摘要

肝細胞癌，簡稱肝癌，是目前最常見的肝臟惡性腫瘤，也是全世界好發的癌症之一。肝癌最理想的是能夠早期發現，早期接受治療。目前臨床上的治療，像是化學治療，放射線治療等都無法根治，主要原因是復發的機會很高。這些療程都無法完全將癌細胞殺死的原因是癌細胞內部嚴重缺氧，癌細胞在缺氧環境的抗藥性高。缺氧會引導抗藥性，主要是因為部分的抗癌藥物會藉由氧氣來產生自由基，這些自由基會攻擊細胞來達到治療效果；在缺氧環境下，無法產生足夠的自由基，進而無法達到預期的療效。因此，我們藉由一產氧系統來減緩癌細胞內部的缺氧情形，降低藥物因為缺氧而造成的治療阻力，進而提升治療效果。此產氧系統是利用褐藻酸钠和鈣離子的交聯來包覆過氧化鈣和過氧化氫酶形成微球體；過氧化鈣與水接觸後會開始分解，過氧化氫在過氧化氫酶的催化下會大量地產生氧氣。此系統採用的是體外實驗 (in vitro)，並在兩個環境下進行細胞實驗，即有氧環境及缺氧環境。此交聯10分鐘的微球體具有長時間釋放氧氣的能力，維持了28個小時的釋放時間。從實驗結果得知缺氧環境確實會降低癌細胞對藥物的敏感度，因此氧氣在癌症的治療上扮演了一個關鍵的角色，氧氣越充足，產生的自由基越多，進而殺死越多的癌細胞。根據實驗結果顯示，隨著微球體的量越多，提供的氧氣越充足，成功地提高了癌細胞在缺氧環境下的致死率，降低了藥物因缺氧而造成的治療阻力，提升化學治療效果。綜合本研究的實驗結果，此產氧系統在癌症治療上是一種頗有潛力的治療方法。



關鍵字: 缺氧、抗藥性、產氧系統、降低治療阻力，提升化療的效果

ABSTRACT

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer. General chemotherapy and radiotherapy offer somewhat unsatisfactory responsiveness, the overall recurrence rate is very high, this could be attributed to the chemotherapeutic resistance that results from tumor hypoxia. Recently, hypoxia has been described as an important factor to chemotherapeutic resistance, owing to the redox state, meaning that oxygen (O2) is required to generate ROS to be maximally cytotoxic. To address the above issue, an oxygen generating system was fabricated using alginate, having calcium peroxide as the oxygen generating source, which decompose hydrogen peroxide and release oxygen when contact with water. The decomposition rate could be increased by the addition of catalase, a common enzyme found in nearly all living organisms. This novel system was performed under two controlled conditions, normoxia and hypoxia. Optimization of microspheres, cytotoxicity of material and condition of the system were carried out specifically based on the responses observed from in vitro studies using Hep3B cells as a candidate. Microspheres are capable of sustainably release oxygen over 24 hours while the best cross-linking time during the gelation was found to be 10 minutes. It was observed that cells maintained high viability under hypoxic condition and found that doxorubicin-induced oxyradicals play a vital role in the development of drug resistance. However, this oxygen generating system has successfully reduced the chemotherapeutic resistance which induced by hypoxia and enhanced the chemotherapeutic efficacy of doxorubicin. These results suggest that the developed oxygen generating system is a highly promising approach in maximizing the therapeutic effectiveness with minimal side effects.






Keywords: hypoxia; chemotherapeutic resistance; oxygen generating system; redox cycle of doxorubicin; enhancement of the chemotherapeutic efficacy

TABLE OF CONTENT

摘要
ABSTRACT
LIST OF FIGURES
LIST OF TABLES
LIST OF EQUATIONS

Chapter 1
Introduction
1.1 Hepatocellular carcinoma and its treatment
1.2 Hypoxia in human tumors and its therapeutic resistance
1.3 Role of hydrogen peroxide and catalase
1.4 Objective and specific aims of this study

Chapter 2
Materials and methods
2.1 Materials
2.2 Preparation and characterization of CaO2-alginate microspheres
2.2.1 Fabrication of CaO2-alginate microspheres
2.2.2 Characterization of CaO2-alginate microspheres
2.2.3 Creation of hypoxic environment
2.2.4 Oxygen release behavior
2.2.5 H2O2 release profile and pH value
2.3 Cytotoxicity assay
2.4 Intracellular accumulation of doxorubicin and therapeutic resistance test
2.5 Intracellular ROS detection
2.6 Enhancing the chemotherapeutic efficacy of doxorubicin
2.7 HIF 1-alpha expression after re-oxygenation
2.8 Statistical analysis

Chapter 3
Results and Discussion
3.1 Morphology and characteristics of CaO2-alginate microspheres
3.1.1 Morphology of CaO2-alginate microspheres
3.1.2 Cryosectioning of CaO2-alginate microspheres
3.1.3 Oxygen release profiles
3.1.4 H2O2 release profile and pH value
3.2 Cytotoxicity of materials
3.3 Intracellular accumulation of Doxorubicin and therapeutic resistance test
3.4 Intracellular ROS detection
3.5 Enhancement of therapeutic efficacy by oxygen generating system
3.6 HIF 1-alpha expression after re-oxygenation

