SRC 티로신 인산화효소 억제제인 PP2와 테모졸로마이드 병용치료의 악성 교종 세포에 대한 in vitro 및 in vivo 효과

학위논문 (박사)-- 서울대학교 대학원 : 의학과, 2014. 8. 김일한.서론: 교모세포종에서 SRC 티로신 인산화효소의 과발현은 종양의 악성도 및 치료에 대한 좋지 않은 예후와 연관되어있다. 테모졸로마이드(TMZ)와 SRC 티로신 인산화효소 억제제인 PP2를 병용치료하는 것이 종양세포의 성장과 침윤을 억제하여 방사선치료의 치료율을 높일 수 있는지 집락형성분석법과 누드마우스를 이용한 뇌종양 이식모델을 이용하여 평가하였다. 방법: U251과 T98G 세포주에 SRC 티로신 인산화효소 억제제인 PP2(10μM)와 TMZ(25μM)를 처리하여 집락형성분석법을 이용한 방사선 감작을 조사하였다. PP2를 방사선 조사 전에 전처리하는 것이 DNA 이중나선 손상의 수복을 저해하는 가에 대해 방사선조사 6시간 후에 γH2AX 형성 분석을 이용하여 평가하였다. PP2가 U251 세포주의 이동이나 침윤에 영향을 미치는 가에 대해서 변형보이든함분석과 상처치유분석를 이용하여 평가하였고, 혈관유사형성 측정을 수행하였다. E-cadherin, MMP2, EphA2 및 VEGF의 단백질 발현을 평가하기 위해서 웨스턴블롯검사를 하였다. 뇌종양 이식모델에서는 3x105 개의 U251 세포주를 누드마우스의 시상 부위에 이식하고 PP2(10mg/kg)와 TMZ(50mg/kg)를 전뇌방사선(9Gy/3Fx) 치료 전, 치료 중간 및 치료 후에 처리하였다. pGL4 루시퍼레이스 정보제공 벡터를 이용하여 생물발광 영상을 얻어서 마우스 내의 종양의 부피를 측정하였다. 또한 뇌종양에 대한 VEGF, CD31, EphA2와 HIF-1a에 대한 면역화학염색을 시행하였다. 결과: PP2를 전처리하는 것은 정상적인 인간 별아교세포에는 명확한 세포독성 없이 방사선치료 후 U251 세포주의 세포독성을 증가시키고 생존분획을 감소시켰다. 생존분획이 0.5일 때 PP2, TMZ, PP2 및 TMZ를 병용처리했을 때의 민감제 증강률는 U251 세포주에서 각각 1.15, 1.41 및 1.54였으며, T98G 세포주에서는 1.16, 1.26 및 1.38였다. PP2를 처리한 경우 U251 세포주의 침윤 및 이동 능력이 떨어졌다. 방사선 단독조사에 비해서 억제제를 처리한 세포들의 상대적 이동 능력은 PP2, TMZ, PP2 와 TMZ를 병용처리했을 때 각각 0.993±0.131 (p = 0.465), 0.252±0.078 (p < 0.001) 및 0.200±0.066 (p <0.001)였다. 억제제를 처리한 세포들의 상대적 침윤 능력은 방사선 단독조사에 비해서 PP2, TMZ, PP2 와 TMZ를 병용처리했을 때 각각 0.992±0.122 (p = 0.461), 0.257±0.050 (p < 0.001) 및 0.116±0.010 (p < 0.001)였다. PP2에 의해 U251 세포주의 혈관유사형성이 억제되었다. PP2를 처리하였을 때 E-cadherin이 과별현되고 MMP2와 VEGF 및 EphA2는 억제되었다. PP2와 TMZ를 병용처리했을 때 방사선치료 6시간 후 γH2AX 초점의 축적이 증가했으며 이러한 결과는 DNA 손상의 수복이 지연됨을 의미하였다. 뇌종양 이식모델에서는 TMZ와 PP2를 병용치료할 때 종양의 부피가 가장 많이 줄었으나 TMZ 단독치료와 비교할 때에 통계적 차이는 없었다. PP2를 처리한 종양에서 방사선 단독치료 또는 방사선과 TMZ 치료를 한 종양에 비해 VEGF와 CD31의 발현이 억제되었다. 이러한 결과는 PP2가 세포실험과 마찬가지로 동물 내 종양에서도 혈관신생을 억제할 수 있음을 시사하였다. EphA2와 HIF-1a의 발현은 PP2 치료에 의해 억제되지 않았다. 결론: SRC 티로신 인산화효소를 억제할 경우 방사선치료에 의한 세포독성이 증가하고 이러한 효과는 MGMT 프로모터의 메틸화와는 무관하였다. 또한 PP2는 U251 세포주의 침윤, 이동 및 혈관유사형성을 억제하였다. PP2는 뇌종양 이식모델에서 VEGF와 CD31의 발현을 억제하여 항혈관신생효과에 대한 가능성을 보였다. 이러한 실험결과는 TMZ와 SRC 티로신 인산화효소 억제제인 PP2를 병용치료하는 것에 대한 실험증거를 제공하며, 이러한 치료가 악성 교종에서 치료효과를 향상시킬 수 있는 가능한 전략임을 나타낸다.Introduction: Overexpression of SRC tyrosine kinase of glioblastoma is associated with aggressiveness of tumors and poor treatment outcomes. We surveyed whether combined treatment of temozolomide (TMZ) and SRC tyrosine kinase inhibitor (TKI), PP2 would inhibit growth and invasion of glioma cells and thus, improve therapeutic efficacy of radiotherapy using in vitro clonogenic assays and an in vivo human brain tumor xenograft model in nude mice. Methods & Materials: The effect of the SRC TKI, PP2 (10 uM) on radiosensitivity of U251 and T98G cells with or without TMZ (25 uM) using in vitro clonogenic assays was investigated. It was also examined whether pretreatment with PP2 before radiotherapy inhibits DNA DSBs repair by measuring γH2AX foci formation 6 h after radiotherapy. The effect of PP2 on migration and invasion ability of U251 cells were evaluated using modified Boyden chamber assay and wound healing assay. Vasculogenic mimicry (VM) formation assay was also performed. Western blot analyses were performed