Low folate metabolic stress reprograms DNA methylation-activated sonic hedgehog signaling to mediate cancer stem cell-like signatures and invasive tumour stage-specific malignancy of human colorectal cancers.

The mechanistic role of colonic low folate metabolic stress (LFMS) in colorectal cancer (CRC) malignancy development remains unknown. Folate analysis on the 99 paired human CRC tissues localized LFMS to the deep invasive T3/T4 staged tumours with hypo-methylated sonic hedgehog (Shh) promoter region and amplified expressions of Shh ligand and Gli1 effector, which coincided with deregulated expressions of the epithelial-mesenchymal transition (EMT) mediators. Colonic folate levels of CRC were inversely correlated with pluripotent expressions of the SOX2, NANOG and OCT4 markers (p < 0.05). Exposure of human colon adenocarcinoma cells to LFMS microenvironment significantly hypomethylated Shh promoter region, activated Shh signaling, induced transcript and protein expressions of the pluripotent markers, promoted trans-differentiation as EMT by deregulation of Snail mediator and epithelial marker E-cadherin, increased MMP2/MMP9 enzymatic digestion on matrix protein for invasion, and promoted self-renewal capability of anchorage-independent tumor-spheroid formation. LFMS-induced cancer stem cell (CSC) signature and CRC invasion is synergized with inhibition of DNA methylation by 5-Aza-2-deoxycytidine (5AZA) in rewiring EMT genotypes, which can be blockade by the Shh inhibitor (cyclopamine). The in vivo and in vitro data corroboratively identify CSC-like molecular targets specific to the LFMS-predisposed invasive CRC through reprogramming DNA methylation-activated Shh signaling. The study highlights CSC targets specific to LFMS-predisposed invasive CRC in optimizing folate co-chemotherapy to minimize tumour metastasis potential of CRC patients.

Optimal Dietary Intake Composition of Choline and Betaine Is Associated with Minimized Visceral Obesity-Related Hepatic Steatosis in a Case-Control Study.

Few studies on humans have comprehensively evaluated the intake composition of methyl-donor nutrients (MDNs: choline, betaine, and folate) in relation to visceral obesity (VOB)-related hepatic steatosis (HS), the hallmark of non-alcoholic fatty liver diseases. In this case-control study, we recruited 105 patients with HS and 104 without HS (controls). HS was diagnosed through ultrasound examination. VOB was measured using a whole-body analyzer. MDN intake was assessed using a validated quantitative food frequency questionnaire. After adjustment for multiple HS risk factors, total choline intake was the most significant dietary determinant of HS in patients with VOB (Beta: -0.41, p = 0.01). Low intake of choline (<6.9 mg/kg body weight), betaine (<3.1 mg/kg body weight), and folate (<8.8 µg/kg body weight) predicted increased odds ratios (ORs) of VOB-related HS (choline: OR: 22, 95% confidence interval [CI]: 6.5-80; betaine: OR: 14, 95% CI: 4.4-50; and folate: OR: 19, 95% CI: 5.2-74). Combined high intake of choline and betaine, but not folate, was associated with an 81% reduction in VOB-related HS (OR: 0.19, 95% CI: 0.05-0.69). Our data suggest that the optimal intake of choline and betaine can minimize the risk of VOB-related HS in a threshold-dependent manner.

Elevated serum vitamin B(12) levels in association with tumor markers as the prognostic factors predictive for poor survival in patients with hepatocellular carcinoma.

Elevated blood vitamin B(12) (VitB(12)) level has recently been identified as a prognostic indicator for advanced cancer patients. The predictive value of blood VitB(12) for survival of patients with hepatocellular carcinoma (HCC) remains unclear. Our objective was to examine the determinants of elevated serum VitB(12) levels and their associations with prognosis of patients with HCC. The cohort study included 90 HCC patients who were consecutively admitted to the Chi-Mei Hospital, Taiwan, from April 2005 to December 2006. Nutrition and clinical pathological data were collected. Serum VitB(12) levels were determined by radioimmunoassay. Survival curves were calculated by the Kaplan-Meier method. Multivariate analysis of outcome predictors was assessed by Cox regression. Elevated serum VitB(12) levels of HCC patients were associated with reduced levels of albumin, hemoglobin, red blood cells count, and glutamate-pyruvate transaminase (GPT) (P < 0.05). Serum VitB(12) levels were positively correlated with alpha-fetal protein (AFP) levels (r = 0.623, P = 0.001) and tumor size (r = 0.630, P = 0.001; Table 3). By univariate analysis, survival was significantly worse in patients with elevated serum AFP (> 200 mu g/l) and VitB(12) levels (> 1,500 ng/l; P < 0.05). In multivariate analysis, both elevated AFP (> 200 vs. < 20; HR 4.4; CI = 1.9-10.3, P = 0.001) and VitB(12) levels (> 699 vs. < or = 699; HR = 2.88; CI = 1.26-6.6, P = 0.012) were found to be favorable predictive factors for HCC survival. This study shows that the determinants of elevated serum VitB(12) levels in HCC patients were in association with malnourishment, liver injuries, and tumor progression. Elevated VitB(12) levels in concurrence with AFP levels serve as the prognostic factors predictive for poor survival of HCC patients.

