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INTERNATIONAL CONGRESS OF

CancerGenetics

• Time- and Dose-Dependent Cytotoxic Effects of Cisplatin on MDA-MB-231 Triple-Negative Breast Cancer Cells: An MTT Assay-Based Evaluation of IC₅₀

• Homa Kazemzadeh,^1,* Seyed Jalal Zargar,² Shahrokh Safarian,³
  1. Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran
  2. Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran
  3. Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran
  
  
  
• Introduction: Breast cancer is one of the most common and lethal malignancies worldwide. Among its subtypes, Triple-Negative Breast Cancer (TNBC) represents a major challenge due to its aggressive behavior and lack of hormone receptor expression, and chemotherapy remains one of the main therapeutic approaches for its treatment. The MDA-MB-231 cell line is a standard and widely used in vitro cellular model for studying this aggressive subtype of breast cancer. Cisplatin is a well-known chemotherapeutic agent that induces apoptosis in cancer cells through the formation of cross-links in DNA. However, the development of drug resistance and dose-dependent adverse effects, such as nephrotoxicity, are among the major challenges associated with its clinical use. Accurate assessment of cellular sensitivity and determination of inhibitory concentrations at different time points may substantially contribute to the optimization of therapeutic regimens. The aim of this study was to quantitatively evaluate the cytotoxic effects of cisplatin on the MDA-MB-231 cell line at 24, 48, and 72 h using the well-established MTT assay and to determine the half-maximal inhibitory concentration (IC50).
• Methods: In this experimental study, the human breast cancer cell line MDA-MB-231 was obtained and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37 C in an atmosphere containing 5% CO2. After reaching the desired cell confluency, the cells were detached using trypsin and seeded into 96-well plates at a density of 1 x 10^4 cells per well. Following 24 h of cell attachment, the cells were treated with cisplatin at concentrations of 5, 10, 15, 20, 30, 60, 80, and 150 µM and incubated for 24, 48, and 72 h. At the end of each treatment period, 20 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for an additional 3 h in the dark. After removal of the supernatant, the resulting formazan crystals were completely dissolved by adding 100 µL of DMSO. Optical density (OD) was measured at 570 and 630 nm using a microplate reader. The OD value measured at 630 nm was subsequently subtracted from the OD value measured at 570 nm. Statistical analysis, dose-response curve fitting, and calculation of IC50 values based on a nonlinear regression model were performed using GraphPad Prism software.
• Results: The results of the MTT assay demonstrated pronounced dose-dependent and time-dependent effects of cisplatin on the viability of MDA-MB-231 cells. Analysis of the nonlinear regression curves clearly showed that increasing cisplatin concentrations resulted in a substantial decrease in cell viability at all evaluated time points. Evaluation of the IC50 values obtained from the dose-response curves showed that, at 24 h after treatment, the IC50 was 56.12 µM, indicating relative cellular resistance during short-term exposure. When the exposure time was extended to 48 h, the IC50 markedly decreased to 14.67 µM, confirming a substantial increase in the cytotoxic effect of cisplatin at this time point. Further extension of the incubation period to 72 h resulted in the highest level of cytotoxicity, with the IC50 reaching its lowest value of 6.197 µM. Comparison of these values demonstrates that the concentration required to inhibit 50% of the cancer cell population after 72 h of exposure was approximately one-ninth of that required during the first 24 h, indicating a marked time-dependent sensitivity of MDA-MB-231 cells to cisplatin.
• Conclusion: The findings of this study demonstrate that cisplatin exerts potent cytotoxic effects on the MDA-MB-231 triple-negative breast cancer cell line and that this effect follows a clear dose- and time-dependent pattern. The marked reduction in IC50 from 56.12 µM at 24 h to 6.197 µM at 72 h indicates that increasing the duration of drug exposure can substantially reduce the need for higher therapeutic concentrations. This pronounced time dependence may represent a valuable strategy in the design of clinical treatment protocols, suggesting that the use of lower doses of cisplatin over longer treatment periods may provide comparable antitumor efficacy while minimizing systemic adverse effects in patients.
• Keywords: Cisplatin; MDA-MB-231; Cytotoxicity; MTT assay; Half-maximal inhibitory concentration (IC50)