Sex difference in cellular retinol- and retinoic acid-binding proteins in human colon adenocarcinomas

Human colon adenocarcinomas and adjacent non-cancerous, normal colon from the same patient were assayed for the presence and amounts of cellular binding proteins for retinol (CRBP) and retinoic acid (CRABP) by sucrose gradient analysis. In male patients, the mean concentrations of both CRBP and CRABP in the colon cancers were statistically significantly higher than in the adjacent normal colon. By contrast, in female colon cancers, the mean levels for both binding proteins were reduced approximately 2-fold, compared to the concentrations in the adjacent normal colon. These findings reveal an unexpected sex difference in the binding proteins for retinol and retinoic acid in human colon malignancies.     

Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis

In order to determine the prognostic significance of proliferation, differentiation, and apoptosis as intermediate markers for colon tumor development, these indices were measured during the promotion phase of tumorigenesis. Two hundred and sixty male Sprague-Dawley rats were provided with one of two fats (corn oil and fish oil) and two fibers (pectin and cellulose) plus or minus the carcinogen azoxymethane (AOM) and killed at two time points (18 and 36 wk) in a 2x2x2x2 factorial design. In vivo cell proliferation was measured immunohistochemically using incorporation of bromodeoxyuridine into DNA. Differentiation was assessed by binding of Dolichos biflorus agglutinin (DBA) to colonocytes. Apoptosis was measured by immunoperoxidase detection of digoxigenin-labeled genomic DNA. Adenocarcinoma incidence results at week 36 were 70.3% for corn oil + AOM and 56.1% for fish oil + AOM treatment (P < 0.05); no main effect of fiber was observed. At week 18, AOM treatment increased the number of cells per crypt column in the proximal colon compared with saline controls (P = 0.0358) and increased the proliferative zone in the distal colon compared with controls (P = 0.0073). However, changes in cell proliferation at week 18 did not predict the beneficial effect of fish oil versus corn oil. In contrast, DBA binding (the marker for differentiation) was higher in fish oil versus corn oil fed animals in both the proximal and distal colon and in each portion of the crypt (P = 0.0001). There were a greater number of apoptotic cells/crypt column in the proximal colon (P = 0.0019) and distal colon (P = 0.0358) with fish oil compared with corn oil, and indices of apoptosis also predicted certain fat/fiber interactions. Measurements of differentiation and apoptosis had greater prognostic value to detect dietary effects on tumor incidence than did measurements of cell proliferation.      

Gut microbiota and probiotics in colon tumorigenesis

In the present study we demonstrate that adeno-associated virus (AAV)-mediated anti-DR5 (death receptor 5) mouse-human chimeric antibody (shorten as Adximab) expression significantly suppressed tumor cell growth by inducing apoptosis both in vitro and in vivo. The viral-expressed and cell-secreted Adximab efficiently bound DR5 with an affinity of 0.7nM and induced apoptosis of various tumor cells, but not normal cells. A single intramuscular injection of recombinant AAV particles resulted in a stable expression of Adximab in mouse serum for at least 70days. AAV-mediated Adximab expression led to a significant suppression of tumor growth in nude mice receiving xenografts of human liver and colon cancer. These data suggest that chimeric antibody gene transfer may provide an alternative strategy for the therapy of varieties of cancers.     

Gut microbiota and probiotics in colon tumorigenesis

The human gastrointestinal tract harbors a complex and abundant microbial community reaching as high as 10¹³–10¹⁴ microorganisms in the colon. This endogenous microbiota forms a symbiotic relationship with their eukaryotic host and this close partnership helps maintain homeostasis by performing essential and non-redundant tasks (e.g. nutrition/energy and, immune system balance, pathogen exclusion). Although this relationship is essential and beneficial to the host, various events (e.g. infection, diet, stress, inflammation) may impact microbial composition, leading to the formation of a dysbiotic microbiota, further impacting on health and disease states. For example, Crohn’s disease and ulcerative colitis, collectively termed inflammatory bowel diseases (IBD), have been associated with the establishment of a dysbiotic microbiota. In addition, extra-intestinal disorders such as obesity and metabolic syndrome are also associated with the development of a dysbiotic microbiota. Consequently, there is an increasing interest in harnessing the power of the microbiome and modulating its composition as a means to alleviate intestinal pathologies/disorders and maintain health status. In this review, we will discuss the emerging relationship between the microbiota and development of colorectal cancer as well as present evidence that microbial manipulation (probiotic, prebiotic) impacts disease development.     

