties.

The cell from a particular cell live when in log

phase a trysinized.

It is counted in a homocytometer and adjusted multiwell plates (96 well plates)

The cells are treated with a various concentration of drug for specified duration

CONT….After MIT 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 the incubator and dark-

blue colored formazan crystal are thoroughly dissolved in DMSO in room temperature.

The plates are then read on a ELISA reader at 570 nm.

To calculate the percent cell viability with respect to control is calculated using,

% cell viability =(OD of treated cells/ OD of control cells) × 100

Sulphorhodamine B Assay

The Sulphorhodamine B assay measures whole-culture protein content, which should be

proportional 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 dead cell either lyse or are lost during procedure, the amount of SRB binding is proportional to the number of life cells left

in a culture after drug exposure.

DMBA-induced Mouse Skin Papillomas

This is a classical to stage experimental carcinogenesis model.

Mouse skin is generally most sensitive to

epidermal carcinogenesis.

SENCAR mice are highly sensitive to DMBA induced skin tumor.

DMBA acts as a inhibitor and 12- O- tetradecanoyl- pharbol- 13 acetate is used as

a promoter to induce skin papillomas and Squamous cell carcinomas.

CONT…

Mice are topically applied a single dose of 2.5 µg DMBA in acetone on the shared back,

followed by 5-10 µg of TPA in 0.2 ml acetone twice weekly on the same site starting one

week 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.

DMBA-induced Rat Mammary Gland Carcinogenic

Female Sprague-Dawley are use for this method.

Rats are given single intragastric injection of 12 mg/kg DMBA at 50 days of age.

This dose results in 80-100% incidence of total mammary tumors within 120 days post

carcinogen.

This model can detect the agents/drugs inhibiting carcinogen activation.

DNBA produced capsulated tumors with high incidence.

Drug efficacy is measured as percent reduction in adenoma incidence, percent increase in

adenocarcinoma latency compared with that of carcinogen control.

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