Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.     

Inhibition of CD95-mediated apoptosis through beta 1 integrin in the HSG epithelial cell line

The HSG cell line serves as a model for salivary gland epithelial progenitor cell differentiation. In order for a progenitor cell to differentiate, the cell must maintain viability within its niche. Studies were designed to elucidate the mechanism for integrin-mediated HSG cell survival. HSG cells, grown on Matrigel®, were resistant to CD95-mediated apoptosis. Western blot analysis showed that Matrigel® induced the expression of bcl-2, bcl-xL, p63, and ΔNp63. This induction occurred by as early as 2 hrs and remained for 24 hrs. CD95-mediated apoptosis resistance was dependent, however, upon the expression of the bcl-2 family. Furthermore, Matrigel® induced bcl-2 family expression was dependent on the transactivation of the EGF receptor pathway since PD98059 and AG1478 inhibited Matrigel® induced bcl-2 family expression and caused HSG cells to be sensitive to CD95-mediated apoptosis. Activation of the EGF receptor pathway, by itself, however, was not sufficient to inhibit apoptosis. Blocking antibody showed that bcl-2 family expression was mediated through β1 integrin. These studies show that salivary progenitor epithelial cell survival is integrin dependent and involves the transactivation of the EGF receptor pathway.     

Arterial structure and function in mild primary hyperparathyroidism is not directly related to parathyroid hormone, calcium, or vitamin D

Objective

Elevated levels of calcium and parathyroid hormone (PTH), characteristics of primary hyperparathyroidism (PHPT), may be associated with cardiovascular morbidity and mortality in the general population. We evaluated the possible vascular effects of these risk factors in patients with mild PHPT by using standard methods and new imaging techniques.

Design

A prospective case-control study.

Subjects and Methods

Forty-eight patients with mild PHPT without any known cardiovascular risk factors were studied at baseline and at one year after parathyroidectomy (PTX) in comparison with 48 healthy age- and gender-matched controls. We measured biochemical variables, augmentation index (AIx), aortic pulse wave velocity (PWV_ao), radial (IMT_rad) and common carotid artery (IMT_cca) intima media thicknesses, and the grayscale median (IM-GSM) of the latter.

Results

No significant differences were observed between PHPT patients and controls at baseline for AIx (28.6±12.2 vs. 27.7±12.8%), IMT_rad (0.271±0.060 vs. 0.255±0.053 mm), IMT_cca (0.688±0.113 vs. 0.680±0.135 mm), or IM-GSM (82.3±17.2 vs. 86.5±15.3), while PWV_ao was slightly higher in patients (8.68±1.50 vs. 8.13±1.55, p<0.05). Systolic blood pressure (SBP), calcium, and PTH were higher in patients compared with controls, and decreased after PTX, while vitamin D was lower in patients and increased after PTX. While AIx, PWV_ao, IMT_rad, and IMT_cca were related to SBP, neither correlated to vitamin D levels. Only PWV_ao correlated weakly to plasma PTH (r = 0.29, p<0.01) and ionized calcium (r = 0.22, p<0.05) but showed no relation when age and SBP were adjusted for.

Conclusion

We found normal arterial function despite high calcium, PTH, and low vitamin D levels, in patients with mild PHPT without cardiovascular risk factors. The cardiovascular risk associated with low vitamin D and/or high PTH and calcium levels may be explained by their coupling to blood pressure and other risk factors rather than direct effects on arterial structure.     

An Investigation on the Effect of Polyethylene Oxide Concentration and Particle Size in Modulating Theophylline Release from Tablet Matrices

Polyethylene oxide has been researched extensively as an alternative polymer to hydroxypropyl methylcellulose (HPMC) in controlled drug delivery due to its desirable swelling properties and its availability in a number of different viscosity grades. Previous studies on HPMC have pointed out the importance of particle size on drug release, but as of yet, no studies have investigated the effect of particle size of polyethylene oxide (polyox) on drug release. The present study explored the relationship between polymer level and particle size to sustain the drug release. Tablets produced contained theophylline as their active ingredient and consisted of different polyethylene oxide particle size fractions (20–45, 45–90, 90–180 and 180–425 μm). It was shown that matrices containing smaller particle sizes of polyox produced harder tablets than when larger polyox particles were used. The release studies showed that matrices consisting of large polyox particles showed a faster release rate than matrices made from smaller particles. Molecular weight (MW) of the polymer was a key determining step in attaining sustained release, with the high MW of polyox resulting in a delayed release profile. The results showed that the effect of particle size on drug release was more detrimental when a low concentration of polyox was used. This indicates that care must be taken when low levels of polyox with different particle size fractions are used. More robust formulations could be obtained when the concentration of polyox is high. Differential scanning calorimetry (DSC) traces showed that particle size had no major effect on the thermal behaviour of polyox particles.KEY WORDS: DSC traces, particle size, polyox, sustained release, theophylline     

