2-(Octadecyloxy) phenethyl (2-(trimethylammonio)ethyl) phosphate induces microtubules stabilizing and mitotic cell death through the inhibition of Wnt/β-catenin signaling pathway in human colorectal cancer cells

박상은 경희대학교 대학원 2022 국내석사

Abnormal activation of the Wnt/β-catenin signaling pathway is important for the pathogenesis and progression of human colorectal cancer cells (CRC). Because of the persistent dependence on the Wnt/β-catenin signaling pathway for growth and survival, inhibition of this pathway is an attractive approach for novel therapies. In this study, synthesized 2-(Octadecyloxy) phenyl (2-trimethylammonio) phosphate, an alkylphospholipid compound that inhibits the proliferation and survival of human colorectal cancer cells. It was found that Ah-yn-1 induced mitotic arrest is due to increased activation of the cdc2/cyclin B1 complex and phosphorylation of Histone H3. In addition, Ah-yn-1 induces the phosphorylation of BUBR1 and association between mitotic arrest deficiency 2 (MAD2) and cell division cycle protein (CDC) 20, indicating activation of the mitotic checkpoint complex (MCC). These results showed that Ah-yn-1 induces α-tubulin polymerization to induce apoptosis, including reduction of BCL-2 levels and enhancement of caspase-3 activation through microtubule stability. Moreover, Ah-yn-1 regulated epithelial-to-mesenchymal transition (EMT)-mediated proteins involving E-cadherin, Claudin-2, and Occludin in maintaining stabilized cell junctions and Suppression of the levels of Wnt3a, GSK3β and β-catenin protein expression, resulting in the downregulation of cyclin D1 and cMyc proteins in HT29 and HCT116 cells. This study demonstrates a novel anticancer activity of Ah-yn-1, which inhibits colorectal cancer cell EMT, thereby reducing cancer metastasis. In addition, it also provides new evidence for the development of Ah-yn-1 as a treatment for metastatic colorectal cancer.

The role of TM7SF2 in colorectal cancer metastasis and recurrence

홍인표 순천향대학교 일반대학원 2023 국내석사

Colorectal cancer (CRC) is a common fatal cancer. Worldwide, it is the fourth most common cancer in men and the third most common in women. CRC has a high survival rate of low stage without metastasis. However, when recurrence and metastasis occur, the survival rate is very low. Therefore, the early detection and treatment of metastasis-related factors can significantly increase survival rates. In this study, the transmembrane 7 superfamily member2 (TM7SF2) gene was validated as a biomarker for predicting recurrence and metastasis. Immunohistochemical staining was performed on a 236 CRC tissues, and the clinicopathological factors of patients with CRC were analyzed. Kaplan-Meier analysis was performed to confirm the relationship between the survival rate of patients with CRC and TM7SF2 expression. Additionally, TM7SF2 knockdown was performed using siRNA in SW480 and SW620 cells. TM7SF2 knockdown was confirmed using RT-PCR and immunoblotting. Functional studies were conducted to investigate the effects of TM7SF2 on cell proliferation, migration, invasion, and colony formation. As a result, clinicopathological evaluation showed, that TM7SF2 expression is associated with the pT (p=0.002), pN (p=0.006), metastasis (p=0.004), and clinical stage (p=0.004). The survival rate decreased when TM7SF2 was overexpressed (p<0.05). RNA expression levels decreased by 90.3 % in SW480 and 75.5 % in SW620, and protein expression levels decreased by 78.4 % in SW480 and 62.2 % in SW620. All functions were significantly suppressed in the CRC cell lines when TM7SF2 was knockdown. Therefore, TM7SF2 has the potential to be used as a biomarker for preventing colorectal cancer recurrence and metastasis.

