1.Induction of 90K-specific Cytotoxic T Lymphocytes for Colon Cancer Immunotherapy.
Ji Hee LEE ; Myung Suk PARK ; Ik Joo CHUNG
Immune Network 2010;10(6):206-211
BACKGROUND: Dendritic cell (DC)-based tumor vaccine is an attractive modality for the treatment of colon cancer because it has been recurred and produced few side effects in patients. Secretory glycoprotein 90K has been found at elevated level in various cancer tissues and sera. We investigated to establish a more effective DC vaccine for the treatment of colon cancer in which the levels of 90K are elevated. METHODS: We obtained the concentrated 90K from 293T cells stably expressing 90K. DCs were cultured from peripheral blood monocytes, and a DC vaccine pulsed with tumor lysate was compared with a DC vaccine pulsed with 90K. We measured the functional activity for CTLs by using IFN-gamma-enzyme linked immunoabsorbent spot (ELISPOT) assay. RESULTS: DCs pulsed with tumor lysate+90K exhibited the enhanced T cell stimulation, polarization of naive T cell toward Th1. The CTLs generated by DCs pulsed with 90K efficiently lysed HCT116 cells. The results indicate that 90K-speicifc-CTLs can recognize 90K proteins naturally presented by colon cancer cells. CONCLUSION: Our study suggests that 90K-specific CTLs generated by 90K-pulsed DCs could be useful effector cells for immunotherapy in colon cancer.
Colon
;
Colonic Neoplasms
;
Dendritic Cells
;
Glycoproteins
;
HCT116 Cells
;
Humans
;
Immunotherapy
;
Monocytes
;
Proteins
;
T-Lymphocytes, Cytotoxic
2.Competing endogenous RNA regulation mechanism and its role in the development and progression of colorectal cancer.
Xian-hua GAO ; Chuan-gang FU ; Xin-yuan LAO ; Zhu-jun TAN
Chinese Journal of Gastrointestinal Surgery 2012;15(12):1318-1321
MicroRNAs are negative regulators of mRNA, and latest studies show that "mRNAs can also inhibit microRNAs". With these reciprocal interactions, different mRNAs with identical "microRNA binding site" cim regulate each other by competitively binding to the same microRNA pool. This is the novel competing endogenous RNA (ceRN A)regulating mechanism. The ceRN A mechanism, which is a totally new regulating mechanism , greatly expands the regulatory network across genes. It has been proved by experimental evidence that, in HCT116 colon cancer cells,KRAS and PTEN , ZEB2 and PTEN can regulate each other by ceRNA mechanism.
Colorectal Neoplasms
;
genetics
;
HCT116 Cells
;
Humans
;
MicroRNAs
;
genetics
;
PTEN Phosphohydrolase
;
RNA, Messenger
3.Effects of Genistein and Daidzein on the Growth of Human Colon Cancer HCT-116 Cells.
Jong Heon SHIN ; Ku Seong KANG ; Joung Ok KIM ; Ghil Suk YOON ; Tae Gyun KWON ; Jung Wan KIM ; Yoon Kyung SOHN
Korean Journal of Pathology 2006;40(1):46-51
BACKGROUND: Genistein and daidzein are two major soybean isoflavones. They have received increasing attention because of their possible roles for cancer prevention. However, their mechanisms of action and molecular targets on the human colon cancer cells are not fully understood. METHODS: Human colon cancer HCT-116 cells were treated with genistein and daidzein to investigate their effects on the cell growth and this was analyzed with MTT assay. TUNEL assay and Hoechst33342 stain were carried out to identify apotosis. RESULTS: Daidzein was able to inhibit cell proliferation and induce apoptosis of the HCT-116 cells, but genistein didn't affect the cell growth. The ER antagonist ICI182780 didn't attenuate the antiproliferative and proapoptotic effects of daidzein: this means the effect of daidzein on the HCT-116 cells may not be dependent on the ER pathway. The other soybean isoflavone, genistein, attenuated the effects of daidzein on the HCT-116 cells and its mechanism should be elucidated. CONCLUSIONS: These data suggest that daidzein may act as a preventive agent on human colon cancer, and its mechanism of action doesn't involve the ER-dependent pathway.
Apoptosis
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Cell Proliferation
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Colon*
;
Colonic Neoplasms*
;
Genistein*
;
HCT116 Cells*
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Humans*
;
In Situ Nick-End Labeling
;
Isoflavones
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Soybeans
4.Impact of lysosome-associated protein transmembrane-4 beta on proliferation and invasion of colorectal cancer.
