NSAIDs may regulate EGR-1-mediated induction of reactive oxygen species and non-steroidal anti-inflammatory drug-induced gene (NAG)-1 to initiate intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer

This study aims to investigate the unclear molecular relationship involved in the activation of intrinsic pathway of apoptosis and NSAID-activated gene-1 (NAG-1) induction as a putative target in NSAIDs-mediated chemoprevention of colorectal cancer. Male Sprague-Dawley rats were administered with a colon-specific pro-carcinogen, 1,2-dimethylhydrazine dihydrochloride to achieve the early stages of colorectal cancer. Histopathological examination was performed for the analysis of neoplastic lesions while flow cytometry was performed for the relative quantification of intracellular reactive oxygen species (ROS), differential mitochondrial membrane potential (MMP or ΔΨ _M), and apoptotic events. Various target biomolecules were analyzed either for their mRNA or protein expression profiles via RT-PCR and quantitative Real-Time PCR, or Western blotting and immunofluorescence, respectively. Enhanced gene as well as protein expression of pro-apoptotic agents was observed with the daily oral administration of two NSAIDs viz. Sulindac (cyclooxygenase (COX)-non-specific) and Celecoxib (a selective COX-2 inhibitor). A significant increase in early growth response-1 (EGR-1) protein expression and nuclear localization in NSAIDs co-administered animals may have positively regulated the expression of NAG-1 with a significant enhancement of intracellular ROS in turn decreasing the ΔΨ _M to initiate apoptosis. In silico molecular docking analysis also showed that Sulindac and Celecoxib can block the active site pocket of B-cell lymphoma-extra large (Bcl-xL, anti-apoptotic transmembrane mitochondrial protein) which could be a putative mechanism followed by these NSAIDs to overcome anti-apoptotic properties of the molecule. NSAIDs-mediated up-regulation of EGR-1 and thereby NAG-1 along with implication of higher ROS load may positively regulate the intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer.     

Effect of regulated expression of the fragile histidine traid gene on cell cycle and proliferation

The mechanism of tumor suppressor action of the fragile histidine triad (FHIT) gene is unknown. Disruption of cell cycle regulation leads to the tumor formation and many tumor suppressor genes suppress tumorigenesis through their effect on cell cycle regulation. We examined the expression of FHIT during the cell cycle, and determined whether overexpression of FHIT affects cell cycle kinetics and apoptosis. The FHIT cDNA was cloned into the ecdysone-inducible expression vector in both the sense and antisense orientations. Overexpression of the sense or antisense construct did not affect cell proliferation, cell cycle distribution or apoptosis in human 293T cells. Analysis of the FHIT expression in 293T cells collected at various cell cycle phases showed that the expression of FHIT is not under cell cycle regulation. These results indicate that the tumor suppressor activity of the FHIT gene may be independent of an effect on the cell cycle and apoptosis mechanisms.     

The p53 tumor suppressor participates in multiple cell cycle checkpoints

The process of cell division is highly ordered and regulated. Checkpoints exist to delay progression into the next cell cycle phase only when the previous step is fully completed. The ultimate goal is to guarantee that the two daughter cells inherit a complete and faithful copy of the genome. Checkpoints can become activated due to DNA damage, exogenous stress signals, defects during the replication of DNA, or failure of chromosomes to attach to the mitotic spindle. Abrogation of cell cycle checkpoints can result in death for a unicellular organism or uncontrolled proliferation and tumorigenesis in metazoans (Nyberg et al., 2002). The tumor suppressor p53 plays a critical role in each of these cell cycle checkpoints and is reviewed here.     

NSAIDs induce apoptosis in nonproliferating ovarian cancer cells and inhibit tumor growth in vivo

Ovarian cancer (OC) is one of the most lethal gynaecological cancers, which usually has a poor prognosis due to late diagnosis. A large percentage of the OC cell population is in a nonproliferating and quiescent stage, which poses a barrier to success when using most chemotherapeutic agents. Recent studies have shown that several nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in the treatment of OC. Furthermore, we have previously described the molecular mechanisms of NSAIDs' induction of cancer apoptosis. In this report, we evaluated various structurally distinct NSAIDs for their efficacies in inducing apoptosis in nonproliferating OC cells. Although several NSAIDs-induced apoptosis, Flufenamic Acid, Flurbiprofen, Finasteride, Celocoxib, and Ibuprofen were the most potent NSAIDs inducing apoptosis. A combination of these agents resulted in an enhanced effect. Furthermore, we demonstrate that the combination of Flurbiprofen, which targets nonproliferative cells, and Sulindac Sulfide, that affects proliferative cells, strongly reduced tumor growth when compared with a single agent treatment. Our data strongly support the hypothesis that drug treatment regimens that target nonproliferating and proliferating cells may have significant efficacy against OC. These results also provide a rationale for employing compounds or even chemically modified NSAIDs, which selectively and efficiently induce apoptosis in cells during different stages of the cell cycle, to design more potent anticancer drugs.     

miR-155 regulates the proliferation and cell cycle of colorectal carcinoma cells by targeting E2F2

MicroRNAs play important roles in carcinogenesis by negatively regulating the expression of target genes. Here we explore the biological function of miR-155 and the underlying mechanism in colorectal carcinoma. We validate, for the first time, that E2F2 is a direct target of miR-155 using western blot and a luciferase reporter assay and that miR-155 regulates the proliferation and cell cycle of colorectal carcinoma cells by targeting E2F2 using siRNA technology. We also found, for the first, time that E2F2 acts as a tumor suppressor in colorectal carcinoma. Overall, miR-155 plays an important role in colorectal carcinoma tumorigenesis by negative regulation of its targets including E2F2 and may be a potential therapeutic target for colorectal carcinoma treatment.     

