Chemotherapeutic effect of Zerumbone on melanoma cells through mitochondria-mediated pathways.

BACKGROUND: Zerumbone (ZER) is a phytochemical that appears to regulate cell proliferation and apoptosis. It has been reported to have an anti-tumour effect in various malignant cells; however, the effect and the mechanism of ZER on melanoma cells needs to be clarified. AIM: To explore whether ZER has an effect on human melanoma cells and to identify the mechanisms involved. METHODS: We determined the chemotherapeutic action of ZER on the human malignant melanoma (MM) A375 cell line by CCK-8 immunohistochemistry, Hoechst 33342 staining and flow cytometry analysis. We also investigated the signalling pathways by which ZER induces apoptosis in A375 cells, using western blotting, reverse transcription PCR and caspase-3 activity analysis. RESULTS: ZER induced significant cytotoxic action in A375 cells. Hoechst 33342 staining and flow cytometry apoptosis analysis further demonstrated that ZER induced apoptosis in A375 cells. Treatment with ZER downregulated Bcl-2 gene and protein levels, upregulated Bax and Cytochrome c gene and protein levels, and activated Caspase-3. CONCLUSIONS: ZER might have a chemotherapeutic effect on human melanoma cells through mitochondria-mediated pathways.

The clinical significance of CA19-9 in ovarian mature cystic teratoma.

OBJECTIVE: To evaluate the clinical significance of CA19-9 in patients with ovarian mature cystic teratoma (MCT). MATERIALS AND METHODS: A retrospective study was performed on 65 patients with pathologically-confirmed MCT and 80 patients with benign epithelial ovarian tumors. Serum tumor markers for all patients and tissue CA19-9 for MCTs were measured. The relationships between clinical characteris- tics of MCTs and CA19-9, as well as the correlation between serum and tissue level of CA19-9 in MCTs, were evaluated. RESULTS: The mean serum level of CA19-9 in MCTs was significantly higher than that in benign ovarian epithelial tumors (49.9 ± 73.4 IU/ml vs. 17.08 ± 24.8 IU/ml). CA19-9 was the only tumor marker with a mean serum level above the cut-off value and the elevation rate was 30.76% in MCTs. The positive tissue expression rate of CA19-9 in MCT patients were 50.9% and were higher than that of preoperative serum levels (50.9% vs. 32.7%). CONCLUSION: Serum CA19-9 has the highest positivity rate among other tumor markers in MCT. Elevated serum CA19-9 is not an uncommon finding MCT and could be used as a marker in the differential diagnosis of MCT in patients with pelvic mass.

Association of the ApoE gene polymorphism and dietary factors with cerebral infarction and circulating lipid concentrations.

The aim of this study was to explore the relationship between the ApoE gene polymorphism and dietary factors with stroke and circulating lipid levels in the Chinese population. We selected 580 patients with stroke and 580 age- and gender-matched healthy controls, and examined their ApoE polymorphism genotype using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. We also analyzed the relationship between the ApoE gene polymorphism and dietary factors as well as plasma lipid concentrations in this cohort. We detected six ApoE genotypes in the study populations, and determined that the E4 allele was positively associated with cerebral infarction (CI), whereas allele E2 was negatively associated with total cholesterol (TC) and low-density lipoprotein-cholesterol levels. The dietary habits of the subjects with descending order of average total TC and triglyceride levels were: subjects addicted to oily food > subjects addicted to sweets > subjects addicted to smoking > subjects addicted to alcohol > subjects following a vegetarian diet (P < 0.05). Our results demonstrated that the ApoE gene polymorphism was associated with a risk for CI in a Chinese population.

Serum and tissue level of YKL-40 in endometrial cancer.

OBJECTIVE: Serum YKL-40 level is elevated in patients with several malignancies. This study was designed to assess the correlation between serum YKL-40 and the corresponding tissue expression in endometrial cancer (EC). MATERIALS AND METHODS: Preoperative serum levels of YKL-40 were measured by enzyme-linked immunosorbent assay (ELISA) from 41 patients with EC, 27 patients with uterine myoma, and 30 healthy women. YKL-40 protein expression in tissue was determined by immunohistochemistry for patients with EC and patients with uterine myoma. RESULTS: Median preoperative serum YKL-40 level was 157.2 microg/l (range 76.0 - 301.2) in EC compared with 86.6 microg/l (range 69.3 - 191.1) in uterine myoma, and 86.2 microg/l (range 52.1 - 201.1) in healthy women (p < 0.05). Of 41 patients with EC, 26 patients with elevated serum YKL-40 level statistically differed from the remaining 15 patients with normal serum YKL-40 level with respect to FIGO Stage, tumor grade, washing cytology, and serum CA125 (p < 0.05). In multivariate analysis, elevated serum YKL-40 significantly correlated with FIGO stage (p < 0.05) and tumor grade (p < 0.01). The percentage of positive YKL-40 tissue staining was higher in EC patients (34.1%, 14/41) than in uterine myoma patients (11.1%, 3/27) (p < 0.05) and was lower than that of elevated serum levels in EC (26/41, 63.4%) (p < 0.05). CONCLUSIONS: The elevated preoperative serum YKL-40 is related to stage and histologic grade of EC. The discordance between serum and tissue level of YKL-40 in EC indicates intrauterine tumor may not be the only source of serum YKL-40.

