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Anti-Cancer Drugs

Screening & Bioassay

Introduction:

Cancer is a term for diseases in which abnormal cells divide without control and can invade nearby

tissues and also spread to other parts of the body.

There are several main types of cancer (Carcinoma/Sarcoma/Leukemia/Lymphoma/CNS cancers).

Cancer is a genetic disease—that is, it is caused by changes to genes that control the way our cells

function, especially how they grow and divide.

Causes:

Changes (mutations) to the DNA within cells.

Errors in the instructions can cause the cell to stop its normal function.

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Anti-cancer Drugs:

There are several major classes of anticancer drugs,Include: (alkylating agents, antimetabolites, natural products, and hormones).

The term chemotherapy frequently is equated with the use of anticancer drugs.

History:

In 1940s Mechlorethamine (alkylating agent) one of the first to treat Lymphoma.

. In 1956 Methotrexate (antimetabolite) became the first drug to cure a solid tumour, and the following year 5-fluorouracil was introduced.

The specificity of anticancer drugs plays an important role in reducing the severity of side effects associated with the drugs’ use.

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Methods of Induction

Examples of Models:

Polycyclic aromatic hydrocarbons induce digestive system cancer in mice.

Mammary tumor virus (MMTV) or other oncogenic viruses and mutations.

Tobacco-specific carcinogenic compounds.

Some chemical agents: (Cadmium/Arsenic/Abestos)

Radiation offers another route of mechanism:

Ultraviolet radiation

Ionizing radiation

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How can we induce cancer in lab rats?

The dominant technique is ―xenografting”,

The site of injection can vary with the particular cancer type.

The lab animal generally has to be immunocompromised.

Advantages & Disadvantages.

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SCREENING OF ANTI CANCER DRUGS

IN VITRO Method :

1)Dye Exclusion Test:

Parameter: the structural integrity of the cells.

The Method.

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1. Cell lines are counted, cultured and innoculated in 96 well plates as above. 2. Cells were incubated with different concentrations of test compounds for 4days. 3. Number of cultured cells in different wells were counted using hemocytometer after staining with suitable dyes. %cell viability = ( no.of viable cell / Total no.of cells (viable+dead) ) ×100

2)Tetrazolium Salt Assay:

Parameters: viability, proliferation and activation of cells.

The Method.

It is performed to determine the Enzymatic properties.

1-Cells from particular cell lines in log phase of growth are trypsinised,

2- It is counted in a hemocytometer and adjusted multi well plates (96 well plates) The cells are treated with a various concentration of drug for specified duration:

3-After MTT dye is added in each well and plates are incubated at 37° C for 4 hrs in a CO2 incubator.

4-The plates are taken out from the incubator and dark- blue colored formazan crystal are thoroughly dissolved in DMSO in room temperature.

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

6-To calculate the percent cell viability with respect to control is calculated

.

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3)The Sulphorhodamine B assay:

Parameter: The whole-culture protein content.

The Method.

1-Cell culture are stained with a protein staining dye, Sulphorhodamine B.

2-SRB is a bright pink anionic dye that binds to basic amino acid of cell.

3- Unbound dye is then removed by washing with acetic acid.

4-During the dead cell either lyse or are lost during procedure,the amount of SRB binding is proportional to the number of live cells left in a culture after drug exposure.

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Advantages of in vitro method .

• Reduce the usage of animals

• Less time consuming, cost effective & easy to manage

• Able to process a larger number of compounds quickly

with minimum quantity.

• Range of concentrations used are comparable to that

expected for in vivo studies.

Disadvantages:

• Difficulty in Maintaining of cultures.

• Show Negative results for the compounds which gets

activated after body metabolism and vice versa.

Impossible to ascertain the Pharmacokinetics..

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In Vivo Methods

1)Chemical carcinogen model:

DMBA induced mouse skin papillomas

Two stage :

› Initiator – DMBA (dimethylbenz[a]anthracene),

› Promotor – TPA (12-O-tetradecanoyl-phorbol-13- acetate)

The Method.

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Method

1- 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 week after DMBA application.

2- Percent tumor incidence and multiplicity of treatment groups is compared

with DMBA control group.

3-Drug under test can be administered either topically or oral route.

4- The tumor incidence in this model is usually about 100% DMBA controls.

5- In repeated topical application of DMBA Alone has also been shown to

induced carcinogenesis.

6-Drug efficacy is measured as percent reduction in carcinoma incidence,

compared with that of carcinogen control.

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2-)VIRAL INFECTION METHOD

Some viruses carry cellular oncogenes

Abelson murine leukemia virus – Abl

Moloneymurine sarcoma virus – Raf

Mouse mammary tumor virus

3-)Transplantation Methods

Ectopic – Implanted into a different organ than the original (typically subcutaneous or kidney capsule)

Orthotopic – Implanted into the analogous organ of the original tumor.

Advantages :

Cheap, fast & easy to use.

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4) IN VIVO Hollow Fiber Assay

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―James P. Allison‖ of the United States and ―Tasuku Honjo‖ of Japan.

Their achievement (key feature of the immune system).

The drugs based on their work ―checkpoint inhibitors‖.

Remarkable remissions in people with lung and skin cancers.

Treatment of cancer patients.

17 2018 Nobel Prize in Medicine:

1. Almeida C.A. (2010) Cancer: basic science and clinical aspects. Wiley Blackwell,

London.

2. Hanahan D., Weinberg R.A. (2000) The hallmarks of cancer. Cell100, 5770.

3. Altman R., Sarg M. (2000) The cancer dictionary. Facts on File, New York.

4. Berg K., Zhai L., Chen M. et al. (1994) The use of watersoluble formazan

complex to quantitate the cell number and mitochondrial function of Leishmania major

promastigotes. Parasitology Research80, 235-239.

5. Malich G., Markovic B., Winder C. (1997)The sensitivity and specificity of the MTS

tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell

lines. Toxicology124, 179-192.

6. Matesic L., Locke J.M., Bremner J.B. et al. (2008) N-phenethyl and N-naphthylmethyl

isatins and analogues as in vitro cytotoxic agents.

7. https://med.stanford.edu/news/all-news/2018/01/cancer-vaccine-eliminates-tumors-

in-mice.html

18 References:

Made By: ID

Marihan Samir 1141157

Norhan saber 1141429

Fady medhat 1141068

Antony sameh 1141094

Kerolos ayman 1141024

Nihal amin 1091100

Nour adly 1091218

Mostafa elgendy 1121304

Hassan elsaed 1121208

Hossam diab 1141087

Youssef samer 1141098

Group: 2B

Under supervision of : Dr : Nahed Raslan

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