Normal Colorectal Epithelial Cells Research Articles

Liensinine perchlorate inhibits colorectal cancer tumorigenesis by inducing mitochondrial dysfunction and apoptosis.

Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options, and there is an urgent need to develop novel therapeutic agents with good treatment efficiency and low toxicity. This study aims to examine the anticancer bioactivity of liensinine, a constituent of Nelumbo nucifera Gaertn, in CRC and investigate the action mechanisms involved. Liensinine was found to induce apoptosis and exert a significant inhibitory effect on the proliferation and colony-forming ability of CRC cells in a dose-dependent manner without any observed cytotoxicity on normal colorectal epithelial cells. Mechanistically, our data from quantitative proteomics, western blot analysis and flow cytometry analyses demonstrated that exposure of CRC cells to liensinine caused cell cycle arrest, mitochondrial dysfunction and apoptosis, accompanied by the activation of the JNK signaling pathway. Furthermore, animal experiments showed that liensinine markedly suppressed the growth of CRC tumor xenografts in nude mice by reducing the Ki-67 proliferation index, but did not damage the vital organs of the animals. This study demonstrated for the first time that liensinine, a food-source natural product, could be a novel therapeutic strategy for treating CRC without obvious side effects.

SATB2/\u03b2-catenin/TCF-LEF pathway induces cellular transformation by generating cancer stem cells in colorectal cancer

Recent studies have demonstrated the involvement of colorectal cancer (CRC) stem cells (CSC) in transformation, cancer progression and metastasis. The main goal of this paper was to examine the molecular mechanisms by which SATB2 induced malignant transformation of colorectal epithelial cells. SATB2 induced malignant transformation and these transformed cells gained the characteristics of CSCs by expressing stem cell markers (CD44, CD133, LGR5 and DCLK1) and transcription factors (c-Myc, Nanog and Sox2). Overexpression of SATB2 in normal colorectal epithelial cells increased cell motility, migration and invasion, which were associated with an increase in N-cadherin and Zeb1, and decrease in E-cadherin expression. SATB2 overexpression also upregulated XIAP and cyclin D1, suggesting its role in cell survival and cell cycle. Furthermore, the expression of SATB2 was positively correlated with β-catenin expression in CRC. In contrary, depletion of SATB2 inhibited cell proliferation, colony formation, cell motility and expression of β-catenin, Snail, Slug, Zeb1 and N-cadherin, and upregulated E-cadherin. Furthermore, SATB2 silencing inhibited the expression of stem cell markers, pluripotency maintaining transcription factors, cell cycle and cell proliferation/survival genes and TCF/LEF targets. Finally, β-catenin/TCF-LEF pathway mediated the biological effects of SATB2 in CSCs. These studies support the role of SATB2/β-catenin/TCF-LEF pathway in transformation and carcinogenesis.

Abstract 3316: Mieap, the mitochondria-eating protein, induces cell death by eating unhealthy mitochondria

Abstract Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria. Mieap suppresses murine intestinal tumor via its mitochondrial quality control (1). Mieap-regulated mitochondrial quality control is frequently inactivated in human colorectal cancer (2). To explore the role of the Mieap-regulated mitochondria quality control function in human colorectal cancer tissues, we examined the Mieap expression in primary colorectal cancer tissues by performing immunohistochemical analysis with anti-human Mieap antibody. Mieap expression was very low in normal colorectal epithelial cells, whereas the strong expression of the Mieap protein was detected in the hypoxic cell death regions of colorectal adenoma and adenocarcinoma, suggesting the role of Mieap in hypoxic cell death. Overexpression of Mieap in vitro induced large vacuole structures (MIV: Mieap-induced vacuole) in HCT116 colorectal cancer cells (Mieap-deficient colorectal cancer cells). MIV engulfed and degraded unhealthy mitochondria, leading to caspase-dependent cancer cell death. Re-expression of Mieap in HCT116 cells resulted in striking suppression of tumor growth in vivo. These results suggest that Mieap plays a critical role in hypoxic tumor cell death via lysosomal degradation of unhealthy mitochondria. (1) Tsuneki et al. Mieap suppresses murine intestinal tumor via its mitochondrial quality control. Sci. Rep. 5: 12472 (2015). (2) Kamino et al. Mieap-regulated mitochondrial quality control is frequently inactivated in human colorectal cancer. Oncogenesis 5: e181 (2016). Citation Format: Yasuyuki Nakamura, Masayuki Tsuneki, Makoto Yamamoto, Hirofumi Arakawa. Mieap, the mitochondria-eating protein, induces cell death by eating unhealthy mitochondria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2017-3316

