Discovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability.

Myeloid cell leukemia 1 (Mcl-1) protein is a key negative regulator of apoptosis, and developing Mcl-1 inhibitors has been an attractive strategy for cancer therapy. Herein, we describe the rational design, synthesis, and structure-activity relationship study of 3,5-dimethyl-4-sulfonyl-1H-pyrrole-based compounds as Mcl-1 inhibitors. Stepwise optimizations of hit compound 11 with primary Mcl-1 inhibition (52%@30 µM) led to the discovery of the most potent compound 40 with high affinity (Kd = 0.23 nM) and superior selectivity over other Bcl-2 family proteins (>40,000 folds). Mechanistic studies revealed that 40 could activate the apoptosis signal pathway in an Mcl-1-dependent manner. 40 exhibited favorable physicochemical properties and pharmacokinetic profiles (F% = 41.3%). Furthermore, oral administration of 40 was well tolerated to effectively inhibit tumor growth (T/C = 37.3%) in MV4-11 xenograft models. Collectively, these findings implicate that compound 40 is a promising antitumor agent that deserves further preclinical evaluations.

Regulation of Nrf2 by phosphorylation: Consequences for biological function and therapeutic implications.

The transcription factor nuclear factor erythroid-derived 2-like 2 (NRF2) participates in the activation of the antioxidant cytoprotective pathway and other important physiological processes to maintain cellular homeostasis. The dysregulation of NRF2 activity plays a role in various diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. Thus, NRF2 activity is tightly regulated through multiple mechanisms, among which phosphorylation by kinases is critical in the posttranslational regulation of NRF2. For instance, PKC, casein kinase 2, and AMP-activated kinase positively, while GSK-3 negatively regulates NRF2 activity through phosphorylation of different sites. Here, we provide an overview of the phosphorylation regulation pattern of NRF2 and discuss the therapeutic potential of interventions targeting NRF2 phosphorylation.

A comparison of 3D-CTA and 4D-CE-MRA for the dynamic monitoring of angiogenesis in a rabbit VX2 tumor.

PURPOSE: To compare three-dimensional computed tomography angiography (3D-CTA) and four-dimensional contrast-enhanced magnetic resonance angiography (4D-CE-MRA) for the in vivo monitoring of tumor angiogenesis. MATERIALS AND METHODS: VX2 tumors were implanted into the right thigh muscle of 30 New Zealand white rabbits. The animals were randomly assigned to 5 groups, which, respectively, were scanned by 3D-CTA and 4D-CE-MRA on day 4, 7, 10, 13, or 16 after tumor implantation. After scanning, tumors were resected and processed for conventional histology and CD-31 immunohistochemistry. Tumor volume measurements derived from CT and MR imaging were compared with histopathological data. The minimum tumor diameter and the number of new tumor blood vessels detectable by 3D-CTA and 4D-CE-MRA were also compared. RESULTS: There were no significant differences in the tumor volume measurements derived from CT, MR, and histological analysis. The minimum diameter of tumor vessels detectable by 3D-CTA (0.68 ± 0.07 mm) was significantly less than that by 4D-CE-MRA (0.85 ± 0.12 mm) (P=0.005). The number of tumor vessels detected by each imaging method was not significantly different until day 13 after implantation, when 3D-CTA detected a greater number (P<0.001). The morphologic process of tumor angiogenesis was demonstrated dynamically by 3D-CTA and 4D-CE-MRA in vivo. CONCLUSIONS: Tumor angiogenesis can be dynamically monitored in vivo by 3D-CTA and 4D-CE-MRA. Of the two methods, 3D-CTA has better spatial resolution, but 4D-CE-MRA allows temporal resolution of tumor angiogenesis.

Studies of pathology and VEGF expression in rabbit cerebrospinal fluid metastasis: application of dynamic contrast-enhanced MRI.

OBJECTIVE: The objective was to analyze the correlation of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with vascular endothelial growth factor (VEGF) protein expression and to assess the potential application of DCE-MRI to the rabbit cerebrospinal fluid (CSF) metastasis model. METHODS: Thirty New Zealand rabbits were divided into experimental and control groups. In the experimental group, VX2 tumor cells were injected into the subarachnoid space at the plane of cisterna magna in 24 rabbits. In the control group, physiological saline was injected into the subarachnoid space at the plane of cisterna magna in six rabbits. DCE-MRI was performed at multiple time points, and several pharmacokinetic parameters, including K(trans), K(ep) and V(e), were calculated. Also, VEGF levels in plasma and CSF were evaluated by enzyme-linked immunosorbent assay prior to DCE-MRI examination. After DCE-MRI examination, the rabbits were sacrificed, and the corresponding tumor specimens were harvested. Hematoxylin-eosin staining and VEGF immunohistochemical staining were carried out, and VEGF expression in the specimens was evaluated by the immunohistochemical scoring system. RESULTS: Vascular endothelial growth factor positive staining was localized in the cytoplasm and cell membranes of tumor cells, as well as in a subset of epithelial cells. Both VEGF immunohistochemical scores and VEGF expression in CSF and plasma exhibited positive correlations with K(trans) and K(ep) values as demonstrated by rank correlation statistical analysis. CONCLUSIONS: Vascular endothelial growth factor expression in plasma and CSF in the CSF metastasis model was higher than in normal tissues. Therefore, DCE-MRI reliably indicated VEGF expression in the rabbit CSF metastasis model.

