In-Vitro Anticancer Activity by SRB Assay

Dept. of Pharmaceutical chemistry, JKKNCP. _{Page | 71} In order to study the anti tumor activity of new compound, it is important to determine the cytotoxicity concentration of the compound. Cytotoxicity tests define the upper limit of the extract concentration, which is non toxic to the cell line. After the addition of the drug, cell death and cell viability was estimated. The result is confirmed by additional metabolic intervention experiment such as SRB assay.

9.3.1. Materials Required SRB Assay 1. Monolayer culture in log phase

2. Drug extracts (different concentration) 3. 0.45 filter

4. 5mL sterile storage vials

5. Tissue paper, Marker pen, Spirit, Cotton, and Gloves 6. 1mL, 2mL pipettes, Micropipettes and tips

7. Discarding Jar with 1% Hypochlorite solution 9.3.2. SRB Assay introduction

The sulforhodamine B (SRB) assay, which was developed in 1990, remains one of the most widely used methods for in vitro cytotoxicity screening. The assay relies on the ability of SRB to bind to protein components of cells that have been fixed to tissue culture plates by trichloroacetic acid (TCA). SRB is a bright-pink aminoxanthene dye with two sulfonic groups that bind to basic amino-acid residues under mild acidic conditions, and dissociate under basic conditions. As the binding of SRB is stoichiometric, the amount of dye extracted from stained cells is directly proportional to the cell mass.

The strong intensity of SRB staining allows the assay to be carried out in a 96- well format showed that the assay can detect densities as low as 1,000–2,000 cells per well. The SRB method has proven to be practical, because after the TCA-fixed and SRB-

Dept. of Pharmaceutical chemistry, JKKNCP. _{Page | 72} SRB-stained cells is also stable. With its high level of sensitivity, adaptability to the 96- well format and endpoint stability, the SRB assay is well suited to large-scale screening applications, as well as research.

This assay has been widely used for drug-toxicity testing against different types of cancerous and non-cancerous cell lines. Other cell-growth assays have been employed to assess drug efficacy against both intracellular pathogens and host cells simultaneously in co-cultures and it is possible to use the SRB method for this purpose. In addition, the SRB method has also been shown to be effective for in vitro testing of cancer cell sensitivity to radiation, and for the study of interactions between radiotherapy and chemotherapy, with sensitivity comparable to that of the standard clonogenic assay.

The application of the SRB assay is limited to manual or semiautomatic screening due to the multiple washing and drying steps, which, at present, are not amenable to automation. This method nevertheless provides an efficient and sensitive tool for screening, especially for use in less well-equipped laboratories.

Calculate the percentage of cell-growth inhibition using the formulae below. For primary screening, we use a threshold of 50% cell-growth inhibition as a cut-off for compound toxicity against cell lines. However, the threshold can be varied according to toxicity criteria defined by the investigators. For IC50 determination, plot a dose–response curve between the compound concentration and percent growth inhibition. IC50 values can be derived using curve-fitting methods with statistical analysis software or IC50 calculation software.

9.3.3. EXPERIMENTAL PROCEDURE FOR SRB ASSAY

Dept. of Pharmaceutical chemistry, JKKNCP. _{Page | 73} serum and 2 mM L-glutamine. For present screening experiment, cells were inoculated into 96 well microtiter plates in 100 µL at plating densities as shown in the study details above, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs.

• Experimental drugs were initially solubilize in dimethyl sulfoxide at 100mg/ml and diluted to 1mg/ml using water and stored frozen prior to use. At the time of drug addition, an aliquote of frozen concentrate (1mg/ml) was thawed and diluted to 100 µg/ml, 200 µg/ml, 400 µg/ml and 800 µg/ml with complete medium

containing test article. Aliquots of 10 µl of these different drug dilutions were added to the appropriate micro titer wells already containing 90 µl of medium, resulting in the required final drug concentrations i.e.10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml.

• After compound addition, plates were incubated at standard conditions for 48 hours and assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of cold 30 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded; the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (50 µl) at 0.4 % (w/v) in 1 % acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing five times with 1 % acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on an plate reader at a wavelength of 540 nm with 690 nm reference wavelength.

Dept. of Pharmaceutical chemistry, JKKNCP. _{Page | 74}

•

control wells. Percent Growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells * 100.

• Using the six absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at the four concentration levels (Ti)], the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition was calculated as:

[Ti/C] x 100 %

• In this study Adriamycin used as standard drug. The table 9.1 shows the SRB Assay of tested compounds against Human Lung Cancer Cell Line A-549. And the table 9.2 represents the LC50, TGI and GI50 values of the tested compound.

Dept. of Pharmaceutical chemistry, JKKNCP. _{Page | 75} Table 9.1. The SRB Assay of tested compounds against Human Lung Cancer Cell Line

A-549

Code

Human Lung Cancer Cell Line A-549 % Control Growth

Drug Concentrations (µg/ml)

Experiment 1 Experiment 2 Experiment 3 Average Values