Loss of Fas expression and high expression of HLA-E promoting the immune escape of early colorectal cancer cells.

Previous studies have investigated the mechanisms of immune evasion of tumor cells in numerous types of advanced solid malignant tumor, and several types of immune preparations have been administered as antitumor adjuvant therapies. However, in the majority of studies, the efficacy of therapies has been revealed to be limited. The present study aimed to investigate the immune evasion mechanisms employed by early colorectal cancer cells and the expression of the molecules associated with immune evasion during the malignant transformation process of normal colorectal epithelial cells to measure the effects of immune intervention for early colorectal cancer, and to improve the efficacy of immunotherapy. A total of 60 colorectal tissues, including 15 normal mucosa, 15 adenoma, 15 early cancer and 15 advanced cancer tissues, from patients undergoing endoscopic procedures in Huadong Hospital Affiliated to Fudan University (Shanghai, China) were collected. A comparison of baseline characteristics among these four groups was performed. The expression levels of human leukocyte antigen-A (HLA-A), apoptosis antigen 1 (Fas), c-c chemokine receptor type 5 (CCR5), Fas ligand (FasL) and HLA-E in each group were detected by immunohistochemical analysis. Furthermore, 15 patients with advanced colorectal cancer were enrolled into the present study. Advanced cancer and paracancer tissues (normal mucosal tissues 3 cm away from the margin of cancer tissues) were collected from each patient by colonoscopic biopsy. The expression levels of HLA-A, Fas, CCR5, FasL and HLA-E in each group were detected by western blot analysis. During the malignant transformation process of normal colorectal epithelial cells, the expression levels of CCR5, FasL and HLA-E increased significantly (P<0.001), whilst the expression levels of Fas reduced significantly (P=0.0271). In the early cancer group, the expression levels of Fas reduced significantly (P=0.0239), whilst the expression levels of HLA-E increased significantly (P<0.001) compared with adenoma group. In conclusion, a loss of Fas expression and high expression levels of HLA-E may promote the immune evasion of early colorectal cancer cells.

Inhibitor of growth 4 suppresses colorectal cancer growth and invasion by inducing G1 arrest, inhibiting tumor angiogenesis and reversing epithelial-mesenchymal transition.

Previous studies have found that inhibitor of growth 4 (ING4), a tumor suppressor, is reduced in human colorectal cancer (CRC), and is inversely correlated with clinical Dukes' stage, histological grade, lymph node metastasis and microvessel density (MVD). However, its underlying mechanism remains undetermined. In the present study, we analyzed ING4 expression in a panel of human CRC cells using low (LS174T and SW480) and high (LoVo and SW620) metastatic cell lines. We demonstrated that both the low and high metastatic CRC cells exhibited a lower level of ING4 compared to the level in normal human colorectal mucous epithelial FHC cells. Furthermore, ING4 expression in high metastatic CRC cells was less than that in low metastatic CRC cells. We then generated a lentivirus construct expressing ING4 and green fluorescent protein (GFP), established a ING4-stably transgenic LoVo CRC cell line, and investigated the effect of lentiviral-mediated ING4 expression on high metastatic LoVo CRC cells. Gain-of-function studies revealed that ING4 significantly inhibited LoVo CRC cell growth and invasion in vitro and induced cell cycle G1 phase arrest. Moreover, ING4 obviously suppressed LoVo CRC subcutaneously xenografted tumor growth and reduced tumor MVD in vivo in athymic BALB/c nude mice. Mechanistically, ING4 markedly upregulated P21 and E-cadherin but downregulated cyclin E, interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), Snail1, N-cadherin and vimentin in the LoVo CRC cells. Our data provide compelling evidence that i) ING4 suppresses CRC growth possibly via induction of G1 phase arrest through upregulation of P21 cyclin-dependent kinase (CDK) inhibitor and downregulation of cyclin E as well as inhibition of tumor angiogenesis through reduction of IL-6, IL-8 and VEGF proangiogenic factors; ii) ING4 inhibits CRC invasion and metastasis probably via a switch from mesenchymal marker N-cadherin to epithelial marker E-cadherin through downregulation of Snail1 epithelial-mesenchymal transition (EMT)-inducing transcription factor (EMT-TF).

