高浓度反全式维甲酸对人舌鳞癌细胞Cal27增殖的影响

目的探讨较高浓度反全式维甲酸对口腔鳞癌Cal27细胞系增殖、凋亡、周期、迁移等生物学过程的影响,为其在口腔鳞癌临床治疗中的作用提供理论支持。方法用不同浓度反全式维甲酸对口腔舌鳞癌Cal27细胞进行刺激,分别用CCK-8法检测增殖,流式检测细胞凋亡、细胞周期,细胞划痕法检测细胞迁移能力。结果反全式维甲酸对舌鳞癌Cal27细胞进行处理,发现不同浓度反全式维甲酸均可抑制Cal27细胞增殖,尤其在100μmol/L时,细胞增殖明显变慢,几乎处于增殖停滞状态,而相同浓度反全式维甲酸对正常皮肤成纤维细胞的增殖未见有显著影响。在迁移实验中,3个浓度梯度均能减慢细胞迁移,60μmol/L与100μmol/L 2个浓度对迁移影响较大,特别是在100μmol/L时,细胞24 h几乎未有任何迁移。凋亡结果显示,不同浓度作用细胞时,均存在凋亡增加的现象,但不同药物浓度之间凋亡细胞数量未见明显差异(P> 0.05)。结论反全式维甲酸可通过增加凋亡,改变细胞周期,进而抑制舌鳞癌细胞的活性,减慢细胞增殖,抑制程度随浓度增高而增强。

Inhibition of all-trans retinoic acid on MDM2 gene expression in astrocytoma cell line SHG-44

Objective To investigate the impact of all-trans retinoic acid （ATRA） on MDM2 gene expression in astrocytoma cell line SHG-44, and to provide basic data for further research on the progression mechanism and gene therapy of human astrocytoma. Methods The differential expressions of MDM2 gene and protein in SHG-44 cells were detected by cDNA microarray and Western blot, respectively, before and after treatment of ATRA. The expressions of MDM2 protein in WHO grade Ⅱ and grade Ⅳ astrocytomas were determined by immunohistochemical streptavidin-peroxidase method. Some differentially expressed genes were selected randomly for Northern blot analysis. Results The intensity ratio of ATRA-treated to untreated SHG-44 cell was 0.37 in the cDNA microarray, suggesting that the expression of MDM2 gene was down-regulated in SHG-44 cells after treatment with ATRA. Some genes differentially expressed in the microarray were confirmed by Northern blot. Western blot demonstrated that the optical density ratios of MDM2 to β-actin in ATRA-treated and untreated SHG-44 were 14.02±0.35 and 21.40±0.58 （t = 24.728, P = 0.000）, respectively, suggesting that the expression of MDM2 protein was inhibited in ATRA-treated SHG-44 cells. Moreover, the percentages of MDM2-positive protein were 24.00% （6/25） and 56.52% （13/23） （x^2 = 5.298, P = 0.021） in WHO grade Ⅱ and grade Ⅳ astrocytomas, respectively, suggesting that the expression of MDM2 protein may increase along with the elevation of astrocytoma malignancy. Conclusion ATRA can inhibit MDM2 gene expression in SHG-44 cells, and MDM2 is related to astrocytoma progression.

The Effect of As_2O_3 on the Epression of Drugresistance Molecule in Malignant Neoplasmas

Objective: To detect the action of arsenic trioxide (As_2O_3) on theexpression of Tumor drug-resistant molecule. Methods: APL cell line MR_2 resistant to all-transretin-oic acid (ATRA ) was put into research, while APL cell line NB_4 was used for control. Theim-munocytochemical assays were used to detect the expressions of P-glycoprotein (P_(gp)) andGluta-thione S-transferase ( GST) . Results: Not only the expression of P_(gp) in MR_2 cell line(30%-40%) was significantly higher than that in NB4 cell line (10%-20% ) (P < 0.001), but also theexpression of GST in MR_2 cell line (60. 4 +- 4.0 )-( 66.5 +- 4.4) was significantly higher thanthat in NB4 cell line (28.3 +- 5.6)-(31.2 +- 5. l)(P < 0.05). As_2O_3 at the concentration of0.5-2.0 μmol/l could significantly decrease the expression of P_(gp) and GSTπ, but could donothing about the expression of GSTα and GSTμ. Conclusion: The lower expression of P_(gp) andGSTπ might be the sensitive indications of frustrating drug-resistance, while GSTα and GSTμ mightnot be the case. ATRA might be the substrates of P_(gp) transmission and GSTπ catalysis .

