P-30: Study of the Effects of AKT Cellular Pathway on Growth and Proliferation of T-47D Breast Cancerous Cell Line in the Present of Sulfthiazole: A Real-Time Study
Objective: One of the most common malignancies in the world is the breast cancer which it has assigned to itself the most cancer deaths in the world after the lung cancer. Sulfonamides have antibacterial as well as anti-carbonic anhydrase and anti-metalloprotease activity and are able to decrease tumor cell growth and proliferation. With respect to this subject, in this paper, inhibitory effects of sulfathiazole on the growth, proliferation and survival of breast cancer cells (T-47D) has been considered. Materials and Methods: The effect of drug (Sulfathiazole or Doxorubicin) on the cell viability/proliferation was determined using MTT assay. Flow cytometry was performed to analyze cell cycle distribution and to quantify drug-induced apoptotic death of cells using DAPI staining and Annexin V-FITC - propodium iodide (PI) staining, respectively. Caspase-3 activity was assayed using a fluorometric immunosorbent enzyme assay kit according to the manufacturer’s instruction. To detect fragmented DNA in the treated cells, apoptotic DNA Ladder kit was used. To compare the expression of the individual genes examined, quantitative RT-PCR and Real-time PCR was performed. Results: We expected that in the presence of drug (LC50) the two main causes of cellular death (apoptosis and necrosis) could be introduced as the two basal factors describing 50 percent reduction of the cell population in the medium. So, observing specific morphological signs of apoptosis and necrosis was being considered using fluorescent microscopy after staining with Annexin-PI. The number of apoptotic and necrotic cells was also determined by flow cytometric technique. The obtained data revealed that just small number of cells involved in apoptosis or necrosis while a significant increase in the activity level of caspase-3 was registered. Interestingly, DNA laddering test also confirmed that no significant level of apoptosis did occur. The flow cytometric graphs drawn after DAPI treatment indicated that no cell cycle arrest occurred in sulfathiazole treated cells. Obtaining of these results motivated us to study the transcription level of several genes involved in apoptosis, cell cycle and cell survival processes using semi-quantitative RT-PCR and real time PCR methods. Conclusion: Overall, our findings showed that in T-47D cells the typical pathway of apoptosis has had a problem on those protein members settling after caspase-3 (e.g. DFF40) and activated by this enzyme. Present study provides new insights related to the cellular and molecular mechanisms of action of sulfonamide drug family (with anticancer activity) in T-47D cell line and perhaps the other types of breast cancer cells.