Major Protein of Carcinoembryonic Antigen Gene Family - CD66c, A Novel Marker in Colon Carcinoma.

In view of rising trend of the incidence of colorectal carcinoma in the Indian population due to adoption of western lifestyles and behaviours, we investigated the expression of the new emerging stem cell biomarker, CD66c in colorectal carcinoma of Indian origin. To study the expression of CD66c in human colorectal carcinoma and to correlate level of marker expression with tumour staging. This hospital based prospective study was conducted on 26 colorectal carcinoma patients in the age group of 20 years to 70 years. Surgically resected tumour specimens along with adjacent normal tissue were collected taking necessary precautions, paraffin embedded sections were prepared and used for histological and immunohistochemical analysis of CD66c. Descriptive statistical measures like mean, standard deviation, percentage was applied. Other inferential statistical tests like Chi-square, Fisher's-exact test and one-way ANOVA was applied to find out the association of CD66c with different stages. The difference were interpreted as statistically significant when p <0.05. CD66c showed differential expression with membrane positivity in normal colorectal epithelial cells and cytoplasmic expression in tumour cells. There was significant correlation between CD66c expression and tumour site (p=0.02) with colon carcinoma showing positive expression compared to the rectal carcinoma. There was no significant correlation between CD66c staining and tumour stage (p=0.947). No significant relationship was observed between CD66c expression and other clinicopathologic variables studied such as sex (p=0.552), age (p=0.713) and tumour grade (p=0.263). CD66c can be specifically used for colon carcinoma and may be a novel marker in colon carcinoma stem cell isolation. The quantification of CD66c can be further verified by flow cytometry and RT-PCR. Further studies can be carried out using CD66c alone or in combination with other markers to develop cancer stem cell directed therapy.

Upregulation of long non-coding RNA PRNCR1 in colorectal cancer promotes cell proliferation and cell cycle progression.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Long non-coding RNAs (lncRNAs) have been confirmed to play a critical regulatory role in various biological processes including carcinogenesis, which indicates that lncRNAs are valuable biomarkers and therapeutic targets. The novel lncRNA prostate cancer non-coding RNA 1 (PRNCR1) is located in the susceptible genomic area of CRC, however the functional role of PRNCR1 remains unknown. Thus, we aimed to investigate the clinical significance and biological function of PRNCR1 in CRC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expression profile of PRNCR1 in CRC tissues and cell lines. An antisense oligonucleotide (ASO) was designed to knock down PRNCR1. In a cohort of 63 patients, PRNCR1 was significantly overexpressed in CRC tissues compared with the expression in adjacent tissues, with an average fold increase of 10.55 (P=0.006). Additionally, a high level of PRNCR1 was associated with large tumor volume (P<0.05). Based on receiver operating characteristic curve (ROC), we found that the area under the curve (AUC) of PRNCR1 was 0.799 while the AUC of conventional biomarker CEA-CA199 was 0.651, indicating that PRNCR1 could be a sensitive diagnostic biomarker of CRC. Compared with the normal human colorectal epithelial cell line (FHC), PRNCR1 was upregulated in most CRC cell lines (HCT116, SW480, LoVo and HT-29). After knockdown of PRNCR1 by ASO, CRC cell proliferation ability was significantly inhibited. We further found that PRNCR1 knockdown induced cell cycle arrest in the G0/G1 phase and a significant decrease in the proportion of cells in the S phases. In contrast, PRNCR1 knockdown did not affect cell apoptosis or invasive ability. Hence, these data indicate that PRNCR1 promotes the proliferation of CRC cells and is a potential oncogene of CRC.

