ICAM-1 expression in vaginal cells as a potential biomarker for inflammatory response

This study aimed to elucidate the mechanisms that may lead to an efficient strategy to induce a suitable host response of the vaginal mucosa upon exposure to intravaginally delivered exogenous compounds. It was hypothesized that the upregulation of intercellular adhesion molecule (ICAM)-1 gene expression may reflect the inflammatory response evoked by exogenous compounds. Major emphasis was placed on ethylenediamine tetraacetic acid (EDTA) which was added as a synergistic agent to conventional spermicidal agents or anti-HIV drugs. The levels of ICAM-1 mRNA were examined as a surrogate marker for inflammatory response in human vaginal epithelial cells upon exposure to EDTA or interleukin (IL)-1β (i.e. positive control, 25 mM). The effects of estrogen on EDTA-induced ICAM-1 expression were also evaluated for the estrogen involvement in the inflammatory process of the vaginal mucosa. ICAM-1 expression in human vaginal cells (VK2/E6E7 cells) increased as EDTA concentration added to human vaginal cell lines increased. The effects of estrogen on EDTA-induced ICAM-1 expression in human vaginal epithelial cells were estrogen-concentration dependent; estrogen at lower concentrations (～1–10 nM) did not affect ICAM-1 expression, whereas estrogen at higher concentrations (～100 nM–1 µM) attenuated ICAM-1 expression. The influence of estrogen in ICAM-1 expression suggests the beneficial effects of estrogen on the regulation of vaginal homeostasis. Identification and quantification of specific surrogate markers for the inflammatory response evoked by exogenous compounds and their regulation by estrogen will lead to an efficient strategy against sexually transmitted diseases including AIDS.     

Feasibility of Histological Scoring and Colony Count for Evaluating Infective Severity in Mouse Vaginal Candidiasis

Qualitative measurement of the infective level is relatively difficult in experimental vaginal candidiasis. Female BALB/c mice aged 8 to 10 weeks were randomly divided into E1, E2 and E0 groups, which received subcutaneous injection of 0.05 mg, 0.1 mg of estradiol benzoate or 0.1 ml soybean oil 3 days before vaginal inoculation, respectively, and hormone treatment continued every other day thereafter. Each group was further divided into infected and noninfected subgroups. The infected mice were inoculated intravaginally with 10 µl (5 × 10⁴ conidia) of Candida albicans suspension, while the noninfected mice were inoculated with 10 µl phosphate-buffered saline. Direct microscopic examination, colony count and vaginal histopathology including infection degree and inflammation extent were performed at 3, 7 and 14 days post inoculation. Estrogen treatment increased the vaginal fungal burden and extent of infection and inflammation compared with the control group, and 0.3 mg/week estrogen generally induced more severe infection and inflammation than 0.15 mg/week estrogen did. Colony count peaked on day 3 and decreased remarkably after 7 days. Infection score increased gradually during the first 7 days and decreased on day 14, while inflammation extent exacerbated progressively over the course of 14 days. This study demonstrates that the modified histological scoring system might be more feasible than colony count for evaluation of infectivity and dynamic change in experimental vaginal candidiasis.     

Imbalanced estrogen metabolism in the brain: possible relevance to the etiology of Parkinson’s disease

Damage to DNA by dopamine quinone and/or catechol estrogen quinones may play a significant role in the initiation of Parkinson’s disease (PD). Depurinating estrogen–DNA adducts are shed from cells and excreted in urine. The aim of this study was to discover whether higher levels of estrogen–DNA adducts are associated with PD. Forty estrogen metabolites, conjugates, and DNA adducts were analyzed in urine samples from 20 PD cases and 40 matched controls by using ultra performance liquid chromatography/tandem mass spectrometry. The levels of adducts in cases versus controls (P?<?0.005) suggest that unbalanced estrogen metabolism could play a causal role in the initiation of PD.     