A study of Circulating immune cell activity changes in Blood of colorectal cancer patients : 대장암 환자의 혈액 안에서 순환성 면역세포의 활성도의 변화에 대한 연구

이지연 건국대학교 대학원 2017 국내석사

Colorectal cancer is the most common forms of a malignant tumor and second leading cause of cancer-related death in the world. The previous study has shown that the immune cell expression in the cancer tissue was associated with cancer progression and overall survival. However, the tumor tissue to confirm the cancer progression could be obtained only after an invasive procedure. Also, the complications during the surgery might influence cancer. On the basis of the relationship between the immune cell expression and cancer progression, the circulating immune cell expression in the blood is expected to have any relationship with the cancer progression but there is no study about it. Therefore, I investigated the association between the circulating immune cells expression in the blood in colorectal cancer patient’s progression. The study was designed to total 57 patients was analyzed. Patient’s progression was classifying as American Joint Committee on Cancer (AJCC), TNM stage. And then subsection was classed as tumor depth, regional lymph node metastasis, and distant metastasis. The levels of CEA, AFP, and CA19-9 in serum were assessed using enzyme-linked immunosorbent assay (ELISA). The expression level of circulating immune cells in the blood, such as cluster of differentiation (CD)4+ including T helper 1 (Th1) and 17 (Th17) cells and CD4+CD39+ cells, CD4+CD73+, regulatory T (Treg) cells and TregCD39+, TregCD73+, CD8+ T cells, and natural killer (NK) cells were assessed using fluorescence-activated cell sorting (FACS). The serum level of CEA, AFP, and CA19-9 and blood levels of CD4+ T cell, CD4+CD39+ cell, CD4+CD73+ cell, Treg cell, TregCD39+, TregCD73+, CD8+ T cell, and NK cells did not significantly differ in tumor depth, regional lymph node metastasis, and distant metastasis. However, the expression of Th1 and Th17 cells did significantly differ between each subsection. Specifically, Th1, stage 4 group had higher expression of stage1 and stage2 and Th17 cells in stage 3 group had higher expression of stage 1 and 2. In the regional lymph node metastasis, expression of Th1 and Th17 cells has significantly increased. In the distant metastasis, Th1 cell had higher expression in M1. Therefore, my result proposes that expression of Th1 and Th17 cells in the blood has affected to cancer progression. Expression of Th1 and Th17 cell in the blood might be effective as prediction markers of lymph node metastasis in the colorectal cancer patient. 대장암은 가장 대표적인 악성 종양이며, 세계에서 암에 의한 사망률이 두 번째로 높은 암이다. 기존의 연구에서 암 조직에서의 면역세포의 발현 정도는 암의 진행과정과 전체 생존율에 연관이 있음이 연구되었다. 그러나 암 조직을 얻기 해서는 암을 제거하는 수술을 통하여만 조직을 얻어 병기 상태를 확인할 수 있다. 또한 복잡한 수술 과정은 암에 다른 영향을 미칠 수도 있다. 이런 면역세포의 발현과 암의 진행과정 사이의 관계에서 환자의 혈액 속에 순환하는 면역세포의 활성도가 암의 진행과정과 연관이 있을 것으로 보았으며, 이에 대한 연구는 되어지지 않았다. 따라서 대장암 환자의 혈액에서 순환하는 면역세포의 발현 정도와 대장암 환자의 암 진행 정도에 대한 연관성을 연구하였다. 이 연구는 모두 57명의 환자가 연구되었고, American Joint Committee on Cancer(AJCC)의 병기분류법에 따라서 TNM stage로 나누었다. 위 병기분류법을 종양의 깊이, 림프절 전이 유무, 원격 전이의 유무 등의 세부항목으로 나누었다. 환자의 혈액에서 확인한 면역세포는 CD4+ including T helper 1 (Th1) and 17 (Th17) cells and CD4+CD39+ cells CD4+CD73+, regulatory T (Treg) cells and TregCD39+, TregCD73+, CD8+ T cells, and natural killer (NK) cells 이며 유 세포 분석기를 사용하여 분석하였다. 대장암 환자의 혈액에서 CD4+ T, CD4+CD39+ cells, CD4+CD73+ cells, Treg, TregCD39+, TregCD73+, CD8+ T, and NK세포는 종양의 깊이, 림프절 전이 유무, 원격 전이 유무에서 유의하게 차이가 나지 않았으며, 도움 T 세포인 Th1 세포와 Th17 세포는 환자의 병기 상태에 따라서 유의하게 달라지는 양상을 보여주었다. 특히 암의 진행 단계에 있어서 Th1 세포와 Th17 세포는 병기 상태가 진행될수록 유의하게 증가하는 양상을 보였다. 종양의 깊이에 따른 분류(T)에서는 유의하게 달라지는 양상을 보이지는 않았지만, 림프절 전이가 있는 상태(N1)에서 두 세포 모두 증가하는 양상을 보였다. 또한 림프절 전이 개수에 따라서 환자를 분류하였을 때, 전이 개수가 증가할수록 두 세포 모두 증가하는 양상을 보여주었다. 다른 장기로의 원격 전이가 있는 상태(M1)에서는 Th1 세포가 유의하게 증가하는 양상을 보여주었다. 결론적으로 혈액 내에 존재하는 순환하는 Th1 세포와 Th17 세포가 암의 진행 상태에 영향을 가질 수 있음을 보았으며, 특히 대장암 환자의 혈액에서 순환하는 Th1세포와 Th17세포의 발현 정도를 통하여 환자의 암의 진행상태 중 림프절로의 전이 유무를 유추할 수 있는 효과적인 표지자로서의 가능성이 있음을 보여줄 수 있다고 보았다.

