Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines.

Temozolomide (TMZ) is the standard chemotherapeutic agent for human malignant glioma, but intrinsic or acquired chemoresistance represents a major obstacle to successful treatment of this highly lethal group of tumours. Obtaining better understanding of the molecular mechanisms underlying TMZ resistance in malignant glioma is important for the development of better treatment strategies. We have successfully established a passage control line (D54-C10) and resistant variants (D54-P5 and D54-P10) from the parental TMZ-sensitive malignant glioma cell line D54-C0. The resistant sub-cell lines showed alterations in cell morphology, enhanced cell adhesion, increased migration capacities, and cell cycle arrests. Proteomic analysis identified a set of proteins that showed gradual changes in expression according to their 50% inhibitory concentration (IC(50)). Successful validation was provided by transcript profiling in another malignant glioma cell line U87-MG and its resistant counterparts. Moreover, three of the identified proteins (vimentin, cathepsin D and prolyl 4-hydroxylase, beta polypeptide) were confirmed to be upregulated in high-grade glioma. Our data suggest that acquired TMZ resistance in human malignant glioma is associated with promotion of malignant phenotypes, and our reported molecular candidates may serve not only as markers of chemoresistance but also as potential therapeutic targets in the treatment of TMZ-resistant human malignant glioma, providing a platform for future investigations.

The accessory subunit of mitochondrial DNA polymerase gamma determines the DNA content of mitochondrial nucleoids in human cultured cells.

The accessory subunit of mitochondrial DNA polymerase gamma, POLGbeta, functions as a processivity factor in vitro. Here we show POLGbeta has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGbeta increased nucleoid numbers, whereas over-expression of POLGbeta reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGbeta altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGbeta preferentially bound to plasmids with a short displacement-loop, in contrast to POLGalpha. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGbeta is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.

The effect of topical lignocaine gel in pain relief for colposcopic assessment and biopsy: is it useful?

We investigated the use of topical ligocaine gel in pain relief for colposcopy and cervical punch biopsy. Ninety women referred for colposcopy due to abnormal cervical cytology were randomised to receive 5 ml of either 2% xylocaine gel or KY jelly to the cervix and the upper part of the vagina for at least 10 minutes prior to the colposcopic procedures. Pain score was obtained at several points of the procedure. Topical lignocaine gel did not significantly relieve pain from cervical punch biopsy and alleviate the stinging sensation from application of acetic acid and Lugol's iodine to cervix and vagina. However, it may be beneficial to a subgroup of women with prior unpleasant experience towards speculum examination.

Elevation of plasma osteopontin level in patients with undifferentiated nasopharyngeal carcinoma.

AIMS: We evaluated the clinicopathologic relevance of plasma osteopontin (OPN) level in nasopharyngeal carcinoma patients. METHODS: Seventy-two plasma samples were collected from patients with undifferentiated nasopharyngeal carcinoma (NPC) before radiotherapy. Plasma OPN level was determined by quantitative sandwich enzyme immunoassay. The plasma OPN level was evaluated for its clinicopathologic relevance. RESULTS: The mean plasma OPN level was significantly higher in NPC patients than in normal controls (184.66 vs 75.89 ng/ml, p<0.001). In addition, high OPN level was found in the patients with advanced cancer and was correlated with neck node metastasis (p<0.05). CONCLUSIONS: Our findings indicated a potential role of OPN in the pathogenesis and nodal metastasis of undifferentiated NPC.

Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds.

Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 microM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.

Clinicopathologic significance of plasma matrix metalloproteinase-2 and -9 levels in patients with undifferentiated nasopharyngeal carcinoma.

UNLABELLED: Increased in plasma pro-MMP2 and pro-MMP9 levels in patients with advanced stage NPC were observed. Plasma pro-MMP2 is a significant independent prognostic marker for undifferentiated NPC. AIM: Upregulation of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) expression is observed in many cancers and high level of these proteins are found in peripheral blood of many cancer patients. In this study, we aimed at evaluating the plasma pro-MMP2 and pro-MMP9 pro-enzymes (pro-MMP2 and pro-MMP9) levels and their clinical significances in patients with undifferentiated nasopharyngeal carcinoma (NPC). METHODS: The plasma pro-MMP2 and pro-MMP9 levels were measured in 40 NPC patients and 40 normal individuals by enzyme linked immunosorbant assay. RESULTS: By using the Cox-regression model, a high pro-MMP2 level was found to be significantly correlated with poorer survival. Patients with plasma pro-MMP2 below 650 ng/ml had higher 5-year survival rate of 89%, compared with 50% for patients with plasma pro-MMP2 above 650 ng/ml. CONCLUSIONS: A high level of plasma pro-MMP2 was associated with poor survival of NPC patients independent of sex, age and stage.

