Flavonoid ingredients of Ginkgo biloba leaf extract regulate lipid metabolism through Sp1-mediated carnitine palmitoyltranferase 1A up-regulation

Background

Lipid accumulation is the primary evidence of non-alcoholic fatty liver disease (NAFLD). Ginkgo biloba extract (GBE) and its flavonoid ingredients (quercetin, kaempferol, and isorhamnetin) could lessen the lipid accumulation associated with up-regulation of the rate-limiting enzyme, carnitine palmitoyltransferase 1A (CPT1A), in the β-oxidation of long-chain fatty acids. In this study, we investigated the mechanisms by which GBE and its flavonoids induced expression of CPT1A.

Results

CPT1A inhibition with RNAi resulted in triglyceride accumulation in HepG2 cells. Through deletion and mutation analysis of CPT1A’s promoter combined with electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments, the CPT1A promoter region (−50 to −5 nt) was determined to contain two putative Sp1 binding sites, namely Sp1a and Sp1b, which might act as the GBE regulation response DNA element. Sp1 might be induced to transfer from cytoplasma to nucleus to bind the promoter region of −50 to −5 nt by GBE. The regulatory effects of GBE on CPT1A were also verified on the flavonoid ingredients quercetin, kaempferol, and isorhamnetin.

Conclusion

Sp1 was crucial in regulating CPT1A expression with GBE and its flavonoid ingredients, and the −50 to −5 nt region of CPT1A promoter played important roles in Sp1 binding.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0087-x) contains supplementary material, which is available to authorized users.     

UDP-Glucuronosyltransferase 1A Compromises Intracellular Accumulation and Anti-Cancer Effect of Tanshinone IIA in Human Colon Cancer Cells

Background and Purpose

NAD(P)H: quinone oxidoreductase 1 (NQO1) mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation is the dominant metabolic pathway of tanshinone IIA (TSA), a promising anti-cancer agent. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. This study aims to explore the role of UGT1A in determining the intracellular accumulation and the resultant apoptotic effect of TSA.

Experimental Approach

We examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We also examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity.

Key Results

Multiple UGT1A isoforms are positively expressed in HT29 but not in HCT116 cells. Cellular S9 fractions prepared from HT29 cells exhibit strong glucuronidation activity towards TSA, which can be inhibited by propofol or UGT1A siRNA interference. TSA intracellular accumulation in HT29 cells is much lower than that in HCT116 cells, which correlates with high expression levels of UGT1A in HT29 cells. Consistently, TSA induces less intracellular ROS, cytotoxicity, and apoptotic effect in HT29 cells than those in HCT116 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol can decrease TSA glucuronidation and simultaneously improve its intracellular accumulation, as well as enhance TSA anti-cancer effect.

Conclusions and Implications

UGT1A can compromise TSA cytotoxicity via reducing its intracellular exposure and switching the NQO1-triggered redox cycle to metabolic elimination. Our study may shed a light in understanding the cellular pharmacokinetic and molecular mechanism by which UGTs determine the chemotherapy effects of drugs that are UGTs’ substrates.     

Demethylation of Circulating Estrogen Receptor Alpha Gene in Cerebral Ischemic Stroke

Background

Estrogen is involved in neuron plasticity and can promote neuronal survival in stroke. Its actions are mostly exerted via estrogen receptor alpha (ERα). Previous animal studies have shown that ERα is upregulated by DNA demethylation following ischemic injury. This study investigated the methylation levels in the ERα promoter in the peripheral blood of ischemic stroke patients.

Methods

The study included 201 ischemic stroke patients, and 217 age- and sex-comparable healthy controls. The quantitative methylation level in the 14 CpG sites of the ERα promoter was measured by pyrosequencing in each participant. Multivariate regression model was used to adjust for stroke traditional risk factors. Stroke subtypes and sex-specific analysis were also conducted.

Results

The results demonstrated that the stroke cases had a lower ERα methylation level than controls in all 14 CpG sites, and site13 and site14 had significant adjusted p-values of 0.035 and 0.026, respectively. Stroke subtypes analysis showed that large-artery atherosclerosis and cardio-embolic subtypes had significantly lower methylation levels than the healthy controls at CpG site5, site9, site12, site13 and site14 with adjusted p = 0.039, 0.009, 0.025, 0.046 and 0.027 respectively. However, the methylation level for the patients with small vessel subtype was not significant. We combined the methylation data from the above five sites for further sex-specific analysis. The results showed that the significant association only existed in women (adjusted p = 0.011), but not in men (adjusted p = 0.300).

Conclusions

Female stroke cases have lower ERα methylation levels than those in the controls, especially in large-artery and cardio-embolic stroke subtypes. The study implies that women suffering from ischemic stroke of specific subtype may undergo different protective mechanisms to reduce the brain injury.     

Impact of Childhood Adversity and Vasopressin receptor 1a Variation on Social Interaction in Adulthood: A Cross-Sectional Study

Background

Arginine vasopressin (AVP) plays a role in social behavior, through receptor AVPR1A. The promoter polymorphism AVPR1A RS3 has been associated with human social behaviors, and with acute response to stress. Here, the relationships between AVPR1A RS3, early-life stressors, and social interaction in adulthood were explored.

Methods

Adult individuals from a Swedish population-based cohort (n = 1871) were assessed for self-reported availability of social integration and social attachment and for experience of childhood adversities. Their DNA samples were genotyped for the microsatellite AVPR1A RS3.

Results

Among males, particularly those homozygous for the long alleles of AVPR1A RS3 were vulnerable to childhood adversity for their social attachment in adulthood. A similar vulnerability to childhood adversity among long allele carriers was found on adulthood social integration, but here both males and females were influenced.

Limitation

Data were self-reported and childhood adversity data were retrospective.

Conclusions

Early-life stress influenced the relationship between AVPR1A genetic variants and social interaction. For social attachment, AVPR1A was of importance in males only. The findings add to previous reports on higher acute vulnerability to stress in persons with long AVPR1A RS3 alleles and increased AVP levels.     

DAPK1 Promoter Methylation and Cervical Cancer Risk: A Systematic Review and a Meta-Analysis

Objective

The Death-Associated Protein Kinase 1 (DAPK1) gene has been frequently investigated in cervical cancer (CC). The aim of the present study was to carry out a systematic review and a meta-analysis in order to evaluate DAPK1 promoter methylation as an epigenetic marker for CC risk.

Methods

A systematic literature search was carried out. The Cochrane software package Review Manager 5.2 was used. The fixed-effects or random-effects models, according to heterogeneity across studies, were used to calculate odds ratios (ORs) and 95% Confidence Intervals (CIs). Furthermore, subgroup analyses were conducted by histological type, assays used to evaluate DAPK1 promoter methylation, and control sample source.

Results

A total of 20 papers, published between 2001 and 2014, on 1929 samples, were included in the meta-analysis. DAPK1 promoter methylation was associated with an increased CC risk based on the random effects model (OR: 21.20; 95%CI = 11.14–40.35). Omitting the most heterogeneous study, the between study heterogeneity decreased and the association increased (OR: 24.13; 95% CI = 15.83–36.78). The association was also confirmed in all the subgroups analyses.

Conclusions

A significant strong association between DAPK1 promoter methylation and CC was shown and confirmed independently by histological tumor type, method used to evaluate methylation and source of control samples. Methylation markers may have value in early detection of CC precursor lesions, provide added reassurances of safety for women who are candidates for less frequent screens, and predict outcomes of women infected with human papilloma virus.