| Colorectal carcinoma (CRC) is one of the most common malignancies in the world and the second most frequent cause of cancer-related death in the United States. At present, the use of combined therapeutic approaches (surgery, chemotherapy, and radiation) for patients with colorectal carcinoma does not evidently improve outcomes. The five-year survival in patients with stage III or IV disease is from 25 percent to 65 percent and from 5 percent to 7 percent, respectively. For many years, the use of chemotherapy and radiotherapy as adjuvant treatment after complete resection of stage II cancers (T3 or T4 with negative lymph nodes, according to the TNM system) or stage III cancers (any T stage with positive lymph nodes) was thought to be ineffective for the treatment dosage was not sufficient to avoid prominent toxic effect and other adverse reactions including diarrhea, nausea, vomiting and bone marrow suppression. With the development of RNA interfering technology, gene therapy has been made significantly progress. But it is impossible that gene therapy is applied to clinical treatment in large scale because of the technological problem of vector construction. Recently, it has been suggested that differentiation inducers may potentially stimulate tumour cell differentiation, alter tumor growth and progression, slow or inhibit metastases, inhibit angiogenesis, and/or effect response to other forms of therapy. Therefore, differentiation therapy for tumors has become the hot spots of anti-cancer research, which regulates the expression of oncogenes and tumor-suppressing genes in tumor cells. With the advent of new genetic and molecular-pathologic techniques, many authors have come to consider the causes and pathogenesis of colorectal carcinoma as multifactorial. All of these factors seem to cooperate in stimulating a disturbance of genes that regulate cell proliferation and programmed cell death. Carcinogenesis and tumor progression are, therefore, thought to be the result of a series of progressive gene alterations, including the activation of oncogenes and the inactivation of tumor-suppressing genes.Homeobox (HOX) genes are defined by the presence of a characteristic 183 base pair DNA sequence (the homeobox) coding for a relatively conserved 61 amino acid section of protein (the homeodomain) containing transcription factors, which are crucial for embryonic development and differentiation. The HOX network contains 39 genes, which are well known as master control genes in embryonic morphogenesis and were first described in organisms in development. However, a link between development and cancer is expected since both processes involve cell proliferation and cell differentiation. Recent research has demonstrated that there is a lot to be learned about the interplay that exists between development, cell cycle, apoptosis and cancer. Improper regulation of HOX genes may result in cancer. These observations have stimulated investigators to attempt to regulate HOX genes expression in cancer cells using biologic substances or chemicals. As a result, this subject aims at studying the altered expression of HOX genes mRNA regulated by phenylacetate (PA), a lead candidate as a cancer differentiating agent, at the levels of cell, subcell and molecule in vitro. Thereby identify further molecular alterations in CRC and screen new diagnostic biomarkers and new treatment targets. This observation will provide valuable insight into the molecularbasis of colorectal carcinoma, and can contribute to cure rate, prolongation of survival, reduction of local rates of recurrence and enhanced quality of life in patients with advanced disease. The main results of the study include: This proliferative inhibition of the colorectal carcinoma cell line HCT-8 treated by PA occurred in a dose-and time-responsive manner. In vitro, PA had been shown to induce tumor cells differentiation as well as acted to strongly inhibit the growth of the colorectal carcinoma cell line HCT-8 by arresting these cells in G1 phase at the transcriptional level. In terms of the regulation of genes expression, PA can regulate the expression of HOX genes, which is the master control gene of this transcription process. To explore the oncogenesis-and/or metastasis-associated HOX genes, we simply quantified the mRNA expression of the 39 class I HOX genes based on reverse transcription-polymerase chain reaction (RT-PCR). Using this system we examined the whole HOX gene network expression profiles in normal human intestinal epithelial cells (IEC) and colorectal carcinoma cell line HCT-8 treated by PA or not. Furthermore, we compared the expression patterns of 39 HOX genes between IEC, HCT-8 cells and PA-treated HCT-8 cells. This observation provided valuable insight intothe molecular basis of colorectal carcinoma as well as identified the specific HOX genes responsive to PA. The study on the effect and mechanisms of HOX genes expression during PA suppressing cell proliferation while inducing cell differentiation in colorectal carcinoma cells was performed. Analysis of our findings in a wider perspective is instructive from several aspects: (1) The clinical application of PA has a bright future. HCT-8 cells were cultured in vitro. Using MTT tetrazolium dye reduction assay, we demonstrated the antitumoral and antiproliferative effects of PA on colorectal carcinoma HCT-8 cells. Studies have demonstrated dose-and time-dependent growth inhibition of HCT-8 cells induced by PA. The inhibitive rate of HCT-8 cells incubated with 0 (control), 1mM, 2mM, 3mM, 4mM, or 5mM PA in culture medium for 24h~72h is 5.1%~24.3%, 16.7%~70%, 30.2%~90.4%, respectively. When HCT-8 cells exposed to 5.0mM PA for 72h, the rate of proliferative inhibition was 90.4%. In vitro studies of PA in colorectal carcinoma cells indicated that PA could induce growth inhibition at concentrations that havebeen achieved in humans with no significant adverse effects. (2) (2) PA can increase the differentiation degree of HCT-8 cells. The extent of tumour cell differentiation was assessed between the 1st to 3rd day