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Introduction: Both EGFR and COX-2 play a role in promoting cellular proliferation and angiogenesis in a variety of human cancer cell lines including HER2/neu positive breast cancer. Our goal is to investigate whether a combined treatment with inhibitors of COX-2 and EGFR will have a more potent antitumor effect than either treatment alone. Methods: The NMF 11.2 cell line, which was derived from the mammary tumors of HER2/neu mice, was used in all experiments. Cells were incubated in serum poor MEM with various concentrations of ZD1839 and/or SC236. At 48 hrs the cells were harvested and cell proliferation was evaluated via trypan blue staining and counting with a hemocytometer. Cell cycle and apoptosis were evaluated by propiduim iodine staining and flow cytometric analysis. In addition, western blotting was preformed to evaluate the protein expression of HER2, COX-2, VEGF, TGF-α, bcl2, bax and PARP. Results: Treatment with ZD1839 or SC236 inhibited cell growth in a dose dependent manner. Furthermore, the combination of these drugs inhibited cell growth in a synergistic fashion. Neither ZD1839 (1 μM) or [ZD1839 (1 μM) + SC236 (20 μM)] effected cell cycle. However, SC236 (20 μM) alone arrested the cells in G 2 /M phase. The combined treatment of ZD1839 + SC236 did have a super additive effect on apoptosis (control = 0.86%; ZD1839 = 2.31%; SC236 = 1.45%; ZD1839 + SC236 = 10.6). Western blot analysis confirmed the findings of increased apoptosis by illustrating the expression of the caspase 3 cleavage component PARP. A decrease in protein expression of both VEGF and TGF-α was also noted. No effect was seen on the expression of EGFR or HER2. Conclusions: ZD1839 and SC236 act in a synergistic manner to inhibit cell growth by means of increased apoptosis. Decreased levels of VEGF and TGF-α may also play a part in cell growth inhibition. Thus the combination of ZD1839 and a COX-2 inhibitor may be an effective adjuvant treatment for breast cancer. |
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