Research on the reactivation of Syk expression caused by the inhibition of DNA promoter methylation in the lung cancer.

The aim of this study was to study the expression of Syk gene and methylation in its promoter region in the lung cancer and to investigate the relationship between silencing of the Syk gene and DNA methylation of the Syk promoter region in lung cancer cell lines. Real-time PCR and immunohistochemistry were used to examine the Syk expression in specimens from 3 lung cancer cell lines and 16 lung cancer patients (tumor tissues and adjacent normal tissues). MSP was used to analyze the methylation status of the Syk promoter region. We also investigated the role of restoring Syk expression by using a DNA methyltransferase inhibitor, 5-aza-CdR, in suppressing invasion of lung cancer cell lines. No expression of the Syk gene was detected in the 3 lung cancer cell lines. In the 16 lung cancer patient samples, Syk expression was significantly lower in the tumor tissues than that in their adjacent normal tissues (P<0.05). Consistently, immunohistochemistry analysis of Syk protein expression showed that in the lung cancer tissues Syk protein expression was also significantly lower than that in their adjacent normal tissues. In the two lung cancer cell lines (NL9980, YTMLC-9) that lack the endogenous Syk expression, 4uM demethylation agent 5-aza-CdR treatment was able to reactivate the Syk gene expression, but not NCI-H446. In conclusion, hypermethylation leads to silencing of the Syk gene in human lung carcinoma cell lines. Methylation of the Syk promoter and loss of Syk expression in lung cancer cell lines are independent biomarkers. Syk may be a potential tumor suppressor in human lung cancer.

Tyrosine hydroxylase, but not dopamine beta-hydroxylase, is increased in rat frontal cortex after traumatic brain injury.

Chronic frontal lobe functional deficits after traumatic brain injury (TBI) may be associated with altered catecholamine systems in the frontal cortex. To test this, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) levels were examined by immunohistochemistry and Western blot at 1, 7, 14, and 28 days after TBI or sham surgery. No alterations in DBH levels were observed by Western blot at any time point examined, but there was a significant increase in TH expression 28 days after TBI (optical density 334 +/- 68% or 3.3-fold, ipsilateral and 218 +/- 39% or 2.2-fold, contralateral) relative to the sham controls. The increase in TH may reflect a compensatory response of dopaminergic neurons to upregulate their synthesizing capacity and increase the efficiency of dopamine neurotransmission chronically after TBI.

Adenovirus-mediated transfer and expression of beta-gal in injured hippocampus after traumatic brain injury in mice.

In models of focal cerebral ischemia, adenoviral gene transfer is often attenuated or delayed versus naive. After controlled cortical impact (CCI)-induced traumatic brain injury in mice, CA1 and CA3 hippocampus exhibit delayed neuronal death by 3 days, with subsequent near complete loss of hippocampus by 21 days. We hypothesized that adenoviral-mediated expression of the reporter gene beta-Galactosidase (beta-Gal) in hippocampus would be attenuated after CCI in mice. C57BL6 mice (n = 16) were subjected to either CCI to left parietal cortex or sham (burr hole). Adenovirus carrying the beta-Gal gene (AdlacZ; 1 x 10(9) plaque-forming units [pfu]/mL) was then injected into left dorsal hippocampus. At 24 or 72 h, beta-Gal expression was quantified (mU/mg protein). Separate mice (n = 10) were used to study beta-Gal spatial distribution in brain sections. Beta-Gal expression in left hippocampus was similar in shams at 24 h (48.4 +/- 4.1) versus 72 h (68.8 +/- 8.8, not significant). CCI did not reduce beta-Gal expression in left hippocampus (68.8 +/- 8.8 versus 88.1 +/- 7.0 at 72 h, sham versus CCI, not significant). In contrast, CCI reduced beta-Gal expression in right (contralateral) hippocampus versus sham (p < 0.05 at both 24 and 72 h). Beta-Gal was seen in many cell types in ipsilateral hippocampus, including CA3 neurons. Despite eventual loss of ipsilateral hippocampus, adenovirus-mediated gene transfer was surprisingly robust early after CCI providing an opportunity to test novel genes targeting delayed hippocampal neuronal death.

Chronic methylphenidate treatment enhances water maze performance following traumatic brain injury in rats.

Methylphenidate (MPH), a central nervous system stimulant with dopaminergic activity, facilitates neurobehavioral outcome following cortical suction ablation injury, but its potential efficacy following experimental traumatic brain injury (TBI) is unknown. Thus, beginning 24 h after controlled cortical impact injury or sham surgery, male Sprague-Dawley rats were injected (i.p.) once daily for 18 days with either MPH (5 mg/kg) or saline vehicle (VEH) and motor function assessed on post-operative days 1-4, followed by Morris water maze training to find a hidden platform on days 14-18. The MPH treatment regimen was ineffective in accelerating beam-balance or beam-walk recovery, but did significantly decrease swim latencies when compared to VEH-treated controls. The results are consistent with published studies showing improved outcome with MPH therapy. Furthermore, this positive finding with delayed treatment suggests that strategies that enhance catecholamine neurotransmission during the chronic post injury phase may be a useful adjunct in ameliorating some of the neurobehavioral sequelae following TBI in humans.

