Nonstructural N- and C-tails of Dbp2 confer the protein full helicase activities.

Human DDX5 and its yeast ortholog Dbp2 are ATP-dependent RNA helicases that play a key role in normal cell processes, cancer development, and viral infection. The crystal structure of the RecA1-like domain of DDX5 is available but the global structure of DDX5/Dbp2 subfamily proteins remains to be elucidated. Here, we report the first X-ray crystal structures of the Dbp2 helicase core alone and in complex with ADP at 3.22 Å and 3.05 Å resolutions, respectively. The structures of the ADP-bound post-hydrolysis state and apo-state demonstrate the conformational changes that occur when the nucleotides are released. Our results showed that the helicase core of Dbp2 shifted between open and closed conformation in solution but the unwinding activity was hindered when the helicase core was restricted to a single conformation. A small-angle X-ray scattering experiment showed that the disordered amino (N) tail and carboxy (C) tails are flexible in solution. Truncation mutations confirmed that the terminal tails were critical for the nucleic acid binding, ATPase, and unwinding activities, with the C-tail being exclusively responsible for the annealing activity. Furthermore, we labeled the terminal tails to observe the conformational changes between the disordered tails and the helicase core upon binding nucleic acid substrates. Specifically, we found that the nonstructural terminal tails bind to RNA substrates and tether them to the helicase core domain, thereby conferring full helicase activities to the Dbp2 protein. This distinct structural characteristic provides new insight into the mechanism of DEAD-box RNA helicases.

CRISPR/Cas9-mediated knockout of LW-opsin reduces the efficiency of phototaxis in the diamondback moth Plutella xylostella.

BACKGROUND: Opsins are crucial for animal vision. The identity and function of opsins in Plutella xylostella remain unknown. The aim of the research is to confirm which opsin gene(s) contribute to phototaxis of P. xylostella. RESULTS: LW-opsin, BL-opsin and UV-opsin, were identified in the P. xylostella genome. LW-opsin was more highly expressed than the other two opsin genes, and all three genes were specifically expressed in the head. Three P. xylostella strains, LW-13 with a 13-bp deletion in LW-opsin, BL + 2 with a 2-bp insertion in BL-opsin, and UV-29 with a 5-bp insertion and a 34-bp deletion in UV-opsin, were established from the strain G88 using the CRISPR/Cas9 system. Among the three opsin-knockout strains, only male and female LW-13 exhibited weaker phototaxis to lights of different wavelengths and white light than G88 at 2.5 lx due to defective locomotion, and LW-13 was defective to sense white, green and infrared lights. The locomotion of LW-13 was reduced compared with G88 at 2.5, 10, 20, 60, 80, 100, and 200 lx under the green light, but the locomotion of LW-13 female was recovered at 80, 100 and 200 lx. The defective phototaxis to the green light of male LW-13 was not affected by light intensity, while the defective phototaxis to the green light of female LW-13 was recovered at 10, 20, 60, 80, 100, and 200 lx. CONCLUSION: LW-opsin is involved in light sensing and locomotion of P. xylostella, providing a potential target gene for controlling the pest. © 2021 Society of Chemical Industry.

Seven new chemical constituents from the underground parts of Eupatorium chinense.

Three new sesquiterpenoids (1-3) and four new benzofuran dimers (+)-4 and (-)-4, (+)-5 and (-)-5, and four known benzofuran dimers (+)-6 and (-)-6, (+)-7 and (-)-7 were isolated from the underground parts of Eupatorium chinense. The enantiomers of racemates (±)-4 ~ (±)-7 were separated by chiral HPLC columns, and their absolute configurations were determined by circular dichroism experiments. The structures of all new compounds were elucidated on the basis of their NMR, and MS data as well as by comparison with literature values. The all of the isolated compounds were tested in vitro for their cytotoxic activities against the Caski, MDA-MB-231 and HepG2 cancer cell lines.

[Effects of split combined application of organic-inorganic fertilizers on plant growth, 15N absorption, utilization and loss of Gala apple tree]. / æœ‰æœºæ— æœºè‚¥åˆ†æ¬¡é æ–½å¯¹å˜Žå•¦è‹¹æžœç”Ÿé•¿ã€15N-å°¿ç´ å¸æ”¶åˆ©ç”¨åŠæŸå¤±çš„å½±å“.