to measure the protein expression including E-cadherin, MMP2, EphA2, and VEGF after PP2 treatment. In in vivo human brain tumor xenograft model in nude mice, 3 x 105 U251 cells were implanted to the thalamus and PP2 (10mg/kg) was treated with or without TMZ (50 mg/kg) before, during and after whole brain radiotherapy of 9 Gy in 3 fractions. Bioluminescence images using pGL4 luciferase reporter vector were taken after inhibitor treatment to measure in vivo tumor volume. Immunohistochemical stains including VEGF, CD31, EphA2, and HIF1a was performed on brain tumors. Results: Pretreatment with PP2 increased cytotoxicity and decreased surviving fraction of U251 cells after radiotherapy without obvious cytotoxic effects on normal human astrocytes. The sensitizer enhancement ratio at a surviving fraction of 0.5 (SER0.5) for PP2, TMZ, and PP2 plus TMZ was 1.15, 1.41, and 1.54 in U251 cells, and 1.16, 1.26, and 1.38 in T98G cells, respectively. Invasion and migration ability of U251 cells were compromised by PP2 treatment. The relative migration of inhibitor-treated cells relative to cells treated with radiotherapy alone was 0.993±0.131 (p = 0.465), 0.252±0.078 (p < 0.001), and 0.200±0.066 (p <0.001) for TMZ, PP2, and TMZ plus PP2, respectively. The relative invasion ability of inhibitor-treated cells relative to cells treated with radiotherapy alone was 0.992±0.122 (p = 0.461), 0.257±0.050 (p < 0.001), and 0.116±0.010 (p < 0.001) for TMZ, PP2, and TMZ plus PP2, respectively. VM formation was also suppressed by PP2. After PP2 treatment, overexpression of E-cadherin, and suppression of MMP2, VEGF, and EphA2 were observed. PP2 treatment combined with TMZ resulted in increased accumulation of γH2AX foci, indicating delayed DNA damage repair. In a human brain tumor xenograft model, combined treatment with PP2 and TMZ showed best tumor volume decrease, but the difference from TMZ alone was not statistically significant. The expression of VEGF and CD31 were down-regulated in PP2-treated tumors relative to RT alone or RT plus TMZ treated tumors, suggesting that PP2 may suppress angiogenesis in in vivo tumor as well as in in vitro cells. The expression of EphA2 and HIF1a of in vivo brain tumors were not influenced by PP2 treatment. Conclusions: These results show that SRC tyrosine kinase inhibition leads to additive cytotoxicity of radiotherapy in malignant glioma cells and this effect was independent of MGMT promoter methylation status. PP2 also decreased invasion, migration, and VM formation of U251 cells. PP2 suppressed VEGF and CD31 expression and thus, showed anti-angiogenic potential in in vivo model using U251 cells. These results provide evidences for the combination therapy of TMZ and SRC TKI, PP2 on malignant glioma cells and present a feasible strategy to improve the therapeutic outcomes.Abstract …………………………………………………………………………i Table of contents ……………………………………………………………iv List of figures …………………………………………………………………v Introduction ……………………………………………………………………1 Materials and Methods ………………………………………………………3 Results …………………………………………………………………………8 Discussion ……………………………………………………………………17 References ……………………………………………………………………20Docto

