Adenomatous Polyposis Coli Gene Expression Research Articles

Anti-colon cancer activities of green-synthesized Moringa oleifera–AgNPs against human colon cancer cells

Abstract The anticancer activity of silver nanoparticles (AgNPs) is well known to be synthesized using green-synthesized methods, although its mechanism of action is not understood fully. Moringa oleifera leaves were used as reducing and stabilizing agents to synthesize AgNPs. Green-synthesized AgNPs were characterized using ultraviolet-visible spectroscopy, dynamic light scattering, transmission electronic microscopy, scanning electronic microscopy, Fourier transform infrared, and energy-dispersive X-ray spectroscopy analyses. The synthesized nanoparticles were then characterized by their anticancer properties by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The real-time polymerase chain reaction was used to check the expression levels of the four genes (β-catenin, adenomatous polyposis coli (APC), and lipoprotein receptor-related proteins 5 and 6 (LRP5/6)). The synthesized nanoparticles were 25 nm on average and spherical in shape and aggregated form. Noteworthy cytotoxicity is how green-synthesized M. oleifera–AgNPs were observed in comparison with the M. oleifera leaf extract against a cancerous cell line. The M. oleifera–AgNPs decreased the expression of CTNNB1 and LRP6 genes, while the LRP5 gene expression increased in both cell lines. With treatment, the APC gene expression decreased in SW480 but increased in HTC116. Our results imply that AgNPs synthesized by M. oleifera extract could be an ideal strategy to combat colon cancer.

Assessment of the Anti-Cancer Efficiency of Silver Moringa oleifera Leaves Nano-extract against Colon Cancer Induced Chemically in Rats.

Background:Colorectal cancer (CRC) categorized as the most common type of gastrointestinal cancers affected both genders equally. Chemotherapeutic drugs became limited due to their deleterious side effects. Therefore, efficiency of M. oleifera leaves extract increased by incorporating silver nanoparticles (Ag-NPs) then studied against colon cancer induced by azoxymethane (AOM) in rats. Methods:Different hematological and biochemical measurements in addition to specific tumor and inflammatory markers were quantified. Histopathological examination for Colonic tissues was performed. Native proteins and isoenzyme patterns were electrophoretically detected in addition to assaying expression of Tumor Protein P53 (TP53) and Adenomatous Polyposis Coli (APC) genes in colonic tissues.Results: M. oleifera nano-extract restored levels of the hematological and biochemical measurements in addition to levels of tumor and inflammatory markers to normalcy in both of nano-extract simult- and post-treated groups. Also, it minimized severity of the histopathological alterations in the simult-treated group and prevented it completely in the post-treated group. The lowest similarity index (SI%) values were noticed with electrophoretic protein (SI=61.54%), lipid (SI=0.00%) and calcium (SI=75.00%) moieties of protein patterns, catalase (SI=85.71%), peroxidase (SI=85.71%), α-esterase (SI=50.00%) and β-esterase (SI=50.00%) isoenzymes in addition to altering the relative quantities of total protein and isoenzyme bands in colon of cancer induced group. Moreover, levels of TP53 and APC gene expression increased significantly (P≤0.05) in colon cancer induced group. The nano-extract prevented the qualitative and quantitative alterations in the different electrophoretic patterns in addition to restoring levels of the gene expressions to normalcy in both of simult- and post-treated groups. Conclusion: M. oleifera nano-extract exhibited ameliorative effect against the biochemical, physiological and molecular alterations induced by AOM in nano-extract simult- and post-treated groups.

Risk Assessment in Hereditary Colorectal Cancer Family by Using APC and MSH2 mRNA Gene Expression and Bayesian Analysis

