Lack of association between ACE ID polymorphism and colorectal cancer in Romanian patients

Introduction
The renin-angiotensin-aldosteron system (RAAS) is an endocrine and paracrine system involved in cell proliferation and apoptosis, immune response, extracellular matrix formation, hemodynamics and angiogenesis. It seems also to be involved in the development or progression of some malignancies (1,2) including gastric (3), colorectal (4,5), breast (6), prostate (7) and endometrial (8) cancers. Several clinical trials and experiments on animal models showed that pharmacological inhibitors of this system have the potential to significantly reduce the cancer risk and the rate of tumor growth and metastases (9,10,11).
The angiotensin I-converting enzyme (ACE), which is a cell surface zinc metallopeptidase plays an important role in the RAAS. The ACE is the rate-limiting enzyme in the conversion of angiotensin I to the powerful vasoconstrictor angiotensin II and also inactivates the bradykinin, substance P and enkephalins. The inter-individual variability of plasma circulating level of ACE was correlated with the insertion/deletion (ACE ID) of a 287-bp Alu repeat in intron 16 of the gene. The individuals who are homozygous for the insertion (II) have lower plasma level than those homozygous for deletion (DD), while ID carriers present an intermediate level (12).
The goal of this study was to analyze the possible association between ACE gene I/D polymorphism and colorectal cancer in a group of Romanian patients.

Materials and methods
Subjects
Between January 2007 and June 2008, blood samples from 108 patients (M:F=64:44) diagnosed with CRC were collected at Cantacuzino Hospital, Bucharest, Romania. Healthy individuals (M:W=84:66) without known family history of malignancies and were selected from healthy persons who attended N. Paulescu Institute (Bucharest) for routine analysis. They were matched for age (63.3±4.5 in cases and 62.3±3.8 in control group) and sex (men vs. women was 64:44 in cases and 84:66 in control). We obtained medical information regarding cancer type, tumor location and clinical evolution for each patient. The CRC was localized in the left (60.9%) or right (15.3%) colon and in rectum (23.8%). This study was approved by the Research Ethics Committee of N. Paulescu Institute. Five ml of blood were collected in a tube containing EDTA after informed consent was obtained from all participants. DNA was extracted from peripheral blood leukocytes using the commercial extraction kit (Wizard Genomic DNA Purification Kit, Promega) according to the manufacturer’s protocol.
Genotyping
The ACE ID gene polymorphism was detected by PCR as described elsewhere (13). Briefly, about 60 ng DNA were amplified in a final volume of 10 mL, containing 1×PCR buffer, 1.5 mmol/L MgCl2, 1 unit Taq DNA polymerase, 100 mmol/L dNTP, and 0.5 mmol/L of each primer (F 5- CTG GAG ACC ACT CCC ATC CTT TCT -3’ and R 5’- GAT GTG GCC ATC ACA TTC GTC AGA T -3’). PCR program consisted in an initial melting step of 2 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C; and a final elongation step of 2 min at 72°C (Corbett research thermocycler). Amplicons were electrophoresed on agarose gel (2%, TBE 1X, 5V / cm). The gel was visualized using Bio-Imaging System after ethidium bromide staining (Fig. 1).
Statistical analysis. Prevalence of alleles and genotypes among cases and control subjects were counted and compared with Hardy–Weinberg equilibrium. Chi-square test (c2) was used to compare the distribution of genotypes and alleles in patients and control groups. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the relevant 2 × 2 contingency table 1. A p value < 0.05 was considered statistically significant. Statistical analysis was performed using SISA programs (14).

Results and discussion
The frequencies of genotypes and alleles of ACE ID in patients with CRC and controls are shown in Table 1. The polymorphism was distributed in accordance to Hardy-Weinberg equilibrium expectation in cancer (c2 = 0.513, p = 0.47) and control (c2 = 0.029, p = 0.86) groups. No statistically significant differences in the distribution of polymorphism between patients and controls have been identified. We observed no differences in distribution of ACE ID genotypes related to gender of patients (II genotype was 20.3% in men and 27.2% in women).
The distribution of the I and D alleles in the control lot is similar to that reported for other European populations (15) or to that referred to another sample of Romanian population (16).
Only two articles referring to the significance of ACE ID polymorphism in CRC biology were found in the documentation process for our paper. After investigation of 92 patients with CRC and 102 healthy controls authors reported that ACE I/D polymorphism is not a risk factor for disease (4). This result is in accordance with the data provided by our study. In the second article, ACE ID polymorphism was tested in 141 CRC patients and 189 healthy controls (5). It has been observed that gender influence the relation between ACE polymorphism and primary tumor size and patient survival. We observed no differences in distribution of ACE ID genotypes related to gender of patients. This difference may be determined by gender distribution in control lots (40% men in their lot and 56% in this study) or by the genetic background of the investigated populations.