Lymphocytic mitochondrial DNA deletions, biochemical folate status and hepatocellular carcinoma susceptibility in a case-control study.

Mitochondrial (mt) DNA deletions and low folate status, proposed characteristics of carcinogenesis, in relation to human hepatocellular carcinoma (HCC) susceptibility are not clearly understood. We hypothesised that low folate status may modify frequencies of mtDNA deletions in humans, both of which could predispose individuals to HCC development. Biochemical folate status of serum and lymphocytes, and frequencies of mtDNA deletions in lymphocytes were determined in ninety HCC cases and ninety cancer-free healthy controls, individually matched by age and sex. The data revealed that HCC patients had lower levels of serum folate (P = 0.0002), lymphocytic folate (P = 0.040) and accumulated higher frequency of lymphocytic mtDNA deletions (P < 0.0001) than the controls. In the total studied subjects, frequencies of lymphocytic mtDNA deletions were associated with hepatitic B infection (P = 0.004) and HCC incidents (P = 0.001), and were correlated with serum folate (r - 0.155; P = 0.041), lymphocyte folate (r - 0.314; P = 0.0001), levels of glutamate-oxaloacetate transaminase (GOT) (r 0.206; P = 0.006), glutamate-pyruvate transaminase (GPT) (r 0.163; P = 0.037) and alpha-fetal protein concentrations (r 0.212; P = 0.005). After adjustment for age, sex, lifestyle and one-carbon metabolite factors, individuals with low blood folate ( < 11.5 nmol/l) or high mtDNA deletions (Delta threshold cycle number (Ct)>5.3) had increased risks for HCC (OR 7.7, 95 % CI 1.9, 29.9, P = 0.003; OR 5.4; 95 % CI 1.7, 16.8, P = 0.003, respectively). When combined with folate deficiency (serum folate < 14 nmol/l), the OR of HCC in individuals with high levels of lymphocytic mtDNA deletions was enhanced (OR 13.3; 95 % CI 1.45, 122; P = 0.008). Further controlling for GOT and GPT levels, however, negated those effects on HCC risk. Taken together, the data suggest that biochemical folate status and liver injuries are important modulators to lymphocytic mtDNA deletions. The mt genetic instability that results from a high rate of mtDNA deletions and/or low folate status increased the risk for HCC, which is mediated by clinical hepatic lesions.

Relationship between folate status and tumour progression in patients with hepatocellular carcinoma.

Previous studies with folate/methyl-deficient rat models proposed the role of folate deficiency in hepatocarcinogenesis and tumour progression. We investigated the relationship between folate status and tumour progression in patients with hepatocellular carcinoma (HCC). Ninety HCC patients (age 62 (sd 10) years) recruited through the Department of Internal Medicine, Chi-Mei Hospital, participated in this cross-sectional study. According to the clinical criteria, 44 % showed marginal folate deficiency (serum folate 7-14 nmol/l; folate intake 278 (sd 212) microg/d), and 16 % were folate deficient ( < 7 nmol/l; 207 (sd 113) microg/d). Serum folate showed inverse correlations with three elements of tumour progression: tumour size (r - 0.29; P = 0.005), tumour multiplicity (r - 0.24; P = 0.018) and metastasis (r - 0.39; P = 0.0001). When HCC progression was categorised into stages I to IV, serum folate decreased as HCC stage progressed (stage I, 24.5 (sd 11.5); stage IV, 10.3 (sd 3.3) nmol/l; P = 0.032). After adjustment for age, sex, lifestyle and dietary factors, patients with low blood folate status (serum folate < 14 nmol/l) had increased risks for advanced tumour progression in large tumours (OR 7.1 (95 % CI 2.27, 21.9); P = 0.0007), tumour multiplicity (OR 3.2 (95 % CI 1.07, 3.51); P = 0.004) and metastasis (OR 4.5 (95 % CI 1.11, 18.4); P = 0.03) relative to those with normal folate status. Further controlling for liver injury, tumour proliferation and tumour stage, however, negated the effect of folate on advanced tumour progression. The data thus suggest that low blood folate status could be a risk factor for tumour progression, which is modulated by clinical lesions present in HCC patients. Future studies with larger sample sizes are warranted to explore the joint effects of low folate and hepatic lesions in human HCC malignancy.

