Determination of cytotoxic activity and apoptosis assays

The cytotoxic effects of the culture extracts or the pure compound were determined against human breast cancer cell line (MCF7) (NCBI Code: C135), colon cancer cell line (SW480) (NCBI Code: C506), and hepatoma G2 cells (HepG2) (NCBI Code: C158), as well as normal cell human umbilical vein endothelial cells (HUVECs) (NCBI Code: C554) by MTT assay (Peng and Zhao 2009). 100 µl of the cell suspensions containing 10⁴ cells were transferred to each well of a 96-well microplate in DMEM or RPMI media supplemented with 10% FBS and 100 mg/l streptomycin and 100 IU/ml penicillin. The microplates were incubated in a humidified atmosphere with 5% CO₂ at 37 °C for 24 h. Then the cultured cell lines were treated with 100 µl of SP 85, crude extract, or doxorubicin at final concentration of 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 µg/ml and incubated for further 36 h. After the end of incubation time, 50 µl of the MTT solution (5 mg/ml) was added to each well and again the plate was incubated for another 4 h. After removing the MTT treated media from the insoluble formazan dye, 100 µl of a DMSO: ethanol (4:1) solution was added to each well and the microplates were shaken in a shaker at 200 rpm for an hour. The optical density (OD) of each well was measured at λ 550 nm using the microplate reader to measure the absorbance of the purple color of the dissolved formazan dye. The cell viability percentage was measured as follows:

The anti-cancer medicine, doxorubicin, was used as the positive control and the absorbance of the culture medium was taken as the blank control (OD_Blank). The concentration of a test compound that causes 50% of the cells are killed or survived (cell viability %) is considered as a measure of cytotoxic potential of the test compound and is expressed as IC₅₀.

Doxorubicin is a 14-hydroxylated derivative of daunorubicin, a tetracyclic polyketide natural product isolated from a Streptomyces species and used as a standard chemotherapy agent for the treatment of several cancers and also used as a positive control. Its cytotoxicity against the human tumor cell lines was compared to SP 85 compound, through IC₅₀ calculation.

In the present study, the induction of apoptosis by SP 85 was determined using DNA fragmentation assay, since it is a hallmark of apoptosis. In apoptotic cells, DNA is cleaved by an endonuclease into nucleosomal units, which are multiples of 180 bp oligomers and appear as a DNA ladder, when run on an agarose gel. For DNA fragmentation assay one of the cancer cell lines, SW480 was treated with 250 μg/ml of SP 85 for 72 h. As the negative control, SW480 was treated with the medium without the addition of SP 85. The lysed cells were centrifuged and the supernatant was monitored for the presence of low molecular weight DNA on an agarose gel (Brady 2004).