背景：腫瘤缺氧會增加對放療及化療的抗性，使腫瘤惡化。大多抑制血管新生藥物作用在血管內皮細胞生長因子 (VEGF) 或其受器，雖可抑制腫瘤生長，但會造成腫瘤缺氧。抗血管新生藥物一號 (Anti-angiogenic drug 01, AAD01) 則是直接作用於血管內皮細胞。最近也有文獻指出，川芎嗪 (Tetramethylpyrazine, TMP) 可以下降VEGF進而抑制血管新生，本實驗將探討這兩種藥物對於治療惡性腦腫瘤有無降低缺氧的影響。
目的：本實驗假設AAD01可能藉由抑制血管內皮細胞增生並改善血流，進而改善惡性腦腫瘤缺氧的情形，並加入TMP藥物治療作為比較是否有類似的效果。
實驗材料與方法：於C6膠質瘤細胞植入雌性Sprague-Dawley成鼠顱內，第五天開始隨機分成三組，開始給與AAD01 (10 µg / 每3 days) 或TMP (2.4 mg/day, for 7 days)，於第13天犧牲動物，以組織染色分析血管及缺氧相關因子，並以西方墨點法定量，並利用單因子變異數分析(one-way ANOVA) 統計分析，p值小於0.05以下為顯著差異。
結果：經由AAD01治療後，腫瘤變小 (60.42%)，體重下降速度明顯趨緩，缺氧基因HIF-1α表現較少，VEGF表現降低，表現VEGF receptor 2 (VEGFR2) 形成血管新枝的細胞減低，腫瘤內總體的血管密度變少，但具有血流循環的血管較多，且腫瘤向外侵潤情況變少；在TMP的組別，血管密度、HIF-1α和VEGF表現量也有下降，但沒有AAD01的效果好，有血流循環的血管也沒有比對照組多。
結論：AAD01除了能抑制不正常血管生成之外，也藉由增加腫瘤的血流循環進而減少缺氧現象，降低新生血管因子的表現，以及腫瘤向外侵潤的能力。

Background: Intra-tumoral hypoxia is associated with malignant progression, tumor invasion and resistance to radiotherapy and chemotherapy of cancers. Anti-angiogenic therapies targeting the vascular endothelial growth factor (VEGF) / VEGF receptor (V EGFR) have shown therapeutic effect for malignant tumors. However, they also increase intra-tumoral hypoxia. Anti-angiogenic drug 01 (AAD01), on the other hand, targets endothelial cells directly. Tetramethylpyrazine (TMP) decreased the expression of VEGF and reduced angiogenesis. This study would test these two drugs to decrease tumor hypoxia in malignant gliomas in rat.
Hypothesis: It is hypothesized that by limiting the rapid proliferation of endothelial cells, AAD01 increase tissue perfusion in gliomas, which in turn result in the decrease of intra-tumoral hypoxia. This study has also evaluated if TMP has similar effect.
Materials and Methods: Suspended 1x107 C6 glioma cells were implanted intracerebrally of the adult female Sprague-Dawley rats. The treatment group received AAD01 (10 µg / every 3 days) or TMP (2.4 mg/day, for 7 days) beginning for the 5th day. At day 13, animals were sacrificed. Tumor hypoxia and angiogenic factors were analyzed by immunohistochemistry and quantified by Western blot. Multiple groups were analyzed by one-way analysis of variance (ANOVA). P-value of less than 0.05 was considered significant.
Results: In the AAD01 treated group, tumor size was decreased (60.42%). These rats also showed less body weight loss. The expression of hypoxic inducible factor-1 α (HIF-1α) and VEGF both decreased significantly in the treatment group than in control group. The sprouting vessel, which is evaluated by staining of VEGFR2 was inhibited in the AAD01 group. The density of total vessels was decreased. The contrast, the density of perfused vessels was increased. The peri-tumor infiltration was less prominent in the AAD01 group. On the hand, the expression of HIF-1α and VEGF in the TMP group was decreased but less effect than AAD01 group. The density of perfused vessels was not significant statistically different from control.
Conclusion: AAD01 reduced tumor hypoxia by increasing tissue perfusion. Otherwise, it can reduce peri-tumor infiltration.

Signature Page i

Thesis Approval Form ii

Acknowledgments iii

Chinese Abstract iv

English Abstract v

Table of contents vi

List of Figures vii

I. Introduction 1

1.1 Malignant Brain Tumor 1

1.2 Treatment Options 2
1.2.2 Radiotherapy 2
1.2.2.1 External beam radiotherapy 2
1.2.2.2 Brachytherapy… 3
1.2.2.3 Whole-brain radiotherapy 3
1.2.2.4 Radioisotope Therapy 4
1.2.3 Chemotherapy 4
1.2.3.1 Nitrosoureas 4
1.2.3.2 Temozolomide 5
1.2.3.3 Epipodophylotoxins 5
1.2.3.4 Topoisomerase I Inhibitors 5
1.2.3.5 New Chemotherapeutic Drugs 6

1.3 The Bottleneck of Therapies in Brain Tumor 8
1.4 Angiogenesis 8
1.4.1 Biology of Angiogenesis in Malignant Gliomas 8
1.4.2 Mechanisms of Antiangiogenic Therapy 10
1.4.3 Kringle domain 10

1.5 Does Anti-Angiogenic Agent Induce Hypoxia? 12

1.6 Hypoxia in Malignant Gliomas 13
1.6.1 Biology of Hypoxia in Malignant Gliomas 13
1.6.2 Defects of Hypoxia in Clinical Trails 14
1.6.3 Tetramethylpyrazine 15

II. Hypothesis 16
2.1 The Purpose in this study 16
2.2 Specific aims 16

III. Materials And Methods 17
3.1 Experiment Animals and Surgical Procedures 17
3.2 Cell Culture 19
3.3 Immunocytochemistry 19
3.4 Perfused Vessels Analysis 21
3.5 Western Blot Assay 21
3.6 Statistical Analysis 23


IV.Results 23
4.1 High-grade malignant C6 gliomas animals’ model23
4.2 AAD01 impairs both angiogenesis and tumor infiltration 24
4.3AAD01 treatment suppressed the hypoxia critical
transcription factor - hypoxia inducible factor-1α (HIF-1α) in malignant C6 gliomas 24
4.4Vascular endothelial growth factor (VEGF), the critical
hypoxia downstream molecular was inhibited in AAD01 group 25
4.5AAD01 inhibits vascular endothelial growth factor
receptor-2 (VEGFR2), the key of angiogenesis 25
4.6The possible mechanism in AAD01 treatment which
Decreased hypoxia 26



V. Discussion 27
5.1 AAD01 and TMP altered the structure of tumor vessels 27
5.2 AAD01 decreased tumor hypoxia and the expression of
VEGF in high-grade malignant gliomas 28
5.3 AAD01 decreased sprouting vessels 29
5.4 AAD01 and other Angiogenic Agents 30
5.5 Conclusions 31

VI. References 32
VII. Appendices 48

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