Yang LUO ; Zhengqian BIAN ; Guangyao YE ; Minhao YU ; Zhengshi WANG ; Shaolan QIN ; Yifei MU ; Ming ZHONG
Chinese Journal of Gastrointestinal Surgery 2015;18(6):606-610
OBJECTIVETo determine whether lysosome-associated protein transmembrane-4 beta (LAPTM4B) over-expression is associated with the proliferation and invasion in colorectal cancer (CRC).
METHODSThirty pairs of CRC tissues, containing carcinoma and adjacent tissues, were used for the examination of LAPTM4B mRNA expression by real-time quantitative PCR (qPCR) assays. Then immunohistochemistry was performed to examine LAPTM4B protein expression in 6 pairs of CRC tissues. Over-expression LAPTM4B and low-expression LAPTM4B cell models were constructed with HCT116 CRC cell lines. CCK8 assay was used to detect the proliferation and Transwell assay was used to detect the invasion of the model cells.
RESULTSqPCR and immunohistochemistry results showed that LAPTM4B expression levels in CRC were higher compared to adjacent tissues (all P<0.01). CCK8 and Transwell assays results showed that LAPTM4B promoted proliferation and invasion of HCT116 cell lines model cells (all P<0.01).
CONCLUSIONLAPTM4B promotes the proliferation and invasion in CRC patients, and may be used as an important potential marker.
Cell Proliferation ; Colorectal Neoplasms ; HCT116 Cells ; Humans ; Immunohistochemistry ; Membrane Proteins ; Neoplasm Invasiveness ; Oncogene Proteins
5.PIG3 Regulates p53 Stability by Suppressing Its MDM2-Mediated Ubiquitination.
Min JIN ; Seon Joo PARK ; Seok Won KIM ; Hye Rim KIM ; Jin Won HYUN ; Jung Hee LEE
Biomolecules & Therapeutics 2017;25(4):396-403
Under normal, non-stressed conditions, intracellular p53 is continually ubiquitinated by MDM2 and targeted for degradation. However, in response to severe genotoxic stress, p53 protein levels are markedly increased and apoptotic cell death is triggered. Inhibiting the ubiquitination of p53 under conditions where DNA damage has occurred is therefore crucial for preventing the development of cancer, because if cells with severely damaged genomes are not removed from the population, uncontrolled growth can result. However, questions remain about the cellular mechanisms underlying the regulation of p53 stability. In this study, we show that p53-inducible gene 3 (PIG3), which is a transcriptional target of p53, regulates p53 stability. Overexpression of PIG3 stabilized both endogenous and transfected wild-type p53, whereas a knockdown of PIG3 lead to a reduction in both endogenous and UV-induced p53 levels in p53-proficient human cancer cells. Using both in vivo and in vitro ubiquitination assays, we found that PIG3 suppressed both ubiquitination- and MDM2-dependent proteasomal degradation of p53. Notably, we demonstrate that PIG3 interacts directly with MDM2 and promoted MDM2 ubiquitination. Moreover, elimination of endogenous PIG3 in p53-proficient HCT116 cells decreased p53 phosphorylation in response to UV irradiation. These results suggest an important role for PIG3 in regulating intracellular p53 levels through the inhibition of p53 ubiquitination.
Apoptosis
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Cell Death
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DNA Damage
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Genome
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HCT116 Cells
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Humans
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In Vitro Techniques
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Phosphorylation
;
Ubiquitin*
;
Ubiquitination*
6.Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system.
Zhiyin ZHOU ; Xiaomei LÜ ; Li ZHU ; Ji ZHOU ; Huidan HUANG ; Chao ZHANG ; Xiaoping LIU
Chinese Journal of Biotechnology 2022;38(3):1074-1085
To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.
CRISPR-Cas Systems/genetics*
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Gene Editing
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Gene Knockout Techniques
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HCT116 Cells
;
Humans
;
RNA, Guide/metabolism*
7.Molecular cloning and preliminary analysis of a fragile site associated gene.
Yi-Wen CAO ; Chuan-Lu JIANG ; Tao JIANG
Biomedical and Environmental Sciences 2006;19(5):392-398
OBJECTIVETo analyze the molecular coining of a fragile site-associated gene.
METHODSGenomic Chinese hamster ovary (CHO) DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions (RT-PCR). The RT-PCR products were cloned into a pCRII TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues.
RESULTSTo investigate the molecular mechanism underlying the initial events of mdr1a amplification, we cloned 1q31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated (FSA) gene. FSA encoded an unusually large mRNA of approximately16 kb. Full-length human FSA cDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder.
CONCLUSIONFSA plays an important role in regulating mammalian epithelial cell growth and differentiation.