New target for Cetuximab Resistance For Colorectal Cancer

김보미 강릉원주대학교 일반대학원 2024 국내석사

Colorectal cancer is still a major cause of death globally. Although targeted therapies, such as cetuximab, are changing the treatment of advanced or metastatic colorectal cancer, drug resistance remains a significant challenge. This study investigates the correlation between cetuximab resistance and S100A4 expression in patients with RAS-wild type metastatic colorectal cancer and exploring the potential to overcome this resistance through the combined treatment of Neferine and Cetuximab. The study aimed to induce cetuximab-resistant SW48R cell lines from RAS-wild type SW48 colorectal cancer cells through repeated cetuximab treatments. Microarray analysis identified a relationship between these resistant cells and the S100A4 gene, which is connected to the Hypoxia pathway. Protein expression analysis across various colorectal cancer cells revealed an increase in S100A4 expression alongside cetuximab resistance, particularly in SW48R cells, indicating increased S100A4 expression through the PI3K/AKT/GSK-3β pathway. Further validation came from Immunohistochemistry (IHC) of colorectal cancer tissues collected from patients at Guro Hospital and subsequent protein expression experiments, both confirming increased expression of S100A4. Treatment with Recombinant S100A4 protein (rS100A4) activated the PI3K/AKT/GSK-3β pathway, elevating S100A4 levels, and resulted in reduced cetuximab sensitivity in SW48R cells, as evident in cell viability and apoptosis assays. Drug screening efforts to overcome cetuximab resistance identified Neferine as an effective compound, demonstrating cell death in cetuximab-resistant cells and decreasing PI3K/AKT/GSK-3β pathway activity and S100A4 protein expression. The combined treatment of Cetuximab and Neferine proved more effective than Cetuximab alone. This research underscores the correlation between overexpression of S100A4 and cetuximab resistance in colorectal cancer, proposing strategies targeting S100A4 to overcome resistance. This approach highlights the potential for improved treatment outcomes in colorectal cancer patients and underscores the importance of strategically targeting S100A4 in drug-resistant colorectal cancer.

Interplay of tumor and microenvironmental states shaping colorectal cancer landscape

