Risk Factors for Colon Cancer in Northeastern Thailand: Interaction of MTHFR Codon 677 and 1298 Genotypes with Environmental Factors

Background

Polymorphisms in methylenetetrahydrofolate reductase (MTHFR), such as MTHFR C677T and A1298C, are associated with several cancers. This study aimed to evaluate the effects of MTHFR polymorphisms on colon cancer risk and possible interactions with environmental factors in a population from northeastern Thailand.

Methods

This hospital-based case–control study was conducted during 2002–2006; 130 colon cancer cases and 130 age- and sex-matched controls were enrolled. Information was collected and blood samples were obtained for assay of MTHFR C677T and A1298C polymorphisms by polymerase chain reaction with restriction fragment length polymorphism techniques. Associations between variables of interest and colon cancer were assessed using conditional logistic regression.

Results

Increased risk of colon cancer was associated with alcohol consumption and bowel habits. Alcohol drinkers who consumed ≤0.50 or >0.50 units of alcohol per day had elevated risks (OR_adj = 3.5; 95% CI: 1.19–10.25 and OR_adj = 1.71; 95% CI: 0.74–3.96, respectively). The risk was also higher in subjects with frequent constipation (11.69; 2.18–62.79) and occasional constipation (3.43; 1.72–6.82). An interaction was observed between the MTHFR C677T polymorphism and freshwater fish consumption on colon cancer risk (P value for interaction = 0.031). Interactions were observed between the MTHFR A1298C polymorphism and bowel habits, family history of cancer, alcohol consumption, and beef consumption on colon cancer risk (P-value for interaction = 0.0005, 0.007, 0.067, 0.003, respectively).

Conclusions

In a Thai population, colon cancer risk was associated with alcohol and beef consumption, bowel habits, and family history of cancer. Interactions between MTHFR polymorphisms and environmental factors were also observed.Key words: colon cancer, MTHFR, polymorphism, environmental factors, Thailand     

Dietary silicon and bone health

Summary  Silicon (Si) is the third most abundant trace element of the human body and is especially associated with connective tissues such as in bone, skin and blood vessels. Dietary Si is predominately derived from plant-foods and, generally, is readily broken down and absorbed in the gastrointestinal tract in the form of orthosilicic acid. Cereals and cereal products (especially beer), green beans and some mineral water are examples of major dietary sources of Si. The majority of absorbed Si is then excreted in urine although some is taken up into tissues. Average Si intakes are around 25 mg/day in the Western World. In this review, dietary sources of Si, its metabolism and evidence for its biological role in bone health are presented.     

Evaluation of health education in the Multi-professional Intervention and Training for Ongoing Volunteer-based Community Health Programme in the north-east of Thailand

This was a survey research conducted in Northestern Thailand during 2009-2010 and designed to evaluate the success of a health education program by comparing levels of health knowledge in the community before and after the launching of a Multi-professional Intervention and Training for Ongoing Volunteer-based Community Health Programme. The survey questionnaire included items about demographic characteristics and health knowledge. The participants were 1,015 members of various communities, who were randomly selected to be included in the survey before launching the intervention, and 1,030 members of the same communities randomly selected to be included in the survey after the intervention was completed. The demographic characteristics of both groups were similar. Overall knowledge and knowledge of all the diseases, except lung and cervical cancer, were significantly higher after the intervention. In conclusion, a Volunteer-based Community Health Programme has advantages for areas where the numbers of health personnel are limited. The use of trained community health volunteers may be one of the best sustainable alternative means for the transfer of health knowledge.     

Preclinical Evaluation of the Antifolate QN254, 5-Chloro- N′6′-(2,5-Dimethoxy-Benzyl)-Quinazoline-2,4,6-Triamine,as an Antimalarial Drug Candidate