Radiation dose-dependent maintenance of G(2) arrest requires retinoblastoma protein

In response to ionizing radiation (IR), cell cycle checkpoints are activated to provide time for DNA repair. Several different checkpoint mechanisms have been elucidated. However, mechanisms that regulate the duration of cell cycle arrest are not understood. Previous studies have shown that the retinoblastoma tumor suppressor protein (RB) is required for radiation-induced G1 arrest. Working with primary fibroblasts derived from Rb+/+ and Rb-/- mouse embryos, we show that RB also regulates the duration of G2 arrest. The initial G2 checkpoint response is enhanced in Rb-/- cells due to a defect in G1 arrest. However, the permanent arrest in G2 induced by higher doses of IR does not occur in Rb-/- cells. Rb-/- cells either resumed proliferation or underwent apoptosis at IR doses that caused the majority of Rb+/+ cells to arrest permanently in G2. The prolongation of G2 arrest in Rb+/+ cells correlated with a gradual accumulation of hypophosphorylated RB. Thus, regulation of the RB function may be an important aspect in the maintenance of cell cycle checkpoints in DNA damage response.     

Gankyrin 与消化系统肿瘤关系的研究进展

消化系统肿瘤与多种肿瘤相关基因的异常表达密切相关,Gankyrin 是一种癌基因,于肝癌、结直肠癌、胰腺癌等消化系统肿 瘤中高表达,并通过依赖泛素的蛋白酶解系统介导多种转录因子、细胞周期调控蛋白以及抑癌蛋白如p53、Rb 等的降解。研究表 明,Gankyrin 在消化系统肿瘤形成及细胞的生长周期调控中发挥重要作用,在消化系统肿瘤的早期诊断及判断预后方面具有一 定的应用前景,同时也可能成为消化系统肿瘤基因治疗的潜在靶点。     

Cdk inhibitors: on the threshold of checkpoints and development

The regulation of cyclin-dependent kinases is at the heart of cell cycle control and, by inference, the control of cell proliferation. Recent advances in regulation of these kinases have uncovered a group of small proteins that bind to and inhibit them, thus preventing cell cycle progression. Linking these proteins to tumor suppressor functions has provided a much sought after connection between cancer and cell cycle control.     

Gankyrin与消化系统肿瘤关系的研究进展

消化系统肿瘤与多种肿瘤相关基因的异常表达密切相关,Gankyrin是一种癌基因,于肝癌、结直肠癌、胰腺癌等消化系统肿瘤中高表达,并通过依赖泛素的蛋白酶解系统介导多种转录因子、细胞周期调控蛋白以及抑癌蛋白如p53、Rb等的降解。研究表明,Gankyrin在消化系统肿瘤形成及细胞的生长周期调控中发挥重要作用,在消化系统肿瘤的早期诊断及判断预后方面具有一定的应用前景,同时也可能成为消化系统肿瘤基因治疗的潜在靶点。     

Pirh2

Ubiquitylation is currently recognized as a major posttranslational modification that regulates diverse cellular processes. Pirh2 is a ubiquitin E3 ligase that regulates the turnover and functionality of several proteins involved in cell proliferation and differentiation, cell cycle checkpoints, and cell death. Here we review the role of Pirh2 as a regulator of the DNA damage response through the ubiquitylation of p53, Chk2, p73, and PolH. By ubiquitylating these proteins, Pirh2 regulates cell cycle checkpoints and cell death in response to DNA double-strand breaks or the formation of bulky DNA lesions. We also discuss how Pirh2 affects cell proliferation and differentiation in unstressed conditions through ubiquitylation and degradation of c-Myc, p63, and p27^kip1. Finally, we link these different functions of Pirh2 to its role as a tumor suppressor in mice and as a prognosis marker in various human cancer subtypes.     

Up-regulation of tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 in human colon cancer Caco-2 cells following repetitive exposure to dietary levels of a polyphenol-rich chokeberry juice

Consumption of berries and red fruits rich in polyphenols may contribute to the reduction of colon cancer through mechanisms not yet understood. In this study, we investigated the response of subconfluent Caco-2 cells (a human colon carcinoma model) to repetitive exposure (2 h a day for a 4-day period) of a subtoxic dose of a chokeberry (Aronia melanocarpa) juice containing mixed polyphenols. To mimic physiological conditions, we subjected the chokeberry juice to in vitro gastric and pancreatic digestion. The effects on viability, proliferation and cell cycle were determined, and changes in the expression of genes in response to the chokeberry treatment were screened using Affymetrix oligonucleotide microarrays. Exposure to the chokeberry juice inhibited Caco-2 cell proliferation by causing G(2)/M cell cycle arrest. We detected changes in the expression of a group of genes involved in cell growth and proliferation and cell cycle regulation, as well as those associated to colorectal cancer. A selection of these genes was further confirmed by quantitative RT-PCR. Among these, the tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), whose expression is known to be reduced in the majority of early adenomas and carcinomas, was up-regulated by the treatment both at the mRNA and protein levels (as shown by flow cytometry analysis). CEACAM1, with a significant regulatory role on cell proliferation of particular interest at early stages of cancer development, may be a potential target for chemoprevention by food components such as those present in polyphenol-rich fruits.     

Radiation-induced genetic instability in vivo depends on p53 status

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.