Optimal treatment strategies for small cell carcinoma of the uterine cervix: A retrospective multi-center study (KROG 19-03)

Objective: This multi-institutional study aimed to identify the optimal treatment strategy for small cell carcinoma of the cervix. Study design: We retrospectively collected the medical records of 166 patients diagnosed with small cell carcinoma of the uterine cervix from January 2000 to December 2015 from 13 institutions of the Korean Radiation Oncology Group. After excluding 18 (10.8 %) patients who initially had distant metastasis, the treatment outcomes of 148 patients were analyzed. Results: After a median 46.4 (1.4-231.9) months of follow-up, the 5-year progression-free survival (PFS) and overall survival (OS) rates of all patients were 45.9 % and 63.5 %, respectively. Distant metastasis was the dominant pattern of failure occurring in 67 patients (45.3 %). We stratified the patients according to the primary local treatment: primary surgery (n = 119), primary radiotherapy (RT) (n = 26), and no local treatment group (n = 3). Although the primary RT group had advanced disease (FIGO stage &gt;= IIB) more frequently than the primary surgery group (80.8 % vs. 47.9 %), the PFS and OS did not differ between the groups in multivariate analysis. Conclusion: Definitive RT is a reasonable local treatment option for small cell cervical cancer, particularly for advanced cases. Given the high rates of distant relapse, an effective systemic therapy protocol is warranted for small cell cervical cancer patients. (C) 2021 Elsevier B.V. All rights reserved

Analysis of 10,811 Cases with Acute Ischemic Stroke from Korean Stroke Registry: Hospital-Based Multicenter Prospective Registration Study

Background: Although several hospital-based stroke studies were published, there has not been any reliable data representing the clinical characteristics of stroke in Korea. We analyzed the clinical characteristics of patients with ischemic stroke registered in the Korean Stroke Registry (KSR), which is the largest prospective hospital-based nation-wide stroke registry in Korea. Methods: The KSR provided standardized protocols for collecting data, which includes the data of demographics, subtypes of stroke, risk factors, and neurological outcome at discharge. The brain imaging studies, including CT or MRI, were performed in all cases. Results: KSR registered 10,811 patients of acute ischemic stroke between Nov. 2002 and Jun. 2004. The large-artery atherosclerosis was the most common subtype (37.3%), followed by small vessel occlusion (30.8%). Hypertension (65.4%) was the most common risk factor, followed by smoking (34.5%) and diabetes (28.3%). Although most of the hypertensive and diabetic patients had been diagnosed before the stroke, less than 45.4% and 32.5% of them were under regular control. The steno-occlusive lesion of extracranial carotid artery was only 29.3% and the ratio of intra- to extracranial artery disease was more than 2 in KSR. Only 20.5% of patients were admitted within 3 hours after stroke onset and 2.1% were treated with intravenous thrombolysis. In-hospital case-fatality was 5.2%, which is relatively comparable to those of previous studies. Conclusions: The KSR provided informative data in understanding the clinical characteristics of ischemic stroke in Korea. Further analysis of KSR will facilitate clinical trials and development of guidelines for the management of stroke patients.N