MTBP promoted the proliferation, migration and invasion of colon cancer cells by activating the expression of ZEB2

Paerhati Shayimu,Aikeremu Yusufu,Aizimaiti Rehemutula,Darebai Redati,Rexida Jiapaer,Rousidan Tuerdi 한국통합생물학회 2021 Animal cells and systems Vol.25 No.3

Colon cancer is a malignant tumor that seriously affects human health. Recently, studies revealed that the expression of MTBP enhanced the proliferation and metastasis of many types of cancer cells. And the data also showed that MTBP has the potential to regulate the expression of ZEB2. However, it is unclear whether MTBP can affect the proliferation, migration and invasion of colon cancer cells by modulating the expression of ZEB2. In this study, we established the MTBP overexpression and knockdown colon cancer cells with the transfection. Next, CCK-8 and transwell assays were carried out to determine the changes of the proliferation and invasion of colon cancer cells, respectively. After that, we overexpressed the ZEB2 in these MTBP knockdown colon cancer cells. Finally, the invasion and migration of these cells were detected with the same methods. We revealed that overexpression of MTBP enhanced the proliferation and invasion of colon cancer cells. Moreover, suppression of MTBP repressed the proliferation, migration and invasion of colon cancer cells. Furthermore, MTBP promoted the expression of ZEB2. The overexpression of ZEB2 abolished the MTBP knockdown induced inhibition of the migration and invasion of colon cancer cells. These results implied that MTBP enhanced the proliferation, migration and invasion of colon cancer cells by activating the expression of ZEB2.

Metastatic colon cancer cell populations contain more cancer stem-like cells with a higher susceptibility to natural killer cell-mediated lysis compared with primary colon cancer cells

KIM, GA RIM,HA, GA-HEE,BAE, JAE-HO,OH, SAE-OCK,KIM, SUN-HEE,KANG, CHI-DUG D.A. Spandidos 2015 Oncology letters Vol.9 No.4

<P>In the present study, the soft agar clonogenicity and the susceptibility of clonogenic cancer cells to natural killer (NK) cells were compared between primary colon cancer cells (KM12C) and metastatic colon cancer cells (KM12L4a and KM12SM) to determine whether the metastatic cancer cells consisted of more cancer stem-like cells and were resistant to NK cell-mediated lysis. The majority of colon cancer cells were positive for putative cancer stem cell markers, including CD44, CD133 and EpCAM, with the exception of KM12C cells, of which only ~55% were positive for CD133. In addition, the expression levels of sex determining region Y-box 2, Nanog and octamer-binding transcription factor 4, which are essential for maintaining self-renewal, were higher in KM12L4a and KM12SM compared with that in KM12C cells. Consistently, an increased clonogenicity of KM12L4a and KM12SM compared with KM12C cells in soft agar was observed. The expression levels of NKG2D ligands, including major histocompatibility complex class I polypeptide-related sequence A/B and UL16 binding protein 2, and of death receptor 5 were significantly higher in KM12L4a and KM12SM than in KM12C cells. Furthermore, the results indicated an increased susceptibility of KM12L4a and KM12SM to NK cell-mediated cytotoxicity in comparison with KM12C cells. These results indicated that metastatic colon cancer cell populations may consist of more cancer stem-like cells, and have greater susceptibility to NK cell-mediated lysis compared with that of primary colon cancers.</P>

Effect of miR27a on Proliferation and Invasion in Colonic Cancer Cells

Gao, Yang,Li, Bao-Dong,Liu, Yong-Gang Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.8

The aim of this study was to detect the expression of miR196a, miR146a, miR27a and miR200a in patients with colon cancer, and investigate the effect of miR27a expression on proliferation and invasion in colonic cancer cells. RT-PCR was employed to detect the expression levels in colon cancers. Then, colon cancer cells were cultured and transfected with 100 nM of miR27a mimics (80 nmol/L) or 80 nM miR27a inhibitors (80 nmol/L) in 24-well plates. Proliferation and invasion of colonic cancer cells were then determined by CCK-8 and Transwell assays, respectively. Our data showed miR27a to be high-expressed in patients with colon cancer. In addition, proliferation and invasion in the miR27a mimic group were significantly higher than in the control group and negative group (P<0.05), while, proliferation and invasion in the miR27a inhibitor group were obviously lowered (P<0.05). In conclusion, high expression of miR27a may play an important role in enhancing proliferation and invasion of colon cancer cells.