BACKGROUND: Some of colorectal cancers (CRCs) are familial, however, heterozygote relatives have approximately 80% lifetime risk of cancer. Risk assessment of CRC’s family could be calculated by direct measurement of mRNA gene expression and Bayesian theorem which is modifying initial background of pedigree risk with additional conditional information. This application has not been reported.METHODS: The cross-sectional translational sequential studies were performed: (1) adenomatous polyposis coli (APC) and MutS homolog (MSH)2 mRNA quantitative RT PCR gene expressions in tissue and whole blood CRC patients; (2) gene expression was determined in matched controls; and (3) pedigree and Bayesian analysis was calculated in the patient’s family of Proband.RESULTS: Fourty CRC and 31 control subjects were enrolled. The mean blood APC level control’s group was 13,261±670 fold-change (fc) and blood MSH2 level was 12,219±756 fc. The cut-off points for hereditary APC was 12,195 fc and MSH2 was 11,059 fc. The mean APC blood level in CRC subject was 11,578±2,638 fc and MSH2 blood level was 11,411±2,912 fc. There were significant differences APC and MSH level between tissue and blood level in CRC. Eight of 40 CRC subjects had a history of familial CRC. Four patients and 10 Probands were available for recurrence risk evaluation of pedigree analysis, RNA PCR quantitative and Bayesian calculation.CONCLUSION: There was determined a cut-off point of hereditary mRNA quantitative expression. The APC and MSH2 levels in CRC subjects were significantly lower than controls. Bayesian analysis allowed for the calculation of relative risk in CRC family members and considered in clinical practice.KEYWORDS: hereditary CRC, APC gene, MSH gene, Bayesian analysis

The Effects of Doxorubicin, Ethanol Extract and Flavonoid-rich Fraction of Euphorbia Splendida Mobayen on the PARP Level, and APC Gene Expression in HT-29 Human Colon Cancer Cell Line

Background: Natural products derived from various sources are being used to develop chemotherapeutic drugs. Euphorbiaceae is widely used to treat different types of cancers. Colorectal cancer (CRC) is the second and third cause of cancer in women and men, respectively. CRC is strongly associated with the deregulation of the Adenomatous polyposis coli (APC) gene and Poly [ADP-ribose] polymerase (PPAR) protein. Objectives: The current study aimed to examine the effect of doxorubicin, ethanol extract, and the flavonoid-rich fraction of Euphorbia Splendida Mobayen on colon cancer HT-29 cell line death, APC gene expression, and PPAR concentration. Methods: Following treatment of cells by Euphorbia ethanol extract, Euphorbia flavonoid-rich fraction, and doxorubicin, cell viability assay was used to investigate the viability status of the HT-29 cell line. Total RNA was isolated from the cell line and converted into cDNA. The expression level of the APC gene was determined by quantitative real-time PCR. Poly (ADP-ribose) polymerase (PPAR) protein was detected by the ELIZA method. Results: We found that Euphorbia ethanol extract, Euphorbia flavonoid-rich fraction, and doxorubicin can stimulate dose-dependent cell death in the HT-29 cell line, increase ACP gene expression (P = 0.001, P = 0.041, P = 0.019), and decrease PARP level (P = 0.001, P = 0.001, P = 0.001, respectively). Conclusions: The findings indicated that doxorubicin, ethanol extract, and the flavonoid-rich fraction of Euphorbia Splendida Mobayen had cytotoxic effects on human colon cancer HT-29 cell line by possibly stimulating apoptosis.

The effect of EBV on WIF1, NLK, and APC gene methylation and expression in gastric carcinoma and nasopharyngeal cancer.

Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p.

Phytochemical remedies: a key strategy towards reversing the aggressive murine colon cancer

Colon cancer accounts for about 500,000 death cases worldwide annually and represents approximately 6.5% of cancers in Egypt. This study employed a murine colon cancer model to identify the mechanism of the therapeutic value of gallic acid or ellagic acid against colon carcinogenesis in vivo. Fifty adult male rats were distributed into five groups. Group (1) was set as a negative control group. Groups (2), (3), (4), and (5) were intrarectally injected with N-methylnitrosourea to induce colon cancer. Group (2) was kept untreated, whereas group (3) and (4) were orally administered gallic acid and ellagic acid, respectively. Group (5) received intraperitoneal injection of 5-fluorouracil. The administration of gallic acid, ellagic acid or 5-fluorouracil harmonized the hazardous effect of N-methylnitrosourea in colon cancer-afflicted rats. This was evidenced by the significant depletion in both of serum tumor associated glycoprotein-72 (TAG72) and galectin-3 (GAL3) levels as well as the significant downregulation in the expression level of frizzled-8 receptor (FRZ-8) gene and significant amplification of adenomatous polyposis coli gene expression level in the colon tissues of all treated groups. Intriguingly, such treatments resulted in a notable improvement in the pathological features of colon tissue sections of N-methylnitrosourea-challenged rats. Generally speaking, the current investigation emphasizes the favorable impact of gallic acid and ellagic acid in monitoring the progression of colon cancer in vivo. We suggest that the mitigation of Wnt signaling pathway may be the probable mechanism by which these compounds can offer their therapeutic actions against colon cancer.