Diffusion MRI and Novel Texture Analysis in Osteosarcoma Xenotransplants Predicts Response to Anti-Checkpoint Therapy

Combinations of targeted drugs have been employed to treat sarcomas, however, response rates have not improved notably, therefore emphasizing the need for novel treatments. In addition, imaging approaches to assess therapeutic response is lacking, as currently measurable indices, such as volume and/or diameter, do not accurately correlate with changes in tumor biology. In this study, quantitative and profound analyses of magnetic resonance imaging (MRI) were developed to evaluate these as imaging biomarkers for MK1775 and Gem in an osteosarcoma xenotransplant model at early time-points following treatment. Notably, we showed that Gem and Gem+MK1775 groups had significantly inhibited tumor growth by day 4, which was presaged by elevations in mean ADC by 24 hours post treatment. Significant differences were also observed at later time points for the Gem+MK1775 combination and MK1775 therapy. ADC distribution and entropy (randomness of ADC values) were also elevated by 24 hours following therapy. Immunohistochemistry demonstrated that these treatment-related increases in ADC correlated with apoptosis and observed cell condensations (dense- and exploded bodies). These findings underline the role of ADC as a quantitative imaging biomarker for therapy-induced response and show promising clinical relevance in the sarcoma patient population.     

Demonstration of a sucrose-derived contrast agent for magnetic resonance imaging of the GI tract

A scaffold bearing eight terminal alkyne groups was synthesized from sucrose, and copies of an azide-terminated Gd–DOTA complex were attached via copper(I)-catalyzed azide-alkyne cycloaddition. The resulting contrast agent (CA) was administered by gavage to C3H mice. Passage of the CA through the gastrointestinal (GI) tract was followed by T₁-weighted magnetic resonance imaging (MRI) over a period of 47 h, by which time the CA had exited the GI tract. No evidence for leakage of the CA from the GI tract was observed. Thus, a new, orally administered CA for MRI of the GI tract has been developed and successfully demonstrated.     

Dose-Dependent Effects of Astaxanthin on Ischemia/Reperfusion Induced Brain Injury in MCAO Model Rat

Excitotoxicity and oxidative stress are central to the pathology of the nervous system, and inhibition of excitotoxicity induced by glutamate is one of the therapeutic goals determined for stroke. The present study aimed to investigate the effects of Astaxanthin, a potent natural antioxidant, on complications caused by acute cerebral stroke. In this research, 60 male Wistar rats were used which were divided into 5 groups as follow: (1) the sham group (vehicle), (2) the ischemic control group (vehicle), and the ischemic groups treated by Astaxanthin with doses of 25, 45, and 65 mg/kg. In the ischemic groups, ischemic model was performed by middle cerebral artery occlusion (MCAO) method, and the Astaxanthin administration was carried out after the artery occlusion and before opening the artery. The obtained results indicated that Astaxanthin could significantly reduce stroke volume, neurological deficits, and lipid peroxidation. Moreover, it was able to restore total oxidant status (TOS) and caspase 3 level to the normal level. The activity of antioxidant enzyme glutathione peroxidase (GPX), and the expression of catalase, GPx and nuclear factor kappa B (NFκb) genes, which were reduced after ischemia, were increased. This phenomenon was particularly pronounced for glutamate transporter 1 (GLT-1). Furthermore, Astaxanthin decreased the augmented pro-apoptotic gene Bax and restored the reduced Bcl2 expression to the normal level. Significant effects on the P53 and PUMA expression were not observed. Overall, the medium dosage of Astaxanthin appears to be more effective in reducing the complications of ischemia, particularly on our major study endpoints (stroke volume and neurological defects). Longer studies with a more frequent administration of Astaxanthin are required to better understand the precise mechanism of Astaxanthin.