Chapter 4
Conclusions

References


LIST OF FIGURES

Chapter 1
Figure 1.1 Redox cycle of DOX. It is possible that DOX- induced oxyradicals play a vital role in the development of drug resistance.
Figure 1.2 H2O2 decomposes to water and oxygen. The decomposition rate at room temperature is, however, immeasurably small. But the rate can be increased by the addition of catalase.
Figure 1.3 H2O2 converted into hydroxyl radical when exposure to ultraviolet light.
Figure 1.4 The catalytic decomposition of H2O2 can be essentially explained by two different mechanisms based on the mutual redox transition Fe (III)/Fe (V) (Kremer-Stein mechanism) and Fe (III)/Fe (II) (Haber and Weiss mechanism).
Figure 1.5 The cell’s most important defense against the excessive production of free radical is the concerted action of three enzymes (superoxide dismutase enzyme, glutathione peroxidase and catalase).
Figure 1.6 (a) pH profile of catalase activity (incubation for 24hr, at 30°C), (b) temperature profile of catalase activity (incubation for 24hr, at pH 7.0). (adopted from reference 27)
Figure 1.7 CaO2 is used as a source of oxygen or H2O2.
Figure 1.8 Schematic diagram displaying the oxygen delivery affects the therapeutic resistance.
Figure 1.9 (a) Chemical structure of alginate, and (b) Mechanism of ionic interaction between alginate and divalent cations. (adopted from reference 36)
Figure 1.10 Schematic illustrations showing (a) Composition and structure of CaO2-alginate microspheres, and (b) Design of oxygen-generating system to enhance chemotherapeutic resistance in solid tumor.
Figure 1.11 Concepts of experimental design.

Chapter 2
Figure 2.1 Fabrication of CaO2-alginate microspheres.
Figure 2.2 C-chamber (hypoxia chamber) from BioSpherix.
Figure 2.3 (a) DO bench meter from HANNA Instruments and (b) DO probe with the apparatus for oxygen detection.
Figure 2.4 SpectraMax M5 components.
Figure 2.5 The experimental setting of chemotherapeutic efficacy study.

Chapter 3
Figure 3.1 Micrograph of (a) CaO2-alginate MS without catalase and (b) CaO2-alginate MS with catalase.
Figure 3.2 Cryosection images of CaO2-alginate MS. (A) A loosen network structure of microsphere under 3 minutes of cross-linking time, size: 2096µm. (B) 10 minutes cross-linked microspheres, size: 2080µm. (C) The compact structure of microspheres that cross-linked under 20 minutes, size: 2090µm.
Figure 3.3 Oxygen release profiles of CaO2-alginate microspheres.
Figure 3.4 The side product effect of CaO2 decomposition. (A) H2O2 released from microspheres (P <0.05, n=6). (B) pH value of medium after cultured with microspheres (n=6).
Figure 3.5 Cytotoxicity of CaO2-alginate MS. (A) Fluorescence microscopic images of live/dead assay of Hep3B cells growing with different amount of microspheres. (B) Quantitative analysis representation of cellular ability using LDH assay (n=6).
Figure 3.6 The intracellular accumulation of doxorubicin. (A) Flow cytometry data for cellular uptake of doxorubicin. Gating was preformed data is representative of 10,000 gated event. (B) Percentage of 10,000 counts, analyzed by flow cytometry (n=6). (C) Fluorescence microscopy of doxorubicin accumulation at a concentration of 10µM.
Figure 3.7 Therapeutic resistance test. (A) Viability results of the cells after they had been treated with free form of doxorubicin (n=6). (B) Confocal images of HIF-1 alpha expression under normoxic and hypoxic conditions.
Figure 3.8 Intracellular level of ROS production using CellROX green reagent. (A) Fluorescence intensities of Hep3B cells under normoxic and hypoxic conditions, determined by flow cytometry (n=6). (B) The FL1 mean in flow cytometry analysis. (C) Confocal images of ROS detection.
Figure 3.9 Result of enhancing therapeutic efficacy by oxygen generating system. (A) Fluorescence microscopy of the qualitative analysis. (B) Quantitative result of the cells treated with CaO2-alginate MS under both normoxic and hypoxic conditions (n=6). (C) Cell survival after different time of re-oxygenation, determined by MTT assay (n=6).
Figure 3.10 Confocal images of HIF-1 alpha after re-oxygenation (environmental) and treated with CaO2-alginate MS.


LIST OF TABLE

Chapter 2
Table 2.1 Specifications of DO bench meter.

Chapter 3
Table 3.1 The pore parameters of CaO2-alginate MS, analyzed by ImageJ software.
Table 3.2 ImageJ software was used to quantify the HIF 1-alpha expression.

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