The roles of different signaling pathways and their related genes in the occurrence and development of colorectal cancer

Colorectal cancer is a kind of malignancies with high incidence in the worldwide, that is seriously harmed human health. So far the pathogenesis of the disease is not fully understood, this causing many difficulties to the diagnosis and treatment of the disease, and resulting in the cure rates of disease is not ideal. With the development of molecular genetics and molecular biology, many oncogenes and tumor suppressor genes have been found to be associated with the disease, and this made it is possible to reveal the pathogenesis of colorectal cancer at the molecular level. However, it is a complex and multi-step process from normal colorectal epithelial cells transformed to colorectal cancer cells, and it is the results of polygenic and multifactorial interactions. Now it is thought that the Wnt, TGF-beta, PI3K/Akt, MAPK and p53 signaling pathways are closely associated with pathogenesis of colorectal cancer. Based on the five kinds of signaling pathways as the main line, this article reviewed the roles of different signaling pathways and their related genes in the pathogenesis of colorectal cancer. Key words: Colorectal neoplasms; Signaling pathways; Gene; Pathogenesis

Guttiferone K Exhibits Selective Cancer Cytotoxicity through ER Stress Aggravation

Guttiferone K (GutK) is a novel phytochemical that was previously reported to show selective cytotoxicity in cancer cells. This study aims to employ different human colorectal cancer (CRC) cell lines as models to confirm endoplasmic reticulum (ER) stress aggravation as the mechanism of GutK to cause apoptosis. Furthermore, normal colorectal epithelial cell line is adopted as a reference to understand how anti‐cancer selectivity occurs.ER stress markers were probed through immunoblotting and RT‐PCR in CRC cell line HCT116 and HT29 after treatment with GutK. Furthermore, prolonged treatment resulted in induction of pro‐apoptotic proteins, CHOP and DR5 expression. These protein were tested whether they could activate apoptosis by monitoring PARP cleavage after siRNA knockdown. Subsequently, realizing ER stress was aggravated by prolonged treatment with low dose of GutK in cancer cells, the same treatment was adopted on normal colon epithelial cell line CCD‐841 CoN to compare cytotoxicity via SrB assay, to check the ER stress state and to confirm selective toxicity through a normal &amp; GFP‐labelled cancer cells co‐culture model by flow cytometry.ER stress activation was confirmed as a result of activation of GRP78 &amp; GRP94, eIF2α and XBP‐1 mRNA splicing after GutK treatment. CHOP and DR5 were induced after prolonged treatment yet effects were abrogated after transfected with siRNA. Normal cells were spared after the same treatment, resulting in only a mild ER stress condition which was insufficient to initiate apoptosis.GutK aggravated ER stress in colorectal cancer cells but not in normal cells to elicit selective cancer cytotoxicity.

Frequent epigenetic silencing of PCDH10 by methylation in human colorectal cancer

Aberrant DNA methylation is common in cancer cells. Epigenetic alterations resulting in the loss of tumor suppression gene functions are frequently involved in tumor development and progression. Recently, methylation of PCDH10 was reported to be associated with multiple hematologic malignancies as well as some solid tumors. Whether the down-regulation of PCDH10 happens in CRC remains unknown. Methylation status of PCDH10 was evaluated by methylation-specific PCR analysis. The effects of PCDH10 re-expression were determined in growth, colony formation, cell cycle, and invasion assays. In this study, we found that 100 % (8 of 8) of colorectal cancer cell lines were silenced for PCDH10, but not normal colorectal epithelial cells. Demethylation treatment confirmed that the reduced expression is associated closely with promoter methylation. Hyper-methylation of PCDH10 was also detected in 85 % of primary colorectal tumors, but not in adjacent normal colorectal tissues. Our results suggest that PCDH10 is an important tumor suppression gene with key roles of suppressing cell proliferation, clonogenicity, and inhibiting cell invasion in the development of colorectal cancer. Thus, PCDH10 methylation may constitute a useful biomarker of colorectal cancer patients.