Epigenetic regulation of APC in the molecular pathogenesis of gallbladder cancer.

Background & objectives:Loss of function of adenomatous polyposis coli (APC) has been reported in cancer. The two promoters of APC, 1A and 1B also have roles in cancer. But, the epigenetic role of APC promoters is not yet clear in gallbladder cancer (GBC) and gallstone diseases (GSD). We undertook this study to determine the epigenetic role of APC in GBC and GSD.Methods:Methylation-specific (MS)-PCR was used to analyze the methylation of APC gene. The expression of APC gene was studied by semi-quantitative PCR, real-time PCR and immunohistochemistry (IHC) in GBC, GSD and adjacent normal tissues.Results:Of the two promoters, APC 1A promoter was found methylated in 96 per cent GBC (P=0.0155) and 80 per cent GSD (P=0.015). Exon 1 was downregulated in grade II (P=0.002) and grade III (P=0.0001) of GBC, while exon 2 was normally expressed. Scoring analysis of IHC revealed 0 or negativity in 34.48 per cent (P=0.057) and 1+ in 24.14 per cent (P=0.005) GBC cases suggesting loss of APC expression.Interpretation & conclusions:The present findings indicate epigenetic silencing of APC in advanced GBC. The methylation pattern, followed by expression analysis of APC may be suggested for diagnostic, prognostic and therapeutic purposes in GBC in future.

APC promoter 1B deletion in familial polyposis—implications for mutation‐negative families

Over 1500 adenomatous polyposis coli (APC) gene mutations have already been identified as causative of familial adenomatous polyposis (FAP). However, routine genetic testing fails to detect mutations in about 10% of classic FAP cases. Recently, it has been shown that a proportion of mutation-negative FAP cases bear molecular changes in deep intronic and regulatory sequences. In this study, we used direct sequencing, followed by multiplex ligation-dependent probe amplification (MLPA) of genomic DNA from family members, affected by classic FAP. We first reported the family as mutation negative. With the launch of a new version of MLPA kit, we retested the family and a novel full deletion of promoter 1B was detected. The exact breakpoints of the deletion were determined by array comparative genomic hybridization (CGH) and long range polymerase chain reaction (PCR), followed by direct sequencing. The total APC expression levels were investigated by quantitative polymerase chain reaction (qPCR) assay and allele-specific expression (ASE) analysis. The APC gene expression was highly reduced, which indicates causative relationship. We suggest that there is a significant possibility that APC promoter 1B mutations could be found in mutation-negative FAP patients. In the light of our findings it seems reasonable to consider targeted genetic re-analysis of APC promoter 1B region in a larger cohort of unsolved cases.

APC gene deletions in gastric adenocarcinomas in a Chinese population: a correlation with tumour progression

The adenomatous polyposis coli (APC) gene encodes a tumor suppressor protein that acts as an antagonist of the Wnt signaling pathway. It has been shown to be involved in genetic instability and to be down-regulated in several human carcinomas. The chromosome locus of APC, 5q21-22, is frequently deleted in gastric cancers (GCs). The functional impact of such regions needs to be extensively investigated in large amount of clinical samples. Case-matched tissues of GC and adjacent normal epithelium (n = 141) were included in this study. Quantitative PCR was carried out to examine the copy number as well as mRNA expression of APC gene in gastric malignancies. Our results showed that copy number deletions of APC were present in a relatively high percentage (25.9%, 34 out of 131) of gastric cancer samples. There was a correlation between APC deletion and tumor progression (p < 0.01) as well as gene expression (p < 0.05) in collected GC samples. On the other hand, mRNA levels of APC were also impaired in GC samples with unaltered copy numbers. Sporadic GCs exhibit different mechanisms of APC regulation.

Detection of APC Gene Deletions in Colorectal Malignancies Using Quantitative PCR in a Chinese Population

The adenomatous polyposis coli (APC) gene has been shown to be involved in genetic instability and to be downregluated in several human carcinomas. The chromosome locus of APC, 5q21-22, is frequently deleted in colorectal cancers (CRCs). The functional impact of such regions needs to be extensively investigated in large amount of clinical samples. Case-matched tissues of CRC and adjacent normal epithelium (n = 134) were included in this study. Quantitative PCR was carried out to examine the copy number as well as mRNA expression of APC gene in colorectal malignancies. Our results showed that copy number deletions of APC were present in a relatively high percentage of colorectal cancer samples (26.1%, 35 out of 134). There was a positive correlation between copy number decrease of APC and tumor progression in CRCs. Furthermore, copy number loss of APC was correlated with decreased mRNA expression. However, mRNA levels of APC were also impaired in CRC samples with unaltered copy numbers, indicating that sporadic CRCs exhibit different mechanisms of APC regulation.