홍유래 성균관대학교 일반대학원 2021 국내박사

Background: Colorectal cancer is one of the most prevalent cancers worldwide, but targeted therapy, including anti-angiogenesis or an immune checkpoint inhibitor, is effective only for subgroups, including mismatch repair-deficient patients. Therefore, elucidation of the cellular and molecular features is crucial for improved targeted therapy. Methods: To understand tumor heterogeneity and the microenvironment in patients with advanced or metastatic colorectal cancer, the single-cell transcriptome profile of 65,352 unsorted single cells from 23 advanced colorectal cancer and 132,098 cells from five synchronous and nine patients with metachronous liver metastatic colorectal cancer were performed. Additionally, genomic and transcriptome profiles were processed to identify associations between genetic alteration and tumor heterogeneity at the single-cell level. Results: In colorectal tumor tissues, cancer cells displayed two discrete molecular features with genetic alterations in TP53, APC, BRAF, KRAS, and SMAD4 from different lineages. Cancer cells with distinct characteristics regulated the immunosuppressive microenvironment directed by myofibroblasts, regulatory T cells, and tumor-associated macrophages. Furthermore, metastatic colorectal tumor tissues had amplified angiogenetic and immune modulation features. Cancer cells in metastatic tumor tissues predominantly expressed the VEGFA gene with a low stemness score, and these were associated with microenvironmental cells, which appeared to be immunosuppressive. Conclusions: The collective view of the cellular and molecular landscape and the intercellular interactions of the tumor and microenvironment in colorectal cancer suggests mechanistic conclusions that could inform the design of efficient therapeutic target strategies. 서론 : 대장암은 전 세계적으로 가장 많이 발생하는 종양 중 하나이지만, 혈관 신생 억제 혹은 면역 관문 억제제와 같은 표적 항암 치료는 불일치 복구 유전자를 가진 환자처럼 특정 그룹에만 효과적이라는 단점이 존재한다. 따라서, 대장암의 표적치료를 위해 세포 및 분자적 특성을 이해하는 것이 매우 중요하다. 방법 : 진행성 및 전이성 대장암 환자의 종양이질성과 종양 미세환경을 이해하기 위해 23명의 진행성 대장암 환자에서 65,352여개의 단일세포 전사체 데이터와 5명의 동시성, 9명의 이시성 간 전이 대장암 환자에 대한 132,098 여개에 대한 단일세포 전사체 데이터 생산을 진행하였다. 추가적으로, 단일세포 수준에서의 종양 이질성과 유전적 변화의 연관성을 확인하기 위해 게놈 및 전사체 프로파일을 생산하였다. 결과 : 대장암 환자 조직에서, 암세포는 각기 다른 상피 세포 그룹으로부터 나타난 돌연변이 그룹 (TP53, APC, 혹은 BRAF, KRAS) 에 따라 두 가지의 분자적 특성으로 분류 가능했다. 또한 암세포는 근섬유아세포, 조절 T세포, 종양 관련 대식세포를 조절함을 확인했다. 더 나아가, 전이성 조직은 혈관 신생 및 면역 조절 기능이 증폭된다는 것을 확인했다. 전이 조직의 종양 세포는 낮은 줄기세포성 특성과 더불어 VEGFA유전자 발현이 많이 일어나며, 이는 면역억제에 관련된 미세환경 세포들과 관련이 있음을 발견했다. 결론 : 이러한 대장암의 세포 및 분자적 청사진과 세포 간 상호작용은 대장암의 효율적인 치료 표적 전략을 제시할 수 있었다.

The Comparison of clinicopathologic characteristics of proximal and distal colorectal cancer with microsatellite instability : Site specific characteristics of colorectal cancer with MSI