Conclusions
This study shows that the ACE ID polymorphism does not confer a risk for CRC and the frequency of I vs. D alleles in our control lot is similar with values reported for other populations.

Acknowledgements
This study was supported by Grant CNCSIS TD 224/2008.

References
1. DESHAYES, F., NAHMIAS, C. - Angiotensin receptors: a new role in cancer? Trends. Endocrinol. Metab., 2005, 16:293.
2. AGER, E.I., NEO, J., CHRISTOPHI, C. - The renin-angiotensin system and malignancy. Carcinogenesis, 2008, 29:1675.
3. EBERT, M.P., LENDECKEL, U., WESTPHAL, S., DIERKES, J., GLAS, J., FOLWACZNY, C., ROESSNER, A., STOLTE, M., MALFERTHEINER, P., RÖCKEN, C. - The angiotensin I-converting enzyme gene insertion/deletion polymorphism is linked to early gastric cancer. Cancer Epidemiol Biomarkers Prev., 2005, 14:2987.
4. NIKITEAS, N., TSIGRIS, C., CHATZITHEOFYLAKTOU, A., YANNOPOULOS, A. - No association with risk for colorectal cancer of the insertion/deletion polymorphism which affects levels of angiotensin-converting enzyme. In Vivo, 2007, 21:1065.
5. RÖCKEN, C., NEUMANN, K., CARL-MCGRATH, S., LAGE, H., EBERT, M.P., DIERKES, J., JACOBI, C.A., KALMUK, S., NEUHAUS, P., NEUMANN, U. - The gene polymorphism of the angiotensin I-converting enzyme correlates with tumor size and patient survival in colorectal cancer patients. Neoplasia, 2007, 9:716.
6. HERR, D., RODEWALD, M., FRASER, H.M., HACK, G., KONRAD, R., KREIENBERG, R., WULFF, C. - Potential role of Renin-Angiotensin-system for tumor angiogenesis in receptor negative breast cancer. Gynecol. Oncol., 2008, 109:418.
7. YIGIT, B., BOZKURT, N., NARTER, F., YILMAZ, H., YUCEBAS, E., ISBIR, T. - Effects of ACE I/D polymorphism on prostate cancer risk, tumor grade and metastatis. Anticancer Res., 2007, 27:933.
8. FREITAS-SILVA, M., PEREIRA, D., COELHO, C., BICHO, M., LOPES, C., MEDEIROS, R. - Angiotensin I-converting enzyme gene insertion/deletion polymorphism and endometrial human cancer in normotensive and hypertensive women. Cancer Genet. Cytogenet., 2004, 155:42.
9. VAN DER KNAAP, R., SIEMES, C., COEBERGH, J.W., VAN DUIJN, C.M., HOFMAN, A., STRICKER, B.H. - Renin-angiotensin system inhibitors, angiotensin I-converting enzyme gene insertion/deletion polymorphism, and cancer: the Rotterdam Study. Cancer, 2008, 112:748.
10. KHURANA, V., BEJJANKI, H.R., CALDITO, G. - Angiotensin converting enzyme inhibitors reduce the incidence of colon cancer: a study of half a million US veterans. Program and abstracts from the Digestive Disease Week, May 20-25, 2006, Los Angeles, California. Abstract S1222.
11. NEO, J.H., MALCONTENTI-WILSON, C., MURALIDHARAN, V., CHRISTOPHI, C. - Effect of ACE inhibitors and angiotensin II receptor antagonists in a mouse model of colorectal cancer liver metastases. J. Gastroenterol. Hepatol., 2007, 22:577.
12. RIGAT, B., HUBERT, C., ALHENC-GELAS, F., CAMBIEN, F., CORVOL, P., SOUBRIER, F. - An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J. Clin. Invest., 1990, 86:1343.
13. RIGAT, B., HUBERT, C., CORVO, P., SOUBRIER, F. - PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res., 1992, 20:1433.
14. UITENBROEK, D.G. - Binomial. SISA. 1997 (http://www. quantitativeskills.com/sisa/distributions/binomial.htm). (Accessed 15.03.2009).
15. ROMUALDI, C., BALDING, D., NASIDZE, I.S., RISCH, G., ROBICHAUX, M., SHERRY, S.T., STONEKING, M., BATZER, M.A., BARBUJANI, G. - Patterns of human diversity, within and among continents, inferred from biallelic DNA polymorphisms. Genome Res., 2002, 12:602.
16. COMAS, D., SCHMID, H., BRAEUER, S., FLAIZ, C., BUSQUETS, A., CALAFELL, F., BERTRANPETIT, J., SCHEIL, H.G., HUCKENBECK, W., EFREMOVSKA, L., SCHMIDT, H. - Alu insertion polymorphisms in the Balkans and the origins of the Aromuns. Ann. Hum. Genet., 2004, 68:120.

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