Identification and preliminary validation of novel biomarkers of acute hepatic ischaemia/reperfusion injury using dual-platform proteomic/degradomic approaches

Hepatic ischaemia/reperfusion (I/R), a major cause of liver damage associated with multiple trauma, haemorrhagic and septic shock, and liver transplantation, contributes significantly to multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver damage is vital for effective management and treatment of ischaemic liver injury. By using high-throughput immunoblotting and cation–anion exchange chromatography/reversed-phase liquid chromatography-tandem mass-spectrometry, we identified several hepatic proteins, including argininosuccinate synthase (ASS) and estrogen sulfotransferase (EST-1), which were degraded in the liver and rapidly released into circulation during I/R injury. ASS accumulated in serum within 10 min, reached a steady state at 30 min, and persisted up until 3 h after reperfusion following 30 min of total hepatic ischaemia. EST-1 appeared rapidly in blood and attained maximum within 1 hour followed by a decline at 3 h of reperfusion. No ASS or EST-1 protein was detected in serum of control or sham operated rats. ASS and EST-1 exhibited greater sensitivity and specificity toward I/R liver injury as compared with alanine aminotransferase (ALT), an established marker of hepatocellular necrosis. In contrast, serum ASS and EST-1 were undetectable in rats with chronic alcoholic liver disease, while the levels of ALT protein were significantly increased. In addition, ASS, but not EST-1 or ALT accumulated in blood only 6 h after treatment with hepatotoxic combination of lipopolysaccharide and D-galactosamine. These data demonstrate the utility of ASS and EST-1 as novel sensitive and specific biomarkers of acute liver ischaemic injury for prospective clinical studies.     

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.     

Preferential recognition of catechol-estrogen modified DNA by circulating autoantibodies in cancer patients

Catecholestrogens [4-hydroxyestradiol (4-OHE₂)] have been implicated in human carcinogenesis, although the mechanism remains unestablished. In this study pUC 18 plasmid DNA was modified with 4-OHE₂ and nitric oxide (NO). The modification induced in native DNA exhibited hyperchromicity, single strand breaks, damage to restriction sites, modification of bases, decrease in Tm and change in ellipticity. Modified DNA was found to be highly immunogenic in experimental animal, eliciting high titer antibodies. Circulating cancer autoantibodies showed preferable recognition of 4-OHE₂-NO-DNA over native form (p < 0.001) and the oxidative epitopes on the DNA isolates from cancer patients were immunochemically detected by using experimentally induced anti-4-OHE₂-NO-DNA antibodies as a probe. Preferential recognition of 4-OHE₂-NO-DNA by cancer autoantibodies coupled with enhanced binding of induced antibodies to DNA isolated from cancer patients is an indicative of oxidative stress induced DNA damage in cancer. Possible involvement of unique epitopes on modified DNA in cancer autoantibody induction has been suggested.     

Sex-dependent correlation between survival and expression of genes related to the circadian oscillator in patients with colorectal cancer

ABSTRACT

Recent evidence supports the important role of the circadian system in cancer progression in humans. The aim of the present study is to evaluate clock (cry1, cry2 and per2) and clock-controlled (vascular endothelial growth factor-a, early growth response protein 1 and estrogen receptor β) gene expression in colorectal cancer and adjacent tissue and identify a possible link between survival of patients and expression of above mentioned genes. The study includes 64 patients of both sexes with previously diagnosed colorectal cancer. RNA was extracted from the tumor tissue and adjacent parts of the resected colon, and real-time PCR was used for detection of clock gene expression. Expression of cry2 and per2 was significantly downregulated in tumor tissue compared to adjacent tissues. After splitting of the cohort according to sex, we detected downregulated levels of cry2 and per2 in male patients, but not in females. Splitting of male and female sub-cohorts according to presence of metastases revealed significant donwregulation of cry2 expression in female patients without distant metastasis. Better survival rate was associated with low expression of cry2 in female patients. Moreover, we observed an increase in cry1 expression in female patients with distant metastases in tumor compared to adjacent tissue. Accordingly, women with high expression of cry1 in tumor tissue displayed worse survival, which was not observed in men. Taken together, expression of clock and clock-controlled genes in tumors of males and females clustered according to presence of distant metastases correlated with survival analysis. Studied clock-controlled genes also showed sex-dependent changes. Low expression of vegf-a in tumor correlated with better survival in men but not in women. High expression of estrogen receptor β mRNA was related to better survival in women but not in men. Low expression of vegf-a, egr1 and estrogen receptor β was associated with worse survival in women compared to men. Our data indicate sex-dependent associations between clock and clock-controlled gene expression in cancer tissue and patient’s survival prognosis.