Laser microdissection and two-dimensional difference gel electrophoresis reveal proteomic intra-tumor heterogeneity in colorectal cancer

We examined the proteins for intra-tumor heterogeneity in colorectal cancer. Tumor cells localized in the ulcer floor, the central area, and the invasive front, as well as normal colorectal epithelial cells were recovered from surgical specimens by laser microdissection. Proteome data were generated by 2D-DIGE and mass spectrometry. By distinguishing the tumor cells based on tissue localization, we observed 88 protein spots at a different intensity in tumor cell groups compared to normal epithelial cells. On the contrary, when the tissue localization of the tumor cells was not taken into account, only 1 protein spot was newly detected with a different intensity. The identified proteins corresponded functionally to the expected phenotypes in that particular region: proteins for stress response were identified in the ulcer floor, glucose metabolism proteins were found in the central area, and apoptosis proteins were associated with the invasive front. These results suggested the following: (1) proteome data may reflect the effects of the tissue microenvironment on tumor cells, (2) the tissue localization of tumor cells should be considered in the sampling process when comparing normal and tumor cells, and (3) we can expect novel findings on the backgrounds of intra-tumor heterogeneity by using the proteomic approach.

High Ran level is correlated with poor prognosis in patients with colorectal cancer

The Ras-like nuclear protein (Ran) is involved in the regulation of nuclear transport, microtubule nucleation and dynamics, and spindle assembly. Its fundamental function is nucleocytoplasmic transport of RNA and proteins. The expression and potential role of Ran in colorectal cancer (CRC) remain unclear. The aim of this study was to investigate the relationship between Ran expression and CRC characteristics. The potential role of Ran as a prognostic indicator was also evaluated. We used immunohistochemistry and western blotting to detect Ran expression in 287 CRC tissues. The relationships between Ran expression and clinicopathological characteristics and overall survival rate were statistically analyzed. CRC tissues had significantly higher Ran expression than normal colorectal epithelial cells. Ran was positively correlated with depth of invasion, lymph node metastases, distant metastases, tumor differentiation, and tumor-node-metastasis stage. However, no correlation was found between Ran expression and patient age or sex. The overall survival rate was consistently and significantly lower in patients with Ran-positive tumors than in those with Ran-negative tumors. Our findings emphasize the important role of Ran in differentiation, disease stage, and metastasis in human CRC. Ran may play an important role in the development of CRC and may serve as a novel prognostic indicator of CRC.

Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers

We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.

Screening Peptides Binding Specifically to Colorectal Cancer Cells from a Phage Random Peptide Library

The aim of this study was to screen for polypeptides binding specifically to LoVo human colorectal cancer cells using a phage-displayed peptide library as a targeting vector for colorectal cancer therapy. Human normal colorectal mucous epithelial cells were applied as absorber cells for subtraction biopanning with a c7c phage display peptide library. Positive phage clones were identified by enzyme-linked immunosorbent assay and immunofluorescence detection; amino acid sequences were deduced by DNA sequencing. After 3 rounds of screening, 5 of 20 phage clones screened positive, showing specific binding to LoVo cells and a conserved RPM motif. Specific peptides against colorectal cancer cells could be obtained from a phage display peptide library and may be used as potential vectors for targeting therapy for colorectal cancer.