윤환식 성균관대학교 2008 국내석사

불안정 microsatellite (부수체)는 대장암의 발생에 중요한 발생 기전으로 설명되고 있다. 최근 불안정 부수체의 대장암에서 근위부와 원위부 사이의 유전자 표현형에 차이가 있다는 사실이 밝혀지고 있다. 하지만 근위부와 원위부의 임상병리학적 차이를 비교한 연구들은 많지 않은 실정이다. 불안정 부수체 대장암의 근위부와 원위부의 임상병리학적 차이를 연구하고자 하였다. 2002년 11월부터 2006년 11월까지, 2203명의 대장암 환자가 부수체 불안정성에 대한 조사를 시행하였다. 이중 182명의 환자가 불안정 부수체를 가진 것으로 확인되었다. 이중 조건을 만족하는 146명을 연구 대상으로 하였으며, 같은 조건을 만족하는 312 명의 안정성 부수체를 가진 대장암 환자를 대조 군으로 포함시켰다. 각 집단의 대장암 위험인자들을 조사하였으며, 불안정 부수체 대장암 환자 112명에서 MLH-1, MSH-2, MSH-6 에 대한 항체를 이용하여 면역조직화학 염색을 실시하였다. 146명의 불안정 부수체 대장암 환자에서 92명 (63%)의 환자가 근위부, 54명 (37%)의 환자가 원위부 대장암 이였다. 근위부 대장암의 평균 발생 연령은 원위부 대장암의 평균 발생 연령에 비해 유의하게 낮았다 (p=0.012). 또한 불안정 부수체 근위부 대장암은 원위부 대장암에 비해 미분화된 조직학적 정도를 보였다. 146명의 불안정 부수체 대장암 환자 중에서, 112명의 환자에서 수선 유전자 (MMR gene)에 대한 면역조직화학 염색을 실시하였다. 78명에서 수선 유전자의 비활성화가 관찰되었다. 55명 (70.5%)의 환자는 hMLH1 불활성화가 있었고, 19명 (17%)에서는 hMSH2 불활성화, 그리고 32명 (28.6%)의 환자에서는 hMSH6 불활성화가 관찰되었다. 평균 발생 연령, 암의 조직학적 특성, hMLH1 유전자의 불활성화 정도 등이 근위부와 원위부 대장암에서 차이가 있는 것을 보였다. 또한 본 연구는 근위부와 원위부의 대장암에서 불안정 부수체의 획득의 원인 유전자가 서로 다르다는 것을 시사하였다. 불안정 부수체 대장암에서 그 기전은 불확실하지만, 근위부와 원위부 암 사이에는 유의한 임상병리학적 차이가 있는 것으로 생각된다. Microsatellite instability (MSI) is recognized as the one of major pathway of colorectal carcinogenesis. It is recognized that there are large regional differences in the gene expression profiles between the sporadic proximal and distal colorectal cancers with MSI. However there have been few studies comparing the clinicopathologic characteristics between proximal and distal colorectal cancer with MSI. We aimed to elucidate whether there was the difference in the clinicopathologic characteristics between the sporadic proximal and distal colorectal cancers with MSI. From December 2002 to December 2006, 2203 patients who underwent surgical resection for CRC in Samsung Medical Center and MSI tests were performed. Out of them, 182 patients were diagnosed to have MSI high (MSI-H) colorectal cancer, and we enrolled these patients. Several patients were excluded for several criteria. Total 146 patients were identified to have MSI-H colorectal cancer. We also enrolled 312 patients with microsatellite stable (MSS) colorectal cancers who met the above mentioned exclusion criteria for control group. We evaluated the risk factors of proximal and distal colorectal cancer in MSI-H and MSS colorectal cancer. In addition immunohistochemical staining using antibody for MLH-1, MSH-2, and MSH-6 was performed for the 112 colorectal cancers with MSI-H. Of the 146 MSI-H colorectal cancer patients, 92 (63%) patients had proximal and 54 (37%) patients had distal colorectal cancer. The median age of patients with proximal MSI-H colorectal cancer was higher than that of patients with distal MSI-H cancer (p=0.012). Patients with proximal MSI-H CRC tended to exhibit a higher histologic grade (i.e. poorly or mucinous or signet ring cell, or undifferentiated) (p=0.015) than the patients with distal MSI-CRC. Out of 146 MSI-H colorectal cancer patients, 112 patients were tested for immunohistochemistry (IHC) of MMR gene. 78 patients were identified as having inactivated MMR genes. 55 (70.5%) patients had hMLH1 inactivation, 19 (17.0%) patients had inactivated hMSH2, and 32 (28.6%) patients had hMSH6 inactivation. The mean age of patients, the histology of cancer, and the frequency of negative expression of MLH-1 were found to have different characteristics between the sporadic proximal and distal MSI-H colorectal cancers. In addition this study showed some different data that there might be different pathway of acquiring MSI between proximal and distal MSI-H colorectal cancer. It is thought that there was significant difference in the clinicopathologic characteristics between the sporadic proximal and distal MSI-H colorectal cancers, although the mechanism of this difference remains unclear.

Neuromedin U contributes to radiation resistance in colorectal cancer via activating YAP/TAZ signaling