Role of Acetylated p53 in Regulating the Expression of map2 in Retinoic Acid-induced P19 Cells

Objective To investigate the regulatory mechanisms of acetylated p53 in the expression of microtubule-associated protein-2(MAP2) in neuronal differentiation of P19 cells induced by all-trans retinoic acid(RA).Methods Neuronal differentiation of P19 cells was initiated with 4-day RA treatment.Immunofluorescence,real-time reverse transcription-polymerase chain reaction(RT-PCR) assay,and map2 promoter driven luciferase assay were performed to detect the expression and relative promoter activity of MAP2 in those RA-treated cells.Real-time PCR-based chromatin immunoprecipitation assay(ChIP) was carried out to reveal the specific recruitment of acetylated p53 onto its binding sites on map2 promoter.Results The expression of MAP2 was markedly increased in RA-induced P19 cells.The map2 mRNA increased 34-fold after 4 days of RA treatment and 730-fold 2 days after the treatment,compared with the cells without RA treatment(control).p53 was recruited to the promoter of map2 gene in acetylated form and thereby enhanced its promoter activity.p300/CBP associated factor(PCAF) was found induced in RA-treated cells and enriched in the nucleus,which might contribute to the acetylation of p53 in the regulation of map2 gene.Conclusions Acetylated p53 may participate in regulating the expression of map2 in RA-induced differentiation of P19 cells.PCAF is possibly involved in this process by mediating the acetylation of p53.

All-trans Retinoic Acid Induced the Differentiation of Human Glioma Cells

OBJECTIVE To observe the effect of all-trans retinoic acid （ATRA） on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.METHODS The expression of glial fibrillary acidic protein （GFAP） was detected by immunocytochemistry staining. The mRNA levels of GFAP, retinoid X receptor α（RXRα）, p21 were examined by semi-quantitative RT-PCR analysis. Luciferase activity assay was performed in the COS-7, MO59K cells to measure p21 promoter transcription activity.RESULTS ATRA could significantly enhance the expression and mRNA level of GFAP by immunostaining and RT-PCR （P〈0.05）. Simultaneously, the mRNA levels of RXRα and p21 were remarkably increased in dose-dependent manner by RT-PCR （P〈0.05）. Furthermore, luciferase assay confirmed that ATRA and RXRα could transactivate p21 promoter in COS-7 and glioma cells （P〈0.05）.CONCLUSION ATRA can induce differentiation of human glioma cells. The RXRα and p21 were activated during ATRAinduced differentiation process. This effect may be caused by directly RXRα-induced p21 gene transactivation. Our findings provide novel evidence for the future studies to explore the molecular mechanism of transcriptional regulation for glioma cell differentiation and cellular therapeutic approaches for glioblastoma.

Ecological Research of Former Brown-coal Quarry-the Most Lake in the Czech Republic

The presented article discusses the results of survey of the Most lake, which was created within hydric recultivation of the site of the former brown-coal mine Most Lezaky in northern Bohemia in the Czech Republic. The survey discovered plants which belong to ruderal, wetland, halophilic and xerothermal species based on their life strategy. The discovered endangered species include Carex secalina, Schoenoplectus tabaernemontani, Salsola call subsp. Rosacea and Tetragonolobus maritimus. A total of 350 species of vascular plants were discovered in the study area. During the monitoring, a total of 146 bird species were discovered. The monitored location can be currently considered as a significant gathering place namely for aquatic and wetland bird species during the winter period and the migration period （e.g., Tachybaptus ruficollis, Podiceps cristatus, Anser anser, Anas platyrhynchos, Anas strepera, Fulica atra and various species of Larus sp.） and as a significant area of occurrence of relatively rare species bound to anthropogenetically disturbed areas of mining and post-mining landscape （e.g., Anthus campestris, Anthus pratensis, Motacillaflava, Oenanthe oenanthe, Saxicola rubetra and Saxicola torquata）. Within the monitoring of the water quality, the saprobic index values （S） ranged in 2013 between 1.62 and 1.92.

