Inhibition of Akt (ser473) phosphorylation and rapamycin-resistant cell growth by knockdown of mammalian target of rapamycin with small interfering RNA in vascular endothelial growth factor receptor-1-targeting vector

Previously we developed dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) for use in small interfering RNA (siRNA) therapy. In the present study, mammalian target of rapamycin (mTOR) expression in cancer cells was silenced with mTOR-siRNA (simTOR) formulated in TEPA-PCL modified with Ala-Pro-Arg-Pro-Gly (APRPG), a peptide having affinity for vascular endothelial growth factor receptor-1 (VEGFR-1). We investigated the effects of inhibition of mTOR, focusing on the differences between cells treated with simTOR and those with rapamycin in terms of Akt (ser473) phosphorylation and antiproliferative effects. Rapamycin treatment is known to induce Akt (ser473) phosphorylation which attenuates the antiproliferative effects of rapamycin. As a result, knockdown of mTOR did not alter or only slightly reduced Akt (ser473) phosphorylation in phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null (LNCaP and MDA-MB-468 cells) and PTEN-positive (DU 145 and MDA-MB-231) cells, although rapamycin induced Akt (ser473) phosphorylation of these cells. Rapamycin suppressed the growth of PTEN-null cells, in which the rapamycin-sensitive mTOR complex 1 (mTORC1) is excessively activated. On the other hand, rapamycin did not suppress the growth of PTEN-positive cells possibly through a negative feedback mechanism via the rapamycin-insensitive mTOR complex 2 (mTORC2) signaling pathway. In contrast, simTOR significantly suppressed the growth of cancer cells regardless of the presence of PTEN, possibly through inhibition of both mTORC1 and mTORC2. These results indicate that mTOR knockdown using APRPG-TEPA-PCL/simTOR is likely to be an effective strategy for cancer siRNA therapy.     

Detection of portal perfusion abnormalities: comparison of 3 ferucarbotran-enhanced magnetic resonance imaging sequences

OBJECTIVE: To estimate the accuracy, sensitivity, and specificity of 3 ferucarbotran-enhanced magnetic resonance (MR) imaging sequences prospectively for the detection of nontumoral portal perfusion abnormalities. METHODS: Thirty-nine noncirrhotic patients with liver metastases underwent computed tomography during arterial portography (CTAP) and MR imaging comprising T1-weighted gradient recalled echo (GRE), T2-weighted fast spin echo (FSE), and T2*-weighted GRE sequences with and without ferucarbotran. Magnetic resonance images were reviewed by 4 blinded observers for rating based on the confidence scale. The accuracy, sensitivity, and specificity for each sequence were measured by receiver operating characteristic analysis. Contrast-to-noise ratio (CNR) and relative signal-to-noise ratio changes were statistically compared. RESULTS: Thirty-nine nontumoral perfusion defects were observed in 22 patients by CTAP. Receiver operating characteristic analysis showed the accuracy was higher for T2*-weighted GRE (0.884) than for T1-weighted GRE (0.572) and T2-weighted FSE (0.597). T2*-weighted imaging achieved the highest sensitivity (81.4%) and the lowest specificity (86.6%). Postenhanced T2*-weighted imaging achieved the highest CNR (19.3 +/- 9.2). CONCLUSIONS: T2*-weighted imaging was the most accurate and sensitive method for detecting portal perfusion abnormalities compared with T1- or T2-weighted imaging, whereas T1- or T2-weighted imaging is superior in specificity to T2*-weighted imaging during ferucarbotran-enhanced MR imaging.     

Hertwig's Epithelial Cells and Multi-root Development of Molars in Mice

Previously, we focused on multi-root development of the upper and lower molars in mice with the aid of three-dimensional (3D) reconstruction technologies. ICR mice, 3 to 28 days old, were used. The upper and lower jaws including the molars were first used for the collection of micro-computed tomography (μCT) data and, consecutively, were processed to prepare paraffin-embedded sagittal serial sections. Timesequential morphogenesis of the upper and lower first molars was monitored using, μCT-3D models, edited in motion view by superimposing 3D models made at different ages. Histology-based 3D reconstruction in conjunction with immunohistochemistry using cytokeratin as a marker of epithelial cells was also emloyed to visualize the Hertwig's epithelial root sheath (HERS)-guided closure of the pulp chamber floor and the latter-stage disintegration and fate of HERS during root development. The results obtained using 3D models verified that the continuous HERS sheet acts as a guide for root canal segregation and, after disintegration with the underlying dentin formation, HERS-derived epithelial cells have diverse fates, including migration into the periodontium and embedding in the cementum. Recent advances in 3D imaging technology allow us to revisit multiple key issues regarding the developmental morphogenesis and pathological entities of teeth and related tissues.     

