NQO1 C609T polymorphism correlated to colon cancer risk in farmers from western region of Inner Mongolia

Introduction

Quinone is a noxious chemical compound, existing generally through nature, in exhaust of automobile, benzene, tobacco smoke, which is involved in development of tumor, mutation and necrosis in mammalian cells. NAD(P)H: quinone oxidoreductase 1 (NQO1) catalyzes two-electron reduction of quinones and nitrogen oxides (1) and turns quinones to hydroquinones directly. Most hydroquinones are more stable and can be metabolized within cells. Therefore, NQO1 can prevent organs from carcinogen-induced tumorigenesis. Previous studies (2,3) showed that NQO1 has protected against carcinogenesis and mutation induced by quinone and its derivants. Studies (4-6) also found that the expression of NQO1 is elevated in many tumor cells and human cancer tissues, suggesting that NQO1 might be the target of drugs for cancer therapy. Polymorphism of NQO1 gene occurs at the site of 609 with cytosine changed into thymine. Although it would not affect synthesis of RNA, the protein product of NQO1 T609 is ubiquitinated and subsequently degraded, which may lead to null NQO1 (7). Our previous study (8) showed that the incidence of NQO1 C609T in gastric cancer patients is higher than that in controls. Individuals with NQO1 (T/C) and NQO1 (T/T) genotypes have higher onset risk with odds ratios (OR) of 2.080 and 3.813 respectively.

Colon cancer is one of the most common cancers, and it is the third cause of cancer related death. Various factors, multiple genes and pathways are involved in colon carcinogenesis and progress. K-ras, adenomatous polyposis coli (APC), tumor protein P53 (TP53) and mismatch repair (MMR) genes (9-12), chromosomal instability, microsatellite instability (13,14) and wnt-hedgehog pathway (15,16) are reported to be involved in initiation and progress of colon cancer. Diet factors and carcinogens play roles in influencing onset risk of colon cancer (17,18). The molecular mechanism of colon cancer has been better understood, however, the prognosis of colon cancer has not been improved too much. Therefore we carried out a case-control study in farmers from western region of Inner Mongolia to examine the association between NQO1 C609T polymorphism and risk of developing colon cancer. Farmers from local region have their unique eating habits. They consume lots of pickled meat and vegetables which contain high levels of salt and nitrite. They are used to have the animal oils. The diet mostly consists of grain and less fresh fruits and vegetables. Most of them are alcohol users. As a result of specific diet of farmers, we study the NQO1 polymorphism and susceptibility to colon cancer, which may also improve the treatment of local colon cancer patients.

Materials and methods

Subjects

All 76 colon cancer patients were hospitalized cases from the Affiliated Hospital of Inner Mongolia Medical College from 2007-2008, which were all diagnosed by histopathological examination, and did not undergo anti-cancer treatment. Of the 76 patients, 46 were male and 30 were female, with a mean age of 60.13±10.96 years (range, 39-79 years). One hundred and sixty healthy individuals who matched the age and sex of the colon cancer patients were selected by routine physical examination. The healthy controls did not have either tumors, tumor-related diseases detected by physical examination, or history of familial colon cancer. All individuals involved in the study gave informed consent.

DNA extraction and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

DNA was extracted from 1 ml venous blood of the investigated objects, and then PCR was performed with the primers of 5'-AAGCCCAGACCAACTTCA-3' and 5'-GCGTTTCTTCCATCCTT-3' and under following conditions: initial denaturation at 94 ℃ for 5 min, followed by 33 circles of denaturation at 94 ℃ for 1min, annealing at 54 ℃ for 45 s and extension at 72 ℃ for 45 s, with a final extension of 10 min at 72 ℃. The PCR products were digested with restriction enzyme Hinf I. The full size of PCR product was 195 bp. There was no enzyme site of HinfI in PCR product of wild-type NQO1 (C/C). If NQO1 C609→T base substitution occurred, the PCR product was digested into two fragments (119 bp and 76 bp). There were 2 fragments in NQO1 (T/T) and 3 fragments in NQO1 (T/C).

Statistical analysis

The frequency of NQO1 (C/C), NQO1 (T/C) and NQO1 (T/T) was compared in control group vs. colon cancer patients and according to clinical characteristics by Chi-square test. OR [95% confidence interval (95% CI)] was calculated. All the statistical analyses were performed by SPSS 11.1 statistic software (SPSS Inc., Chicago, IL, USA).

Results

We investigated the incidence of NQO1 T609 allele in colon cancer patients and control group. We found that the incidence of NQO1 T609 allele in colon cancer patients was 53% which was significantly higher than it in control group (33.75%). The individuals with NQO1 T609 allele had higher risk [2.239 (95% CI: 1.510-3.321) times] to develop colon cancer compared to individuals with NQO1 C609 allele.

Distribution of NQO1 C609T polymorphism was different between colon cancer patients and control group. In the control group, the frequency of NQO1 (C/C), NQO1 (T/C) and NQO1 (T/T) was 48.13%, 36.25% and 15.62%, respectively. While in colon cancer patients, the frequency of NQO1 (C/C), NQO1 (T/C) and NQO1 (T/T) was 27.63%, 38.16%, and 34.21% (Table 1), respectively. There was a significantly increased incidence of NQO1 (T/T) in colon cancer patients compared with control group, (χ2=13.566, P<0.001), while the incidence of NQO1 (T/C) was not increased in colon cancer patients compared with control group. OR analysis showed that individuals with NQO1 (T/T) or NQO1 (T/C) genotype had higher risk [3.813 (95% CI: 1.836-7.920) time and 2.080 (95% CI: 1.026-4.219) time respectively] to develop colon cancer than individuals with wild type NQO1 (C/C) (Table 1).