The study of p16 and p15 gene methylation in head and neck squamous cell carcinoma and their quantitative evaluation in plasma by real-time PCR.

Epigenetic silencing of the p16 and p15 genes by promoter methylation are commonly observed in human epithelial malignancies, including head and neck squamous cell carcinomas (HNSCC). In this study, a methylation-specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the p16 and p15 genes in 73 HNSCC surgical specimens. p16 and p15 gene methylation was also examined in 29 paired metastatic lymph nodes and 29 paired histologically, normal resection margin mucosae. The quantity of cell-free methylated p16 and p15 DNA in the plasma samples of 20 HNSCC patients and 24 healthy controls was also examined using a fluorescence-based real-time PCR assay. The frequencies of p16 and p15 methylation in the primary tumour were 49% and 60%, respectively. Concordant methylation of p16 and p15 in tumour samples and metastatic lymph nodes was found in 59 and 38% of cases, respectively. A significantly higher prevalence of p15 methylation was found in histologically-normal surgical margin epithelia of HNSCC patients with chronic smoking and drinking habits compared with non-smokers and non-drinkers. In addition, methylated p16 and p15 DNA levels were significantly higher in the plasma of HNSCC patients (mean 56 copies/ml plasma and 65 copies/ml plasma, respectively) compared with normal controls (mean 6 copies/ml plasma and 16 copies/ml plasma, respectively). In conclusion, promoter methylation of the p16 and p15 genes is involved in the pathogenesis of HNSCC and may be related to chronic smoking and drinking. The differential levels of methylated p16 and p15 DNA in plasma might be potential useful markers in screening high-risk populations for early HNSCC and monitoring their treatment response.

Uptake of beta-carotene by ovarian and uterine tissues and effects on steroidogenesis during the estrous cycle in cats.

OBJECTIVES: To determine uptake of beta-carotene by ovarian and uterine tissues and influence of dietary beta-carotene on steroidogenesis and production of uterine protein during the estrous cycle in cats. ANIMALS: 56 female cats. PROCEDURE: Cats were fed diets containing 0, 0.4, 2, or 10 mg of beta-carotene daily for 8 weeks prior to detection of estrus. At time of observed estrus, all cats were manually induced to ovulate. Blood samples were obtained at estrus and every 2 days until day 14 after ovulation. On that day, cats underment laparotomy, and the ovaries and uterus were removed. Uterine contents were flushed, and luteal and endometrial tissues were obtained. RESULTS: Concentrations of beta-carotene in plasma and luteal and endometrial tissues increased in a dose-dependent manner. Concentrations of plasma progesterone were higher between days 6 and 10 after ovulation in cats fed diets containing beta-carotene and continued to increase through day 14 after ovulation in cats fed a diet containing 10 mg of beta-carotene. Plasma concentration of estradiol-17beta also was higher between days 0 and 4 after ovulation in cats fed diets containing beta-carotene. Cats fed a diet containing 10 mg of beta-carotene had the highest plasma estradiol concentration. Total uterine protein concentration was higher in cats fed beta-carotene, compared with values for cats fed an unsupplemented diet. CONCLUSION AND CLINICAL RELEVANCE: Cats readily absorb beta-carotene. Increased concentrations of progesterone, estradiol, and uterine protein may provide more optimal ovarian function or a better uterine environment for embryonic survival and development.

Dietary beta-carotene stimulates cell-mediated and humoral immune response in dogs.