Drug resistance against dihydrofolate reductase (DHFR) inhibitors—such as pyrimethamine (PM)—has now spread to almost all regions where malaria is endemic, rendering antifolate-based malaria treatments highly ineffective. We have previously shown that the di-amino quinazoline QN254 [5-chloro-N′6′-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine] is active against the highly PM-resistant Plasmodium falciparum V1S strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence of antifolate resistance. Here, we further demonstrate that QN254 is highly active against Plasmodium falciparum clinical isolates, displaying various levels of antifolate drug resistance, and we provide biochemical and structural evidence that QN254 binds and inhibits the function of both the wild-type and the quadruple-mutant (V1S) forms of the DHFR enzyme. In addition, we have assessed QN254 oral bioavailability, efficacy, and safety in vivo. The compound displays favorable pharmacokinetic properties after oral administration in rodents. The drug was remarkably efficacious against Plasmodium berghei and could fully cure infected mice with three daily oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered some dose limiting toxicity at higher doses that was confirmed in rats. Thus, despite its relative in vitro selectivity toward the Plasmodium DHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a single agent.Malaria control is a global public health priority that has been hampered by the rapid development and spread of resistance against antimalarials. As a consequence, the World Health Organization (WHO) recommends the use of artemisinin-containing combination therapies (ACTs) as a first-line treatment for malaria. Although ACTs are designed to reduce the chance of artemisinin drug resistance development, there are considerable concerns that this may already have occurred. For instance, there is now mounting evidence that the efficacy of artemisinin derivatives is reduced in Southeast Asia, where artemisinin derivatives have been used for a long time as monotherapies (7, 28, 53). This is a cause of concern since the spread of artemisinin resistance will compromise the usefulness of ACTs globally. Thus, there is an urgent need to discover and develop new alternative drugs.For several decades, dihydrofolate reductase (DHFR) has been targeted with different classes of chemical entities for the development of new therapies for a broad range of therapeutic indications, including several parasitic diseases (13). DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which is an essential cofactor in the biosynthesis of deoxythymidylate monophosphate (dTMP), a metabolite essential to DNA synthesis and cell replication.Pyrimethamine (PM) is a potent inhibitor of the Plasmodium DHFR enzyme, and this compound has been widely used in combination with the dihydropteroate synthetase (DHPS) inhibitor sulfadoxine. Unfortunately, PM resistance is now common, rendering this drug ineffective (30). One of the documented mechanisms of antifolate resistance is through the mutation of the target itself—the Plasmodium falciparum dhfr-ts (Pfdhfr-ts) gene, encoding for a bifunctional enzyme which possesses both DHFR and thymidylate synthase (TS) activities carried by two distinct subdomains (29). PM resistance is associated with the mutation of the amino acid Ser to Asn at codon 108 of DHFR (S108N). Ancillary mutations of N51I and C59R are associated with an increase in resistance, and the presence of the mutation I164L results in an even higher level of PM resistance (14, 47). The presence of these mutations significantly decreases the sensitivity of the PfDHFR enzyme to PM inhibition in a biochemical assay (48).The antifolate triazine WR99210 (3, 22) is potent against P. falciparum bearing quadruple mutations of DHFR at S108N, N51I, C59R, and I164L (QM PfDHFR) (20). However, WR99210 has shown limited efficacy in vivo due to poor oral bioavailability and displayed some gastrointestinal toxicity. Attempts have been made to circumvent these issues and a prodrug form of WR99210 known as PS-15 has been shown to be orally active (2). The resolution of the three-dimensional structure of wild-type (WT) and QM PfDHFR—with either PM or WR99210 bound to its active site—provided structural insights into DHFR PM resistance mechanisms, as well as some understanding of the structural features of WR99210 that allow this compound to retain affinity for QM PfDHFR (55, 56).The quinazoline pharmacophore has been successfully used to design drugs for the treatment of cancer and other human diseases. For example, the DHFR inhibitor, trimetrexate (5-methyl-6-[(3,4,5-trimethoxy-phenylamino)-methyl]-quinazoline-2,4-diamine) has been developed to treat various cancers in human patients (15, 32, 33). In a previous study, a series of quinazoline derivatives was tested against the highly PM-resistant P. falciparum strain (V1S) and the DHFR inhibitor 2,4-diamino-5-chloro-6-[N-(2,5-dimethoxybenzyl)-amino]quinazoline (or QN254 here and compound 1 in reference 34) was found to have potent activity—with an IC₅₀ (i.e., the inhibitory concentration that reduces parasite growth by 50% in vitro) of 9 nM (34). Considering the increasingly widespread PM drug resistance, we set out to perform the experiments described here with the goal of further assessing the potential of QN254 as a candidate antimalarial to replace the failing PM. Collectively, our data demonstrate that QN254 (i) binds and inhibits the QM PfDHFR enzyme, (ii) is active on drug-resistant clinical isolates, and (iii) displays pharmacological properties compatible with an oral antimalarial drug candidate. However, preliminary toxicological findings indicate that QN254 does not show a therapeutic window sufficiently large to warrant its progression to the next development stage.     