심명규 Graduate School, Korea University 2022 국내석사

Resistance to radiation therapy remains an obstacle in the treatment of patients with high-risk colorectal cancer. Recent studies have indicated that neuromedin U (NMU) is upregulated in various cancers and contributes significantly to cancer growth and development. However, the impact of NMU on colorectal cancer remains unknown. In this study, NMU was identified as one of the differentially expressed genes in radiation-resistant colorectal cancer through RNAseq and real-time PCR analysis of colorectal cancer organoids derived from tissues of patients. Following the knockdown of NMU using siRNA, the colorectal cancer cells became sensitive to radiation and showed decreased biological functions such as proliferation and migration. In addition, NMU depletion increased radiation-induced apoptosis, as confirmed via TUNEL staining, and inhibited the nuclear translocation of yes associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) through phosphorylation of Hippo pathway proteins. Conversely, overexpression of NMU in colorectal cancer cells revealed the opposite phenotypes. NMU overexpression increased radiation resistance, proliferation, and migration in colorectal cancer cells, whereas it decreased radiation-induced apoptosis. Additionally, phosphorylation of Hippo pathway proteins was decreased and nuclear translocation of YAP and TAZ was increased following NMU overexpression in colorectal cancer cells. Therefore, in this study, NMU contributed to the radiation-resistant phenotypes of colorectal cancer via the activation of YAP and TAZ. Accordingly, it is suggested that NMU can be a potential biomarker and therapeutic target for radiation resistance in patients with high-risk colorectal cancer. 뉴로메딘 U 는 YAP/TAZ 신호 활성화를 통해 대장암의 방사선 저항성에 기여한다. 방사선 치료에 대한 저항성은 위험도가 높은 대장암 환자의 치료에 여러 장애 요소로 남아있다. 최근의 연구에서 뉴로메딘 U (NMU) 는 다양한 암에서 상향 조절되며 암의 성장과 발달에 크게 기여한다고 알려져 있다. 하지만 NMU 가 대장암에 미치는 영향은 아직 거의 알려지지 않았다 . 환자들 의 대장암 조직으 로부터 유래 된 오가노이드를 이용 한 RNAseq 및 실시간 PCR 분석에 의해 NMU 는 대장암의 방사선 저항성을 나타낼 수 있는 바이오마커 후보 중 하나로 분류되었다 . 대장암 세포주에서 NMU 를 감소시키자 세포의 방사선에 대한 반응성이 증가하고 증식 및 이동과 같은 생물학적 기능이 감소하였다. 그리고 NMU 의 감소는 TUNEL 염색을 통해 확인된 방사선 유도 세포자살을 증가시켰고, 히포 신호전달 경로에 관련된 단백질의 인산화를 통해서 YAP 과 TAZ 의 핵으로의 전위를 억제하였다 . 대조적으로, 대장암 세포주에서 NMU 를 과발현 시키자 반대되는 표현형이 관찰되었다 . NMU 의 과발현 은 대장암 세포 의 방사선에 대한 저항성, 증식 및 이동 을 증가 시켰 고 방사선으로 유도된 세포자살 을 감소 시켰다. 또한 N MU 의 과발현에 따라 히포 신호전달 경로 에 관련된 단백질 들 의 인산화가 감소되어 YAP 과 TAZ 의 핵으로의 전위가 증가되었다 . 따라서 본 연구에서는 NMU 는 YAP 과 TAZ 의 활성화를 통해서 대장암의 방사선 저항성에 관련된 표현형에 관여한다는 것을 증명하였다. 그러 므로 NMU 는 대장암의 방사선 저항성을 나타내는 잠재적 인 바이오마커가 될 수 있고, 방사선에 대한 저항성을 갖는 대장암 치료의 표적이 될 것이라 제안한다.

In vivo 실험을 통한 Salicornia europaea의 항암 활성과 숙취 완화 능력에 대한 누에 분말의 인체 적용 시험

차지현 가천대학교 일반대학원 2021 국내석사

Probiotics and prebiotics are tools for improving the gut environment that benefit the host's health. It serves to help humans digest and absorb nutrients that cannot be digested and have been reported to be associated with metabolism, immune system, and disease. Intestinal microbiota may inhibit colitis-related colorectal cancer (CAC) in a colorectal cancer mouse model. Here, in a colitis model induced by azoxymethane (AOM) / sodium dextran sulfate (DSS), Salicornia europaea grind (SG) and mixture of Salicornia europaea and Lactobacillus (MSL) were used to manipulate the environment of the intestinal microflora into the colon. It was found to have an improvement effect on cancer. At 9 weeks after AOM injection, mice were sacrificed to harvest the colon. The length of the harvested colon was measured and the polyps were divided by size and counted. Afterward, 10% formalin was fixed for 24 hours and preserved in 70% ethanol to make a paraffin block. The tissue made of paraffin blocks was sectioned and made into slides. Slides were cut into 5μm thick and stained with hematoxylin and eosin. PCNA and Ki-67 were stained to confirm the cell's proliferation and apoptosis ability, and the degree of transcription of c-Myc due to the accumulation of β-catenin was confirmed through Immunohistochemistry. In mice fed SG and MSL, the length of the colon decreased and the number of high-risk polyps larger than 4mm decreased. Similarly, when examined through H&E staining, the number of big size polyps in the SG and MSL groups was small and the degree of inflammation was slow. The proliferative markers examined through IHC were also expressed at low levels. Our findings suggest that SG and MSL are effective in inhibiting the growth of large-size polyp, which can progress to carcinoma in AOM/DSS-induced colorectal cancer model mice. This is predicted as an effect due to SG and SGF improving the intestinal microbial environment. Hangover causes symptoms such as vomiting, dizziness, headache, and muscle pain through digestive and cognitive functions that are caused by the process of metabolizing and acting on certain substances in alcohol in the body after drinking alcohol. This is mainly caused by acetaldehyde from alcohol metabolism. We chose steamed and freeze-dried mature silkworm larval powder (SMSP) as a natural product for hangovers. While sleep is the best protein nutrient, it contains a variety of nutrients and has the effect of preventing alcoholic liver disease and hangovers. So, we used sookjam, which contains sleep and yeast, as a test food. In this experiment, people aged 19 to 40 years of age who had a hangover after drinking alcohol were designed as a study composition virus, double-blind, randomized, placebo-controlled, cross-design human application test. As a result, it was found that the concentration of acetaldehyde in the intake of sookjam was statistically significantly reduced at all experimental times, acting as a hangover reliever. In addition, the peak blood acetaldehyde concentration (Cmax) was also significantly reduced in the test group. In particular, you can see the effect of relieving hangovers 0.5 hours after alcohol consumption. Because of this, we confirmed the existence of sookjam containing sleep and yeast and revealed that it can act as a hangover reliever. Particularly, when proven in advance, a significant hangover relief effect is expected from 0.5 hours after drinking.