APC Mutation Spectra in Sporadic Colon Cancer Tissue among Korean

Purpose: APC (adenomatous polyposis coli) gene is one of the tumor-suppressor genes that acts in the early stages of cancer. Among general colon cancer patients, normal APC gene expression is deficient in 80%. It seems that APC is the most important gene in the development of colon cancer. This study was performed to analyze the mutation spectra of APC gene in sporadic colon cancer tissue from Korean patients with colon cancer. Methods: A total of 38 patients with sporadic colon cancer were enrolled. Colon cancer tissues were analyzed for the determination of APC gene mutation spectra by multiplex ligation-dependent probe amplification (MLPA) method using SALSA MLPA P403 APC kit (MRC-Holland, Amsterdam, NL). Results: APC gene mutations showing deletion/duplication in one or more exons were detected in 23 (60.5%) patients. Duplication in 13 patients (56.5%), duplication and deletion in 7 patients (30.4%), and deletion in 3 patients (13.1%) was detected. The incidence of APC gene mutation found in this study was highest in exon 3. From this study, no significant differences were observed with respect to clinicopathologic findings and the presence or absence of APC mutations. Conclusion: The frequency of APC gene mutation was about 61% in Korean patients with colon cancer, it showed concordance with the previous reports on the frequency of APC gene mutation from Caucasian patients with sporadic colon cancer. However, in contrast to these reports, the frequency of duplication disclosed much higher than those of western countries.

Hydralazine inhibits human cervical cancer cell growth in vitro in association with APC demethylation and re-expression

The tumor suppressor adenomatous polyposis coli (APC) is frequently silenced by promoter hypermethylation in human cervical cancer. Clinically, it has been approved that DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (5-Aza-dC), can reverse APC promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. Hydralazine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. The purpose of this study was to evaluate the effects of hydralazine on APC reactivation and the inhibition of human cervical cancer cells in vitro. Expression of APC gene, and methylation status were analyzed by RT-PCR, quantitative real time RT-PCR, and methylation-specific PCR methods. beta-Catenin protein that correlates closely with APC was detected by immunohistochemistry method after treatment with hydralazine. MTT and FCM assays were used to observe the changes of proliferation activity, cell cycle, and apoptosis of the cells. Methylated APC was not expressed in HeLa cell, hemimethylated APC was expressed in CaSki cells, and unmethylated APC was expressed normally in SiHa cells. Hydralazine induces APC expression and promotes demethylation in HeLa and CaSki cells. After treatment with 40 mumol/L hydralazine for 72 h, growth inhibitive rates (%) of HeLa, CaSki, and SiHa cell lines were 52.12 +/- 3.78, 44.31 +/- 2.59, and 47.73 +/- 4.73, respectively. On the contrary, the normal cell ECV304 growth inhibitory rate was only 27.18 +/- 0.79. The expression of APC mRNA in HeLa, CaSki, and SiHa cell lines increased 10.35-, 11.40-, and 0.73-fold, respectively. HeLa and CaSki cells were arrested in S phase of the cell cycle by hydralazine, and the percentage of apoptotic cells in the two cell lines treated with hydralazine was increased significantly compared to the untreated cells (P < 0.01). The expression of beta-catenin protein in the cell membrane was observed after the treatment with hydralazine. Hydralazine, an effective inhibitor of APC methylation and promoter of APC re-expression, can inhibit cell growth in human cervical cancer in vitro and be potentially used for the clinical treatment of human cervical cancer.