Fifteen-year-old 'Gala'/M. hupehensis Rehd. trees and 15N trace technique were used to explore the effects of split combined application of organic-inorganic fertilizers on plant growth, 15N absorption, utilization and loss. The main results were as follows: compared to control, combined application of organic-inorganic fertilizers significantly increased the root-shoot ratio, chlorophyll content, total nitrogen content of leaves and mean fruit mass. The effects of split combined application of organic-inorganic fertilizers treatment were more obvious than one time combined application. Combined application of organic-inorganic fertilizers treatment improved the capacity of 15N derived from fertilizer (Ndff) of different organs, with the effects of split combined application of organic-inorganic fertilizers treatment being more significant. The Ndff value of fruits in different treatments were the highest, followed by leaves, biennial branches, fine roots, large roots and perennial branches, and lowest in trunks. Total N content of plant and 15N-urea utilization rate of the split combined application of organic-inorganic fertilizers treatment were 395.39 g and 28.4% respectively, which were obviously higher than the treatments of one time combined application (342.77 g and 21.1%) and no organic fertilizer application (296.41 g and 14.6%), while 15N loss rate was 51.3%, which was obviously lower than the treatments of one time combined application (57.5%) and no organic fertilizer application (60.6%).

[Effects of moxibustion on the structure and function of blood brain barrier in Alzheimer's disease model rats].

OBJECTIVE: To investigate the effects of moxibustion on the structure and function of blood-brain barrier in Alzheimer's disease (AD) model rats. METHODS: Forty-eight SD rats were randomly divided into 4 groups: normal control group, sham operation group, model group, moxibustion group. Model group and moxibustion group rats were injected with aggregated Aß25-35 by bilateral hippocampus. In the rat model, the sham-operated group was injected with the same amount of normal saline in the bilateral hippocampus, and the normal group was not treated. After successful modeling, the moxibustion treatment was given at 2～3 cm above the Baihui, Shenshu and Yintang points of the moxibustion group rats, each time for 10 min, once a day, continuous treatment for 21 d. The Morris water maze test was used to evaluate the learning and memory ability of rats in each group. The Evans blue method was used to detect the permeability of blood-brain barrier. The ultrastructure of blood-brain barrier was observed under electron microscope. The matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) positive cells in hippocampus were detected by immunohistochemistry. RESULTS: Compared with the sham operation group, the escape latency was significantly increased (P＜0.01), and the space exploration time was decreased (P＜0.01), the learning and memory function in model group was impaired seriously, the Evans blue content in the brain was increased significantly (P＜0.01), the perivascular edema became larger, and the blood-brain barrier structure function was impaired. At the same time, the positive expressions of MMP-2 and MMP-9 in hippocampus were increased significantly (P＜0.01). Compared with model group, the learning and memory ability in moxibustion group rats was enhanced (P＜ 0.05), the content of Evans blue in the brain was decreased (P＜0.05), the degree of perivascular edema was reduced, and the damage of blood-brain barrier was improved. Positive expressions of MMP-2 and MMP-9 in hippocampus were decreased (P＜0.05 or P＜ 0.01). CONCLUSION: Moxibustion can decrease the expressions of MMP-2 and MMP-9, and reduce the damage of the structure and function of blood-brain barrier, thereby improving the learning and memory ability of AD model rats, and exerting therapeutic effects.

Prevalence of human papillomavirus infection among 71,435 women in Jiangxi Province, China.

Cervical cancer is the third most common cancer in women worldwide. Human papillomavirus (HPV) has been identified as an etiological factor for cervical cancer. Data on the prevalence and subtype distribution of HPV infection in Jiangxi Province are incomplete. In this study, we investigated HPV subtype distribution and prevalence in Jiangxi Province between August 1, 2010, and December 31, 2015. A total of 71,435 individuals ranging in age from 16 to 77 years were recruited. Cervicovaginal swabs were collected from each participant, and HPV screening was performed. Our results showed that the HPV prevalence was 22.49% in Jiangxi Province. Overall, 14.99% of individuals were positive for a single HPV type, and 7.49% were positive for multiple types. The most frequently detected low-risk genotypes were HPV-6, and high-risk genotypes were HPV-16, -18, -33, -52, and -58. The prevalence and type distribution of HPV infection exhibits regional and age differences; Yingtan had the highest incidence for high-risk HPV infection (32.00%), and peaks in the frequencies of HPV infections were seen for patients under 20 and over 60 years of age. In conclusion, we present data showing that the HPV prevalence varies significantly with age and regions in Jiangxi Province. These results can serve as valuable reference to guide Jiangxi cervical cancer screening and HPV vaccination programs.