Discovery of DNA methylation biomarkers and development of a diagnostic model for liquid biopsy in colorectal cancer

정준기 성균관대학교 일반대학원 2020 국내석사

Background In 2017, 28,111 people were diagnosed with colorectal cancer in South Korea and 8,691 people died of it; the number of people diagnosed with colorectal cancer is also increasing every year. Colorectal cancer is associated with somatic mutation and genetic instability in the process of carcinogenesis, and most colorectal cancers develop pathologically from polyps. Abnormal DNA methylation is one of the major factors influencing the regulation of gene expression and occurs mainly in the early stages of colorectal cancer. It is known that DNA methylation changes in genes such as MGMT, CDKN2A, and MLH1 are associated with the development of colorectal cancer. The aim of this study was to develop candidate methods of liquid biopsy for diagnosing colorectal cancer patients using methylation markers. For this purpose, I produced simulation data using DNA bisulfite sequencing data. After that, I identified DNA methylation markers that can diagnose colorectal cancer through simulation data and developed a logistic regression model. Method The analysis was performed using 118 colon cancer tissues and 118 normal tissue samples obtained from 118 patients and 31 FACS sorted blood cell samples from CRC patients operated on at Samsung Seoul Hospital. I performed statistical analysis, after which I developed a machine learning model for the diagnosis of CRC patients using simulation data. A validation dataset assuming that a few DNA fragments of colorectal cancer are present in blood was produced, and the validity of the developed model was confirmed using a validation dataset to verify the usefulness of the methylation biomarkers. Results A total of 1050 markers were identified, which can distinguish CRC tissue from normal tissue and blood samples. A classifier model was developed using 5% ctDNA simulation data produced through methylation value of CRC tissue and blood samples. Subsequently, the importance of the model was confirmed by using a simulated validation dataset consisting of tumor sequencing reads present at rates of less than 1% (1%: AUC = 1; 0.75%: AUC = 0.937; 0.5%: AUC = 0.875). Conclusion The developed model showed significant performance even if the proportion of colorectal cancer tissue sequencing reads was 0.75% in the simulation data. I expect that this model could distinguish early CRC patients with low ctDNA. If the validity of this model is verified and it is improved to fit actual cfDNA, this model can be used for the clinical diagnosis of patients. Furthermore, I expect the analytical procedures presented in this paper to be available for other cancers. 연구배경 대장암은 한국에서 가장 높은 발병률을 가지는 암 중 하나로 2017년 한해 한국에서는 28,111명이 대장암으로 진단받았으며 8,691명이 대장암으로 사망할 정도로 대장암으로 진단받는 사람의 수가 매년 증가하고 있다. 대장암 환자들에게서 관찰할 수 있는 요소인 DNA methylation의 이상은 체세포 변이, 유전자 불안정성, 유전자 발현과 연관이 있는 요소로서 암 발생 초기에 주로 일어난다. 