The role of beta-carotene on immune response in domestic dogs is not known. Female Beagle dogs were fed 0, 2, 20 or 50 mg beta-carotene/d; blood was sampled at wk 0, 1, 2, 4 and 8 for analysis of the following: lymphoproliferation, leukocyte subpopulations and concentrations of interleukin-2 (IL-2), immunoglobulin (Ig)G and IgM. Delayed-type hypersensitivity (DTH) response was assessed at wk 0, 3 and 7. beta-Carotene supplementation increased plasma beta-carotene concentrations in a dose-dependent manner. Compared with unsupplemented dogs, those fed 20 or 50 mg of beta-carotene had higher CD4+ cell numbers and CD4:CD8 ratio. However, there was no treatment difference in CD8+, CD21+ and major histocompatability complex (MHC) class II+ cells. Plasma IgG, but not IgM concentration was higher in dogs fed beta-carotene throughout the study period. The DTH response to phytohemagglutinin (PHA) and vaccine was heightened in beta-carotene-supplemented dogs. beta-Carotene feeding did not influence mitogen-induced lymphocyte proliferation or IL-2 production. Immune response was impaired in dogs classified as low beta-carotene absorbers compared with similar dogs fed the same amount of beta-carotene. Therefore, dietary beta-carotene heightened cell-mediated and humoral immune responses in dogs.

Dietary beta-carotene absorption by blood plasma and leukocytes in domestic cats.

Three experiments were conducted to study the uptake of oral beta-carotene by blood plasma and leukocytes in domestic cats. In Experiment 1, mature female Tabby cats (12 mo old) were given once orally 0, 10, 20 or 50 mg of beta-carotene and blood taken at 0, 12, 24, 30, 36, 42, 48 and 72 h after dosing. Concentrations of plasma beta-carotene increased in a dose-dependent manner. Peak concentrations were observed at 12-24 h and declined gradually thereafter. The half-life of plasma beta-carotene was 12-30 h. In Experiment 2, cats were dosed daily for six consecutive days with 0, 1, 2, 5 or 10 mg beta-carotene. Blood was sampled once daily at 12 h after each feeding. Daily dosing of cats with beta-carotene for 6 d resulted in a dose-dependent increase in circulating beta-carotene. Experiment 3 was designed to study the uptake of beta-carotene by blood leukocytes. Cats were fed 0, 5 or 10 mg of beta-carotene daily for 14 d. Blood leukocytes were obtained on d 7 and 14 to determine beta-carotene content in whole lymphocytes and in subcellular fractions. Blood lymphocytes took up large amounts of beta-carotene by d 7 of feeding. Furthermore, beta-carotene accumulated mainly in the mitochondria (40-52%), with lower amounts accumulating in the microsomes (20-35%), cytosol (15-34%), and nuclei (1.5-6%). Therefore, domestic cats readily absorb beta-carotene across the intestinal mucosa and transfer the beta-carotene into peripheral blood leukocytes and their subcellular organelles. beta-Carotene uptake kinetics show that some aspects of beta-carotene absorption and metabolism in cats are similar to those of humans.

Dietary beta-carotene is taken up by blood plasma and leukocytes in dogs.

beta-Carotene uptake by blood plasma and leukocytes was studied in mature beagle dogs. In expt. 1, dogs were fed once orally with 0, 50, 100 or 200 mg of beta-carotene and their blood was sampled at 0, 1. 5, 3, 6, 10, 18 and 24 h. Plasma beta-carotene concentrations increased dose-dependently to peak at 6 h postfeeding. Concentrations decreased rapidly thereafter, showing a half-life of 3 to 4 h. In expt. 2, dogs were given daily doses for seven consecutive days with 0, 12.5, 25, 50 or 100 mg beta-carotene. Plasma beta-carotene concentrations increased dose-dependently; concentrations after the last dose were two- to fourfold higher than after the first dose. In expt. 3, dogs were fed 0, 50 or 100 mg beta-carotene daily for 30 d. beta-Carotene was elevated in lymphocytes and neutrophils in supplemented dogs. Furthermore, beta-carotene was taken up by the cytosol, mitochondria, microsomes (lymphocytes and neutrophils) and nuclei (lymphocytes only), proving that dogs can absorb beta-carotene. beta-Carotene is taken up by subcellular organelles of blood lymphocytes and neutrophils and in the plasma and leukocytes beta-carotene may have physiological importance as it relates to immunity in dogs. Uptake kinetics indicated that dogs are not an appropriate animal model for studying beta-carotene absorption and metabolism in humans.