Effect of Hydralazine on demethylation status and expression of APC gene, proliferation and apoptosis of human cervical cancer cell lines

To investigate the correlation between methylation status and gene expression of APC (adenomatous polyposis coli) gene in HeLa, CaSki and SiHa cell lines of cervical carcinoma, and explore the effect of hydralazine on the transcription regulation of the 5'CpG island demethylation of APC gene and the proliferation and apoptosis of the cell lines. Methylation status and the expression of APC gene were analyzed using methylated specific PCR, RT-PCR and FQ-PCR methods. The expression of beta-catenin protein which correlates closely with APC was detected by SP method after treatment with Hydralazine. MTT and FCM assays were used to observe the changes of proliferation activity and apoptosis of the cells after Hydralazine treatment. (1) APC gene was methylated or hemimethylated respectively in HeLa and CaSki cell lines, at the same time, APC gene was not methylated in SiHa cell. (2) After having been treated by 40 micromol/L Hydralazine for 72 hours, growth inhibitory ratios of HeLa, CaSki and SiHa cell lines were (52.12 +/- 3.78)%, (44.31 +/- 2.59)% and (47.73 +/- 4.73)% respectively, on the contrary, normal cell HECV's growth inhibitory ratio was only (27.18 +/- 0.79)%. APC gene in HeLa and CaSki cell lines which were treated by 40 micromol/L Hydralazine for 72 hours was demethylated and expressed positively, the expression of APC mRNA in HeLa, CaSki and SiHa cell lines increased to 10.35, 11.40 and 0.73 times respectively. (3) Hydralazine, when used at the concentration of 40 micromol/L for 72 hours, induced S phase and G2/M phase arrest and apoptosis in HeLa and CaSki cells. beta-catenin protein can be expressed in cell membrane after treatment with Hydralazine. APC gene methylation plays an important role in the carcinogenesis of cervical cells and can re-express after the treatment with Hydralazine which also could inhibit the growth of the cervical cancer cells.

Dietary fat and risk of colon and rectal cancer with aberrant MLH1 expression, APC or KRAS genes

ObjectiveTo investigate baseline fat intake and the risk of colon and rectal tumors lacking MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2) repair gene expression and harboring mutations in the APC (adenomatous polyposis coli) tumor suppressor gene and in the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) oncogene.MethodsAfter 7.3 years of follow-up of the Netherlands Cohort Study (n = 120,852), adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed, based on 401 colon and 130 rectal cancer patients.ResultsTotal, saturated and monounsaturated fat were not associated with the risk of colon or rectal cancer, or different molecular subgroups. There was also no association between polyunsaturated fat and the risk of overall or subgroups of rectal cancer. Linoleic acid, the most abundant polyunsaturated fatty acid in the diet, was associated with increased risk of colon tumors with only a KRAS mutation and no additional truncating APC mutation or lack of MLH1 expression (RR = 1.41, 95% CI 1.18–1.69 for one standard deviation (i.e., 7.5 g/day) increase in intake, p-trend over the quartiles of intake <0.001). Linoleic acid intake was not associated with risk of colon tumors without any of the gene defects, or with tumors harboring aberrations in either MLH1 or APC.ConclusionLinoleic acid intake is associated with colon tumors with an aberrant KRAS gene, but an intact APC gene and MLH1 expression, suggesting a unique etiology of tumors with specific genetic aberrations.

Tumor Suppressor APC Blocks DNA Polymerase β-dependent Strand Displacement Synthesis during Long Patch but Not Short Patch Base Excision Repair and Increases Sensitivity to Methylmethane Sulfonate

In the present investigation, we report a previously unsuspected function of the tumor suppressor protein, APC (adenomatous polyposis coli), in the regulation of base excision repair (BER). We identified a proliferating cell nuclear antigen-interacting protein-like box sequence in APC that binds DNA polymerase beta and blocks DNA polymerase beta-mediated strand-displacement synthesis in long patch BER without affecting short patch BER. We further showed that the colon cancer cell line expressing the wild-type APC gene was more sensitive to a DNA-methylating agent due to decreased DNA repair by long patch BER than the cell line expressing the mutant APC gene lacking the proliferating cell nuclear antigen-interacting protein-like box. Experiments based on RNA interference showed that the wild-type APC gene expression is required for DNA methylation-induced sensitivity of colon cancer cells. Thus, APC may play a critical role in determining utilization of long versus short patch BER pathways and affect the susceptibility of colon cancer cells to carcinogenic and chemotherapeutic agents.

