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Studies on endometrial cancer : cell deaths and cross talks with endometrial stromal cells

Studies on endometrial cancer : cell deaths and cross talks with endometrial stromal cells

초록/요약

자궁내막암은 부인암 악성종양 중 하나로 여성호르몬인 난포호르몬 (estrogen)의 지속적 영향이 주요 암 발생원이다. 자궁내막암의 발생 및 진행은 난포호르몬의 지속적인 노출에 의한 돌연변이의 축적으로 진행된다. 최근 항암연구는 암미세환경에 대한 연구가 활발히 이루어지고 있다. 본 연구는 암미세환경을 통해 암의 세포사멸을 유도하는 실험을 수행하였고 이를 통해 암세포와 기질세포간의 상관관계를 연구하였다. 암세포는 활성산소종에 대한 민감성이 높다. 암은 정상세포에 비해 활성산소종의 비율이 높다. 그 결과 암세포의 생존, 세포분열, 혈관 신생이 일어난다. 그러나 암세포는 이미 높은 농도의 활성산소종의 비율 때문에 외부에서 도입한 활성산소종에 대한 감수성이 높다. 따라서 외부에서 도입한 활성산소종은 항암치료의 요인으로 작용할 수 있다. 본 연구에서는 활성산소종의 일종인 산화질소 (NO)를 선택적으로 생성할 수 있는 저온 대기압 플라즈마 장치를 개발하여 산화질소를 자궁내막암과 자궁기질세포에 노출했다. 그 결과 높은 농도의 산화질소에서 자궁내막암의 선택적인 세포사멸이 일어났다. 이 결과를 바탕으로 높은 농도의 산화질소를 제어하여 항암치료에 적용할 수 있는 가능성을 제시하였다. 암미세환경을 통한 항암연구에 있어서 면역세포 연구는 중요하다. 따라서 면역세포의 활성화를 통한 암세포사멸을 연구하였다. 자연살해세포 (natural killer cells)는 선천면역세포로서 암세포를 직접 사멸할 수 있는 기능이 있다. 자연살해세포의 암세포를 죽이는 원리는 두 가지로 암세포를 인지하는 것과 암세포의 세포사멸유도하는 신호를 전달하는 것이다. Perforin, Granzyme B와 같은 단백질로 표적 세포인 암세포를 사멸할 수 있다. 자연살해세포를 활성화하기 위해서 피톤치드 (phytoncide)를 처리하면서 생쥐를 이용하여 이종종양이식 실험을 진행하였다. 실험 결과 암세포를 이식한 생쥐에서 피톤치드 향을 맡게 한면 암세포의 성장이 대조군에 비해 44.18% 저해되었다. 그리고 비장에서 추출한 자연살해세포로 세포독성을 분석한 결과 피톤치드 향을 처리한 생쥐에서 유의하게 자연살해포의 세포독성이 증가하였다. 피톤치드는 식물에서 나오는 정유성분으로 여러 가지 성분들로 구성 되어 있다. 따라서 동물모델에서 쓰인 피톤치드 향을 기체크로마토그래피분석법을 이용하여 물질의 구성 성분을 분석하여 각각의 성분 물질을 자연살해세포주에 직접 처리하였다. 