Overexpression of Matrix Metalloproteinase-21 is Associated with Poor Overall Survival of Patients with Colorectal Cancer

IntroductionMatrix metalloproteinase-21 (MMP-21) is a member of the MMP family, which is overexpressed in some solid tumors and is thought to enhance the tumor invasion and metastasis ability. The aim of the present study is to examine the MMP-21 expression in human colorectal cancer and normal colorectal tissue using tissue microarray technique and to determine its association with clinicopathological characteristics and prognostic value. Materials and MethodsFour array blocks including 256 cases of colorectal cancer and adjacent normal tissues were investigated by immunohistochemistry assay. Staining evaluation results were analyzed statistically in relation to various clinicopathological characters and overall survival. ResultsHigh level of MMP-21 expression was detected in colorectal cancer, significantly more than in normal colorectal epithelial cells. In colorectal cancer, MMP-21 was significantly positively correlated with depth of invasion, lymph node metastasis, and distant metastasis. The overall survival rate was significantly lower for patients with MMP-21 positive than those with MMP-21 negative tumors. However, no correlations between MMP-21 expression and patients’ age, sex tumor location, or differentiation status were detected. ConclusionOur findings emphasize the important role of MMP-21 in the invasion and metastasis process in human colorectal cancer. It might also serve as a novel prognostic marker that is independent of, and additive to, the TNM staging system.

Matrix Metalloproteinase-9 Is Associated with Relapse and Prognosis of Patients with Colorectal Cancer

Matrix metalloproteinase-9 is a member of the MMP family, which is overexpressed in some solid tumors and is thought to enhance tumor invasion and metastasis ability. The present study aims to examine MMP-9 expression in human colorectal cancer and to determine its association with clinicopathological characteristics and prognosis. Colorectal cancer and adjacent normal tissues from 192 patients were investigated by immunohistochemical assay. Staining evaluation results were analyzed statistically in relation to various clinicopathological characters, disease-free survival, and overall survival. High level of MMP-9 expression was detected in colorectal cancer, significantly more than in normal colorectal epithelial cells. In colorectal cancer, MMP-9 was significantly positively correlated with depth of invasion, lymph node metastasis, and distant metastasis. However, no correlations between MMP-9 expression and patient age, sex, tumor location or differentiation status were detected. Disease-free and overall survival were significantly poorer for patients with positive MMP-9 staining than for those with MMP-9-negative tumors. Our findings emphasize the important role of MMP-9 in the invasion and metastasis process in human colorectal cancer. It could also serve as a novel prognostic marker that is independent of, and additive to, the tumor-node-metastasis (TNM) staging system.

High Level of Notch1 Protein is Associated with Poor Overall Survival in Colorectal Cancer

Notch1 regulates cell proliferation, development, and apoptosis. Aberrant expression of Notch1 has been discovered in many types of tumors. We examined Notch1 expression in colorectal cancer to assess its role as a prognostic indicator. Notch1 protein expression was examined by immunohistochemistry in 223 surgically resected specimens of colorectal cancer and adjacent tissues. The relationship between various clinicopathological features and overall patient survival rate was analyzed. The association of Notch1 expression with the colorectal cancer survival rate was assessed by Kaplan-Meier and Cox proportional-hazards regression. Significantly high Notch1 expression was found in colorectal cancer cells compared with that of normal colorectal epithelial cells. Notch1 was positively correlated with depth of invasion (P = 0.005), lymph node metastases (P = 0.03), and tumor-node-metastasis (TNM) stage (P < 0.001). Consistently, the overall survival rate was significantly lower for patients with Notch1-positive than those with Notch1-negative tumors. However, no correlation between Notch1 expression and patient age, sex or tumor location was found. Notch1 might serve as a novel prognostic marker that is independent of, and additive to, the TNM staging system.