Suppression of rat liver tumorigenesis by 25-hydroxycholesterol and all-trans retinoic acid: differentiation therapy for hepatocellular carcinoma.

To determine whether differentiation therapy is useful in treating patients with hepatoma, we assayed the effects of 25-hydroxycholesterol (25-OH) and/or all-trans retinoic acid (ATRA) on rat AH136B ascites hepatoma cells. Flow cytometric DNA analysis showed that treatment of cells with 25-OH alone induced entry into the sub-G1 phase in a dose-dependent manner. While ATRA alone was ineffective, it enhanced the activity of 25-OH. Condensed and fragmented nuclei occurred mainly in cells treated with 25-OH (4 microg/ml). When cells treated with 25-OH (4 microg/ml), or 25-OH (4 microg/ml) + ATRA (1 microM) were transplanted into Donryu rats, we found that tumor development was completely inhibited; in contrast, rats administered the methanol-treated AH136B cells developed tumors. These findings suggest that AH136B cells in the sub-G1 phase can not recover tumorigenicity, and that the administration of a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, such as 25-OH, and ATRA may be effective in treating patients with hepatoma.     

Presence of cellular rentinol and retinoic acid binding proteins in experimental rumors.

Cellular retinol and retinoic acid binding proteins were detected in mouse skin papillomas, human adenocarcinoma HAD-1, Dunning Leukemia, Walker 256 carcinosarcoma and mammary adenocarcinoma MAC-1. A chondrosarcoma and Sarcoma 180 apparently contain only the cellular retinoic acid binding protein. Neither protein could be detected in Ehrlich carcinoma and L1210 leukemia. The presence of these proteins might be necessary for sensitivity to retinoid therapy.     

Id proteins in cell growth and tumorigenesis

Since the gene encoding Id1 was cloned in 1990, Id proteins have been implicated in regulating a variety of cellular processes, including cellular growth, senescence, differentiation, apoptosis, angiogenesis, and neoplastic transformation. The development of knockout and transgenic animal models for many members of the Id gene family has been particularly useful in sorting out the biologic relevance of these genes and their expression during normal development, malignant transformation, and tumor progression. Here we review the current understanding of Id gene function, the biologic consequences of Id gene expression, and the implications for Id gene regulation of cell growth and tumorigenesis.     

Methylation and regulation of expression of different retinoic acid receptor beta isoforms in human colon cancer

Tumor suppressor genes can become inactivated in cancer via hypermethylation of their promoter. The retinoic acid receptor beta (RARbeta) gene is expressed from two distinct promoters, both of which have CpG islands. RARbeta1 is expressed primarily during embryogenesis, whereas RARbeta2 is expressed in adult tissues and hypermethylated in a number of cancer cells. We used combined bisulfite restriction analysis to evaluate their methylation in colorectal mucosa and tumors. Methylation of RARbeta1 was detected, with a mean of 2% in normal colon tissues in young subjects (< 32 years), and 16% in older subjects (> 75 years) (P < 0.001). Using paired normal/tumor tissue samples, we found higher mean methylation rate in tumors than in adjacent normal tissue (mean, 46% versus 16%; P < 0.001) and hypermethylation of RARbeta1 in all eight cell lines examined. By RT-PCR, RARbeta1 was not expressed in normal adult colon tissues and its expression could not be efficiently activated in most cell lines by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR). RARbeta2 methylation was also observed in normal colon tissues and was lower in young individuals than in older ones (mean, 11% versus 23%; P < 0.05). Among paired samples, RARbeta2 methylation was higher in tumor tissue than in normal tissue in 14 cases, vice versa in 7 cases, and equal in 6 cases. All eight cell lines were hypermethylated and did not express RARbeta2, but RARbeta2 expression could be reactivated easily by 5-Aza-CdR. We suggest that the embryonic RARbeta1 isoform is readily hypermethylated in aging colon mucosa and all colorectal cancers because of its lack of expression in normal tissues. The adult RARbeta2 isoform also shows age-related methylation in normal tissues but more variable methylation in colorectal cancer, perhaps because its expression offers continued protection against methylation or its silencing does not provide a selective advantage in the early stages of the disease.     

