Resistance to topoisomerase II inhibitors in human glioma cell lines overexpressing multidrug resistant associated protein (MRP) 2

For understanding of the resistance to topoisomerase II inhibitors, 50 sublines were isolated as single clones from parental glioma cell lines by exposure to VP-16 or m-AMSA. The quantitative aspects of topoisomerase IIα,multi drug resistant gene (MDR)-1,breast cancer resistance protein (BCRP), and multidrug resistant associated protein (MRP)1-5were studied by Northern blotting in 50 resistant cell lines. By understanding the function of MRP2, we picked up three drug resistant sublines (T98G-m1, T98G-m2, and gli36-VP1) that overexpressed MRP2, but did not overexpress MDR-1 orMRP1-5 except 2. Moreover, in the results of northern blot analysis of mRNA for topoisomerase IIα identical results are observed in parental cell lines and their resistant cell lines, suggesting that alterations in topoisomerase II do not account for the resistance in these cells. To determine whether the cellular sensitivity to anticancer agents was closely associated with the cellular levels of MRP2, we established cell lines with the same levels of MRP2 as their parental cells by introducing the MRP2 antisense expression plasmid into resistant cells. Etoposide (VP-16) accumulation and efflux studies were carried out in the parental cell lines and their drug resistant cell lines. Decreases in the H3-VP-16 accumulation and increases in the efflux were observed in these drug resistant cell lines. In the cytotoxicity assay, these drug resistant cell lines were resistant to multiple topoisomerase II inhibitors with little cross resistance to vincristine, and display efflux of VP-16. We found that the resistant cells transfected with MRP2 antisense cDNA displayed increased cellular levels of VP-16 and enhanced sensitivities to topoisomerase II inhibitors. In this study on the T98G-m1, T98G-m2, and gli36-VP1 cell lines, we showed a high correlation between MRP2 mRNA and VP-16 efflux, suggesting that MRP2 could be a new transporter for topoisomerase II inhibitors

Technetium-99m sestamibi single photon emission computed tomography findings correlated with P-glycoprotein expression in pituitary adenoma.

The aim of this study is to evaluate whether the technetium-99m sestamibi (99mTc-MIBI) single photon emission computed tomography (SPECT) characteristics of pituitary adenomas might be correlated with cavernous sinus invasion, proliferative potential or the multidrug-resistance (MDR-1) gene product P-glycoprotein (Pgp) expression in pituitary adenomas. Fifteen patients with pituitary adenomas, including 10 nonfunctioning adenomas, two prolactinomas, two GH producing adenomas, and one ACTH producing adenomas was investigated for this study. SPECT images with 99mTc-MIBI were acquired 15 minutes (early) and 3 hours (delayed) after injection. The tumor-to-normal brain ratio was calculated both early (ER) and delayed (DR) images. Retention index (RI) was calculated using the following formula : (DR-ER)/ER×100%. The pituitary adenomas specimens were examined by immunohistochemistry using anti-Pgp and MIB-1 monoclonal antibodies. 99mTc-MIBI SPECT findings were not related to MIB-1 labeling index or cavernous sinus invasion. 99mTc-MIBI SPECT RI (-38.55±20.77) of the Pgp-positive group was significantly lower than that (-15.78±19.40) of Pgp-negative group (p=0.0494). No siginificant difference was observed in the ER and DR of 99mTc-MIBI SPECT between Pgp-positive and negative groups. Our study suggests that although99mTc-MIBI SPECT is not useful to evaluate the proliferative potential or cavernous sinus invasion of pituitary adenomas. 99mTc-MIBI SPECT could predict anti-cancer drug resistance related to the expression of Pgp in pituitary adenomas

Reduction of expression of the multidrug resistance protein (MRP)1 in glioma cells by antisense phosphorothioate oligonucleotides

The tumor cells’ acquisition of resistance to multiple drugs due to overexpression of the multidrug resistance protein(MRP) 1gene is one of major obstacles in cancer chemotherapy. We have attempted to reverse the multidrug resistance (MDR) phenotype by treating etoposide resistant glioma cell lines (T98G-VP and Gli36-VP) with MRP1 antisense oligonucleotides. 20-mer phosphorothioate oligodeoxynucleotide (0.3μM), complementary to the coding region in the MRP cDNA sequence, could significantly inhibit the growth of multidrug resistant cell lines, T98G-VP and Gli36-VP, cultured in etoposide containing medium. No such effect was observed for the parental T98G and Gli36 cell lines. Further investigations by the reverse transcription-polymerase chain reaction and immunoblotting revealed that antisense oligomer could result in a reduction in the level of MRP1 mRNA, probably through hindering MRP1 gene transcription. This study demonstrates that the antisense oligonucleotides can increase the sensitivity of the tumor cells to the anticancer drug by decreasing the expression of the MRP gene. This strategy may be applicable to cure cancer patients with MRP mediated MDR phenotype

Differences in the neuronal stem cells survival, neuronal differentiation and neurological improvement after transplantation of neural stem cells between mild and severe experimental traumatic brain injury

We developed a novel protocol for generation and selective amplification of neural progenitor cells regionally specified to the rostral brain but not the spinal cord from mouse embryonic stem cells (ESCs). The neural progenitors could differentiate in vitro and in vivo into many cholinergic and a few GABAergic neurons but rarely into astrocytes. The transplanted neurospheres could survive in the hippocampus (CA3) of animals with mild traumatic brain injury (TBI). Twelve weeks after transplantation (a week after the behavioral test), we found significant cholinergic differentiation recognized as ChAT immunoreactivity in the eGFP+ transplanted cells. Moreover, the grafts contained a few GAD67+cells. However, we barely found GFAP+ astrocytes within the grafts. Furthermore, presynaptic formations of graft-derived neurons were recognized by immunohistochemistry of near the grafts aroundCA3. However, these findings were not observed in severe TBI group. So, we examined NGF, BDNF, and FGF-2 mRNA by RT-PCR in 12 mice including normal, mild TBI and severe TBI group. Increases in the neurotrophic factors’ mRNA were evident in the hippocampus on the ipsilateral side in the mild TBI group. Statistical analysis revealed significant differences between the mild and severe TBI groups. The data also revealed significant differences between the mild TBI and normal groups. The transplanted neurospheres could survive in the mild TBI animals, but not in the severe TBI group