Combination of vascular endothelial growth factor antisense oligonucleotide therapy and radiotherapy increases the curative effects against maxillofacial VX2 tumors in rabbits.

PURPOSE: To study the effects of combination of vascular endothelial growth factor (VEGF) antisense oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits. METHODS: We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of 150 µg of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined with radiotherapy group (group C), treated with an injection of 150 µg of VEGF antisense oligonucleotide into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with an injection of 300 µl 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF. RESULTS: In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference was statistically different as compared to that before treatment, on day 3 after treatment and other groups (P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before treatment (P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group C was significantly different from groups A and D respectively (P < 0.05). CONCLUSION: Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve as a reliable technique for in vivo monitoring.

[MRI monitoring of cerebral spinal fluid metastasis in rabbit model].

OBJECTIVE: To establish a rabbit model of cerebral spinal flow metastasis, to analyze the growth rate of tumor, and to investigate the value of MRI in monitoring the biology of tumor compared with pathology. METHODS: Twenty-four New Zealand white rabbits were inoculated with suspension of VX(2) tumor cells in the subarachnoid space via the foramen magnum (experimental group), and 6 rabbits were inoculated with normal saline (control group). MRI examination, including non-enhanced T(1)WI, T(2)WI, and FLAIR sequences and then T(1)WI, FLAIR after dynamic contrast enhanced with Gd-DTPA were done 7 approximately 22 days after inoculation with a 3-day interval. The rabbits were killed after the last MRI scan with their spinal cords, spinal meninges, and tumor taken out to undergo microscopy. RESULTS: (1) MRI plain scan showed that in the experimental group 2 nodi in the medulla and 1 nodes in the cervical spinal cord were found with low signal on T(1)WI and high signal on T(2)WI; and FLAIR imaging showed local lesions with medial signal in 6 rabbits (25%). And no abnormal signs were seen in the control group. (2) MRI enhancement showed that in the experimental group the images of 15 rabbit models were enhanced markedly with irregular thickening of meninges or nodules at the subarachnoid space on T(1)WI, positive signs were confirmed on FLAIR sequence in 16 of the 24 rabbits, and positive signs were noted on DCE-MRI scanning in 18 of the 24 rabbits (75%). In the control group 5 of the 6 rabbits were negative in images. Microscopy showed thickened of meninges and spinal meninges in 20 of the 24 rabbits of the experimental group and spinal cord metastasis in 22 rabbits. No pathological changes were seen in the control group. Statistics showed a CSF metastasis rate of 91.67%. There were significant difference between the plain scan and T(1)WI with enhancement (P < 0.01) and between FLAIR scan and FLAIR enhancement scans. There was a significant difference between T(1)WI and FLAIR enhancement and pathological findings (P < 0.05). There was no significant difference between DCE-MRI method and pathological results (P > 0.05). CONCLUSION: Gd-DTPA enhanced MRI scan sequences has a high sensitivity and specificity and can be used in monitoring the growth of CSF metastasis. There is a disparity between the MRI signs and pathological findings. It is a key that to improve the spatial resolution of machine and to investigate the best method for detecting early metastasis.

[MRI monitoring ultra-small superparamagnetic iron oxide (USPIO) particle labeling C6 rat glioma cells].

OBJECTIVE: To monitor the effects of labeling C6 rat glioma cells with different concentrations of USPIO in vivo and in vitro. METHODS: C6 rat glioma cells of 1 x 10(6), 2 x 10(6) and 1 x 10(7) were labeled with 0 microg/ml, 25 microg/ml, 50 microg/ml USPIO, The signal intensity of cells were evaluated by MRI with T(1)WI, T(2)WI and GRE/30 degrees sequences in vitro. 1 x 10(6) of C6 glioma cells were labeled with 0 microg/ml, 25 microg/ml, 50 microg/ml USPIO and inoculated into the right frontal lobe of 2 rats under stereotaxis apparatus respectively (total 6 rats), Same MRI parameters were used just as above. Iron particle density and cells was measured by HE and Prussian blue stain under microscopy. RESULTS: Different cell population was cultured with 0 microg/ml, 25 microg/ml, 50 microg/ml USPIO about 12 hours. The MR signal intensity of labeling cells were inversely correlated with the different concentration of USPIO groups in T(2)W and GRE/30 degrees imaging (t = 4.19, 3.38, P < 0.05) in vitro. There was an inversely correlation between the labeling cell population and the signal intensity at the same concentration of USPIO (t = 5.16, 2.35, 4.41; P < 0.05). Dyeing degree of labeling cells stained by Prussian blue gradually deepened from 25 microg/ml to 50 microg/ml by microscopy. In vivo MRI can clearly show the cells labeled with 25 microg/ml USPIO. CONCLUSIONS: Iron particle density in the rat glioma cells were gradually increased with the concentration of USPIO. The MR signal intensity was inversely correlated with the cell population at the same condition. 25 microg/ml USPIO labeling rat glioma cells were enough for in vivo monitoring by MRI.