All-trans retinoic acid in pulmonary vascular structural remodeling in rats with pulmonary hypertension induced by monocrotaline

Objective To determine whether all-trans retinoic acid (atR A) exerts an inhibitory effect on rats with pulmonary hypertension induced by monocrotaline.Methods All rats were given a single subcutaneous injection of either monocrotaline (60 mg/kg) or saline.Monocrotaline-injected rats received either atRA (30 mg· kg-1· day-1) or saline through oral-gastro intubation. On Days 7, 14, 21, and 28 respectively after monocrotaline injection, cardiovascular catheters were inserted to examine the mean pulmonary artery pressure of rats in each group. Meanwhile, the matrix metalloproteinase-1 (MMP-1) mRNA expression and hydroxyproline content in the main pulmonary artery were determined by RT-PCR and chromornetry, respectively.Results The mean pulmonary artery pressure of rats in the model group increased significantly on day 21 and reached a peak on Day 28 compared with the control group (25.7+4.3 mm Hg vs 15.1 ± 1.5 mm Hg and 38.5 ± 6.4 mm Hg vs 16.4 ± 2.0 mm Hg, P ＜ 0.01 ). MMP-1 mRNA overexpression was present on Day 14 (0.72 ± 0.15 vs 0.39 ± 0.08, P ＜ 0.01 ) and was rapidly down-regulated on Day 21 and 28 compared with Day 14, but was still higher than that in the control. The hydroxyoroline content of the main pulmonary artery dropped significantly on Day 14 (4.01 ± 1.13 μg/mg vs 5.10 ± 0.91 μg/mg, P ＜ 0.05)and increased significantly on Days 21 and 28 compared with the control, atRA inhibited the MMP-1 mRNA overexpression from Day 14 to Day 28 and reduced the hydroxyproline content (5.59 ± 0.70 μg/mg vs 7.96 ± 1.13 μg/mg and 7.77 ± 0.96 μg/mg vs 9.93 ± 1.27μg/mg, P ＜ 0.01 ) and the mean pulmonary artery pressure compared with the model group ( 19.6 ± 3.2 mm Hg vs 25.7 ± 4.3 mm Hg and 26.3 ± 4.6 mm Hg vs 38.5±6.4 mm Hg, P＜0.01).Conclusion atRA inhibits MMP-1 overexpression and the accumulation of collagen, which might elicit favorable geometric remodeling in rat pulmonary hypertension induced by monocrotaline.

All-trans retinoic acid as a single agent induces complete remission in a patient with acute leukemia of M_(2a) subtype

Objective To present a special case with the karyotype and molecular marker of acute myeloid leukemia (AML)-M 2 who was induced to complete remission by all-trans retinoic acid (ATRA) alone.Methods A recently hospitalized young female patient with acute leukemia was initially diagnosed as M 3 subtype based on morphological French-American-British (FAB) classification. Karyotype analysis using standard G and R banding techniques and RT-PCR were applied to further define the diagnosis. After primarily cultured bone marrow cells from the iliac aspiration were tested for in vitro induced differentiation, the patient was treated with oral all-trans retinoic acid alone, 60?mg per day until complete remission was achieved. Peripheral blood and bone marrow changes were monitored over the whole treatment course.Results The characteristic chromosomal aberration for M 3, the t(15;17) reciprocal translocation, was not found while a t(8;21) translocation was verified. Furthermore, an amplified product of the AML-1/ETO fusion gene instead of the PML/RARα fusion gene was detected by RT-PCR and the diagnosis was corrected from M 3 to M 2. Primary cultured bone marrow cells can be fully induced to terminal differentiation after 4 days exposure to ATRA. A hematological complete remission was achieved after 40 days treatment with ATRA as a single therapeutic agent, suggesting an alternative pathway mediating ATRA-induced myeloid differentiation. Conclusion A leukemia patient with a subtype other than M 3, such as M 2 in this case, may also be induced to complete remission by the mechanism of ATRA-induced terminal differentiation. This implies that there may be a pathway other than PML/RARα fusion gene product which mediates ATRA-induced myeloid maturation in leukemia cells.

All-trans-retinoic acid generation is an antidotal clearance pathway for all-trans-retinal in the retina

The present study was designed to analyze the metabolites of all-trans-retinal(atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid(atRA) in human retinal pigment epithelial(RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol(atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful(91 vs. 29 pmol/mL), suggesting that atRA conversion ? facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26 b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species(ROS) overproduction, heme oxygenase-1(HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1(PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4-/-RDH8-/-mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4-/-RDH8-/-mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.