상피성 난소암 세포에서 MEK 억제제 AZD6244의 세포증식 저해 효과
Anti-proliferative effect in epithelial ovarian cancer cells by MEK Inhibitor AZD6244
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목적: 상피성 난소암 세포에서 세포증식과 생존에 관여하는 ERK 경로의 기초 활성도를 조사하고, MEK 억제제인 AZD6244가 난소암 세포의 증식과 세포주기, 염증성 싸이토카인의 생성에 미치는 영향과 AZD6244를 파클리탁셀과 병합치료를 하였을 때 세포독성 효과의 증강이 있는 지를 알아보고자 하였다. 연구 방법: 난소암 환자의 복수에서 분리한 일차세포와 확립되어진 난소암 세포주를 각각 AZD6244 (0.1-10 μM)를 24, 48, 72시간동안 치료하였다. Cell viability는 CellTiter 96 AQueous One Solution Cell Proliferation Assay를 통해서, caspase 활성도는 Caspase-Glo assay를 이용하였다. 싸이토카인의 분석은 세포배양 후 배양액의 상층액을 채취하여 Luminex 200 system을 이용하여 측정하였고, flow cytometry를 이용해 세포주기를 분석하였으며, p-ERK와 cyclin D1은 western blot 분석을 하였다. 결과: AZD6244는 기초 p-ERK 활성도가 높은 세포나 낮은 세포 모두에서 p-ERK를 억제하였다. AZD6244는 p-ERK의 고발현 세포 (A2780, 01-28, R454)에서 치료농도와 치료기간에 비례하여 효과적으로 증식억제를 보였다. 하지만 p-ERK의 저발현 세포 (CP70, R182)에서는 큰 차이를 보이지 않았다. AZD6244에 민감하게 세포증식의 억제반응을 보인 A2780 세포에서도 caspase-3/7 활성도는 유의한 증가가 관찰되지 않았다. AZD6244는 IL-6, IL-8, MCP-1, GM-CSF를 감소시켰다. 파클리탁셀과 AZD6244의 병합요법은 파클리탁셀에 의한 ERK 경로의 활성화를 AZD6244에 의해 효과적으로 상쇄시키며, 세포독성 효과의 유의한 증강이 나타났다. 결론: AZD6244는 p-ERK의 고발현 세포에서 효과적으로 세포증식 억제효과를 보이며, 파클리탁셀과 AZD6244의 병합요법은 난소암 세포에서 유의한 세포고사를 유도하며 AZD6244에 의한 ERK 경로의 억제가 파클리탁셀의 세포독성 효과를 증강시키는 데에 관여함을 보여준다.
Abstract
Objective: The objectives of this study were to determine the efficacy of AZD6244, a potent, selective MEK inhibitor, in epithelial ovarian cancer (EOC) cells and to determine the enhanced cell death by combined treatment of paclitaxel and AZD6244. Methods: EOC cells were treated with tenfold dilutions of AZD6244 (0.1 to 10 μM) for 24, 48 and 72 hours. Cell viability was determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay. The apoptotic cascade was assessed by Caspase-Glo assays. ERK activation was evaluated by Western blot analyses. Cytokine profiling was performed from culture supernatants using the Luminex 200 system. Results: In vitro cell viability showed that ovarian cancer cells with high p-ERK activities (A2780, R454, 01-28) exhibited significant growth inhibition. Cells with low p-ERK activities (R182, CP70), however, were resistant to AZD6244. Caspase-3 was not activated during AZD6244-induced growth inhibition. AZD6244 significantly inhibited p-ERK1/2 in both cell types regardless of constitutive levels of p-ERK. Proinflammatory cytokines including IL-6, IL-8, MCP-1 and GM-CSF were significantly decreased. Paclitaxel activated the p-ERK levels in paclitaxel-resistant R182 cells with low basal p-ERK level. MEK inhibition by AZD6244 enhanced paclitaxel-induced apoptosis significantly in R182 cells. Conclusion: These results demonstrate that AZD6244 has a potent growth inhibitory effect in ovarian cancer cells with high p-ERK activities. In addition, targeted inhibition of the extracellular signal-regulated kinase pathway with AZD6244 can enhance the anti-tumor efficacy of the cytotoxic paclitaxel.
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