after exposure to 1.0,2.0,4.0mM PA, using electronic microscope. It was found that PA could change the nucleus of HCT-8 cells from irregular shape to round shape with large number of rough endoplasmic reticulum hyperplcosia in cytoplasm arranging ribosome orderly, which facilitates the transportation of inherent message in an almost normal manner. (3) FCM analysis revealed that suppression of cell growth by PA was due to G1 arrest. When HCT-8 cells treated with 5.0mM PA for 72h, cell cycle analysis with flow cytometry showed that the percentage of HCT-8 cells in G0/G1 phase and (G2+M) phase reduced significantly and S phase increased relatively. RNA and nuclear protein are synthesized in G1 phase increased relatively. The main content of RNA is mRNA, and the late G1 phase is the beginning of mRNA transcription. For these reasons, it is postulated that PA acts on the transcription process of colorectal carcinoma HCT-8 cells affecting the expression of oncogenes mRNA and suppressing the total quality of RNA in cancer cells.(4) HOX genes, the master control gene of transcription process, respond to PA. Twenty-two kinds of HOX genes were divided into 3 groups (P1, P2, and P3) according to their primer sequence, and cells samples were analyzed for HOX genes mRNA expression by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). PCR products were electrophoresed in a 2.5% agarose gel and intensity of the bands was observed under a UV illuminator. The mRNA expression level of HOX genes was expressed as the expression ratio of HOX gene/β-actin by GIS. P1 group mRNA expression was (0.5781 ±0.0836) after HCT-8 cells treated with PA and significantly lower than (0.7701 ±0.0883) in HCT-8 cells. Both mRNA expression of P2 (0.3941 ±0.0819) and P3 (0.5601 ±0.0736) in PA treated HCT-8 cells were significantly higher than (0.1221 ±0.0782) and (0.1806 ±0.0811) in HCT-8 cells. It suggested that PA could regulate the mRNA expression of HOX genes groups. (5) Specific HOX genes responding to PA have been preliminarily screened. Because PA can induce HCT-8 cells differentiation, we next explored whether the pattern of HOX genes expression is altered as a result of change in the differentiation. We investigated the expressionprofiles of 39 known HOX genes in IEC, the human adenocarcinoma cell line HCT-8 and cells treated with PA to determine whether PA regulates expression of HOX genes in colorectal carcinoma cell line HCT-8. Primer sequences for amplification of 39 HOX genes and β-actin were designed with the use of Primer Express 1.5 (Applied Biosystems). Each gene sequence was obtained from GenBank. Using the RT-PCR technique, we observed that 30 out of the 39 HOX genes are expressed in IEC cells, similar to the levels of expression (31/39) in either HCT-8 cells or the PA-treated HCT-8 cells. HOX A3 was not expressed in HCT-8 cell line. HOXA4 was not expressed in IEC. HOXC10 was not expressed in IEC and HCT-8 treated with PA. Using the semi-quantitative RT-PCR in combination with qualitative analysis, we chose the HOX genes, the levels of mRNA expression of which have significant differences in IEC versus HCT-8 cells and PA-treated versus untreated cells, as the target genes in colorectal carcinoma. In contrast, HOXA1, A2, B1, B2, B4, D1, and D12 HOX genes were silent in all materials examined. In contrast, the HCT-8 cell line demonstrated a low level of HOXA3, A5, A7, A11, B9, B13, C4, C6, D9 and D11 as well as a high level of HOXA4, B3, B8, C8, C9, C10, D4 and D13 genes expression, a pattern typical to the IEC.Our results showed that HOXB8, C8, C10, D4 and D13 genes mRNA expression were down-regulated after HCT-8 cells treated with PA as well as HOXA3, A5, A7, A11, B13, C4, C6, D9 and D11 expression were up-regulated. Our data suggest that the deregulated expression of HOXA3, A4, A5, A7, A11, B3, B8, B9, B13, C4, C6, C8, C9, C10, D4, D9, D11 and D13 genes might be involved in colorectal carcinoma development. The punctual difference in HOX pattern expression between normal human colon IEC and HCT-8 cells appeals for further investigations in the understanding of HOX genes involvement in colorectal carcinoma cells transformation. Taken together, these studies have confirmed that the mechanism of PA action is its effect on the transcription process of HCT-8 cells as well as G1 cell cycle arrest plays a key role in reversing the characteristic cellular dedifferentiation of tumor cells and inhibiting their growth. HOX genes in response to differentiation-inducing conditions correlate with anti-proliferative and differentiation-inducing effects in colorectal carcinoma cells. The creativity of the research is: PA can regulate the mRNA expression of HOX genes during PA-mediated cell growth arrest and celldifferentiation induction in colorectal carcinoma cells; we assayed the expression profiles of 39 HOX genes in IEC and colorectal carcinoma cell line HCT-8 treated by PA or not; application of HOX target-based screens might preliminarily identify some HOX genes which have an oncogenic or a tumour suppressor gene potential in colorectal carcinoma. Since 1997, reports about the effects of HOX genes on colorectal carcinoma genesis and progress abroad have been occurred. These studies have confirmed HOXB6, B8, C8 and C9 were up-regulated in colorectal carcinoma cell lines. HOXB13 could down-regulate the expression of TCF-4 and its responsive genes, C-myc and cyclin D1. These HOX genes may represent a new nuclear effector gene network, responding in the nucleus to the Ras and Src common downstream signal transduction pathways. The new finding of this research is that PA has a regulative effect on the mRNA expression of HOX genes. The new opinion of the research is that PA mainly acts at both the transcriptional and post transcriptional levels in colorectal carcinoma cells. Proved by Medline retrieve, the result that PA regulates the mRNA expression of HOX genes is a creative study at home and abroad. The whole domestic reports about the anti-tumor effect and mechanism of PA...
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