Identification of transcripts involved in digestion, detoxification and immune response from transcriptome of Empoasca vitis (Hemiptera: Cicadellidae) nymphs.

Tea production has been significantly impacted by the false-eye leafhopper, Empoasca vitis (Göthe), around Asia. To identify the key genes which are responsible for nutrition absorption, xenobiotic metabolism and immune response, the transcriptome of either alimentary tracts or bodies minus alimentary tract of E. vitis was sequenced and analyzed. Over 31 million reads were obtained from Illumina sequencing. De novo sequence assembly resulted in 52,182 unigenes with a mean size of 848nt. The assembled unigenes were then annotated using various databases. Transcripts of at least 566 digestion-, 224 detoxification-, and 288 immune-related putative genes in E. vitis were identified. In addition, relative expression of highly abundant transcripts was verified through quantitative real-time PCR. Results from this investigation provide genomic information about E. vitis, which will be helpful in further study of E. vitis biology and in the development of novel strategies to control this devastating pest.

[Analysis of TERT and TIN2 Gene Expression in Patients with Acquired Aplastic Anemia and Their Correlation with Pathogenesis].

OBJECTIVE: To detect the mRNA expression levels of TERT and TIN2 in peripheral blood mononuclear cells of acquired aplastic anemia(AAA) patients, and to explore their correlation with pathogenesis of acquired aplastic anemia. METHODS: Peripheral blood mononuclear cells of 40 cases of AAA including 33 cases of non-severe aplastic anemia(NSAA), 7 cases of severe aplastic anemia (SAA) and 20 subjects as control group were collected to detect mRNA expression of TERT and TIN2 by using real-time quantitative polymerase chain reaction(RT-qPCR), the correlation of TERT and TIN2 mRNA expression levels with classification of peripheral blood cells were analyzed. RESULTS: The expression levels of TERT and TIN2 mRNA in patients with AAA were lower significantly than those in control group (P<0.05), and the SAA (P<0.01). The expression levels of TERT and TIN2 mRNA in patients with SAA were all lower significantly than those in patients with NSAA (P<0.05). The expression levels of TIN2 mRNA in patients with NSAA were lower significantly than those in control group (P<0.01). There were no significant difference in the expression level of TERT mRNA between patients with NSAA and control group (P=0.082). There was significant correlation between the expression level of TERT mRNA and red blood cells count (r=0.437, P=0.029), and hemoglobin level (r=0.522, P=0.007). There was significant correlation between the expression levels of TIN2 mRNA and the lymphocyte percentage (r=-0.404, P=0.045). CONCLUSION: The expression level of TERT mRNA may be associated with the red blood cells and hemoglobin level. The expression level of TIN2 mRNA may be associated with the lymphocyte percentage.

Draft Genome Sequence of the Bacterium Lysobacter capsici X2-3, with a Broad Spectrum of Antimicrobial Activity against Multiple Plant-Pathogenic Microbes.

Lysobacter capsici strain X2-3 was isolated from the wheat rhizosphere in China and exhibits a remarkable capacity to inhibit the growth of multiple pathogens. Here, we report the draft genome sequence of L. capsici strain X2-3 in China.

Prognostic factors influencing clinical outcomes of malignant glioblastoma multiforme: clinical, immunophenotypic, and fluorescence in situ hybridization findings for 1p19q in 816 chinese cases.

Malignant glioblastoma multiforme (GBM) is the most malignant brain tumor and despite recent advances in diagnostics and treatment prognosis remains poor. In this retrospective study, we assessed the clinical and radiological parameters, as well as fluorescence in situ hybridization (FISH) of 1p19q deletion, in a series of cases. A total of 816 patients with GBM who received surgery and radiation between January 2010 and May 2014 were included in this study. Kaplan-Meier survival analysis and Cox regression analysis were used to find the factors independently influencing patient progression free survival (PFS) and overall survival (OS). Age at diagnosis, preoperative Karnofsky Performance Scale (KPS) score, KPS score change at 2 weeks after operation, neurological deficit symptoms, tumor resection extent, maximal tumor diameter, involvement of eloquent cortex or deep structure, involvement of brain lobe, Ki-67 and MMP9 expression level and adjuvant chemotherapy were statistically significant factors (p<0.05) for both PFS and OS in the univariate analysis. Cox proportional hazards modeling revealed that age ≤50 years, preoperative KPS score ≥80, KPS score change after operation ≥0, involvement of single frontal lobe, deep structure involvement, low Ki-67 and MMP9 expression and adjuvant chemotherapy were independent favorable factors (p<0.05) for patient clinical outcomes.