热休克蛋白在肿瘤发生发展中的作用

热休克蛋白(HSP)在一系列肿瘤中呈高表达,其家族各成员不仅促进了肿瘤细胞的增殖,而且抑制了其死亡途径,对于肿瘤的发生发展十分重要,并与肿瘤免疫、耐药和不良预后关系密切.因而,HSP已成为新抗癌策略的靶向.     

维甲酸诱导甲状腺滤泡状癌FIE-133细胞凋亡前后的蛋白质差异表达

目的：建立双向电泳和质谱技术探索维甲酸诱导甲状腺滤泡状癌凋亡的蛋白差异表达,为肿瘤治疗提供分子生物学证据。方法：无菌的含10%胎牛血清1640培养基培养甲状腺滤泡状癌细胞株FIE-133,分为正常对照组和实验组,实验组用维甲酸刺激24小时共同培养,提取对照组和实验组全蛋白,双向电泳分离蛋白质,采用PDQuest2-DE软件分析两组间差异表达的蛋白质斑点,用基质辅助激光解吸电离飞行时间串联质谱进行鉴定。结果：双向电泳图谱显示：实验组（维甲酸刺激组）与对照组的蛋白组存在显著性差异,质谱初步鉴定了5种主要蛋白。结论：双向电泳和质谱鉴定技术可以有效分离和分析维甲酸诱导甲状腺滤泡状癌FIE-133细胞凋亡的蛋白差异表达,为探索维甲酸治疗甲状腺滤泡状癌提供分子生物学的证据。     

维甲酸诱导甲状腺滤泡状癌FIE-133细胞凋亡前后的蛋白质差异表达

目的:建立双向电泳和质谱技术探索维甲酸诱导甲状腺滤泡状癌凋亡的蛋白差异表达,为肿瘤治疗提供分子生物学证据。方法:无菌的含10%胎牛血清1640培养基培养甲状腺滤泡状癌细胞株FIE-133,分为正常对照组和实验组,实验组用维甲酸刺激24小时共同培养,提取对照组和实验组全蛋白,双向电泳分离蛋白质,采用PDQuest2-DE软件分析两组间差异表达的蛋白质斑点,用基质辅助激光解吸电离飞行时间串联质谱进行鉴定。结果:双向电泳图谱显示:实验组(维甲酸刺激组)与对照组的蛋白组存在显著性差异,质谱初步鉴定了5种主要蛋白。结论:双向电泳和质谱鉴定技术可以有效分离和分析维甲酸诱导甲状腺滤泡状癌FIE-133细胞凋亡的蛋白差异表达,为探索维甲酸治疗甲状腺滤泡状癌提供分子生物学的证据。     

Phytic acid and minerals: effect on early markers of risk for mammary and colon carcinogenesis

This study determined the effect of inositol hexaphosphate or phytic acid (PA; 1.2%), Ca (1.5%) and Fe (535 p.p.m.) alone, and PA in combination with Ca or Fe in a high-fat diet (25%) on the labeling (LI) and mitotic (MI) cell proliferation indices, nuclear aberration (NA) and intraductal proliferation (IDP) in the mammary gland, as well as the LI in colonic epithelial cells. Diet supplementation with PA alone caused reductions (P less than 0.05) in the colon LI by 18%, and in the LI and NA in the total mammary gland structures of mice by 29 and 30% respectively. Supplementation with Fe or particularly Ca caused increases in the colon LI and in the mammary LI, MI, NA and IDP but these were reduced by 25-53% (P less than 0.05) in the presence of PA. These results support the hypothesis that PA may reduce the risk for both colon and mammary cancer and its effect is related to its mineral binding ability. Furthermore, significant relationships (P less than 0.01) were observed between the LI and MI or NA in the total structures of the mammary gland. The number of IDPs also related (P less than 0.05) to LI or NA in the terminal end bud structure of the mammary gland, suggesting that highly proliferating mammary cells, particularly in the terminal end bud structure, are of greater risk for nuclear damage and development to IDP. A significant relationship (P less than 0.01) was observed between the cell proliferation in the mammary gland and that in the colon, indicating that both tissues can be influenced similarly by dietary constituents.     