Differences in biological features of gastric dysplasia, indefinite dysplasia, reactive hyperplasia and discriminant analysis of these lesions

To investigate the differences in biological features of gastric dysplasia (Dys), indefinite dysplasia (IDys) and reactive hyperplasia (RH) by studying the biomarker alterations in cell proliferation, cell differentiation, cell cycle control and the expression of house-keeping genes, and further to search for markers which could be used in guiding the pathological diagnosis of three lesions. Expressions of MUC5AC, MUC6, adenomatous polyposis coli (APC), p53, Ki-67, proliferation cell nuclear antigen (PCNA) and EGFR were studied by immunohistochemistry with a standard Envision technique in formalin-fixed and paraffin-embedded specimens from 43 RH, 35 IDys, 35 Dys and 36 intestinal type gastric carcinomas (IGC). In addition, Bayes discriminant analysis was used to investigate the value of markers studied in differential diagnosis of RH, IDys, Dys and IGC. The MUC5AC and MUC6 antigen expressions in RH, IDys, Dys and IGC decreased gradually (MUC5AC: 86.04%, 77.14%, 28.57%, 6.67%; MUC6: 65.15%, 54.29%, 20.00%, 25.00%, respectively). The expressions of the two markers had no significant difference between RH and IDys, but were all significantly higher than those of the other two lesions (MUC5AC: chi2=27.607, 38.027 and 17.33, 26.092; MUC6: chi2=16.54, 12.665 and 9.282, 6.737, P<0.01). There was no significant difference between RH and IDys, Dys and IGC in MUC6 expression. The APC gene expression in the four lesions had a similar decreasing tendency (RH 69.76%, IDys 68.57%, Dys 39.39%, IGC 22.86%), and it was significantly higher in the first two lesions than in the last two (chi2=7.011, 16.995 and 14.737, 19.817, P<0.05). The p53 expression in RH, IDys, Dys and IGC was 6.98%, 20%, 57.14% and 50%, respectively. There was no significant difference between RH and IDys or Dys and IGC, but the p53 expression in RH and IDys was significantly lower than that in Dys and IGC (chi2=7.011, 16.995 and 14.737, 19.817, P<0.01). The Ki-67 label index was significantly different among four lesions (RH: 0.298+/-8.92%, IDys: 0.358+/-9.25%, Dys: 0.498+/-9.03%, IGC: 0.620+/-10.8%, P<0.001). Positive immunostaining of PCNA was though observed in all specimens, significant differences were detected among four lesions (F=95.318, P<0.01). In addition, we used Bayes discriminant analysis to investigate molecular pathological classification of the lesions, and obtained the best result with the combination of MUC5AC, Ki-67 and PCNA. The overall rate of correct classification was 67.4% (RH), 68.6% (IDys), 70.6% (Dys) and 84.8% (IGC), respectively. Dys has neoplastic biological characteristics, while RH and IDys display hyperplastic characteristics. MUC5AC and proliferation-related biomarkers (Ki-67, PCNA) are more specific in distinguishing Dys from RH and IDys.

Analysis of adenomatous polyposis coli gene expression, APC locus-microsatellite instability and APC promoter methylation in the progression of melanocytic tumours

Adenomatous polyposis coli gene (APC) defects have been demonstrated for the first time in familial adenomatous polyposis. Recent reports indicate that the APC gene is an intermediary between cell adhesion molecules and the cytoskeleton and that it may function as a gatekeeper of colonic epithelial proliferation. The objective of this study was to analyse APC's presence in lentigos, primary melanomas and melanoma metastases. By immunohistochemistry, APC was demonstrated in all lentigos, in 75 out of 88 primary melanomas and in 16 out of 28 melanoma lymphatic metastases. The percentage of immunolabelled tumour cells (APC index) in lentigos ranged between 5 and 69%, in primary melanomas between 0 and 98% and in melanoma metastases between 0 and 52%. Statistically significant differences between lentigos and primary melanomas and between lentigos and metastases in APC expression were found. In a multivariate analysis, APC showed an independent prognostic impact. Analysis of microsatellite instability in the APC locus was performed on 29 melanomas. Microsatellite instability was found in 5/29 melanomas and loss of heterozygosity in 1/29 melanomas. Promoter methylation of APC was found in 6/10 APC-negative primary melanomas and in 9/10 APC-negative melanoma lymphatic metastases investigated. We conclude about important role of APC alterations for melanoma progression.