그 결과 표적 세포를 혈액암 세포주로 하였을 때에 자연살해세포의 세포독성이 α-pinene에서 가장 증가하였다. α-pinene을 자연살해세포에 처리하고, 인간 자궁내막암 세포를 표적으로 한 세포독성 결과도 혈액암 세포주와 같이 세포독성이 증가하였다. α-pinene에 의한 자연살해세포의 활성화의 세포내 신호전달경로를 알기 위해 실험한 결과 Erk와 Akt 신호전달 단백질이 강화되었다. 이 결과를 통해 피톤치드를 통한 자연살해세포의 활성화가 자궁내막암 세포사멸을 강화시키는 것을 밝혔다. 암세포가 단계적으로 성장하기 위해서는 암미세환경의 영향을 받게 된다. 암세포는 암세포를 둘러싸는 다양한 종류의 세포를 사이토카인을 통해 특성을 바꾼다. 암세포에 의해서 특성이 바뀐 주변 세포들은 다시 암세포의 생존, 세포분열, 혈관 신생에 도움이 되는 신호를 전달한다. 마지막 단계에서는 암세포는 침윤과 전이가 일어난다. 이와 같은 암세포와 암 주변 세포 간의 상호작용을 연구하기 위해서 자궁내막암과 자궁내막기질세포를 공배양하여 사이토카인의 발현 양상을 어레이방법으로 분석하였다. 그 결과 33개의 사이토카인의 발현양이 증가하였고 15개의 사이토카인의 발현양이 감소하였다. 난포호르몬 조건과 공배양의 결과로 자궁내막암의 이동 능력을 분석한 결과 유의하게 자궁내막암의 이동 능력이 증가하였다. 특히, CCL2와 GDF15의 역할을 알아보기 위해서 두 단백질을 자궁내막암 세포주에서 과 발현하였다. 그 결과 epithelial-mesenchymal transition (EMT)와 관련된 단백질의 발현양이 증가하였다. α-SMA, vimentin, twist 1, twist 2, MMP3, MMP9와 같은 대표적인 EMT관련 단백질들의 발현이 증가하였다. 자궁내막세포에 TNFα를 처리하여 CCL2가 발현하는 것을 확인하였다. 따라서 자궁내막암이 난포호르몬의 자극을 받아 TNFα가 발현하고 TNFα에 의해 자궁내막세포에서 CCL2의 발현이 증가하였다. CCL2는 다시 자궁내막암에 영향을 주어 자궁내막암에서 EMT관련 단백질이 증가하였다. 이 결과를 통해 자궁내막암과 자궁내막세포 간의 상호작용이 일어나는 것과, TNFα와 CCL2를 통한 상호작용이 자궁내막암의 이동 능력을 증가시키는 것과 EMT가 일어나는 것을 밝혔다. 결론적으로 암미세환경의 조절을 통해 암세포 사멸을 효과적으로 유도할 수 있었다. 또한 자궁내막암과 자궁내막세포와의 상호작용의 기능을 알게 되었다. 고농도의 산화질소를 통해 자궁내막암세포를 선택적으로 사멸을 유도할 수 있음을 밝혔다. 피톤치드를 이용하여 자연살해세포의 활성화를 증가시켜 자궁내막암의 세포사멸을 효과적으로 유도하였다. 마지막으로 자궁내막암과 자궁내막세포와의 공배양을 통한 분석으로 TNFα, CCL2 발현이 자궁내막암의 이동 능력과 EMT를 증가시키는 것을 확인하였다. 이와 같이 암미세환경에 대한 이해와 조절을 통해 더욱 강력하고 효과적인 항암제개발을 할 수 있을 것으로 기대할 수 있다.