Xanthone glycosides from Swertia bimaculata with α-glucosidase inhibitory activity.

Seven new xanthone glycosides (1-7) were isolated from the n-butanol extract of Swertia bimaculata, together with six known compounds (8-13). Their structures were elucidated on the basis of extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, UV, and IR) and comparison with data reported in the literature. All the compounds were evaluated for their α-glucosidase inhibitory activities in vitro, and compounds 3, 4, and 7 exhibited significant activities to inhibit α-glucosidase. Meanwhile the effects of different substitutions on the α-glucosidase inhibitory activity of xanthone glycosides from S. bimaculata are also discussed.

[Study on Quality Standard for Panax japonicus Rhizome].

OBJECTIVE: To establish a quality standard for Panax japonicus rhizome. METHODS: Ginsenoside Ro and Chikusetsusaponin IVa were used as reference substances in the TLC identification and HPLC method. Additionally, acid insoluble ash and moisture were determined according to the procedures recorded in the Appendix of Chinese Pharmacopeia (2010 edition). RESULTS: The TLC identification with GF, showed a good resolution with clear spots and its optimum developer was the underlayer of chloroform-methanol-formic acid-water = 4.5:1.5: 0.1:0.3. The content of Ginsenoside Ro and Chikusetsusaponin N a were determined by HPLC. The mixture of acetonitrile and water (0.15% phosphate) with gradient elution as the mobile phase was used at a flow rate of 1.0 mL/min, the detection wavelength at 203 nm and the column temperature at 40 °C. The calibration curve was linear in the range of 31.25-2,000 g/mL for Ginsenoside Ro and Chikusetsusaponin IVa (r = 0.9999, respectively). The average recovery was 101.19% and 102.50%, and RSD was 1.59% and 1.80% respectively. The content of Ginsenoside Ro and Chikusetsusaponin IVa was no less than 1.5% respectively. An average content of moisture was 7.36% and acid insoluble ash was 0.84%. CONCLUSION: These methods are producible, sensitive and simple, which can be used to control the quality of Panax japonicus rhizome.

Holotransferrin enhances selective anticancer activity of artemisinin against human hepatocellular carcinoma cells.

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine artemesia annua L. (the blue-green herb) in the early 1970s, which has been confirmed for effectively treating malaria. Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill cancer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransferrin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling (TUNEL) and colony formation assay, respectively. The results showed that artemisinin at various concentrations significantly inhibited growth, colony formation and cell viability of SMMC-7721 cells (P<0.05), likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P<0.01). Our data suggest that treatment with artemisinin leads to inhibition of viability and proliferation, and apoptosis of SMMC-7721 cells. Furthermore, we observed that holotransferrin significantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

Antidiabetic effects of Swertia macrosperma extracts in diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Swertia macrosperma is a traditional folk medicine used for its anti-hepatitis, antipyretic and antidotal effects as "Dida" or "Zangyinchen" in Tibet, Yunnan and Guizhou province for a long time, and it has been reported for its anti-diabetic effects in a Chinese patent. Swertia macrosperma was reported rich in xanthones, iridoids, seco-iridoids and their glycosides, several of which had been documented as potential antidiabetic agents. The objective of this study was to investigate the antidiabetic effect of Swertia macrosperma in diabetic rats. MATERIALS AND METHODS: This study was designed firstly to evaluate the effect of Swertia macrosperma on glucose consumption in HepG2 cells. Based on the result in HepG2 cells, the antidiabetic effect of ethanol extract (EE) and n-butanol extract (BE) were investigated in diabetic rats induced by high fat fed and streptozotocin. The effects of EE and BE on fasting blood glucose, oral glucose tolerance test, serum insulin, glycosylated hemoglobin, serum lipid level, serum antioxidant parameters, glucokinase, glucose-6-phosphatase activities and glycogen content in liver tissue were measured, histology examination of pancreatic tissue was also carried out. RESULTS: After 4 weeks treatment with EE and BE, apparently decreased fasting blood glucose concentrations were observed in these treated groups, compared with the diabetic control groups. Additionally, improvement in serum antioxidant parameters and lipid profile were evidenced clearly. Moreover, EE and BE had effects of protecting the pancreatic ß-cells and stimulating insulin secretion from the remaining pancreatic ß-cells, evidenced by pancreatic histology examination. Increased glucokinase activity and decreased glucose-6-phosphatase activity were observed in liver. CONCLUSION: The results of in vivo and in vitro experiment suggested that EE and BE of Swertia macrosperma had excellent effects on controlling the hyperglycemia and hyperlipidemia in diabetic rats.

