Invitro Screening of Anti-Cancer Drugs.

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Various models for screening anticancer compounds by invitro method.

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Invitro Screening of Anti-Cancer Drugs.

  1. 1. 1 J.K.K. Munirajah Medical Research Foundation College of Pharmacy, B.Komarapalayam, Nammakal. 2 PGP College Of Pharmaceutical Sciences And Institute Nammakal, Tamilnadu
  2. 2. Cancer is a term used for diseases in which abnormal cells divide without control and are able to invade the adjacent and distant tissues from the normal cells of the body. Due to lack of its pathophysiology, the challenging task at this moment is to identify the novel methods which can identify and develop molecules. Even though in vivo models provide more predictable results, the NCI has planned an in vitro screening prior to in vivo testing of a therapeutic agent for reducing the consumption, usage & death of animals and for testing a large number of compounds in small quantities. Those screened compounds will be recommended for the next stage of testing in animals. <ul><li>Carcinoma </li></ul><ul><li>Sarcoma </li></ul><ul><li>Leukemia </li></ul><ul><li>Lymphoma and myeloma </li></ul><ul><li>Central nervous system cancers </li></ul>
  3. 3. PROPERTIES ASSAYS ENZYMATIC PROPERTY MTT ASSAY MEMBRANE INTEGRITY DYE EXCLUSION TEST DNA CONTENT FLUORESCENT ASSAY PROTEIN CONTENT SRB ASSAY CLOGENIC PROPERTY CLOGENIC ASSAY
  4. 4. <ul><li>Tumor cell lines derived from several cancer types </li></ul><ul><li>lung, colon, melanoma, renal, ovarian, brain, and leukemia. </li></ul><ul><li>Quality-assurance criteria </li></ul><ul><li>Adaptable to a suitable growth medium </li></ul><ul><li>Show reproducible profiles for growth and drug sensitivity </li></ul><ul><li>The lines were prepared and cryopreserved using reagents such as DMSO( di methyl sulfoxide ) which preserve the cell during freezing. </li></ul><ul><li>DMSO is toxic at room temperature. </li></ul><ul><li>THAWING OF THE CELLS </li></ul><ul><li>Bringing the freezed ampoule to room temperature by slow agitation </li></ul><ul><li>The freezed cryovials plunged into the water bath & is rapidly thawed until it gets liquefied. </li></ul><ul><li>The solution is centrifuged with saline for 10 minutes to remove the DMSO </li></ul><ul><li>The saline is discarded and aliquot is taken for cell counting ,cell viability </li></ul><ul><li>and for sub culturing. </li></ul>
  5. 5. <ul><li>MTT assay a quantitative colorimetric assay for measuring cellular growth, cell survival and cell Proliferation based on the ability of living cells. </li></ul><ul><li>Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) a tetrazolium salt is reduced to purple formazan by mitochondrial dehydrogenase of living cells in which tetrazolium ring gets cleaved in mitochondria . </li></ul><ul><li>PROCEDURE </li></ul><ul><li>Cells from particular cell lines in log phase of growth are trypsinised, Check the cell viability through haemocytometer. </li></ul><ul><li>Adjust to appropriate density in suitable medium and inoculated in multiwell plates (usually 96 well microtiter plates). </li></ul><ul><li>Cells were treated with various Conc. of test compounds and Incubated the plate at 37 °C in 5% CO 2 /95% humidified air (1-4 days). </li></ul><ul><li>Cultures were taken out and 10 μl of MTT was added (5 mg/ml) into each well and incubated for 4hrs. </li></ul>
  6. 6. 5. Centrifuge the plate, discard the supernatant and precipitated formazan salt was dissolved in 100 μl of isopropenol/DMSO. 6. The plate samples were read at 570 nm microtiter plate reader. IC 50 of drugs can be determined by counting the viable cells. % Cell viability Absorbance of treated cells (%MTT reduction) Absorbance of untreated cells 100
  7. 7. <ul><li>This assays relayed on the structural integrity of the cells. </li></ul><ul><li>Live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells would have lost membrane integrity. </li></ul><ul><li>Hence they would take up the dyes while the live cells exclude it. </li></ul><ul><li>Cell lines are counted, cultured and innoculated in 96 well plates as above. </li></ul><ul><li>Cells were incubated with different concentrations of test compounds for 4days. </li></ul><ul><li>Number of cultured cells in different wells were counted using hemocytometer after staining with suitable dyes . </li></ul>No. of viable cells Total no of cells (viable+dead) 100 % Cell viability A fluorescent stain used for labeling DNA in which the replicating DNA will incorporate the dye. Resulting fluorescence is measured by flow cytometry/ florescent microscopy.
  8. 9. <ul><li>SULPHORHADAMINE B assay measures whole protein content which is proportional to the cell number. </li></ul><ul><li>SRB – a bright pink anionic protein staining dye that binds to the basic amino acids of the cellular proteins. </li></ul><ul><li>Cell lines are counted, cultured and innoculated in 96 well plates. </li></ul><ul><li>After incubation with different concentrations of test compounds, the cell cultures are stained with SRB dye. </li></ul><ul><li>Washing with CH 3 COOH removes the unbound dye and the protein bounded dye is extracted using Triss base and optical density is determined by 96-well plate reader. </li></ul>such as CLONOGENIC assay which measures the growth inhibiting activity by counting the Tumour colonies of a single cell suspension grown in Agar/methyl cellulose medium. CELL COUNTING assay , by counting the tumor cells using haemocytometer.
  9. 10. <ul><li>Reduce the usage of animals. </li></ul><ul><li>Testing the ability of the compound to kill the cells by taking the advantage of various properties of cell. </li></ul><ul><li>Able to process a larger number of compounds quickly with minimum quantity. </li></ul><ul><li>Range of concentrations used are comparable to that expected for in vivo studies. </li></ul><ul><li>Difficulty in Maintaining of cultures. </li></ul><ul><li>Show Negative results for the compounds which gets activated after body metabolism and vice versa. </li></ul><ul><li>Impossible to ascertain the Pharmacokinetics. </li></ul>

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