Differential effects of traumatic brain injury on vesicular acetylcholine transporter and M2 muscarinic receptor mRNA and protein in rat.

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may result from presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and the M2 muscarinic receptor subtype can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2 muscarinic receptor subtype protein and medial septal mRNA levels at 4 weeks following injury. Rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 2.5 to 2.9 mm in depth) and sham surgery. Animals were sacrificed, and coronal sections (35 microm thick) were cut through the dorsal hippocampus for VAChT and M2 immunohistochemistry. Semiquantitative measurements of VAChT and M2 protein in hippocampal homogenates from injured and sham rats were assessed with Western blot analysis. Changes in VAChT and M2 mRNA levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). At 4 weeks after injury, both immunohistochemical and Western blot methods demonstrated an increase in hippocampal VAChT protein. An increase in VAChT mRNA was also observed. Immunohistochemistry demonstrated a loss of M2; however, there was no significant change in M2 mRNA levels in comparison with sham controls. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI through differential transcriptional regulation.

Chronic effects of traumatic brain injury on hippocampal vesicular acetylcholine transporter and M2 muscarinic receptor protein in rats.

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may be due to presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and M2 receptors can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2 muscarinic subtype receptor protein levels at four time points: 1 day, 1 week, 2 weeks, and 4 weeks following injury. Rats were anesthetized and surgically prepared for controlled cortical impact injury (4 m/s, 2.5- to 2.9-mm depth) and sham surgery. Animals were sacrificed and coronal sections (35 micro(m) thick) were cut through the dorsal hippocampus for VAChT and M2 immunohistochemistry. Semiquantitative measurements of VAChT and M2 protein in hippocampal homogenates from injured and sham rats were assessed using Western blot analysis. Immunohistochemistry showed no obvious changes in VAChT and M2 immunoreactivity at 1 day and 1 week postinjury. At 2 and 4 weeks postinjury, an increase in hippocampal VAChT protein and a corresponding loss of hippocampal M2 protein was observed compared to sham controls. Consistent with these results, Western blot analyses at 4 weeks postinjury demonstrated a 40-50% increase in VAChT and a 25-30% decrease in M2. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI, by upregulating the storage capacity and subsequent release of ACh and downregulating presynaptic inhibitory receptors.

Expression of interleukin (IL)-1 beta, IL-6 and their respective receptors in the normal rat brain and after injury.

The expression of interleukin (IL)-1 beta, IL-6 and their respective receptors has been studied in the rat brain before and up to 24 h after injury. Messenger RNA transcripts of these four genes were detected by in situ hybridization (ISH) in different structures of the intact brain. The distribution was very similar for IL-1 beta, IL-6 and IL-6 receptor (IL-6R). The expression of IL-1R was more widespread. Within hours after injury, an increased expression of IL-1 beta, and thereafter of IL-6 was documented. The expression of IL-1R and IL-6R was also increased. This expression was bilateral and not restricted to the injured area. Within 24 h, all ISH patterns had returned to normal. The molecular data were confirmed by protein data. Indeed, the distribution of IL-6 (detected by immunocytochemistry) agreed with the ISH patterns for IL-6. Furthermore, extracellular fluid was collected by microdialysis at the site of the lesion during 12 h and successive fractions were assayed for the presence of bioactive IL-1 and IL-6. Increases in IL-1 and later in IL-6 levels were detected. The rapid and concomitant increased expression of IL-1 beta, IL-6 and their receptors after injury stresses their possible early role in inflammatory mechanisms also in the brain, before any recruitment of inflammatory cells from remote nervous and not nervous areas.

Human T-cell lymphotropic virus infection among blood donors in south Florida. The Transfusion Safety Study Group.

Knowledge of the epidemiologic pattern of human T-lymphotropic virus (HTLV) in the United States is being enlarged by blood donor screening. We tested stored sera from 29,937 donations made in South Florida in 1984-1985. Twenty-three donors were confirmed as seropositive, a prevalence of 0.8 per 1,000 donations. Specificity was supported by serologic retesting and virus culture of 11 donors located for follow-up. Sex- and age-specific prevalences did not differ significantly; blacks, however, accounted for 65% of seropositive donations. Within South Florida, one section of Miami had a prevalence of 4.5 per 1,000 donations, significantly above the 0.1 to 1.1 per 1,000 rates for other parts. An epidemiologic association with known HTLV-I endemic areas could account for most infections; all seven typed isolates were characterized as HTLV-I. Exposures, however, were diverse, sometimes multiple, and had no necessary relationship to personal lifestyle. This finding suggests that sources of infection were varied. Seropositive family members emphasize familial clustering of HTLV-I infection.