Animals ; CHO Cells ; Cell Line ; Chromosome Fragile Sites ; genetics ; Cloning, Molecular ; Cricetinae ; Cricetulus ; HCT116 Cells ; HT29 Cells ; Humans
8.Characterization of sphere-forming HCT116 clones by whole RNA sequencing.
Eunkyung CHUNG ; Inkyung OH ; Kil Yeon LEE
Annals of Surgical Treatment and Research 2016;90(4):183-193
PURPOSE: To determine CD133+ cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133+ clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential. METHODS: Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133- clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0.4 Gy γ-irradiation. RESULTS: Interestingly, there were no differences between HCT116 parental and HCT116 CD133+ clones when the cells comprised 0.5% of the total cells, and CD133- clone demonstrated radiosensitive changes compared with parental and CD133+ clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133+ clones. CONCLUSION: Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133+, and CD133- increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions.
Cell Separation
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Clinical Coding
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Clone Cells*
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Colonic Neoplasms
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HCT116 Cells
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Humans
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Neoplastic Stem Cells
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Parents
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RNA*
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Sequence Analysis, RNA*
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Transcriptome
9.Down regulation of multidrug resistance-associated protein 4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro.
Zhi-qi YU ; Chang ZHANG ; Rui CAI ; Xin-yuan LAO ; Hao WANG ; Xian-hua GAO ; Yi-fang HAN ; Xiao-qing ZHANG ; Guang-wen CAO ; Chuan-gang FU
Chinese Journal of Gastrointestinal Surgery 2012;15(1):67-71
OBJECTIVETo investigate the effect of multidrug resistance-associated protein 4 (MRP4) expression on the radiosensitivity of colorectal carcinoma cell lines in vitro.
METHODSThe vector of shRNA for RNA interference was constructed and then transfected into HCT116 cell line to steadily down-regulate the expression of MRP4. HCT116 cells were divided into 3 groups including the CON group(non-transfected), NC group (negative control virus was added), and KD group (RNAi target was added for transfection). To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow cytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTT assay at different time points.
RESULTSShRNA plasmid was successfully constructed. Transfection of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression (HCT116-KD). MRP4 mRNA and protein expression were significantly down-regulated following RNA interference(P<0.05). Twenty-four hours after radiation, the apoptosis rate of KD cell line was (71.7±0.8)%, significantly higher than that in the CON group [(56.1±0.9)%] and NC group[(59.8±0.8)%](P<0.05). Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups(P<0.05).
CONCLUSIONSExpression of MRP4 is closely related to radio-tolerance of colorectal carcinoma. Down-regulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.
Colorectal Neoplasms ; genetics ; metabolism ; Down-Regulation ; HCT116 Cells ; Humans ; Multidrug Resistance-Associated Proteins ; genetics ; RNA Interference ; Radiation Tolerance ; genetics
10.Effects of sodium selenite on the expressions of beta-catenin and its target cyclin D1 in colorectal cancer cells HCT 116 and SW480.
Hui LUO ; Yang YANG ; Cai-Min XU
Acta Academiae Medicinae Sinicae 2011;33(6):654-658
OBJECTIVETo explore the effects of sodium selenite on the expressions of beta-catenin and cyclin D1 in colorectal cancer cells HCT 116 and SW480.
METHODSHCT 116 and SW480 cells were treated by 10 micromol/L sodium selenite at different time points. The expressions and transcription of beta-catenin and cyclin D1 were detected by Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Meanwhile, the impact of MG132 (a proteasome inhibitor) pretreatment on the expressions of beta-catenin and cyclin D1 was observed through Western blot analysis. The interaction between beta-catenin and T cell factor 4 (TCF4) after selenite treatment was evaluated using co-immunoprecipitation assay.
RESULTSSodium selenite inhibited the expression of beta-catenin and transcription of its target such as cyclin D1. MG132 pretreatment prevented the inhibition of beta-catenin signaling triggered by selenite in HCT 116 and SW480 cells. Furthermore, selenite treatment disrupted the interaction between beta-catenin and TCF4 in HCT 116 and SW480 cells.
CONCLUSIONSSodium selenite can lower the expression levels of beta-catenin and its target cyclin D1, during which the proteasome-mediated degradative pathway may be involved. The decreased interaction between beta-catenin and TCF4 due to sodium selenite may be also involved in the regulation of beta-catenin signaling.
Cell Line, Tumor ; Colorectal Neoplasms ; metabolism ; Cyclin D1 ; metabolism ; HCT116 Cells ; Humans ; Sodium Selenite ; pharmacology ; beta Catenin ; metabolism