대장암 조직에서 survivin의 발현에 관한 연구

세포자멸사는 조직의 발달과 기관의 형성에 필수적인 기전이다. 이 과정이 억제되면 세포의 생명주기가 연장되므로 돌연변이가 있거나 비정상적인 형태를 가진 세포의 축적을 유도하여 암종을 발생시키고 항암제에 대한 세포의 저항성을 증가 시킨다고 알려져 있다. Survivin은 최근에 밝혀진 IAP군(Inhibitor of Apoptosis family)으로서 세포자멸사를 억제하는 효과를 내는 것으로 알려져 있으며 이 유전자는 염색체 17q25에 위치한다. Survivin은 태생기에는 발현되나 정상적인 상인의 조직에서는 발현되지 않으며 여러 종류의 암종이나 변형이 일어난 세포에서만 발현되는 유전자로 그 전사 단백질은 16.5kDa이다. 여러 암종에서 survivin의 단백질의 존재에 대한 연구가 시작되고 있으나 아직도 그 역할이나 조절기전에 대해서는 거의 알려진 바가 없다. 이에 따라 인간의 대장암 조직에서 survivin의 유전자가 실제로 발현하고 있는지 확인하고 기존에 알려진 세포자멸사의 최종 효과자인 caspase 3와 그 기질인 PARP와 survivin과의 연관성을 알아보고자 본 연구를 실시하였다. 양성 대조군은 survivin 발현이 확인되었던 HL60, Daudi, THP1 md의 세포주와 그외 COLO 201, ZR-75-1, WiDr 등의 세포주를 이용하였고 음성 대조군은 정상 대장 조직 6개를 대상으로 하였다. 대장암 36예의 수술 환자의 냉동조직에서 RNA를 추출하여 survivin과 caspase 3에 대한 역전사 연쇄중합효소반응을 실시하였다. 파라핀 포매조직의 확보가 가능하였던 20예의 대장암 조직에서 caspase 3와 PARP에 대한 면역조직화학 염색을 실시하여 그 발현도를 양성과 음성으로 구분하였다. 또, caspase 3에 대한 웨스턴 영동을 실시하여 각 조직에서 caspase 3의 단백질 발현이 어느정도인지 확인하고자 하였다. 역전사 중합효소반응 결과 survivin은 36예의 대장암 조직 중 22예(61.1%), caspase 3는 27예(75%)에서 양성이었다. 면역조직화학 염색의 결과 caspase 3, PARPdml 경우 각각 13예(65%), 17예(85%)에서 정상에 비해 발현도가 저하되었거나 소실되어 있었다. 웨스턴 영동의 결과 정상 대장조직과 암조직에서 모두 32kDa의 비활성형의 띠를 확인할 수 있었으며 암조직의 경우 16예(80%)에서 정상조직보다 과발현되어 있었다. 임상, 병리 소견과 survivin의 발현도를 비교한 결과 통계적인 의의가 없었으며 caspase 3, PARPdml 발현도와도 유의한 차이가 없었다. 본 연구의 결과 대장암 조직에서 survivin은 61.1%의 매우 높은 비율로 발현되고 있음을 알 수 있었고 이는 대장암에서 세포자멸사가 억제되어 있으며 이 과정에 survivin이 작용하고 있을 가능성을 시사하는 소견이다. Caspase 3는 대표적인 세포자멸사의 최종 효과자이고 세포자멸사가 진행되면 32kDa의 비활성형이 분리되어 활성형으로 변화하는것으로 알려져 있으며 caspase의 활성도는 단백질단계에서 결정되므로 웨스턴 영동의 결과가 더 정확하다. 웨스턴 영동의 결과 암조직에서 caspase 3에 대한 비활성형의 따가 두렷하게 과발현되는 것을 확인할 수 있었으나 이 결과와 survivine의 발현여부와의 관련성은 없었다. 기존의 연구 결과와 같이 caspase 3은 불활성 형태로 존재하여 대장암 조직에서 세포자멸사가 감소되어 있는 것으로 생각되고, 암조직에서 세포자멸사 억제인자인 survivin의 mRNA가 비정상적으로 발현되고 있는 것으로 보아 survivne이 암종에서의 mRNA가 비정상적으로 발현되고 있는 것으로 보아 survivine이 암종에서의 세포자멸사 억제기전에 작용하는 것으로 보이며 추후 심도있는 연구가 필요할 것으로 생각된다. ; Apoptosis is a programmed cell death that is essential step for tissue development and morphogenesis of organs. When apoptosis is suppressed, the cell life span is delayed, thus accumulation of genetic mutation is induced resulting in the development of neoplasm. Survivin, the recently discovered apoptosis inhibitor of IAP family, is located in chromosome 17q25. It is found only in fetal tissue and transformed tissue but never found in normal adult tissue. Several authors reported survivin expression in various cancer tissues, that suggested the role of survivin in cancer development. In this study, the author intended to find the degree of survivin expression and caspase 3 that is the know substances related to apoptosis using the RT-PCR technology. That the cell lines were known to have survivin expression such as HL60, Daudi, THP1, COLO 201, ZR-75-1 and WiDr were used for the positive control. For the negative control, normal colon tissues were included. The total RNAs from the frozen tissue of 6 normal colon, 36 specimens of colorectal cancer and cell lines were used in RT-PCR. After the primers for survivin and caspase 3 were produced, PCR amplification was done by thermal cycler. Next, the 20 paraffin embedded tissues among the above patients were used in immunohistochemical study to find out the degree of protein expression of caspase 3 and PARP. The results were interpreted in either positive or negative. Using same tissues, western blotting was conducted for caspase 3 as usual manners. The expression rates of survivin were compared with the result of the immunohistochemical staining, western blotting of caspase 3 and PARP. The survivin expression by RT-PCR techniques was found at the 22 cases(61.1%). As a result of the immunohistochemical staining, 13 cases(65%) in caspase 3, 17 cases(85%) in PARP showed reduced staining. In western blotting, 32kDa inactive form of caspase 3 was expressed in 16 cases(80%), but band of active split form was not found. No significant relationship was found between survivin expression and clinicopathologic datas of colorectal carcinoma or expression of caspase 3 and PARP. In conclusion, survivin is expressed highly in mRNA level and caspase 3 protein is reduced and its inactive form is overexpressed in human colorectal cancer tissues. These result suggest that apoptosis mechanism is depressed in colorectal cancer tissues and survivin plays a role in the inhibition of apoptosis of colorectal cancer. More profound study will be needed in this field so that the apoptosis mechanism in tumorigenesis can be confirmed.논문개요 ------------------------------------------------------------ ⅶ I. 서론 ------------------------------------------------------------- 1 II. 연구대상 및 방법 ------------------------------------------------ 4 A. 연구대상 -------------------------------------------------------- 4 B. 연구방법 -------------------------------------------------------- 4 1. 역전사 연쇄중합효소반응 ---------------------------------------- 5 2. 면역 조직화학 염색 --------------------------------------------- 7 3. 웨스턴 영동 ---------------------------------------------------- 7 4. 통계분석 ------------------------------------------------------- 9 III. 결과 ----------------------------------------------------------- 12 A. 대장암 환자의 임상소견 및 병리소견의 분석 ----------------------- 12 B. 대장암조직에서 survivin의 발현 양상 ----------------------------- 12 C. Caspase 3의 발현 결과 ------------------------------------------- 13 D. PARP의 발현과 결과 ---------------------------------------------- 13 IV. 고찰 ------------------------------------------------------------ 19 V. 결론 ------------------------------------------------------------- 24 참고문헌 ------------------------------------------------------------ 25 영문초록 ------------------------------------------------------------ 3

