Aneurysmal Subarachnoid Hemorrhage Onset Alters Pyruvate Metabolism in Poor-Grade Patients and Clinical Outcome Depends on More: A Cerebrospinal Fluid Metabolomic Study.

Cerebral metabolism alterations influence cerebrospinal fluid (CSF) composition and are sensitive to brain injury. In subarachnoid hemorrhage (SAH) patients, Fisher scale, Hunt-Hess scale, and World Federation of Neurological Societies (WFNS) grading scale evaluating SAH severity are inadequate to predict long-term outcome; therefore, in an effort to determine metabolite pattern disparity and discover corresponding biomarkers, we designed an untargeted CSF metabolomic study covering a broad range of metabolites of SAH patients with different severity and outcome. The present study demonstrated the SAH altered the cerebrospinal fluid metabolome involving carbohydrate, lipid, and amino acid metabolism. Pyruvate metabolism was enhanced in SAH patients with Hunt-Hess scale above III, and the CSF pyruvate level was significantly associated with WFNS grading scale above III. There is no significant variation among CSF metabolome in SAH patients with merely different amounts and distribution of bleeding. SAH patients with unfavorable outcome present upregulated CSF amino acids level and enhanced lipid biosynthesis. The present study provides a novel possibility of early identification of patients who might possess unfavorable outcome and further clarification of the underlying pathophysiology.

[Relationship between miR-218 and CDK6 expression and their biological impact on glioma cell proliferation and apoptosis].

OBJECTIVES: To investigate the relationship between the expression of miR-218 and CDK6 in glioma cells, and their biological impacts on the tumor cell proliferation and apoptosis. METHODS: Expression levels of miR-218 as well as CDK6 and Ki-67 proteins were analyzed in 60 cases of gliomas with various grades and 10 control brain tissue samples by tissue microarray, locked oligonucleotide probe in situ hybridization and immunohistochemistry. Glioblastoma multiform cell line (U87MG) was transfected with miR-218 mimics (mimics group) and a control sequence (control group), followed by qRT-PCR detection of miR-218 and immunocytochemical stain of CDK6 and Ki-67, respectively. Single cell gel electrophoresis was used to detect the presence of apoptotic cell. RESULTS: The miR-218 labeling indexes (LI) were statistically different (P<0.05) among all groups including control (22.45 +/- 0.59) and various glioma groups (grades I - II 4.00 +/- 1.07, grade III 1.87 +/- 1.06 and grade IV 0.94 +/- 0.78, respectively). The CDK6 LI of the four groups was 7.25 +/- 1.20, 16.71 +/- 0.80, 24.43 +/- 0.62 and 32.05 +/- 0.43, respectively. Significant differences existed between the control group and the glioma groups, and between grade IV and grades I - II glioma groups (P<0.01). Ki-67 positive cell densities of the above four groups (0.00 +/- 0.00, 9.30 +/- 3.48, 31.15 +/- 9.44 and 60.15 +/- 13.60) were significantly different from one and another (P<0.01). The expression of miR-218 negatively correlated with CDK-6 LI (r = -0.480, P<0. 01) and Ki-67 positive cell density (r = - 0.534, P<0.01), while the latter two positively correlated with each other (r = 0.530, P<0.01). U87MG transfection experiment showed that the miR-218 level of the mimics group was significantly higher than that of the control group (P<0.01). CDK6 and Ki-67 LI of the mimics group (14.74 +/- 1.19 and 30.88 +/- 3.31) were significantly lower than those of the control group (79.06 +/- 2.07 and 64.94 +/- 3.96, P<0.01), whilst its apoptotic index (AI) (68.44 +/- 7.05) was significantly higher than that of the control group (13.04 +/- 0.97, P<0.01). CONCLUSIONS: The expression level of miR-218 is an important reference indicator for the assessment of the grade of gliomas. An aberrant decrease of its expression may lead to an increase of the CDK6 expression and proliferative activity of giloma cells. Introducing exogenous miR-218 may effectively down-regulate the CDK6 expression, inhibit cell proliferation and induce apoptosis of malignant giloma cells. These findings imply that miR-218 may serve as a therapeutic agent against malignant glioma.

[Effects of azidothymidine on p33ING1b expression, apoptosis and senescence of TJ905 human glioblastoma cell line].

