| Background and objectivesColorectal cancer (CRC) is one of the most common malignant tumors in the world. Based on the estimates from American Cancer Society in 2011, about 141,120 new cancer cases and 49,380 deaths from colorectal cancer are projected to occur in the United States in 2011. Colorectal cancer is the third leading cause of cancer death and the most frequent diagnosed cancer in Digestive System Neoplasms in the Western world. Meanwhile, the prevalence of colorectal carcinoma has increased year by year in China in the past several years. Until now, the colorectal cancer incidence rates has been ranked the fourth, and mortality rate has been ranked the fifth in China. It is even higher in large cities such as Beijing and Shanghai. Among the various approaches of clinical treatment including surgery, radiotherapy and chemotherapy, neoadjuvant chemotherapy, molecular targeted therapy, none of them could give an satisfactory treatment result with low 5 years survival rate due to the fact that most Colorectal cancer patients are in the terminal stages of cancer when they receive treatments. CRC is known as a disease with multiple phases in which a series of pathophysiological response and aberrant signal transduction pathways are dominantly involved. Hence, molecular targeted therapy has become one of the hot spots in aspect of anti-carcinogenesis and anticancer activity.Human APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper motifl) gene, also known as DIP 13a (DCC-interacting protein 13a), is an intracellular adaptor protein which was initially identified as an Akt2-binding protein in a yeast-two hybrid screen by Yasuhiro Mitsuuchi in 1999. APPL1 contains three multifunctional domains, including BAR, PH and PTB. It associates with a diverse set of membrane receptors and signaling proteins and acts as a signaling adaptor mediating several different signaling ways which are related to cell survival, growth, proliferation, apoptosis and differentiation. Recently, a few study have showed that APPL1 expresses widely in various kind of cancer cells including colon cancer, prostate cancer, ovarian cancer, and hepatoma. Whether APPL1 is tumor suppressor genes or cancer-promoting genes and what kinds of mechanisms are mediated by APPL1 in these carcinomas remains unclear. In this study, we preliminarily confirmed that APPL1 was expressed in human colorectal cancer tissues and investigated the relationships between the expression of APPL1 and clinicopathological parameters of colorectal cancer, and discussed the role and possible mechanisms of APPL1 during the development and progression in colorectal carcinoma.Materials and Methods1 Materials:35 cases of colorectal cancer specimens were collected from the First Affiliated Hospital of Zhengzhou University between March 2011 and December 2011 by surgical resection including 25 men and 10 women who aged 39 to 82 (average 57.8±11.9 years) and pathologically indentified. The colorectal cancer patients were composed of 27 tubular adenocarcinoma,3 papillary adenocarcinoma,3 mucinous adenocarcinoma and 2 signet-ring cell adenocarcinoma. With regard to the differentiation grades,4 were graded as high,23 were graded as middle and 8 as low. According to staging system referring to UICC TNM Classification criterion, version sixth (2002),8 patients were staged as I,22 patients were staged as II,5 patients were staged asⅢand 0 patients were staged asⅣ. In addition,27 cases of normal mucosa tissue were collected from hemorrhoid patients. All of the specimens were divided into two parts. One part of specimens were fixed in 10% formaldehyde, paraffin embedded,4 u m serial section for SP immunohistochemistry and the others were conserved in liquid nitrogen using for RT-PCR after surgeries within 30 minutes. ALL patients did not undergo the radiotherapy, chemotherapy and targeted therapy for cancer preoperative before surgery.2 Main reagent:polyclonal rabbit antihuman APPL1 antibody (BBI company), S-P agent box (Invitrogen company), DAB color kit(Beijing Zhongshan Golden Bridge Biotechnology company), Total RNA purification kit (BBI company), RevertAid-TM First Stand cDNA Synthesis Kit and DreamTaq GreenPCR Master MIX (Fermentas company).3 Primer design:To get APPL1 protein, two primers were designed as follows: forward primer base sequence 5’-CATCCAGAAAGAAACAACACCA-3’ and reverse primer base sequence 5’-CATTAAGGTATCCAGCCTTTCG-3’ Meanwhile, to obtain human p-actin protein, another two primers were synthesized including forward primer base sequence 5’-TGACGTGGACATCCGCAAAG-3’ and reverse primer base sequence 5’-CTGGAAGGTGGACAGCGAGG-3’. The primers above were designed by Sangon Biotech company.4 Methods4.1 Immunohistochemistry APPL1 was performed on cases using APPL1 antibody at 1:100 ratio and PBS was used as a negative control.4.2 RT-PCR (reverse transcription-polymerase chain reaction) First of all, the total RNA was extracted from specimens in one-step pillar extraction with RNA purification kit. Second, do the reverse transcription reaction and polymerase chain reaction according to kit instruction.5 Statistical analysis All statistical analyses were performed using the statistical package SPSS version 17.0. Wilcoxon Mann-Whitney test was adopt to rank data. APPL1mRNA expression levels of colorectal cancer were compared with those of normal mucosa using Independent Sample T-test. The data which no correspondence of homogenetiy of variance, was evaluated using Kruskal-Wallis test. Results were considerable statistically significant at the level ofα=0.05.Results1 Immunohistochemistry analysis showed that APPL1 was wildly expressed in all colorectal carcinoma tissues and normal mucose tissues and mainly located in cytoplasm, which was pale-yellow, tan-yellow, brown grain. The positive rate of the expression of APPL1 including positive and strong positve rate was 88.7%(30/35) in colorectal carcinoma tissues and 51.8%(14/27) in normal mucosa tissues, and the difference between two groups is statistically significant.(P<0.05) 2 The relationships between the expression of APPL1 and clinicopathological features were examined. The expression levels of APPL1 proteins in colorectal carcinoma tissues were correlated with tumor differentiation degree, lymph node metastasis and TNM stage (all P<0.05), but no significance relation with gender, age, or tumor size (all,P>0.05).3 RT-PCR analysis showed that APPL1mRNA expressed in all resected specimens, and its levels were significantly higher in tumor tissues than that in normal mucosa (P<0.05).ConclusionsThe expression levels of APPL1 protein is increased in colorectal neoplasm and is closely associated with tumor differentiation degrees, lymph node metastasis and TNM stage. In a word, APPL1 may be a potential therapeutic target for CRC. |
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