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초록/요약

Endometrial cancer is one of the malignant tumors of gynecological cancer. The main cause of cancer is constant effects of the female hormone estrogen. The development and progression of endometrial cancer proceed with the accumulation of mutations due to the continuous exposure to estrogen. Recently, cancer research has been actively conducted focusing on the tumor microenvironment. This study was aimed to induce the cell death of endometrial cancer through the manipulation of the tumor microenvironment focusing on the humoral communication between cancer cells and stromal cells. Cancer cells are known to be highly sensitive to reactive oxygen species (ROS). Cancer cells, in general, contain a higher amount of ROS than normal cells. Decent levels of reactive oxygen species induce cancer cell survival, cell division, and angiogenesis. However, cancer cells are highly sensitive to externally introduced ROS because cancer cells have already high concentrations of ROS. Therefore, ROS introduced from outside may act as a chemotherapeutic agent. In this study, we have manufactured a low-temperature atmospheric plasma generating device that can selectively produce nitric oxide (NO), a type of ROS, and exposed endometrial cancer cells to nitric oxide. The result was consistent with a selective cell death of endometrial cancer cells at high concentrations of nitric oxide. Based on these results, a high concentration of nitric oxide in a controllable mode can be applied to cancer chemotherapy. Immune cell research is an essential part of the cancer research through the tumor microenvironment. Therefore, cancer cell death through the activation of immune cells was studied. Natural killer (NK) cells are innate immune cells that can destry cancer cells directly. There are two basic principles of killing cancer cells by NK cells: the first of which is the recognition of cancer cells by NK cells and the second is the transmission of signals that induce cancer cell death. Proteins such as perforin and granzyme B can use the killing of cancer cells. Cancer-xenographic experiments were conducted using phytoncides to activate NK cells. Activated NK cells were expected to kill tumors more effectively. As a result of the experiment, phytoncide scents was applied to mice which carried endometrial tumor xenografts. Our results showed that the volume of the tumor xenografts was reduced by 44.18% compared to that of control group. The cytotoxicity of NK cells extracted from the spleen of mice was significantly increased in the phytoncide scent-treated mice. Phytoncides are essential oils derived from plants consisting of various components. Therefore, phytoncide scents used in mice was analyzed by gas chromatography. Then, each component was separately applied to mouse NK cell line to determine which component of phytoncides was effective in activating NK cells. As a result, the cytotoxicity of NK cells was the highest in α-pinene when target cells were hematologic cancer cell line. When α-pinene was applied to NK cells and targeted to endometrial cancer cells, the cytotoxicity of NK cells was significantly enhanced to the same extent as K562 cells. Examining the intracellular signaling pathway resulting from the activation of NK cells by α-pinene revealed that Erk- Akt signaling pathway was enhanced. Cancer progression can be divided into five parts. tumor initiation, proliferation, angiogenesis, invasion and tumor metastasis. From the tumor proliferation stage, cancer cells are affected by the surrounding called tumor microenvironment. Cancer cells exchange, thus communicate with various types of surrounding cells with a mediation of cytokines. Surrounding cells reprogrammed by cancer cells retransmit signals that help cancer cells survive, and proliferate with angiogenesis. To this regard, cytokine array analysis was performed to explore the interaction between endometrial cancer cells and endometrial stromal cells. Endometrial cancer cells and endometrial stromal cells were co-cultured and analyzed for cytokine array. Results showed that the expression levels of 33 cytokines were increased while the expression levels of 15 cytokines were decreased. As a result of co-culture, the motility of endometrial cancer was significantly increased through co-culture in an estrogen-dependent manner. Among several cytokines, the roles of CCL2 and GDF15, according to the cytokine array results, were further studied. Two proteins were overexpressed in human endometrial cancer cell line HEC-1A cells. As a result, the expression levels of these proteins were comparable to epithelial-mesenchymal transition (EMT). Expression of representative EMT related proteins such as α-SMA, vimentin, Twist 1, Twist 2, MMP3, and MMP9 were increased. Treatment of endometrial stromal cells with TNFα was confirmed to induce the expression of CCL2. Therefore, endometrial cancer was stimulated by estrogen, and TNFα was expressed, and TNF alpha increased CCL2 expression in endometrial cells. CCL2 again affected endometrial cancer, resulting in increased EMT-related proteins in endometrial cancer. The results revealed that the cross-talk between endometrial cancer and endometrial cells, the cross-talk between TNF alpha and CCL2 increased motility and EMT of endometrial cancer. In conclusion, the control of the tumor microenvironment effectively induced the cancer cell death. In addition, we have revealed the function of cross-talks bewteen endometrial cancer cells and endometrial stromal cells through cytokines. High levels of nitric oxide have been shown to selectively induce endometrial cancer cells death. Phytoncides increased the activation of NK cells, thus effectively induced the apoptosis of endometrial cancer cells. Finally, the co-culture of endometrial cancer cells and endometrial stromal cells revealed that expression of TNFα and CCL2 increased the mobility and EMT of endometrial cancer cells. Thus, through the understanding and control of the tumor microenvironment, it can be expected to develop more powerful and effective anti-cancer drugs.