Unusual accumulation of sulfated glycosphingolipids in colon cancer cells

The structures of glycosphingolipids from highly purified colorectal cancer cells and normal colorectal epithelial cells of 16 patients have been analyzed in fine detail (Misonou Y, Shida K, Korekane H, Seki Y, Noura S, Ohue M, Miyamoto Y. 2009. Comprehensive Clinico-Glycomic Study of 16 Colorectal Cancer Specimens: Elucidation of aberrant glycosylation and ts mechanistic causes in colorectal cancer cells. J Proteome Res. 8:2990-3005). Further structural analyses demonstrated that colon cancer cells from two patients accumulated unusual glycosphingolipids which were not observed in either colorectal cancer cells or normal colorectal epithelial cells from the other patients. Mass spectrometry analyses revealed that the unusual structures include sulfated oligosaccharides. The structures of the glycosphingolipids of the cancer cells from these two cases were analyzed by methods which include enzymatic release of carbohydrate moieties, fluorescent labeling with aminopyridine and identification using two-dimensional mapping, enzymatic digestion and mass spectrometry together with methanolysis, and the use of newly synthesized sulfo-fucosylated oligosaccharides as standards. The colon cancer cells from one of the patients demonstrate a variety of oligosaccharides as major components which are sulfated at the C6 position of subterminal GlcNAc and at C3 positions of terminal galactose with or without sialylation or fucosylation. These include 6-sulfo Le(x), 6'-sialyl 6-sulfo lactosamine, and 3'-sialyl 6-sulfo Le(x), in addition to sialylated or fucosylated derivatives of type-1 and type-2 hybrid oligosaccharides. The colon cancer cells from the other patient have two kinds of sulfated oligosaccharides, a 6-sulfo Le(x) structure and a 3'-sulfo Le(x) structure, as minor components. Taking into consideration the clinical features of the two patients, the biological significance of sulfated glycosphingolipids on cancer cells is discussed.

Clinicopathological significance of stanniocalcin 2 gene expression in colorectal cancer

Laser microdissection (LMD) and microarray were used to identify genes associated with colorectal cancer. Stanniocalcin 2 (STC2) expression and clinicopathological significance in 139 clinical colorectal cancer samples were specifically investigated using real-time quantitative reverse transcription-polymerase chain reaction. A number of genes upregulated in colorectal cancer cells compared to normal colorectal epithelial cells were identified including STC2. STC2 gene expression in cancer tissue was higher than in corresponding normal colorectal epithelial tissue in 124 of 139 cases (89.2%, p < 0.01). Tumors with high STC2 expression showed higher frequencies of lymph node metastasis, lymphatic invasion, tumor depth, tumor size and AJCC Stage classification (p < 0.01). Patients with high STC2 expression also showed significantly worse overall survival rates than those with low STC2 expression (p < 0.01). Furthermore, STC2 gene appeared to be associated with colorectal cancer progression and may be a useful prognostic indicator for colorectal cancer.

Comprehensive Clinico-Glycomic Study of 16 Colorectal Cancer Specimens: Elucidation of Aberrant Glycosylation and Its Mechanistic Causes in Colorectal Cancer Cells

The structures of neutral and acidic glycosphingolipids from both normal colorectal epithelial cells and colorectal cancer cells, which were highly purified with the epithelial cell marker CD326, have been analyzed. The analysis was performed on samples from 16 patients. The carbohydrate moieties from glycosphingolipids were released by endoglycoceramidase II, labeled by pyridylamination, and identified using two-dimensional mapping and mass spectrometry. The structures from normal colorectal epithelial cells are characterized by dominant expression of neutral type-1 chain oligosaccharides. Three specific alterations were observed in malignant transformation; increased ratios of type-2 oligosaccharides, increased alpha2-3 and/or alpha2-6 sialylation and increased alpha1-2 fucosylation. Although the degree of alteration varies case to case, we found that two characteristic alterations tend to be associated with clinical features. One is a shift from type-1 dominant normal colorectal epithelial cells to type-2 dominant colorectal cancer cells. This shift was found in 5 patients having hepatic metastasis. The other is specific elevation of alpha2-3 sialylation observed in 2 cases exhibiting high serum levels of CA19-9. Examination of the activities of the related glycosyltransferases revealed that while some alterations could be accounted for by changes in the activities of related glycosyltransferases others could not. Although the number of cases analyzed is small, these findings provide valuable information which will help in the elucidation of the mechanism of synthesis of aberrant glycosylation and its involvement in cancer malignancy.