대부분의 대장암은 용종이 발생한 후 병리학적인 발달과정을 거처 암이 발생하게 되는데 대장암의 발생에 영향을 주는 유전적 요인에 대해서는 MGMT, CDKN2A, MLH1과 같은 유전자 영역에서의 DNA methylation 변화가 대장암의 발생과 연관이 있음이 알려져 있다. 본 연구의 목표는 액체생검을 통해 대장암을 진단할 수 있는 DNA methylation marker를 발굴하고 진단 모델을 개발하였다. 이를 위하여 ctDNA의 비율이 낮은 경우를 가정한 시뮬레이션 데이터를 생산해 대장암을 구분할 수 있는 methylation 마커를 발굴하였으며 발굴한 마커를 사용해 logistic regression model을 개발하고 이를 검증하였다. 연구 방법 삼성서울병원에서 대장암을 진단받고 수술을 받은 대장암 환자 118명에게서 얻은 118건의 대장암 조직과 118건의 정상조직 샘플 그리고 31건의 FACS sorted cell data를 활용하여 분석을 진행하였다. 나는 통계검정을 통하여 마커를 발굴한 후 118개의 대장암 암 조직 샘플과 48건의 WBC 샘플을 활용하여 소량의 대장암 조직의 DNA가 혈액 내에 존재하는 것을 가정한 시뮬레이션 데이터셋을 제작하였으며 시뮬레이션 데이터를 사용해 진단 모델을 개발한 후 모델의 유용성을 검증하였다. 연구 결과 총 1050개의 마커를 발굴하였으며 이들 마커를 통해 대장암조직과 정상조직, 그리고 혈액 샘플을 구분할 수 있음을 확인하였다. 대장암 조직과 혈액 샘플의 methylation 값을 사용해5% ctDNA를 가정한 시뮬레이션 데이터를 생산하여 모델을 개발하였으며 이후 독립적인 시뮬레이션 데이터로 모델을 검증하였다. 1%의 시뮬레이션 데이터로 검증하였을 시 AUC=1의 성능을 얻었으며 0.75%에서는 AUC=0.937, 0.5%에서는 AUC=0.875의 성능을 얻을 수 있었다. 결론 본 연구에서 개발한 모델을 통해서 0.75%의 시뮬레이션 데이터에서 좋은 성능을 얻어 초기 대장암 환자에게 활용될 수 있으리라 기대한다. 임상적 활용을 위해서는 실제 cfDNA를 사용해 고도화시키는 과정이 필요하지만 개발된 모델은 초기 대장암 환자를 진단할 수 있을 것이다. 더욱이 이 논문에서 대장암 데이터데 적용한 분석과정을 다른 암종에도 적용함으로써 향후의 유용성이 기대된다.

Thymosin β4가 인간 대장암세포의 이동과 침윤에 미치는영향

박정일 전남대학교 대학원 2015 국내박사

Background: Colorectal cancer is the third most commonly diagnosed cancer in the world. With an early detection and improved treatment technique, colorectal cancer death rates have been steadily decreasing over the past decades. The five-year survival rate for patient with an early stage increases to about 90 %, but only 39 % of colorectal cancers are diagnosed at this stage. However, high mortality of this disease with a late stage and/or metastasized cancer reflects the reduced understanding of its etiological factors and pathogenesis model. Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes, including angiogenesis, wound healing, the inflammatory response, invasion, and metastasis. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. Methods: The expression of Tβ4 mRNA was evaluated by semi-quantitative RT-PCR and immunohistochemical staining in colorectal cancer tissue samples. Overexpression of Tβ4 in colorectal cancer cell line was established by transfection of Tβ4 expression plasmid, and knockdown of Tβ4 was performed using siRNA. Cell proliferation, migration, and invasion assay was used to elucidate the biological function of Tβ4. Results: Tβ4 mRNA expression was found to be significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues in 26 (81%) of 32 cases examined. High levels of Tβ4 expression in the colorectal cancer tissues closely correlated with LN metastasis and TNM stage of colorectal cancer. Upregulation of Tβ4 in colorectal cancer cell lines induced morphological changes and the EMT. Furthermore, Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cell lines showed decreased activities, but no change in proliferation activity. It was also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. Conclusion: Our results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment. Keywords: Thymosin β4, invasion, migration, β-catenin, colorectal cancer