적정 구성 배양 HCT-8 기반 대장암 스페로이드의 암 줄기세포능 및 항암제 내성 평가의 비교 평가 연구

이승준(Seung Joon Lee),김형갑(Hyoung-Kab Kim),이향범(Hyang Burm Lee),문유석(Yuseok Moon) 한국생명과학회 2016 생명과학회지 Vol.26 No.11

암은 비균질적으로 구성된 세포집합체로 간질세포 및 세포 외 기질로 구성된 미세환경과 상호작용에 의해 발병, 전이, 심화되는 복잡한 질병이다. 하지만, 기존의 2차원 배양 세포 기반 플랫폼이 3차원적 생체 환경과 암의 비균질성을 대표하기 힘든 한계를 극복하기 위해 스페로이드 배양 세포를 비롯한 다양한 플랫폼 개발이 활발해지고 있다. 본 연구에서는 특히 감염, 염증 및 식이적 환경성 영향력에 민감한 HCT-8 대장암 세포주를 기반으로하여 3차원 스페로이드 배양법을 보다 효과적인 방법으로 개선하고, 대장암 스페로이드 세포를 기반으로 암의 비균질적인 특질과 항암내성 연구의 간단하고 개선된 플랫폼을 제시고자 하였다. 3차원 배양법 최적화를 위해 물리적 배양환경 조성과 배양배지 구성에 따른 스페로이드 형태형성을 비교 분석하고 암 줄기세포군의 증가 양상을 확인한 결과, 필수요소로 구성된 제한 배지와 균일한 형태의 비부착성 표면 배양접시에서 배양된 스페로이드가 균일한 형태의 구형을 형성하고 암 줄기세포군이 증가함을 확인하였다. 대장암 스페로이드 세포를 기반으로 대장암 치료제인 5-Fluorouracil (5-FU)에 대한 화학적 감응성 변화를 측정한 결과, 암 줄기세포가 5-FU에 대한 화학적 감수성 저해의 원인이 되며, 최적배양 조건에서 암 줄기세포의 약제 내성의 표현이 증대되었다. 이는 암줄기세포의 항암제 내성에 대한 잠재적 위험성을 내포하는 것으로, 이 방법론은 감염, 염증 및 식이적 요인과 연관된 대장암 스페로이드 세포 기반 항암제 약물반응을 검증하기 위해 효과적이면서 간소한 시험법으로 활용될 수 있을 것이다. Cancer is a complex disease heterogeneously composed of various types of cells including cancer stem-like cells responsible for relapse and chemoresistance in the tumor microenvironment. The conventional two-dimensional cell culture-based platform has critical limitations for representing the heterogeneity of cancer cells in the three-dimensional tumor niche in vivo. To overcome this insufficiency, three-dimensional cell culture methods in a scaffold-dependent or –free physical environment have been developed. In this study, we improved and simplified the HCT-8 colon cancer cell-based spheroid culture protocol and evaluated the relationship between cancer stemness and responses of chemosensitivity to 5- Fluorouracil (5-FU), a representative anticancer agent against colon cancer. Supplementation with defined growth factors in the medium and the culture dish of the regular surface with low attachment were required for the formation of constant-sized spheroids containing CD44<SUP>+</SUP> and CD133<SUP>+</SUP> colon cancer stem cells. The chemo-sensitivities of CD44<SUP>+</SUP> cancer stem cells in the spheroids were much lower than those of CD44<SUP>-</SUP> non-stem-like cancer cells, indicating that the chemoresistance to 5-FU is due to the stemness of colon cancer cells. Taken together, the inflammation and oncogenic gut environment-sensitive HCT-8 cell-based colon cancer spheroid culture and comparative evaluation using the simplified model would be an efficient and applicable way to estimate colon cancer stemness and pharmaceutical response to anticancer drugs in the realistic tumor niche.

Array-비교유전체보합법을 이용한 결장암세포주에서의 유전체 변이 연구

김미진(Mi Jin Kim),박수연(Soo Yeun Park),한후재(Hoo Jae Hann) 대한해부학회 2009 Anatomy & Cell Biology Vol.42 No.4