Usefulness of combining testing for p16 protein and human papillomavirus (HPV) in cervical carcinoma screening

OBJECTIVE: To evaluate the value of the combination of p16 and HPV detection in the screening for cervical cancer. METHODS: 186 patients with previous abnormal cervical lesion were studied. After colposcopic examination, two conventional Pap slides were prepared: the first was Papanicolaou-stained and examined by cytologist; the second was immunocytochemically stained for p16. Cervical cells were collected by brush using for HPV detection by Hybrid Capture II. Biopsy of any colposcopically abnormal lesions was performed. RESULTS: The 186 cervical samples were classified cytologically as normal (148), ASCUS (13), low-grade (11), high-grade (12) dysplasia and squamous cell carcinoma (2). P16 and HPV were found in all high-grade dysplasia and SCC, and in 64% and 27% of low-grade dysplasia, 62% and 0% of ASCUS and 7.4% and 3.4% of normal, respectively. 18 of p16-positive cases (11%) were HPV-negative, 14 of them in the ASCUS and normal group. Compared to histological results, all of the p16-positive cases of squamous metaplasia, CIN II/III and SCC were HR-HPV-positive. Therefore, the cases that were positive for both with normal cytology (5 cases) or low-grade dysplasia (3 cases) may comprise a high-risk group for neoplastic change. CONCLUSION: The combination of p16 and HPV detection may be useful in cervical cancer screening to identify high-risk patients requiring early and proper management.     

Effect of human papillomavirus 16 oncoproteins on oncostatin M upregulation in oral squamous cell carcinoma

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.     

Behavioural risk factors for cervical cancer from a prospective study in Khon Kaen, Northeast Thailand

Fifty seven cases of cancer of the cervix were identified within a cohort of 16,648 women enrolled in a cohort study of lifestyle and cancer in a rural population of Northeast Thailand. They were compared with 228 matched controls from the same cohort, with respect to demographic, behavioural and reproductive factors potentially associated with risk of the disease. Number of pregnancies and age at having first child were associated with the risk of cervical cancer, but in contrast to studies elsewhere, age at having first sexual intercourse, number of sexual partners and sexually-transmitted infections were not associated with risk. The lack of effect may be simply due to the small size of the study. But it is also likely that these aspects of female sexual behaviour are not very relevant in a relatively conservative rural population. These aspects may be investigated further in future, as new incident cancers are accrued into the cohort.     

Combined spatial limitation around residues 16 and 108 of Plasmodium falciparum dihydrofolate reductase explains resistance to cycloguanil

Natural mutations of Plasmodium falciparum dihydrofolate reductase (PfDHFR) at A16V and S108T specifically confer resistance to cycloguanil (CYC) but not to pyrimethamine (PYR). In order to understand the nature of CYC resistance, the effects of various mutations at A16 on substrate and inhibitor binding were examined. Three series of mutations at A16 with or without the S108T/N mutation were generated. Only three mutants with small side chains at residue 16 (G, C, and S) were viable from bacterial complementation assay in the S108 series, whereas these three and an additional four mutants (T, V, M, and I) with slightly larger side chains were viable with simultaneous S108T mutation. Among these combinations, the A16V+S108T mutant was the most CYC resistant, and all of the S108T series ranged from being highly to moderately sensitive to PYR. In the S108N series, a strict requirement for alanine was observed at position 16. Crystal structure analyses reveal that in PfDHFR-TS variant T9/94 (A16V+S108T) complexed with CYC, the ligand has substantial steric conflicts with the side chains of both A16V and S108T, whereas in the complex with PYR, the ligand only showed mild conflict with S108T. CYC analogs designed to avoid such conflicts improved the binding affinity of the mutant enzymes. These results show that there is greater spatial limitation around the S108T/N residue when combined with the limitation imposed by A16V. The limitation of mutation of this series provides opportunities for drug design and development against antifolate-resistant malaria.     

Hormonal Contraceptive Use and Breast Cancer in Thai Women

Background

Breast cancer is the most common cancer in women worldwide. We investigated the association of hormonal contraceptive use and breast cancer in Thai women.

Methods

A cohort study was conducted in Khon Kaen, Thailand. There were 70 cases of histologically confirmed breast cancer among 11 414 women aged 30 to 69 years who were recruited as participants in the cohort study during the period from 1990 through 2001. The study population was followed-up until December 31, 2011. To identify factors associated with incidence of breast cancer, hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using a Cox proportional hazards model.

Results

The 11 414 women provided a total observation time of 157 200 person-years. Breast cancer risk among women with a history of hormonal contraceptive use was 1.31 times that of women without such a history, but the difference was not statistically significant (95% CI, 0.65–2.65). No type of hormonal contraceptive was associated with a significant increase in breast cancer risk as compared with women who had never used hormonal contraceptives (oral contraception: HR = 1.35, 95% CI, 0.65–2.78; injection contraception: HR = 1.25, 95% CI, 0.56–2.80), and there was no relationship between duration of hormonal contraceptive use and breast cancer.

Conclusions

There was no association between hormonal contraceptive use and breast cancer; however, this finding should be viewed with caution due to the small number of cases.Key words: hormonal contraceptive, breast cancer, Thai women