Table 1 Association between NQO1 C609T polymorphism and colon cancer onset risk
Full table

We investigated the distribution of NQO1 C609T polymorphism in the differentiation of colon cancer. The frequency of NQO1 (T/T) was found that it is increased in colon cancer patients with the increased malignance (32.00% in well-differentiated colon cancer patients, 40.00% in moderately-differentiated colon cancer patients and 42.00% in poorly-differentiated colon cancer patients). In well-differentiated colon cancer patients, NQO1 (T/T) gene carriers had 2.541 times (95% CI: 0.990-6.522 times) higher chance in developing colon cancer than NQO1 (T/C) and NQO1 (C/C) gene carriers, which was more than the risk in control group (χ2=3.956, P<0.05). As for moderately-differentiated colon cancer patients, individuals with NQO1 (T/T) genotype had a higher onset risk than individuals with NQO1 (T/C) or NQO1 (C/C) genotype with OR of 3.713 (95% CI: 1.542-8.935), which was more risk than the control group (χ2=9.374, P<0.01). In poorly-differentiated colon cancer patients, NQO1 (T/T) gene carriers had much higher onset risk than NQO1 (C/C) and NQO1 (T/C) gene carriers with OR of 3.471 (95% CI: 1.356-8.886), which was significantly more risk than the control group (χ2=7.345, P<0.01) (Table 2). With the decreasing differentiation of colon cancer, the patients with NQO1 (T/T) genotype were increased, indicating that NQO1 C609T polymorphism may be involved in the progress of colon cancer.

Table 2 Association between NQO1 C609T polymorphism and differentiation of colon cancer
Full table

Discussion

Studies about the association between NQO1 C609T polymorphism and cancers have been carried out. The results are controversial. Some studies showed that NQO1 C609T polymorphism is correlated with several types of cancers, including esophageal (19,20), gastric (19,21), renal (22), breast (23), cervical (24), bladder(25) and urothelial (22-26) cancers. In contrast, there is no correlation between NQO1 C609T polymorphism and esophageal (27), gastric (27), renal (28), breast (27,29), hepatocellular (30), head and neck (31) cancers. NQO1 *2/*2 is associated with poor survival in non-small cell lung cancer patients (32). These results indicated that the inactive NQO1 may affect the risk of developing cancers depending on the type of cancer, ethnic group, diet factors and carcinogen exposure.

Previous studies (2,3) showed that NQO1 could inhibit colon carcinogenesis at both initiation and post initiation stages in carcinogens induced Sprague-Dawley rat colon cancer model. There was an increase frequency of NQO1*2/*2 genotype in colon cancer patients compared with control group. However, the frequency of NQO1*1/*2 did not show increasing in colon cancer patients compared with control group. As far as tobacco and alcohol use concerned, the incidence of NQO1*2/*2 genotype was almost same in tobacco and alcohol users and nonusers, indicating that the consumption of tobacco and alcohol was not associated with NQO1 C609T polymorphism. Using colon cancer patients and age, gender matched healthy individuals from local region, our study found that NQO1 C609T polymorphism was correlated with colon cancer onset risk. Our result was consistent with other studies (33-35).

To further elucidate the association between NQO1 C609T polymorphism and colon cancer, we investigated the correlation between NQO1 C609T polymorphism and the differentiation of colon cancer. We found that the frequency of NQO1 (T/T) was increased in colon cancer patients with the increased malignance, and the patients with NQO1 (T/T) had higher onset risk to develop colon cancer in well-differentiated, moderately-differentiated and poorly-differentiated colon cancer, which suggested that NQO1 may play an role in prevention colon carcinogenesis and progress. The increasing trend of NQO1 (T/T) frequency did not show any significant difference between differentiation of colon cancer, which may due to the small numbers of colon cancer patients involved. Therefore, larger numbers of patients will be enrolled to demonstrate our result in the future study.

Hypoxia of tumor cells is one of the most significant reasons in failure of radiotherapy and chemotherapy. Drugs such as reductants have a specific cytotoxicity to hypoxia cells. This kind of chemicals forms the anionic clusters which can get electrons after reduction in cells. These anionic clusters have high reaction with oxygen. Under hypoxia, these anionic clusters have toxicity, or develop into metabolic products with toxicity. Lots of anticancer drugs containing quinone are used in clinical treatment such as “mitomycin” (36). Our study finds that the control group has 15.62% NQO1 (T/T) genotype, while colon cancer patients have 34.21% NQO1 (T/T) genotype. Our studies indicated that over one third of patients have no NQO1 activity. It is necessary to identify the right population of cancer patients to be treated with the drug containing quinone. Alternatively we also can treat this part of patients with other kinds of anticancer drugs. It is reported that “lochnerol” and dimethylfumarate (DFM) can induce the activity of enzymes like NQO1 (37,38), so we also can treat cancer patients with NQO1 (T/C) genotype with “lochnerol” and special dietary combined with other anticancer drugs to improve the effect of chemotherapy. Based on our data, identifying the polymorphism of NQO1 for cancer patients has an important role in choosing anticancer drugs for clinical treatment.

In conclusion, after studying 160 healthy controls and 76 colon cancer patients, we found that there was a significantly increased incidence of NQO1 T609 allele in patients with colon carcinoma compared with it in control group. NQO1 T609 allele carriers had higher risk than NQO1 C 609 allele carriers to develop colon carcinoma.

Acknowledgements

Our research is supported by the Autonomous region Natural Science Foundation of China, No. 200711020906.

Disclosure: The authors declare no conflict of interest.

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