Question

Doctor Amanda Abraham is an expert cancer biologist. She received a 50 mg of newly synthesised chemical compound from an international collaborator to test its in vitro anticancer activity. Collaborator provided following information about the compound:

Physical state: odourless white crystalline solid Molecular weight: 500 Da
Solubility: very little solubility in water , but the compound is highly soluble in Dimethyl Sulfoxide ( DMSO )

Please help Amanda in advising on following 7 aspects:

1. Suggest an assay to be used for assessment of cytotoxicity of compound and provide reason for this selection.
2. What cells/cell lines should be used?
3. What experimental controls should be used and why?
4. What treatments doses should be tested and why?
5. What exposure times should be tested and why?
6. How would you know if the compound possess any anticancer activity or not?
7. Please outline the proposed assay plan for vitro cytotoxicity/anticancer activity.

Answer 1

1. Cytotoxicity assay such as MTT can be used to to study cytotoxicity of the compounds. This is very simple, quick, affordable and reliable assay to study anti cancerous activity of compounds.

2. Depending upon availability, various cancerous cell lines such as leukemia (K-562, MOLT-4,REH. Nalm-6 etc) Lung (A549, A-427) and Kidney (A498) can be used.

3. Experiment control

A. Toxicity assay only with DMSO: As drug is dissolved in the DMSO therefore it is important to study effect of only DMSO to the cell line.

B. Without DMSO and drugs: To check the viability of cells under experimental conditions.

4. and 5. Treatment doses and exposure time is depends on the half life of the drug. In lab, generally two treatment doses after every 24 hours for the 48 hours is used. However, it varies from drug to drug.

6. You can compare the growth and proliferation of cancerous cell lines in the presence and absence of drug (control). If compound possess anti cancerous activity then one must expect the decrease in the proliferation and growth as compared to the control cell line

7. Followings steps can be proposed for the MTT assay

1. Incubate the cell line in the presence and absence (control) of drug for 24 hrs

2. Monitor under microscope, if the drug is toxic then cells will die and will float on the media.

3. Add the MTT reagent and incubate for 15 min in dark

4. If the compound is toxic then lower number of cell will grow. If the drug is non toxic but showing anticancer activity, then active cells will be present but their number is less than control cell (cells grown without drug) and more than if the drug is toxic.

5. In MTT assay, enzyme dehydrogenases break down the MTT into violet colour crystal which will be dissolved in DMSO. The intensity of the violet colour is directly proportional to the live cell number.

6. Measure the intensity of violet colour under spectrophotometer.