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목차

TABLE OF CONTENTS
ABSTRACT (English) i
CHAPTER 1. Cell death induction of endometrial cancer cells by nitric oxide 1
1.1. INTRODUCTION 2
1.2. MATERIALS&METHODS 4
1.2.1 Cell culture and Reagent 4
1.2.2 Nitric oxide generation and quantification 4
1.2.3 Nitric oxide Griess assay 4
1.2.4 Cell viability assay 5
1.2.5 Immunoblot assay 5
1.2.6 Annexin V and PI staining assay 6
1.2.7 Measurement of intracellular reactive oxygen species level 6
1.2.8 Statistical analysis 6
1.3. RESULTS 7
1.3.1 The plasma device generates nitric oxide gas whose concentrations depend on the O2 gas level. 7
1.3.2 Concentrations of nitric oxide dissolved in the cell culture media linearly depends on NO gas applied to the media 7
1.3.3. Nitric oxide reduces endometrial cancer cell viability but not endometrial stromal cells. 7
1.3.4 Nitric oxide induces apoptosis of HEC-1A cells. 8
1.3.5. Nitric oxide increases cell death signaling favoring apoptosis. 9
1.3.6. Nitric oxide induces ROS-mediated apoptosis in HEC-1A cells. 9
1.4. DISCUSSION 11
1.5. FIGURES LEGENDS 14
1.6. REFERENCES 25
CHAPTER 2. Phytoncides enhance anti-cancer activity of natural killer cells 29
2.1. INTRODUCTION 30
2.2. MATERIALS&METHODS 32
2.2.1 Cell culture and reagent 32
2.2.2 Mice tumor xenografts and phytoncide treatment regimens 32
2.2.3 Tumor volume estimation 33
2.2.4 Mice natural killer cell isolation and measurement of cytotoxicity 33
2.2.5 Component analysis of phytoncide scents by gas chromatography mass spectrometry 34
2.2.6 Natural Killer cell NK-92mi activation 34
2.2.7 Reverse transcription and real-time PCR 35
2.2.8 Natural killer cell cytotoxicity assay 35
2.2.9 Immunoblot assay 36
2.2.10 Statistical analysis 36
2.3. RESULTS 38
2.3.1. Phytoncide inhibited the growth of CT-26 colon cancer cell xenografts in balb/c mice. 38
2.3.2. Phytoncide enhances mouse spleen Natural Killer activity. 38
2.3.3. Ingredient analysis of phytoncide scents. 39
2.3.4. Phytoncides elevate the expression of cytotoxic effector proteins in NK-92mi cells 39
2.3.5. α-pinene increase NK-92mi cell cytotoxicity. 39
2.3.6. α -pinene induce Natural killer cytotoxicity activation through Erk, Akt pathways 40
2.4. DISCUSSION 41
2.5. FIGURES LEGENDS 43
2.6. TABLES LEGENDS 46
2.7. REFERENCES 59
CHAPTER 3. Endometrial cancer cell and endometrial stromal cell cross-talk induces the epithelial to mesenchymal transition via cytokines 65
3.1. INTRODUCTION 66
3.2. MATERIALS&METHODS 68
3.2.1 Cell culture and reagent 68
3.2.2 Wound healing assay 68
3.2.3 Reverse transcription PCR and real-time PCR 69
3.2.4 Human Cytokine assay 70
3.2.5 Overexpression of growth differentiation factor-15 (GDF15) and chemokine (C-C motif) ligand 2 (CCL2) in HEC-1A cells 70
3.2.6 Statistical analysis 71
3.3. RESULTS 72
3.3.1 Co-cultured endometrial cancer cells cross-talk with endometrial stromal cells via cytokines. 72
3.3.2 Co-culture of endometrial cancer cells increases its motility 72
3.3.3 Cytokine expression profiles in conditioned media 73
3.3.4 The overexpression GDF15 and CCL2 induces epithelial-mesenchymal transition (EMT) in HEC-1A cells 73
3.3.5 The estrogen-TNFα-CCL2 axis plays a role in cancer invasion/metastasis by amplifying its EMT 74
3.4. DISCUSSION 75
3.5. FIGURES LEGENDS 79
3.6. TABLES LEGENDS 82
3.7. REFERENCES 94
ABSTRACT (Korean) 101

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