Preventive effects of polysaccharides from Liriope spicata var. prolifera on diabetic nephropathy in rats.

This study was undertaken to investigate preventive effects of polysaccharides (LSP) from Liriope spicata var. prolifera on diabetic nephropathy in rats, which were induced by high fat-fed and low-dose streptozotocin (STZ). The levels of fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) in diabetic rats were significantly decreased after treated with LSP for 28 days. Additional, the glucose tolerance of diabetes rats showed improvement after administration of LSP. The results also indicated that LSP were able to normalize hyperlipidemia, ameliorate oxidative stress, improve renal function parameters, inhibit the structural damages of kidney tissue and down-regulate the system of advanced glycation end products - receptor for advanced glycation end products (AGE-RAGE). In conclusion, LSP had potential preventive effects on diabetic nephropathy in diabetic rats.

[Clinicopathologic features of papillary tumors of the pineal region].

OBJECTIVE: To study the clinicopathologic features of papillary tumor of the pineal region (PTPR). METHOD: Three hundred and eighty six cases of pineal region and posterior third ventricle tumors, two newborn and two adult pineal glands were analyzed by HE, PAS and immunohistochemistry of 16 antibodies (EnVision method). RESULTS: Five cases of PTPR were diagnosed with mixed papillary features and densely cellular areas, and included one recurrent case. In the papillary areas, the vessels were lined by one or several layers of cuboidal/columnar cells; the vessel wall was hyalinized. In the densely cellular areas, sheets or nests of tumor cells were seen. The tumor cells of these five cases were immunoreactive to CK, CK8/18, synaptophysin, MAP2, nestin, S-100, and vimentin. Four cases were immunoreactive to NSE and CgA; and 2 cases were immunoreactive to NF. All five cases were negative for EMA, CK5/6, CEA, and NeuN. Ki-67 labeling index ranged from 1% to 6%.Three patients were alive, and the recurrent one died. CONCLUSIONS: PTPR occurs in patients with over a wide age range, from children to adults, and is more commonly found in male than female. PTPR is composed of both papillary and solid areas, characterized by epithelial cytology, and needs to be differentiated from ependymoma. PTPR may originate from the specialized ependymocytes of the subcommissural organ. The prognostic factors are early diagnosis, complete surgical resection and radiotherapy.

[Expression of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia].

This study was aimed to investigate the expression levels of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia, and to explore their relation with onset of acquired aplastic anemia. Peripheral blood mononuclear cells of 40 patients with acquired aplastic anemia and 20 normal subjects as control were collected to detect mRNA expression of TRF1, TRF2 and RAP1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of TRF1 and RAP1 in peripheral blood mononuclear cells of patients with acquired aplastic anemia were significantly higher than that in normal controls (P < 0.05), while the expression level of TRF2 was lower than that in normal controls (P < 0.01). There was significant correlation between TRF2 and RAP1 expressions level (r = 0.522, P = 0.001). It is concluded that the changes in expression levels of TRF1, TRF2 and RAP1 may play a role in the pathogenesis of acquired aplastic anemia.

Hyperglycemia induces the variations of 11ß-hydroxysteroid dehydrogenase type 1 and peroxisome proliferator-activated receptor-γ expression in hippocampus and hypothalamus of diabetic rats.

In this paper, we first observed that there were differences in expressions of 11ß-HSD1 and PPAR-γ, in hippocampi and hypothalami, among constant hyperglycemia group, control group and the fluctuant glycemia group, using Immunohistochemical analysis. However, whether in expression o f 11ß-HSD1 or PPAR-γ, there were no statistic differences between the control group or the fluctuant glycemia group. So, we removed the fluctuant glycemia group, retaining only constant hyperglycemia group and control group, being fed for 8 weeks. After 8 weeks of induction, 11ß-HSD1 expression increased and PPAR-γ expression decreased in the constant hyperglycemia group compared with control group, both in hippocampi and hypothalami, by Western Blot. The constant hyperglycemia group also showed impaired cognition in MORRIS watermaze, lower serum corticosterone level, and higher Serum ACTH concentration after 8 weeks. We inferred that the cognition impairment may be related to the abnormal expression of 11ß-HSD1 and PPAR-γ in central nerves system. As for 11ß-HSD1 is a regulating enzyme, converting the inactive 11-dehydrocorticosterone into the active glucocorticoid corticosterone, thus amplifying GC action in local tissues. It is also well known that high local GC levels can affect the cognitive function. In addition, PPAR-a protective receptor, which is related to cognition.