OBJECTIVES: To investigate the pharmacological effects of azidothymidine (AZT) on p33ING1b expression, senescence and apoptosis of TJ905 glioblastoma cells. METHODS: TJ905 cells were treated with AZT at a serial concentrations of 50, 100 and 200 µmol/L. Semi-quantitative RT-PCR and cytochemical staining of senescence related-galactosidase (sß-Gal) were used to evaluate the expression of p33ING1b mRNA and to label the senescent cells at the 1st, 3rd and 6th generations, respectively. In situ cell death detection and single cell gel electrophoresis were used to detect the apoptosis at the 3rd and 6th generations. RESULTS: AZT induced the expression of p33ING1b mRNA and senescence of the tumor cells of the 1st generation in a dosage and time dependent manner. At the 6th generation, the relative amount of p33ING1b RT-PCR product (1.44±0.23) and sß-Gal labeling index of 200 µmol/L group (45.62±6.74) were significantly higher than those of the 1st (0.95±0.13 and 7.82±2.40) and the 3rd generation cells (1.35±0.23, 26.27±7.17) of the same group, and cells of the same generation in the 50 µmol/L (0.85±0.24, 27.37±6.41) and 100 µmol/L groups (1.23±0.34, 35.49±5.12, P<0.01). There was a significant positive correlation between the p33ING1b mRNA expression and the labeling index of sß-Gal. Pro-apoptotic effects of AZT became obvious at the 6th generation. CONCLUSION: AZT upregulates the expression of p33ING1b, a possible mechanism in regulating senescence and apoptosis of the TJ905 cells.

[Detection of chromosomal DNA imbalance in medulloblastoma by comparative genomic hybridization].

OBJECTIVE: To investigate the relationship between chromosomal genomic DNA imbalance in medulloblastoma (MB), and the age and gender. METHODS: The gains and losses of chromosomal genomic DNA in 16 MBs were analyzed using comparative genomic hybridization. RESULTS: The gains and(or) losses were found in 15 of the 16 cases. There was not significant difference (P > 0.05) between the total gains (10/16) and losses (11/16). Both of their differences had also no significance between different age and gender groups (P > 0.05). In 15 cases with gains and(or) losses, single-, two-, three- and multi-chromosome genomic DNA imbalances were 3/15, 4/15, 1/15 and 7/15 respectively. Eleven gain zones (+5q, +6q, +7q, +11q, +15q, +17p, +17q, +19q, +20q, +21q, +Xp) and twenty-five loss zones (-1p, -1q, -2p, -2q, -3q, -4p, -6p, -6q, -8p, -8q, -10p, -10q, -11p, -14q, -16p, -16q, -17p, -18p, -18q, -19p, -19q, -20p, -20q, -Xp, -Xq) were detected in those tumors. +7q (6/16), +17q (6/16), -14q (5/16) and -10q (3/16) were the most frequent, but -14q only occurred in the cases of > 10-year-old. CONCLUSIONS: Most MBs have chromosomal genomic DNA imbalances. The frequent imbalance zones are mainly at the long arms of some chromosomes. +7q, +17q, -14q and -10q correlate closely to development of the tumors. -14q is important factor to result in MBs of > 10-year-old group. MB has possibly different molecular genetics subtype.

[Detection of chromosomal imbalance in ependymoma by comparative genomic hybridization].

OBJECTIVE: To investigate genomic DNA imbalances in ependymomas (EDMs) and their correlations with the tumor histological types, grades, locations, patients' gender and age. METHODS: Chromosomal gains and losses in 16 cases of EDM were analyzed using comparative genomic hybridization. RESULTS: Chromosomal regional gain and loss were found in 15 and 13 of 16 EDM cases respectively including totally 24 regional gains and 19 regional losses in all the tumors studied. Both regional gains and losses were mostly seen in myxopapillary EDMs (MPE, WHO grade I), more commonly seen in cellular EDMs (CE, WHO grade II) and tanycytic EDMs (TE, WHO grade II) than in anaplastic EDMs (AE, WHO grade III). Some of the regional gains and losses appeared only in one subtype of MPE, CE, TE and AE cases resulting in development of specific imbalance profiles of certain subtype in these cases. MPE, CE and TE often had +7. Chromosomal +5 occurred only in MPE and CE, and -22q was only seen in CE and TE. AE frequently had +1q, but none had +5, +7, -4q, -19q and -22q. The frequencies of any regional gain or loss were not affected by patients' genders (P > 0.05). Chromosomal +1q and +7p happened predominantly in intracranial EDMs with an averagely onset age of

[Azidothymidine inhibition of telomerase activity and proliferation of TJ905 human glioblastoma cells].