Resistance to retinoic acid and altered cytokeratin expression of human papillomavirus type 16-immortalized endocervical cells after tumorigenesis

Human papillomaviruses (HPVs) and cigarette smoking are epidemiologically associated with cervical cancer. We recently found that HEN-16 and HEN-16-2 HPV type 16-immortalized endocervical cells form tumors after treatment with cigarette smoke condensate and derived 2 tumor cell line cultures, HEN-16T and HEN-16-2T, respectively. Here, we examine the molecular pathologic effect of tumorigenesis. HEN-16T and HEN-16-2T exhibit unchanged status and expression of integrated HPV 16 DNA. However, the expression of the cytokeratin CK7 and CK13 endocervical cell markers is more homogeneous in monolayer and organotypic raft cultures after tumorigenesis. For the effect of retinoic acid on monolayers for growth inhibition, HEN-16T were significantly less sensitive than the normal and immortalized non-tumorigenic cells. HEN-16-2T were completely resistant. Moreover, the rafts from both tumorigenic cell line cultures were resistant to retinoic acid and continued to display thick rafts and homogeneous severe dysplasia/carcinoma in situ. In contrast, the non-malignant HEN-16 and HEN-16-2 rafts were thinner, and treatment with retinoic acid blocked the formation of severe dysplasia, reconstructing an epithelium resembling that of the normal endocervix. Our results support the significance of non-viral factors in the mechanism by which cigarette smoking induces tumorigenesis in the late stages of HPV-initiated progression to cervical cancer. Importantly, our data indicate that the sensitivity to retinoic acid of the HPV-containing endocervical cells is lost following tumorigenesis in vitro and possibly in women. © 1996 Wiley-Liss, Inc.     

Tight junction proteins: From barrier to tumorigenesis

The tight junction is a multi-protein complex and is the apical most junctional complex in certain epithelial and endothelial cells. A great deal of attention has been devoted to the understanding of these proteins in contributing to the barrier function – that is, regulating the paracellular flux or permeability between adjacent cells. However, tight junction proteins are now recognized as having functions beyond the barrier. The focus of this review is to discuss the barrier function of the tight junction and to summarize the literature with a focus on the role of tight junction proteins in proliferation, transformation, and metastasis.     

Biochemical markers in bone metastasis

Certain types of cancers have a strong propensity to metastasize to bone, which requires combination of multiple factors responsible for the different steps of metastasis. Bone metabolic markers are now widely used in clinical practice and give useful information on the ongoing bone metabolism, reflecting the activity of bone-resorbing osteoclasts and bone-forming osteoblasts. Bone markers have a potential as diagnostic tools for bone metastasis, and are useful in monitoring the response to anticancer as well as antiresorptive therapies. Since bone metabolic markers alone are insufficient for the diagnosis and assessment of bone metastasis, it is important to combine bone markers with tumor-related markers and imaging studies such as scintigraphy and MRI. More recently, soluble receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated as markers for osteoclastogenic activity. Serum levels of these factors and/or their ratios may provide additional information on the severity of bone disease and the prognosis.     

Modulation of different clusterin isoforms in human colon tumorigenesis

Clusterin is a ubiquitous secretory heterodimeric disulfide-linked glycoprotein, which is implicated in several physiological processes, including immune regulation, cell adhesion and morphological transformation, lipid transportation, tissue remodelling, membrane recycling and cell-cell interactions. A large number of studies have focused their interest on clusterin gene products as mediators of cell cycle progression and cell death induction, although data on the different isoforms and their role in the different cell processes are still obscure. Recently, an increased clusterin expression in breast cancer has been reported. In order to elucidate the role of clusterin in tumor progression and whether one of its isoforms is preferentially expressed in tumorigenesis, we examined its presence throughout the different steps of human colon carcinoma, one of the best-characterized models of human tumor progression. The immunohistochemical observation of 30 bioptic and surgical colon specimens demonstrated a cell compartment clusterin translocation from the nucleus to the cytoplasm directly related to tumor progression. In fact, a nuclear localization found in healthy colonic mucosa is consistent with the involvement of the proapoptotic nuclear form in the regulation of cell cycle progression and in cell death induction. The progression towards high-grade and metastatic carcinoma leads to cytoplasmic clusterin distribution. Protein extracts from freshly isolated cells of the same patients confirm in high-grade carcinomas with metastatic nodes the complete loss of the proapoptotic nuclear form and a cytoplasmic overexpression of the highly glycosylated form. Data obtained from in vitro experiments confirm that this form is released in the extracellular space and corresponded to the fully glycosylated one. These data suggest that the controversial data on clusterin function in tumors may be related to the pattern shift of its isoform production. As the secreted form of clusterin is correlated to cell matrix formation, cell membrane remodeling and cell-cell adhesion, the overexpression of this form in highly aggressive tumors and metastatic nodes could be a potential new prognostic and predictive marker for colon carcinoma aggressiveness.     

