MTT assay

The effect of 23,24-dihydrocucurbitacin B (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) on the viability of different cervical cancer cell lines and normal fR2 and HCerEpiC cells was evaluated using an MTT assay. Cells were seeded at 1×10⁶ cells/well in 96-well plates for 12 h and then treated with different concentrations of 23,24-dihydrocucurbitacin B (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 and 200 µM) for 24 h. A total of 20 µl MTT solution (2.5 mg/ml) for 24 h was then added to each well. The medium was removed and 500 µl dimethyl sulfoxide was added to each well to dissolve formazan crystals. Optical density was recorded using an ELISA plate reader at a wavelength of 570 nm. 23,24-dihydrocucurbitacin B exhibited marked anticancer activity against all cell lines; however, further experiments were performed on the HeLa cancer cell line alone as the lowest MIC was observed against this cell line.