APC Haploinsufficiency, but Not CTNNB1 or CDH1 Gene Mutations, Accounts for a Fraction of Familial Adenomatous Polyposis Patients Without APC Truncating Mutations

Familial adenomatous polyposis (FAP) is an autosomal dominant condition characterized by the development of hundreds to thousands of colorectal adenomatous polyps. In addition to the classic form, there is also attenuated polyposis (attenuated adenomatous polyposis coli; AAPC), which is characterized by a milder phenotype. FAP/AAPC is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Very recently, germline mutations in the base-excision repair gene MYH have been associated with recessive inheritance of multiple colorectal adenomas in a subset of patients. APC pathogenic alterations are mostly (>95%) represented by frameshift or nonsense mutations leading to the synthesis of a truncated protein. We identified 20 APC truncating mutation carriers out of 30 FAP/AAPC patients from different Italian kindreds. In the remaining 10 patients, we searched for alterations other than truncating mutations by enzymatic mutation detection, real-time quantitative RT-PCR, and genotyping of polymorphic markers encompassing the APC locus. Moreover, to assess whether mutations of genes interacting with APC can substitute or act in association with APC alterations, we sequenced both CTNNB1 (β-catenin) and CDH1 (E-cadherin) genes. No CTNNB1 or CDH1 mutations were found. On the contrary, four patients showed a reduced APC gene expression compared with healthy subjects. In three of the four cases, genotyping results were compatible with a constitutive allelic deletion. In one case this conclusion was confirmed by haplotype segregation analysis. Our results support the notion that FAP/AAPC can result from APC constitutive haploinsufficiency, with gene deletion being a possible cause of reduced gene expression.

Reduction of Intestinal Neoplasia With Adenomatous Polyposis Coli Gene Replacement and COX-2 Inhibition Is Additive

Mutations of the adenomatous polyposis coli (APC) gene are implicated early in colorectal tumorigenesis. Restoration of normal APC expression through gene therapy may prevent or reduce intestinal neoplasia. Furthermore, the relationship between colorectal tumors and increased cyclooxygenase-2 (COX-2) activity provides a rationale for the use of selective COX-2 inhibitors such as rofecoxib (Vioxx) to prevent the formation of polyps. This study was performed to determine the effects of liposome-mediated APC gene therapy and a selective COX-2 inhibitor on intestinal neoplasia in vivo. Five-week-old Min mice weaned on a 30% high-fat diet were randomized to receive no treatment (control), APC only, Vioxx only, and APC/Vioxx. APC-treated mice received a plasmid containing the human APC cDNA (pCMV-APC) mixed with a liposome preparation that was administered biweekly. Vioxx was administered at 200 ppm in the high-fat rodent chow. The control mice were treated similarly with a plasmid construct lacking the APC gene. Confirmation of exogenous APC gene expression was determined by Western blot analysis. After 2 months, there was a 54% and 70% reduction in the total number of intestinal polyps after APC and Vioxx treatment, respectively. Combined APC/Vioxx therapy reduced polyp formation by 87%. The reduction of intestinal neoplasia by APC gene replacement and COX-2 inhibition suggests their separate roles in intestinal tumorigenesis. Each modality, both individually and together, may prove therapeutic and therefore contribute to new strategies in the prevention and treatment of colorectal cancer. ( J Gastrointest Surg 2002;6:563–568.)

Upstream stimulating factor-1 (USF1) and USF2 bind to and activate the promoter of the adenomatous polyposis coli (APC) tumor suppressor gene.

The adenomatous polyposis coli (APC) gene product is involved in cell cycle arrest and apoptosis, and loss of function is associated with the development of colorectal carcinogenesis. Although it has been demonstrated that the APC gene is inducible, its transcriptional regulation has not been elucidated. Therefore, we characterized the promoter region of the APC gene and transcription factors required for basal expression. The APC gene has a TATA-less promoter and contains consensus binding sites for Octamer, AP2, Sp1, a CAAT-box, and three nucleotide sequences for E-box A, B, and M. The E-boxes are functional in several cancer cell lines and upstream stimulating factor-1 (USF1) and USF2 interact with these sites, with a preferred sequence-specificity for the B site. Analysis of activation of the cloned APC promoter by USF1 and USF2 in transient transfection assays in HCT-116 cells demonstrated that mutation of the E-box B site completely abolished the basal promoter activity. Further, the ectopic USF1 and USF2 expression in HCT-116 cells with deletion mutations of E-box A, B, and M sites showed that these E-boxes contribute to USF1- and USF2-mediated transcriptional activation of the APC promoter, with maximum promoter activity being associated with the E-box B site. Thus, USF1 and USF2 transcription factors are critical for APC gene expression.