악성 중피종과 폐암에서 Pemetrexed와 Simvastatin 병합요법에 의한 세포고사의 배가효과 : Synergistic Effect of Apoptosis by Pemetrexed and Simvastatin in Malignant Mesothelioma and Lung Cancer

국문 초록 (Abstract)

목적 : Pemetrexed는 multitargeted antifolate 제제로 악성중피종과 비소세포폐암의 치료제로 사용된다. Statin은 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor로 고지혈증 치료제로 사용되며, 항증식효과와 세포고사유도 효과가 밝혀진 바 있다. 그러나 Pemetrexed에 의한 세포고사에 Simvastatin의 병합 효과는 연구된 바 없으며, 본 연구를 통해 저자들은 Pemetrexed와 Simvastatin의 병합요법이 단독 요법에 비해 악성중피중과 폐의 악성 종양 세포에 세포고사능을 배가시키는지 알아보고자 하였다.
방법 : Pemetrexed와 Simvastatin에 의해 각각 그리고 병합하여 악성중피종 세포주인 MSTO-211와 비소세포폐암 세포주인 A549를 치료하였다. 세포 생존능은 MTT 분석을 통해 측정하였다. Pemetrexed와 Simvastatin에 대한 반응으로 나타난 세포고사의 표지자들은 Western blotting을 통한 방법으로 확인하였다. 세포고사 세포의 비율은 annexin V-propidium iodide staining으로 확인하였다. Reactive oxygen species (ROS) 생성은 H2DCFDA and MitoSOX Red를 이용한 flow cytometry로 측정하였으며, N-acetylcysteine 전처리하여 그 효과를 확인하였다. 세포고사는 reactive oxygen species (ROS), 미토콘드리아 기능 부전 표지자, Bcl-2 family members의 분석을 통해 좀 더 구체적인 확인이 이루어졌다. Bim siRNA를 통해 Pemetrexed와 Simvastatin에 의한 세포고사에서 그 역할을 알아보도록 하였다.
결과 : Pemetrexed는 caspase 에 의한 세포고사와 세포내 ROS 생성을 유도하여, MSTO-211과 A549 세포주에서 S1RT1을 감소시킨다. Pemetrexed와 Simvastatin 병합 요법은 MSTO-211와 A549 세포주에서 각각을 단독 처리했을 때보다, caspase에 의한 세포고사를 광범위하게 일으킨다. 뿐만 아니라, Pemetrexed와 Simvastatin 병합 요법을 처리하였을 때 세포 내 reactive oxygen species (ROS) 생성이 각각을 단독 처리했을 때보다, 저명하게 증가하였다. 병합요법은 또한, 미토콘드리아 막전위의 소실을 증가시켜, cytochrome c의 세포질로의 분비를 유도하였고, 세포고사와 관련된 단백인 inhibitor of apoptosis proteins (IAP) 와 B-cell lymphoma-2 (Bcl-2)의 발현을 변화시켰다. 반면, n-acetyl cysteine (NAC)으로 처리한 결과 Pemetrexed와 Simvastatin에 의한 세포고사와 미토콘드리아 기능부전 효과를 상쇄시켰다. Pemetrexed와 Simvastatin에 의한 세포고사는 Bim siRNA에서 거의 나타나지 않았다.
결론 : 악성중피종과 폐암 모델에서 Pemetrexed와 simvastatin의 병합요법은 세포고사능을 배가시키며, 이는 ROS의한 미토콘드리아 기능 부전과 Bim 유도를 통해 이루어진다.

다국어 초록 (Multilingual Abstract)

Purpose : Pemetrexed is a multitargeted antifolate used for the treatment of malignant mesothelioma and non-small cell lung cancer (NSCLC). Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors used primarily for hyperlipidemia, have been studied for their antiproliferative and proapoptotic effects. However, the effects of simvastatin on pemetrexed-induced apoptosis have not been investigated. In this study, we investigated whether combination treatment with pemetrexed and simvastatin potentiates the apoptotic activity above that is seen with either drug alone in malignant mesothelioma and lung cancer cells.
Method & materials : Malignant mesothelioma cells MSTO-211 and NSCLC cells A549 were treated with pemetrexed and simvastatatin alone and in combination. Cell viability was measured by the MTT assay. The expression of apoptotic markers in response to pemetrexed and simvastatin was examined by western blotting. The proportion of apoptotic cells was determined using annexin V-propidium iodide staining. Reactive oxygen species (ROS) generation was measured by flow cytometry using H2DCFDAandMitoSOX Red, and the effects of pretreatment with N-acetylcysteine were tested. Apoptosis was further characterized by determination of the levels of reactive oxygen species (ROS), mitochondrial dysfunction markers, and Bcl-2 family members. Bim siRNA was used to identify a role in apoptosis induced by pemetrexed and simvastatin.
Results : Pemetrexed enhanced caspase-dependent apoptosis, induced intracellular ROS generation, and down-regulated SIRT1 in MSTO-211 and A549 cells. The combination of pemetrexed and simvastatin induced more extensive caspase-dependent apoptosis than either drug alone in MSTO-211 and A549 cells. In addition, reactive oxygen species (ROS) generation in cells treated with both pemetrexed and simvastatin was markedly increased compared to cells treated with either pemetrexed or simvastatin alone. Combination treatment also increased the loss of mitochondrial membrane potential, increased cytosolic release of cytochrome c, and altered expression of inhibitor of apoptosis proteins (IAP) and B-cell lymphoma-2 (Bcl-2) families of apoptosis related proteins. On the other hand, pretreatment with n-acetyl cysteine (NAC) prevented apoptosis and mitochondrial dysfunction by pemetrexed and simvastatin. In addition, Bim siRNA conferred protection against apoptosis induced by pemetrexed and simvastatin.
Conclusion : Combination of pemetrexed and simvastatin potentiates their apoptotic activity beyond that of either drug alone in malignant mesothelioma and lung cancer cells. This activity is mediated through ROS-dependent mitochondrial dysfunction and Bim induction.

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and methods 3
• 1. Reagents 3
• 2. Cell culture and viability test 4
• 3. Annexin V assay for the assessment of apoptosis 4
• 4. Measurement of the mitochondrial membrane potential (ΔΨm) 5
• 5. Western blotting 6
• 6. Preparation of cytosolic and mitochondrial fractions 7
• 7. Gene silencing 7
• 8. Statistical analysis 8
• Ⅲ. Results 9
• 1. Effect of pemetrexed on the apoptotic activity of malignant mesothelioma and lung cancer cells 9
• 2. Effect of pemetrexed and simvastatin, alone and in combination on the growth of malignant mesothelioma and lung cancer cells 10
• 3. Combination of pemetrexed and simvastatin enhances caspase-dependent apoptosis 10
• 4. Combination of pemetrexed and simvastatin enhances intracellular ROS production 11
• 5. Combination of pemetrexed and simvastatin leads to mitochondrial dysfunction 12
• 6. Combination of pemetrexed and simvastatin induces changes in IAP and Bcl-2 families 12
• 7. Pretreatment with NAC prevents apoptosis induced by pemetrexed and simvastatin 13
• 8. Combination of pemetrexed and simvastatin induces apoptosis by up-regulation of Bim expression 14
• Ⅳ. Discussion 16
• V. Conclusion 19