Surgical outcomes of Korean ulcerative colitis patients with and without colitis-associated cancer

AIM: to determine the clinicopathologic characteristics of surgically treated ulcerative colitis (UC) patients, and to compare the characteristics of UC patients with colitis-associated cancer (CAC) to those without CAC. METHODS: Clinical data on UC patients who underwent abdominal surgery from 1980 to 2013 were collected from 11 medical institutions. Data were analyzed to compare the clinical features of patients with CAC and those of patients without CAC. RESULTS: Among 415 UC patients, 383 (92.2%) underwent total proctocolectomy, and of these, 342 (89%) were subjected to ileal pouch-anal anastomosis. CAC was found in 47 patients (11.3%). Adenocarcinoma was found in 45 patients, and the others had either neuroendocrine carcinoma or lymphoma. Comparing the UC patients with and without CAC, the UC patients with CAC were characteristically older at the time of diagnosis, had longer disease duration, underwent frequent laparoscopic surgery, and were infrequently given preoperative steroid therapy (P < 0.001-0.035). During the 37 mo mean follow-up period, the 3-year overall survival rate was 82.2%. CONCLUSION: Most Korean UC patients experience early disease exacerbation or complications. Approximately 10% of UC patients had CAC, and UC patients with CAC had a later diagnosis, a longer disease duration, and less steroid treatment than UC patients without CAC. © The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved

New hope for wound healing after bowel resection

[No abstract available

SiRNA targeting livin decreases tumor in a xenograft model

AIM: To evaluate the effect of silencing Livin gene expression with siRNA to apoptosis and proliferation in a colon cancer cell line. METHODS: To investigate the anticancer effect of silencing Livin gene expression, we established an siRNA transfected cell line using the HCT116 colon cancer cell line. After confirming the successful transfection, MTT assay, flow cytometry and annexin V staining were employed to evaluate the antiapoptotic effect. To confirm the in vivo effect of Livin-siRNA, different doses of LivinsiRNA were injected into xenografted tumors in BALB/c nude mice model. RESULTS: Livin expression was dramatically decreased after siRNA transfection, especially at 25 μmol/L of siRNA, but this suppression was not dose-dependent. The cell count at 18 h after transfection was significantly reduced as compared with controls (P < 0.01), but tended not to decrease proportionally depending on transfected dose or time. MTT assay revealed that silencing the Livin gene suppressed cellular proliferation at 18 h after transfection (P = 0.04); however, the inhibitory effect disappeared thereafter. Also, there was no significant difference in cellular proliferation depending on siRNA dose. The rate of apoptosis also increased with silencing of the Livin gene. In vivo, the tumor size significantly decreased after LivinsiRNA injection at 20 μmol/L concentration (P = 0.03). There were no significant body weight changes of mice after siRNA injection. Histologic examination revealed no significant toxic reaction in kidney, liver and brain of mice. CONCLUSION: SiRNA-mediated downregulation of Livin expression can induce apoptosis in colon cancer in vitro and in vivo, which suggests the possibility of new cancer therapeutics using siRNA. © 2011 Baishideng

Hemoglobin induces colon cancer cell proliferation by release of reactive oxygen species

Aim: To study whether hemoglobin could amplify colon cancer cell proliferation via reactive oxygen species (ROS) production. Methods: Colon cancer cell line HT-29 was grown in the conventional method using RPMI1640 media. The viability of the cells was measured using the colorimetric MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay after adding hemoglobin. We determined reactive oxygen species levels to be indicators of oxidative stress in HT 29 cell lines with and without hemoglobin and/or 5-fluorouracil (5-FU), 5′-deoxy-5-fluorouridine (5-DFUR) using fluorometric dichlorofluorescin diacetate (DCFH-DA) assay. Results: Cellular proliferation was increased with hemoglobin in a concentration-dependent manner. A significant increment on ROS levels was found in HT 29 cells following hemoglobin incubation. The cytotoxic effects of 5-FU and 5-DFUR were significantly blunted by administration of hemoglobin. There was a slight increase of peroxiredoxin 1, superoxide dismutase 1 concentration according to different hemoglobin concentrations. Conclusion: Hemoglobin has a cellular proliferative effect on HT-29 colon cancer cell line by production of ROS. Also, hemoglobin abates cytotoxic effects of chemotherapeutic agents such as 5-FU and 5-DFUR. © 2006 The WJG Press. All rights reserved

Extensive bowel ischemia with heavy alcohol consumption: Report of a case

Alcohol is well-recognized systemic toxin that causes numerous adverse effects, including psychosocial problems, fatal myocardial infarction, stroke and atherosclerosis. The intra-abdominal complications caused by acute alcohol consumption have not been defined. We report an 80-year-old man with sub-acute small bowel and colonic ischemia after heavy alcohol intake in one sitting. We performed a resection of gangrenous bowel segments. Microscopically, there were diffuse infarction, with vasoconstriction of the mesenteric vessels in the ileum and colon without any thrombotic occlusion of the intestinal vessels. The clinicians should always be assured by confirmation of a history of recent substance abuse in patients with unexplained abdominal pain, and mesenteric ischemia should be considered in the differential diagnosis of acute or chronic abdominal pain in consumers of alcohol. © 2012 The Korean Society of Coloproctology

The synchronous prevalence of colorectal neoplasms in patients with stomach cancer