Induction of intensive tumor suppression by antiangiogenic photodynamic therapy using polycation-modified liposomal photosensitizer

BACKGROUND: The authors previously observed that antiangiogenic scheduling of photodynamic therapy (PDT) was effective in causing tumor regression through hemostasis. It would thus be expected that photosensitizer entrapped in polycation liposomes (PCLs) would be efficiently taken up in tumor-derived angiogenic vascular endothelial cells due to the strong electrostatic adhesion between the polycation and the plasma membrane, thus resulting in enhanced phototherapeutic efficacy. METHODS: Tumors and angiogenesis were induced by subcutaneous injection of Meth-A sarcoma cells into 5-week-old male BALB/c mice. PDT treatment was performed by an intravenous (i.v.) injection of benzoporphyrin derivative monoacid ring A (BPD-MA)-entrapped liposomes or the PCLs (0.25 mg/kg in terms of BPD-MA), followed by exposure to a laser light of 689 nm with 150 J/cm(2) of fluence 15 minutes post injection. RESULTS: As a result of PDT on angiogenesis-model mice prepared by the dorsal air sac technique, neovascular destruction after laser irradiation was observed when BPD-MA entrapped in PCLs was used. Furthermore, strong suppression of tumor growth was identified by the PCL-mediated PDT treatment along with a prolonged life span for the mice. Destruction of angiogenic vessels and subsequent tumor cell apoptosis were observed after PCL-mediated PDT treatment in an immunofluorescence study. Interestingly, the biodistribution of the injected BPD-MA that was delivered by PCLs indicated invariable photosensitization levels in tumor tissues. CONCLUSIONS: The study revealed that antiangiogenic PDT treatment using a low dose of BPD-MA entrapped in PCLs efficiently induced the destruction of angiogenic vessels and subsequent tumor suppression by vessel occlusion.     

Experimental and clinical evaluation of functional electrical stimulation of the anal sphincter]

To determine the most effective parameter of functional electrical stimulation of the anal sphincter (FES), the present study was carried out in female mongrel dogs anesthetized with alpha-chloralose urethane. When spontaneous and rhythmic micturition contractions of the bladder were present, they were more effectively inhibited by the stimulation with low frequency (5 to 10 Hz). Based on the results of this experiment 18 patients with urge incontinence were treated by maximal electrical stimulation with the following parameters. The duration for each stimulus was 0.2 msec, frequency 5 Hz, amplitude 30 to 150 volts. Every patient received ten treatments for two weeks, each lasting for 30 minutes. A clinical cure for urge incontinence was noted in 12 patients. As for urodynamic studies, FES increased significantly the volumes of the first desire to void (FDV) and maximum desire to void (MDV); however, it did not increase significantly the maximum urethral closure pressure or residual urine volume. Eighteen patients were divided into two groups; an unstable bladder group and a neurogenic bladder group. In the latter, the increases in volumes of FDV and MDV were significant. Second, 18 patients were divided into two groups according to the administration of lack of anticholinergic agents. For subjective symptoms, the rate of improvement of urge incontinence was significantly higher in the group administered the agents. These findings suggested that FES was very useful for the treatment of urge incontinence, with its efficacy augmented by the administration of anticholinergic agents.     

Experimental venous thrombi: MRI characteristics with histopathological correlation

Objectives: The purpose of this study was to evaluate the MRI characteristics of venous thrombus over set time thresholds with histopathological correlation in a porcine model. Methods: Inferior vena cava thrombi were induced in 12 pigs. MRI was performed in three pigs 2 h, 1 day, 3 days and 2 weeks after thrombus induction. Results: The MRI characteristics were analysed in correlation with histopathological findings. The thrombi after 2 hours, which consisted of red blood cells (RBCs), showed isointensity on T(1 )weighted images (T(1)WIs) and hyperintensity on both T(2 )weighted images (T(2)WIs) and diffusion-weighted images (DWIs). The mean apparent diffusion coefficient (ADC) value was 1.93 × 10(-3) mm(2) s(-1). The thrombi after Day 1, which consisted of RBCs and migrating neutrophils at the periphery, showed isointensity on T(1)WIs, slight hyperintensity on T(2)WIs and hypointensity on DWIs. The mean ADC value was 1.62 × 10(-3) mm(2) s(-1) [corrected]. The thrombi after Day 3, which consisted of RBCs and peripheral inflammatory cells including macrophages, showed isointensity with peripheral hyperintense regions on T(1)WIs and hypointensity on both T(2)WIs and DWIs. The mean ADC value was 1.67 × 10(-3) mm(2) s(-1). After 2 weeks, the thrombi, which revealed RBC lysis surrounded by granulation tissues, showed isointensity on T(1)WIs and hyperintensity on T(2)WIs and DWIs. The mean ADC value was 2.48 × 10(-3) mm(2) s(-1). Conclusion: The temporal MRI characteristics seemed to be related to chemical and physical changes in RBC and organisation of granulation tissues. Free radicals generated by macrophages might also be related to some extent.