Beta-carotene uptake and changes in ovarian steroids and uterine proteins during the estrous cycle in the canine.

The uptake of beta-carotene by reproductive tissues and the effects of beta-carotene on reproductive function in the dog are unknown. We studied the uptake of beta-carotene by blood, corpus luteum, and uterine endometrium and the role of dietary beta-carotene in influencing ovarian steroid and uterine protein production during the estrous cycle in the dog. Mature female Beagle dogs (n = 56) were fed diets containing 0, 2, 20, or 50 mg of beta-carotene daily for approximately 6 wk before estrus detection. Blood was sampled at regular intervals from estrus through d 45 after ovulation (d 0 = ovulation), when laparotomy was performed. The ovaries were obtained for the isolation of corpus luteum. The uterus was flushed with phosphate-buffered saline and the endometrium obtained by scraping. Beta-carotene was not detectable in plasma, corpus luteum, or endometrium of unsupplemented dogs. However, beta-carotene and alpha-carotene in plasma, corpus luteum, and uterine endometrium increased in a dose-dependent manner. Alpha-carotene made up a high percentage of total carotenoids even though the alpha-carotene content in the dietary source was very low. Dogs fed 50 mg of beta-carotene had significantly higher concentrations of plasma progesterone between d 12 and 26 compared with unsupplemented dogs. Dietary beta-carotene did not influence plasma estradiol-17beta and total uterine proteins. Therefore, beta-carotene is absorbed into plasma, corpus luteum, and uterine endometrium of dogs. Furthermore, dietary beta-carotene increased plasma progesterone concentrations during the estrous cycle. It is possible that dietary beta-carotene may improve reproductive function in the canine.

Dietary lutein stimulates immune response in the canine.

The possible immuno-modulatory action of dietary lutein in dogs is not known. Female Beagle dogs (17-18-month old; 11.4+/-0.4kg body weight) were supplemented daily with 0, 5, 10 or 20mg lutein for 12 weeks. Delayed-type hypersensitivity (DTH) response to saline, phytohemagglutinin (PHA) and a polyvalent vaccine was assessed on Weeks 0, 6 and 12. Blood was sampled on Weeks 0, 2, 4, 8 and 12 to assess (1) lymphocyte proliferative response to PHA, concanavalin A (Con A), and pokeweed mitogen (PWM), (2) changes in peripheral blood mononuclear cell (PBMC) populations, (3) interleukin-2 (IL-2) production and (4) IgG and IgM production. After the completion of 12-week study, we continued to collect the blood weekly up to 17 weeks to evaluate the changes in immunoglobulin production upon first and second antigenic challenges on Weeks 13 and 15. Plasma lutein+zeaxanthin was undetectable in unsupplemented dogs but concentrations increased (P<0.05) rapidly on Week 2 in lutein-supplemented dogs. Thereafter, concentrations generally continued to increase in dose-dependent manner, albeit at a much slower rate. Dogs fed lutein had heightened DTH response to PHA and vaccine by Week 6. Dietary lutein increased (P<0.05) lymphocyte proliferative response to all three mitogens and increased the percentages of cells expressing CD5, CD4, CD8 and major histocompatibility complex class II (MHC II) molecules. The production of IgG increased (P<0.05) in lutein-fed dogs after the second antigenic challenge. Lutein did not influence the expression of CD21 lymphocyte marker, plasma IgM or IL-2 production. Therefore, dietary lutein stimulated both cell-mediated and humoral immune responses in the domestic canine.

Modulation of humoral and cell-mediated immune responses by dietary lutein in cats.