Expression and significance of P53 protein and MDM-2 protein in human gliomas.

BACKGROUND: P53 is one of the most studied tumor suppressors in the cancer research, and over 50% of human tumors carry P53 mutations. MDM-2 is amplified and/or overexpressed in a variety of human tumors of diverse tissue origin. The aim of this study was to examine the expression of P53 protein and MDM-2 protein in gliomas, and to investigate the relationship between the expression of the two proteins and the histopathological grades of glioma. The relationship between MDM-2 protein expression and P53 protein expression was also analyzed. METHODS: The expression of P53 protein and MDM-2 protein was immunohistochemically detected using monoclonal antibodies in 242 paraffin embedded tissues, including 30 normal brain tissues from patients with craniocerebral injury and 212 tissues from patients with primary glioma (grade I - II group: 5 cases of grade I, 119 cases of grade II; and grade III--IV group: 53 cases of grade III, and 35 cases of grade IV). RESULTS: The P53 positive rate was significantly higher in the glioma groups than in the control group (P < 0.0001). The P53 positive rate was significantly higher in glioma tissues of grade III - IV than in glioma tissues of grade I - II group (P = 0.001). The MDM-2 positive rate was significantly higher in glioma groups than in the control group (P < 0.0001). There was no significant difference in the MDM-2 positive rate between the two glioma groups (P = 0.936). The expression of P53 protein was not related to expression of MDM-2 protein (P = 0.069) CONCLUSIONS: Overexpression of P53 protein might be related to the occurrence and progression of glioma. Overexpression of MDM-2 protein may play an important role in glioma tumorigenesis, but may not be involved in glioma progression. The overexpression of MDM-2 protein was an early event in malignant transformation of glioma. MDM-2 may be a key player in glioma in its own right.

[Regulation mechanism of breast cancer resistance protein by toremifene to reverse BCRP-mediated multidrug resistance in breast cancer cells].

OBJECTIVE: To explore the regulation mechanism of the reversal of breast cancer resistance protein-mediated multidrug resistance by toremifene. METHODS: Two recombinant plasmids (pcDNA3-promoter-BCRP and pcDNA3-CMV-BCRP) were designed to express the wild-type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a CMV promoter as control, respectively. Two recombinant plasmids were transfected into ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines. Four kinds of BCRP expressing cell lines of MCF-7/Promoter-BCRP, MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP were established in which BCRP was promoted by the BCRP promoter and a CMV promoter as control, respectively. The drug resistant cells were treated with toremifene. Then RT-PCR, Western blot, mitoxantrone efflux assays and cytotoxicity assay were performed to detect the reversal function of BCRP by toremifene on the drug resistance cell lines. RESULTS: Toremifene significantly downregulated BCRP mRNA levels in a dose-dependent manner in ERα-positive MCF-7/Promoter-BCRP cells than that of untreated control cells. In MCF-7/Promoter-BCRP cells, toremifene at the dose of 0.1, 1 and 10 µmol/L decreased BCRP mRNA expression by 29.5% (P < 0.05), 68.1% (P < 0.01) and 97.4% (P < 0.01), respectively. After being treated with toremifene and 17ß-estradiol, the BCRP mRNA level in MCF-7/Promoter-BCRP cells was 64.2% ± 1.3%, significantly higher than that of toremifene treatment control cells (3.8% ± 0.2%,P < 0.01). Furthermore, the effect of toremifene on BCRP protein is similar in BCRP mRNA. Toremifene obviously increased the mitoxantrone fluorescence intensity and decreased the efflux activity by 47.3% (P < 0.05) in MCF-7/promoter-BCRP cells when compared with the untreated control, whereas intracellular accumulation of mitoxantrone obviously decreased and the efflux activity increased by 61.5% were observed in combination with 17ß-estradiol when compared with toremifene treatment alone. The results therefore suggested that toremifene reversed mitoxantrone resistance in MCF-7/Promoter-BCRP cells. However, in MCF-7/CMV-BCRP, MDA-MB-231/Promoter-BCRP and MDA-MB-231/CMV-BCRP cells, toremifene or in combination with 17ß-estradiol did not affect intracellular mitoxantrone uptake. CONCLUSION: Taken together, our findings indicate that expression of BCRP is downregulated by toremifene, via a novel transcriptional mechanism which might be involved in the ERE of BCRP promoter through ER-mediated to inactivate the transcription of BCRP gene.