한국인 결장암세포주 세 개를 대상으로 array-비교유전체 보합법을 시행한 결과 다양한 염색체 이상 및 유전자의 증폭과 결실이 발견되었다. 기존의 결장암 연구에서 알려져 있는 염색체 1p, 1q, 2q, 8p, 8q 부위의 증가와 염색체 4q, 12q, 20p 부위의 감소를 본 연구에서도 확인하였고, 14q32.33, 16p13.3, 16q24.3 부위의 증가와 9q13, Yq11.233 부위의 감소는 본 연구를 통하여 새롭게 발견되었다. 또한 본 연구를 통하여 다양한 유전자의 변화를 확인하였다. 세 개의 결장암세포구 모두에서 공통적으로 증가한 유전자는 1q22, 1q32.1, 2q35, 8p12, 8q22.3, 14q32.33, 16p13.3, 16q24.3에 위치하고, 감소한 유전자는 9q13, 14q32.33, 20p12.1, Yq11.223에 위치한다. 다른 대장암 연구에서도 확인된 ELF 3, AAMP 유전자의 증가를 본 연구에서도 확인하였고, 다른 암 연구에서 암을 유발하는 것으로 알려진 GON4L, TIMM17Q, RNPEP, TMBIM1, GPBAR1, PPP1R13B 그리고 SOX8 유전자를 본 연구를 통해 결장암에서도 증가하는 것으로 처음 확인하였다. 또한 암발생과 관련된 염색체 부위에서 확인되었지만, 그 기능이 밝혀지지 않은 PNKD, ODF1, CBWD3, C20orf133그리고 RBMY2SP 유전자를 확인하였다. 새롭게 밝혀진 이들 유전자들은 결장암의 발생 또는 진행과 관련된 후보 유전자일 것으로 판단되며, 향후 기능적 연구를 통해 그 관련성 규명이 요구된다. Cancer development is accompanied by genetic events like losses, gains amplification of certain chromosome regions or alterations of chromatin structure. Array-based CGH(Array-CGH) is a highly comprehensive, sensitive and fast technique to allow investigation of general changes in target oncogenes and tumor suppressor genes. Recently, the prevalence of colon cancer is rapidly increasing in Korea an now it is the fourth leading cause of cancer death. So, the purpose of this study is to examine genomic alterations in colon cancer cell lines and to search novel genes which might be related to the development of colon cancer. In this study, genomic alterations are analyzed by using array-CGH in three colon cell lines from Korean, SNU-81, SNU-407 and SNU-1047. We observed numerous chromosomal imbalances from all cell lines. The common chromosomal gains were observed in 1p36.33, 1q22, 1q32.1, 2q35, 8p22.3, 14q32.33, 16p13.3, and 16q24. Common chromosomal losses were found in 4q22.1, 9q13, 14q21.1, 14q32.33, 20p12.1, Xq21.1, and Yq11.223. Gains of 1p, 2q, 8p, and 8q or losses of 4q, 14q and 20p are already known to be associated with the colon cancer development. For gene alterations, we could see gains of some genes such as ELF3 and AAMP, which were already reported to be associated with colon cancer. Also, we could find some gene alterations which were known to be associated with other cancer types. These genes were GON4L, RNPEP, TMBIM1, TIMM17A, GPBAR1, PPP1R13B and SOX8. Besides, we found alterations of new genes such as PKND and LEPROTL1. The association of these genes with colon cancer is first demonstrated here. These genes may be the novel candidate genes functioning in the development of colon cancer. In conclusion, array-CGH demonstrated the complexity of genetic aberrations in several colon cell lines. These data about the patterns of genomic alterations could be a basic step for understanding more detailed genetic events in the carcinogenesis and also provide information about possible target genes for diagnosis and treatment in colon cancer.

STK899704 inhibits stemness of cancer stem cells and migration via the FAK-MEK-ERK pathway in HT29 cells

( Hui-ju Jang ),( Yesol Bak ),( Thu-huyen Pham ),( Sae-bom Kwon ),( Bo-yeon Kim ),( Jintae Hong ),( Do-young Yoon ) 생화학분자생물학회 2018 BMB Reports Vol.51 No.11

Colon cancer is one of the most lethal and common malignancies worldwide. STK899704, a novel synthetic agent, has been reported to exhibit anticancer effects towards numerous cancer cells. However, the effect of STK899704 on the biological properties of colon cancer, including cancer cell migration and cancer stem cells (CSCs), remains unknown. Here, we examined the inhibitory effect of STK899704 on cell migration and CSC stemness. In the wound healing assay, STK899704 significantly inhibited the motility of colon cancer cells. Furthermore, STK899704 downregulated the mRNA expression levels of the cell migration mediator focal adhesion kinase (FAK). STK899704 also suppressed mitogen-activated protein kinase kinase and extracellular signal-regulated kinase, which are downstream signaling molecules of FAK. Additionally, STK899704 inhibited stemness gene expression and sphere formation in colon cancer stem cells. These results suggest that STK899704 can be used to treat human colon cancer. [BMB Reports 2018; 51(11): 596-601]

COX-2- and endoplasmic reticulum stress-independent induction of ULBP-1 and enhancement of sensitivity to NK cell-mediated cytotoxicity by celecoxib in colon cancer cells

Kim, So-Jung,Ha, Ga-Hee,Bae, Jae-Ho,Kim, Ga Rim,Son, Cheol-Hun,Park, You-Soo,Yang, Kwangmo,Oh, Sae-Ock,Kim, Sun-Hee,Kang, Chi-Dug Elsevier 2015 Experimental cell research Vol.330 No.2