The immuno-modulatory role of dietary lutein in domestic cats is unknown. Female Tabby cats (10-month old; n=56) were supplemented daily for 12 weeks with 0, 1, 5 or 10mg lutein. Blood was collected on Weeks 0, 2, 4, 8 and 12 to assess the following: (1) mitogen-induced peripheral blood mononuclear cells (PBMCs) proliferation, (2) changes in PBMC subpopulations, (3) interleukin-2 (IL-2) production and (4) plasma immunoglobulin (Ig)G production. In addition, delayed-type hypersensitivity (DTH) response to concanavalin A (Con A) or a polyvalent vaccine was performed on Weeks 0, 6 and 12. Dietary lutein increased plasma lutein concentrations in a dose-dependent manner (p<0.001) and concentrations had not reached steady state after 12 weeks of feeding in cats given 5 or 10mg lutein. Concentrations of plasma retinol and alpha-tocopherol were not influenced by diet. The DTH response to vaccine but not to Con A increased (p<0.05) in a dose-dependent manner on Week 6. Compared to control, cats fed lutein also showed enhanced Con A- and pokeweed mitogen-stimulated PBMCs proliferation. Dietary lutein also increased the percentages of CD4+ and CD21+ lymphocytes on Week 12 but had no significant effect on pan T, CD8 and MHC class II markers. Plasma IgG was higher (p<0.05) in cats fed 10mg lutein on Weeks 8 and 12. These results support the immuno-modulatory action of lutein in domestic cats.

A comparison of the anticancer activities of dietary beta-carotene, canthaxanthin and astaxanthin in mice in vivo.

The anticancer activities of beta-carotene, astaxanthin and canthaxanthin against the growth of mammary tumors were studied in female eight-wk-old BALB/c mice. The mice were fed a synthetic diet containing 0, 0.1 or 0.4% beta-carotene, astaxanthin or canthaxanthin. After 3 weeks, all mice were inoculated with 1 x 10(6) WAZ-2T tumor cells into the mammary fat pad. All animals were killed on 45 d after inoculation with the tumor cells. No carotenoids were detectable in the plasma or tumor tissues of unsupplemented mice. Concentrations of plasma astaxanthin (20 to 28 mumol/L) were greater (P < 0.05) than that of beta-carotene (0.1 to 0.2 mumol/L) and canthaxanthin (3 to 6 mmol/L). However, in tumor tissues, the concentration of canthaxanthin (4.9 to 6.0 nmol/g) was higher than that of beta-carotene (0.2 to 0.5 nmol/g) and astaxanthin (1.2 to 2.7 nmol/g). In general, all three carotenoids decreased mammary tumor volume. Mammary tumor growth inhibition by astaxanthin was dose-dependent and was higher than that of canthaxanthin and beta-carotene. Mice fed 0.4% beta-carotene or canthaxanthin did not show further increases in tumor growth inhibition compared to those fed 0.1% of each carotenoid. Lipid peroxidation activity in tumors was lower (P < 0.05) in mice fed 0.4% astaxanthin, but not in those fed beta-carotene and canthaxanthin. Therefore, beta-carotene, canthaxanthin and especially astaxanthin inhibit the growth of mammary tumors in mice; their anti-tumor activity is also influenced by the supplemental dose.

Dietary lutein but not astaxanthin or beta-carotene increases pim-1 gene expression in murine lymphocytes.

This study investigates the effect of dietary carotenoids on pim-1 gene expression in mouse splenocytes. Female BALB/c mice were fed 0%, 0.02%, or 0.4% astaxanthin, beta-carotene, and lutein for two weeks. Plasma and liver were obtained for the analysis of carotenoids. Splenocytes were isolated and cultured in the presence of concanavalin A, and the level of pim-1 mRNA was determined by Northern blot analysis. None of the carotenoids were detectable in the plasma and liver of unsupplemented mice. In plasma the concentration of astaxanthin (4.9-54.7 mumol/l) was dramatically higher than that of lutein (1.4-2.0 mumol/l) and beta-carotene (0.1-0.7 mumol/l). Carotenoid uptake by the spleen but not the liver reflected that observed in plasma. In mice fed 0.4% of each carotenoid, the absolute concentration of the carotenoid in the liver was highest for astaxanthin (24 nmol/g) followed by beta-carotene (7.5 nmol/g) and lutein (1.58 nmol/g). Mice fed lutein showed a dose-related increase in pim-1 mRNA expression. The steady-state level of pim-1 mRNA in mice fed 0.4% lutein was sixfold higher than in mice fed 0.02% lutein. In contrast, dietary astaxanthin and beta-carotene did not affect pim-1 expression. Therefore, an increase in pim-1 mRNA was observed in splenocytes stimulated with concanavalin A in lutein-fed mice. This appears to be a unique effect of lutein and may be associated with its antitumor activity observed in vivo.