[No abstract available

Epidermal growth factor receptor mutations in colorectal cancer patients

Purpose: The epidermal growth factor receptor (EGFR) plays an important role in tumorigenesis and tumor progression of colorectal cancer and leads to the activation of intracellular signaling pathways. The use of anti-EGFR-targeted therapy has increased for patients with colorectal cancer, but patients with EGFR mutations will be resistant to anti-EGFR-targeted therapy. The identification of gene mutations is critical in cancer treatment; therefore, the aim of this study is to investigate the incidences of EGFR mutations in colorectal cancer patients in Korea. Methods: We retrospectively reviewed 58 colorectal cancer patients who underwent surgery between 2003 and 2006. We analyzed their EGFR mutations in four loci by DNA sequencing. In addition, we analyzed the correlation between the presence of EGFR mutation and patients' clinicopathologic features. Results: Of the 58 patients, 35 patients were male and 23 were female. Their mean age was 63.28 ± 11.18 years. Two patients (3.45%) were diagnosed as stage Tis, 7 patients (12.07%) as stage 1,24 patients (41.38%) as stage II, 20 patients (34.48%) as stage III, and 5 patients (8.62%) as stage IV. As a result of mutational analysis, EGFR mutations on exon 20 were detected in 13 patients (22.41%, G→A transitions). No EGFR mutations were detected on exons 18, 19, and 21. EGFR mutation was increased in the earlier stage and in the absence of lymph node metastasis (P = 0.028). Conclusion: The incidence of EGFR mutation in Korean colorectal cancer patients is 22.41%. In addition, EGFR mutation was significantly increased in the earlier stage and in the absence of lymph node metastasis. © 2011 The Korean Society of Coloproctology

Early experience with Doppler-guided hemorrhoidal artery ligation

Purpose: Doppler-guided hemorrhoidal artery ligation (HAL) is an alternative technique to the standard Milligan-Morgan hemorrhoidectomy. The purpose of this pilot study is to introduce the HAL technique for grade 2-3 internal hemorrhoids and to evaluate the efficacy of this technique in Korea in terms of results and patient satisfaction. Methods: The HAL procedure was performed on 29 patients with grade 2 or 3 internal hemorrhoids. Twenty-eight procedures were performed under local anesthesia with lidocaine, and one procedure was performed under general anesthesia due to synchronous surgery for gallstones. With the lithotomy position, the pulsation of the hemorrhoidal artery was localized using a doppler probe, and 3-6 branches of the hemorrhoidal artery were ligated with vicryl 2-0. Patient course was evaluated before and after the procedure by using questionnaires with a visual analog scale. Results: The mean age of the patients was 44±24 yr. There were no significant complications with this procedure. At 3 mo after the operation, symptom scores of anal pain, anal bleeding, and anal prolapse were significantly improved (0.4, 1.0, and 2.4, respectively) compared to the symptom scores before the operation (3.4, 4.6, and 5.9, respectively). The postoperative satisfaction score was 8.1, and the recommendation score was 8.5. Conclusion: HAL is a safe and effective technique to relieve anal pain, bleeding, and prolapse of internal hemorrhoids. A comparative study with other procedures and a long-term follow-up after HAL should be the basis for valdating the efficacy of this procedure. ©2009 The Korean Society of Coloproctology

Clinical applications of radio-frequency ablation in liver metastasis of colorectal cancer

Purpose: The aim of this study is to evaluate long-term survival and prognostic factors for radio-frequency ablation (RFA) in colorectal liver metastases. Methods: We retrospectively reviewed 35 colorectal liver metastases patients who underwent RFA between 2004 and 2008. We analyzed survival after RFA and prognostic factors for survival. Results: Of the 35 patients, 23 patients were male and 12 were female. Their mean age was 62.40 ± 12.52 years. Mean overall survival was 38.8 ± 4.6 months, and mean progression free survival was 19.9 ± 3.4 months. Three- and 5-year overall survival rates were 42.7 ± 0.1% and 26.0 ± 0.1%, respectively. Three- and 5-year progression-free survival rates were 19.6 ± 0.1% and 4.9 ± 0.04%, respectively. Overall survival and progression-free survival were significantly improved in male and in patients with carcinoembryonic antigen (CEA) ≤ 100 ng/mL, carbohydrate antigen (CA) 19-9 ≤ 100 ng/mL, absence of extrahepatic disease, and a unilobar hepatic lesion. In addition, progression-free survival was improved in patients with a solitary hepatic lesion. On the multivariate analysis, significant survival factors were the absence of extrahepatic disease and the presence of a unilobar hepatic lesion. Conclusion: RFA for colorectal liver metastases is an effective treatment option in male patients and in patients with CEA or CA19-9 ≤ 100, absence of extrahepatic disease, a solitary hepatic lesion, and a unilobar hepatic lesion. © 2011 The Korean Society of Coloproctology