OBJECTIVE: To investigate the pharmacological effects and underlying mechanism of azidothymidine (AZT) on human glioblastoma cells in vitro. METHODS: The telomerase activity of human glioblastoma TJ905 cells was determined by TRAP assay after 24 hrs' incubation with 50, 100, 200 micromol/L AZT and control vehicle solution. Colony formation efficiencies of the cells were recorded. Cells of the 1st, 3rd and 6th generations were harvested, followed by evaluations of cyclin A protein expression by Western blot, cell cycle distribution by flow cytometry, apoptotic level by single cell gel electrophoresis and proliferation index by Ki-67 immunocytochemical staining. RESULTS: AZT inhibited telomerase activity of TJ905 cells. Cyclin A expression levels in the cells treated with 50 and 100 micromol/L AZT were significantly lower than controls (P < 0.01), and down-regulation of the expression was in a dose- and time-dependent manner. Compared with controls, G(0)/G(1) phase cells were obviously decreased (P < 0.05 approximately 0.01) and S phase cells significantly increased (P < 0.05 approximately 0.01) after treatment with 50, 100 and 200 micromol/L AZT. The cell numbers of G(0)/G(1) and S phases at the 1st generation of above three treated groups changed in a dose-dependent manner, whereas S phase cells increases in all AZT treatment groups and G(0)/G(1) phase cell decrease in group treated with 50 micromol/L AZT were also in a time-dependent manner. Both the apoptotic cells of the 1st and 6th generations of all AZT treatment groups were significantly more than controls (P < 0.05 approximately 0.01), their numbers of the 6th generations of the three groups increased with AZT concentration (P < 0.05 approximately 0.01), and all of them were more than the 1st and 3rd generations of the same dosage group (P < 0.05 approximately 0.01). Colony formation efficiencies and Ki-67 labeling indexes of the three AZT treatment groups were distinctly lower than controls (P < 0.01), and they were also decreased with the elevation of AZT concentration and/or the elongation of the incubating time. The difference of any above parameter had no significance among the 1st, 3rd and 6th generations of control group (P > 0.05). CONCLUSION: AZT blocks S/G(2) conversion of TJ905 cells by inhibition of telomerase activity and cyclin A expression, leading to an enhancement of apoptosis and suppression of cell proliferation.

Expression and significance of Survivin mRNA in lung cancer tissue microarray detected by FISH.

OBJECTIVE: To investigate the expression of Survivin mRNA in lung cancer tissue microarray (TMA) by fluorescence in situ hybridization (FISH) method, and determine the role and significance of it in lung cancer genesis and progress. METHODS: The expression of Survivin mRNA was detected by FISH method and TMA technology. Fifty-four cases of lung cancer and 10 cases of normal lung tissue were examined. RESULTS: Survivin mRNA was expressed in 66.7% (36/54) of lung cancer; the positive ratio of lung cancer was significantly higher than that of normal lung tissue (0/10; chi2 = 15.238, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in poor differentiated cancer (20/24, 83.3%) than moderate and well differentiated cancer (16/30, 53.3%; chi2 = 5.40, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in group with lymph node metastasis (27/32, 84.4%) than without lymph node metastasis (9/22, 40.9%; chi2 = 11.084, P < 0.05). The positive ratio of Survivin mRNA was significantly higher in stage III-IV(12/13, 92.3%) than stage I - II (24/41, 58.5%; chi2 = 5.066, P < 0.05). CONCLUSION: Survivin mRNA highly expresses in lung cancer, which is related to the progress and malignant behavior. Survivin may play a promoting role in lung cancer genesis and progress and provide a basis for estimating prognosis and treatment.

[Expression of KAI1, MRP-1, and FAK proteins in lung cancer detected by high-density tissue microarray].

BACKGROUND & OBJECTIVE: The genesis, development, invasion, and metastasis of tumor are closely correlate with alterations of multi-genes. At present, the roles of Kang-ai-1 (KAI1), motility-related protein-1 (MRP-1), and focal adhesion kinase (FAK) in lung cancer have seldom been reported. This study was designed to investigate the roles of KAI1, MRP-1, and FAK in tumorigenesis and development of lung cancer, and their values in diagnosis and predicting the prognosis of lung cancer. METHODS: The expression of KAI1, MRP-1, and FAK proteins in a high-density tissue microarray containing 240 spots were detected by SP immunohistochemistry. RESULTS: The positive rates of KAI1 and MRP-1 were significantly lower in primary lung cancer than in normal lung tissue (25.9% vs. 100%, 42.6% vs. 100%, P<0.05). The positive rate of FAK was significantly higher in primary lung cancer than in normal lung tissue (44.4% vs. 10.0%, P<0.05). The expression of KAI1, FAK, and MRP-1 in primary lung cancer had no correlation with age and gender of the patients, and macroscopic and histological type of tumor, but had correlations with tumor differentiation, clinical stage, and lymph node metastasis. In addition, the expression of MRP-1 had correlation with histological type of tumor; the positive rate of MRP-1 was significantly lower in small cell lung cancer than in non-small cell lung cancer (0 vs. 50.0%, P<0.05). KAI1 expression was negatively correlated to FAK expression (rs=-0.458, P<0.05); MRP-1 expression was positively correlated with KAI1 expression (rs=0.535, P<0.05), and negatively correlated with FAK expression (rs=-0.438, P<0.05). CONCLUSIONS: The abnormal expression of KAI1, MRP-1, and FAK proteins are related to invasion and metastasis of lung cancer. Combined detection of the 3 proteins may be useful in predicting the development and prognosis of lung cancer.