Dietary choline and folate relationships with serum hepatic inflammatory injury markers in Taiwanese adults.

BACKGROUND AND OBJECTIVES: The relationships of dietary choline and folate intake with hepatic function have yet to be established in the Taiwanese population. We investigated the associations of choline and folate intake with hepatic inflammatory injury in Taiwanese adults. METHODS AND STUDY DESIGN: Blood samples and data on dietary choline components and folate intake from 548 Taiwanese adults without pathological liver disease were collected. Dietary intake was derived using a semiquantitative food-frequency questionnaire. Serum liver injury markers of alanine transaminase, aspartate transaminase, and hepatitis viral infection were measured. RESULTS: Elevated serum hepatic injury markers (>40 U/L) were associated with low folate and free choline intake (p<0.05). Folate intake was the most significant dietary determinant of serum aspartate transaminase concentration (beta=-0.05, p=0.04), followed by free choline intake (beta=-0.249, p=0.055). Folate intake exceeding the median level (268 µg/d) was correlated with a reduced rate of hepatitis viral infection (p=0.032) and with normalized serum aspartate transaminase (odds ratio [OR]=0.998, 95% confidence interval [CI]=0.996-1, p=0.042) and alanine transaminase (OR=0.998, 95% CI=0.007-1, p=0.019). Total choline intake exceeding the median level (233 mg/d) was associated with normalized serum aspartate transaminase (OR=0.518, 95% CI=0.360-0.745, p=0.018). CONCLUSIONS: The newly established relationships of dietary intake of total choline and folate with normalized hepatic inflammatory markers can guide the development of dietary choline and folate intake recommendations for Taiwanese adults.

Interrelationships among genetic C677T polymorphism of 5,10-methylenetetrahydrofolate reductase, biochemical folate status, and lymphocytic p53 oxidative damage in association with tumor malignancy and survivals of patients with hepatocellular carcinoma.

SCOPE: Metabolic genotypes of 5,10-methylenetetrahydrofolate reductase (MTHFR) and folate status on oxidative DNA lesions in hepatocellular carcinoma (HCC) has not been elucidated. The aims of the study were to investigate the folate-polymorphic interactions on genetic oxidative damage in association with advanced HCC malignancy and prognosis. METHODS AND RESULTS: The study included 232 HCC patients with folate nutrition, MTHFR C677T polymorphic, p53 genetic and tumour pathological data collected and analyzed for their survivals after a 7.8-years following up. By adjustment for oxidative risk factors of HCC, the compound CT and TT genotypes in relative to the CC wild-type were associated with 83% reduced lymphocytic p53 oxidative lesions of HCC patients with RBC folate lower than 688 ng/mL (OR: 0.17, 95%CI: 0.07-0.43). Such genetic protective effects by the CT/TT genotypes were 2-fold enhanced among those with high RBC folate (OR: 0.08, 95% CI: 0.03-0.21, P for interaction < 0.001). For those with non-folate-deficient status, the compound CT and TT vs. CC genotypes were associated with 80% reduced risks of advanced HCC stages (III&IV) (OR: 0.2, 95%CI: 0.08-0.56). Such protection was negated either by adjustment of lymphocytic p53 oxidative lesions or by 3-fold increased risks among those with high RBC status (OR: 0.6, 95%CI; 0.31-1.41, P for interaction = 0.009). Multivariate Cox proportional hazards analysis showed that the CT/TT genotypes vs. CC wild-type were the independent predictable factor for better survival outcome of HCC patients (HR: 0.48, CI = 0.30-0.79). For CC homozygote, the second vs. the bottom tertile levels of RBC status were associated with 2-fold increased mortality rate of HCC patients (HR: 2.05, CI = 1.0-4.1). CONCLUSION: Our data demonstrated that reduced MTHFR activities associated with the MTHFR T allele may interact with RBC folate as the risk modifiers of lymphocytic p53 oxidative lesions of HCC patients. The CT/TT genotypes correlated with lower risks of late-stage HCC and a favorable survival of HCC patients, depending on p53 oxidative lesions or RBC folate status.