<P><B>Abstract</B></P> <P>In the present study, we investigated whether celecoxib could induce the expression of NKG2D ligands in clonogenic colon cancer cells, and increase their susceptibility to NK cell-mediated cell death. Celecoxib and its non-coxib analog, 2,5-dimethyl celecoxib, induced ULBP-1 and DR5 in both COX-2 negative HCT-15 cells and COX-2 positive HT-29 cells. Celecoxib increased their susceptibility to NK92 cells in both DELFIA assay and soft agar colony forming assay. The inducibility of ULBP-1 and DR5 by celecoxib was not different between CD44- and CD44+ HCT-15 cells, and CD133- and CD133+ HT-29 cells. Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells, at least comparable to less clonogenic CD44- HCT-15 and CD133- HT-29 cells, respectively. In addition, celecoxib induced CHOP, and thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5 but not ULBP1 in HCT-15. Taken together, these findings suggest that celecoxib induces the expression of ULBP-1 as well as DR5 in clonogenic colon cancer cells via COX-2 and ER stress-independent pathways, and increases their susceptibility to NK cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Celecoxib induced ULBP-1 and DR5 in both HCT-15 cells and HT-29 cells, and subsequently increased their susceptibility to NK92 cells. </LI> <LI> Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells. </LI> <LI> 2,5-Dimethyl celecoxib also induced ULBP-1 and DR5 in both HTC-15 and HT-29 cells similarly with celecoxib. </LI> <LI> Treatment of HCT-15 cells with thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5, but not ULBP-1. </LI> </UL> </P>

Regulated Mesenchymal Stem Cells Mediated Colon Cancer Therapy Assessed by Reporter Gene Based Optical Imaging

Kalimuthu, Senthilkumar,Zhu, Liya,Oh, Ji Min,Lee, Ho Won,Gangadaran, Prakash,Rajendran, Ramya Lakshmi,Baek, Se Hwan,Jeon, Yong Hyun,Jeong, Shin Young,Lee, Sang-Woo,Lee, Jaetae,Ahn, Byeong-Cheol MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.4

<P>Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (<I>p</I> < 0.05 and 0.01) whereas no significant changes were observed in DOX(−) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(−) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.</P>

Growth Inhibition of Colon Cancer through Inactivation of STAT3 Pathway by IL-10 and IL-1ra Released from Murine Macrophage

원도희,권순미,조미란,남상윤,이범준,윤영원,오기완,한상배,홍진태 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.4

The objective of this study was to know the effect of macrophage on human colon cancer cell growth. The results showed that co-culture of colon cancer cells with macrophage inhibited the growth of colon cancer cells (HCT116 and SW620) depending on the number of macrophage, RAW 264.7 cells as well as activated THP-1 cells accompanied with down regulation of pSTAT3 in cancer cells. We also found that expression and release of cancer cell growth inhibitory cytokines; IL-1 receptor antagonist (IL-1ra), and IL-10 were increased in macrophages. Blocking of STAT3 pathway with specific inhibitor and siRNA of STAT3 abolished cancer cell growth of colon cancer, and expression of IL-1ra and IL-10. Moreover, neutralization of IL-1ra and IL-10 with antibodies, reversed macrophages-induced cancer cell growth inhibition. These data showed that IL-1ra and IL-10 released from macrophage inhibits colon cancer cell growth through inhibition of STAT3 pathway.

Growth Inhibition of Colon Cancer through Inactivation of STAT3 Pathway by IL-10 and IL-1ra Released from Murine Macrophage

Jin Tae Hong, Dohee Won1, Mi Hee Park, Sun Mi Kown, Miran Jo, Sang-Yoon Nam, Beom Jun Lee, Young Won Yun, Ki-Wan Oh, Sang Bae Han 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.4

The objective of this study was to determine the effect of macrophages on growth of human colon cancer cells. The results showed that co-culture of colon cancer cells with macrophages inhibited the growth of colon cancer cells (HCT116 and SW620) depending on the number of macrophages, RAW 264.7 cells, and activated THP-1 cells accompanied by down regulation of pSTAT3 in cancer cells. We also found that expression and release of cancer cell growth inhibitory cytokines, IL-1 receptor antagonist (IL-1ra) and IL-10, was increased in macrophages. Blocking of the STAT3 pathway with specific inhibitor and siRNA of STAT3 abolished the growth of colon cancer cells and expression of IL-1ra and IL-10. In addition, neutralization of IL-1ra and IL-10 with antibodies resulted in reversal of macrophage-induced inhibition of cancer cell growth. These data showed that IL-1ra and IL-10 released from macrophages inhibit growth of colon cancer cells through inhibition of the STAT3 pathway