Colonoscopic colostomy model in rats for colon tumorigenesis studies

A colonoscopic colostomy model for colorectal carcinogenesisand chemoprevention was developed in F344 rats. The colon wastransected at the middle of the transverse colon and suturedto two openings. The proximal opening, located on the middleline of the upper abdominal wall, was for the exit of feces.The distal one located on the left back was the colostomy forthe lower part of colorectum, which was approximately 9 cm inlength and isolated from the feces. The two openings of thecolostomy were completely separated with at least 4 cm distancebetween them. The animals were treated with 1,2-dimethylhydrazine(DMH) or methylazoxymethanol acetate (MAMAc) systemically ortopically. Colon tumors in the isolated colorectum were observedby colonoscopy. Twenty six tumors in the isolated colorectumwere found by colonoscopy with a mean latent period of 25.6weeks in 10/15 (66.6%) animals treated with DMH (30 mg/kg subcutaneously,weekly for 30 weeks). Twelve tumors were found by colonoscopywith a mean latent period of 32.8 weeks in 6/17 (35.3%) animalstreated with MAMAc (5 mg/kg enema, weekly for 35 weeks). Tumorswith 0.5 mm diameter were detected as early as 21 weeks followingthe first dose of DMH and 28 weeks following the first doseof MAMAc respectively. Video camera-assisted endoscopic examinationdetected suspected small tumors 6 weeks earlier than endoscopicevaluation alone. The number, size and location of the tumorsobserved by colonoscopy were significantly correlated with thoseobserved at necropsy. The tumor growth rate was monitored weekly.The tumor growth curve was expressed as the mean tumor diametersand calculated tumor volumes. Histological study at necropsyshowed that 48.1% of the tumors were adenomas and 51.9% wereadenocarcinomas. With a complete fecal diversion and colonoscopy,the model is potentially useful to study colon carcinogenesisand the inhibition of colon carcinogenesis.     

R-etodolac induces E-cadherin and suppresses colitis-related mouse colon tumorigenesis

Colorectal cancer is one of the most serious complications of ulcerative colitis (UC), and the risk of UC-associated neoplasia increases as the region and duration of the disease increase. Selective cyclooxygenase (COX)-2 inhibitors effectively diminish carcinogenesis in a murine UC model. However, this may exacerbate colitis. The selective COX-2 inhibitor etodolac is marketed as a racemic mixture of the R- and S-enantiomers. The biochemical and pharmacological effects of etodolac are caused by the S-enantiomer, while the R-enantiomer lacks COX-inhibitory activity. In this study, we evaluated the effect of R-etodolac on colitis-related mouse colon tumorigenesis. The mice received 1,2-dimethlhydrazine (DMH), and then chronic colitis was induced by administration of two cycles of DSS (each cycle: 3% DSS for 7 days followed by distilled water for 14 days). The mice were sacrificed 28 days after the completion of both cycles. Mice were divided into the following groups: group A served as a disease control; group B received a low (2-mg/kg) dose of R-etodolac every 3 days during the entire period; group C received a high (10-mg/kg) dose of R-etodolac on the same schedule as group B; and group D served as a normal control. Administration of R-etodolac decreased the disease activity index during the DSS administration cycle. The mean number of tumors was 17.8, 15.2, 6.0, and 0 in groups A-D, respectively. In group C, R-etodolac significantly suppressed the occurrence of neoplasia (p<0.05). Although R-etodolac treatment did not affect COX-2 expression, it significantly enhanced expression of E-cadherin in both neoplastic lesions and background mucosa (i.e., lesion-free colon). Thus, administration of R-etodolac exerts a suppressive effect on the development of neoplasia in a murine model of DSS-induced colitis without exacerbation of the colitis. These results suggest that R-etodolac could be useful in the prevention of UC-associated neoplasia.