Exogenous Basic Fibroblast Growth Factor and Nerve Growth Factor Enhance Sprouting of Acetylcholinesterase Positive Fibers in Denervated Rat Hippocampus

Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) were administered into the rat brain following unilateral fimbria-fornix transection. Both bFGF and NGF stimulated the sprouting of acetylcholinesterase (AChE) positive fibers in the hippocampus on the lesioned side. Furthermore, a small number of AChE-positive fibers were regenerated even when only the vehicle was administered. Rats treated with NGF as well as control group had only thin fibers, whereas those treated with bFGF had not only thin fibers but also thick fibers. These results indicate that intrinsic NGF is released and acts on damaged neurons directly, while bFGF acts them on directly and/or indirectly after brain injury.</p

Detection of subependymal veins using high-resolution magnetic resonance venography

High-resolution magnetic resonance venography (HR-MRV) of intracranial subependymal veins using a two-dimensional Fourier-transform time-of-flight technique was performed on normal volunteers and clinical cases of cerebral disease. For the pulse sequence, fast-field-echo sequence was used with the following parameters: TR/TE/ flip angle = 34ms/12ms/50deg., 256 x 256 matrix, 1 mm effective slice thickness, 150mm field of view, and one signal acquisition. Sequential vertical coronal sections were taken against the skull base. The anterior septal vein, the medial atrial vein, the anterior caudate vein and thalamostriate vein were detected in all subjects. In all clinical cases, HR-MRV was equal in diagnostic capability to conventional cerebral angiography.</p

Matrix metalloproteinase 2 and 9 expression correlated with cavernous sinus invasion of pituitary adenomas

Object :Matrix metalloproteinase (MMP) 2 and 9 are important for tissue breakdown in the process of tumor invasion. The aim of this study is to evaluate the relationship between the expression of MMP-2, MMP-9,MIB-1 LI and cavernous sinus invasion in pituitary adenomas. Methods : Tissue samples from54 patients with pituitary adenomas were studied. Expression of MMP-2, MMP-9, and MIB-1 labeling index (LI) were evaluated by immunohistochemical method. In sixteen cases, the expression of MMP-2 and MMP-9 mRNA was also examined by RT-PCR assay. Results : Thirty-four patients were women and 20 were men, with a mean age of 49.9 years old (range 18-76 years). There were 12 cases with cavernous sinus invasion, and 42 were noninvasive cases. MMP-2 and MMP-9 score of invasive case (3.9±0.5, 4.1±0.4)were significantly higher than those (2.3±0.2 p＜0.01 2.6±0.2 p＜0.01) without invasion. The MIB-1 LI of this study presented no significantly difference between the invasive and noninvasive pituitary adenomas. The percentage of MMP-2 mRNA/β-actin mRNA and MMP-9 mRNA/β-actin mRNA were also observed significantly higher in invasive pituitary adenomas (68.2±15.3 % 59.7±12.5 %) than noninvasive pituitary adenomas (21.8±8.2 % , p＜0.05 33.3±5.4 % , p＜0.05). Conclusions : Our study suggests that the expression of MMP-2 and MMP-9 may have a value to assess the invasive pituitary adenomas, and proliferation and invasion of pituitary adenomas may present a different mechanism

Neural stem cells transplantation in cortex in a mouse model of Alzheimer's disease

Objective. The goal of this study was to elucidate the effect of neurospheres (NS) on dementia in the mouse model of nucleus basalis of Meynert (NBM) lesion. Methods. Mouse embryonic stem cell (ES) derived neurospheres were transplanted into the frontal association cortex and barrel field of S1 cortex of C57BL/6mice 4weeks after including a lesion of NBM by ibotenic acid, while other healthy mice that received ES cells served as control. Behavioral tests by 8-armradial maze were conducted 8 weeks after transplantation, and double staining of choline acetyltransferase (ChAT), serotonin, amyloid-βprotein (AP) and green fluorescent protein(GFP)12 weeks after transplantation.We found that the neurospheres transplanted into the mouse cortex survived and produced many ChAT-positive neurons and a few serotoninpositive neurons in and around the grafts. The working memory error decreased significantly in the mice grafted with neurospheres. In contrast, the ES cells developed into teratomas in all of the control mice and expressed no neurons, and the working memory deteriorated remarkably. Conclusions. Transplantation of neurospheres, but not ES cells, into the prefrontal and parietal cortices, dramatically alleviated the cholinergic deficits and recent memory disruption in the NBM lesioned mice

Plasma xanthine oxidoreductase activity in Japanese patients with type 2 diabetes across hospitalized treatment

Yusuke Kawachi; Yuya Fujishima; Hitoshi Nishizawa; Hirofumi Nagao; Takashi Nakamura; Seigo Akari; Takayo Murase; Naohiro Taya; Kazuo Omori; Akimitsu Miyake; Shiro Fukuda; Mitsuyoshi Takahara; Shunbun Kita; Naoto Katakami; Norikazu Maeda; Iichiro Shimomura. Plasma xanthine oxidoreductase activity in Japanese patients with type 2 diabetes across hospitalized treatment. J Diabetes Investig. 2020