Critical Role of p38 in Spinal Cord Injury by Regulating Inflammation and Apoptosis in a Rat Model.

STUDY DESIGN: To evaluate the effect of p38 pathway on spinal cord injury (SCI), a rat model of SCI was performed. OBJECTIVE: We determined the effect of p38 on SCI and SCI related inflammation, apoptosis, and autophagy. SUMMARY OF BACKGROUND DATA: SCI is a severe clinical problem worldwide. It is difficult to prevent cell necroptosis and promote the survival of residual neurons after SCI. p38, a class of mitogen-activated protein kinases, its effect on SCI and SCI related inflammation, apoptosis, and autophagy have not been studied very well. METHODS: The rats were randomly divided into the following four groups: the sham-operated (sham) group, the SCI group, the SCI + vehicle group, and the SCI + SB203580 (10 mg/kg) group. The p38 inhibitor SB203580 was administered by oral (10 mg/kg/d) gavage once per day for 14 days. Neurological recovery was assessed using the Basso, Beattie, and Bresnahan locomotion rating scale. Apoptosis, autophagy, and inflammation related proteins were measured by enzyme-linked immunosorbent assay kits or western blotting. RESULTS: Our results showed that p38 was upregulated after SCI from day 3, which was paralleled with the levels of its proteins ATF-2, suggesting an increase in p38 activity. Our results showed administration of SB203580 attenuated histopathology and promoted locomotion recovery in rats after SCI. SB203580 administration significantly inhibited inflammatory cytokines levels as well as the inflammation signaling pathway. SB203580 administration also modulated the apoptosis and autophagy signaling pathway. CONCLUSION: Our findings suggest that p38 inhibitor SB203580 treatment alleviates secondary SCI by inhibiting inflammation and apoptosis, thereby promoting neurological and locomoter functional recovery, thus suggest the important role of p38 in neuronal protection after SCI. LEVEL OF EVIDENCE: N/A.

A dual-targeting liposome conjugated with transferrin and arginine-glycine-aspartic acid peptide for glioma-targeting therapy.

The treatment of a brain glioma remains one of the most difficult challenges in oncology. In the present study a delivery system was developed for targeted drug delivery across the blood-brain barrier (BBB) to the brain cancer cells. A cyclic arginine-glycine-aspartic acid (RGD) peptide and transferrin (TF) were utilized as targeting ligands. Cyclic RGD peptides are specific targeting ligands of cancer cells and TFs are ligands that specifically target the BBB and cancer cells. Liposome (LP) was used to conjugate the cyclic RGD and TFs to establish the brain glioma cascade delivery system (RGD/TF-LP). The LPs were prepared by the thin film hydration method and physicochemical characterization was conducted. In vitro cell uptake and three-dimensional tumor spheroid penetration studies demonstrated that the system could target endothelial and tumor cells, as well as penetrate the tumor cells to reach the core of the tumor spheroids. The results of the in vivo imaging further demonstrated that the RGD/TF-LP provided the highest brain distribution. As a result, the paclitaxel-loaded RGD/TF-LP presents the best antiproliferative activity against C6 cells and tumor spheroids. In conclusion, the RGD/TF-LP may precisely target brain glioma, which may be valuable for glioma imaging and therapy.

[Efficacy of delayed administration of etanercept after spinal cord injury].

OBJECTIVE: To observe the effect of delayed administration of etanercept on the motor function, the expression of apoptosis-related genes and the pathological alterations of spinal cord in vivo in experimental murine model of spinal cord injury (SCI). METHODS: Seventy-two male adult SD rats were randomly divided into 6 groups, which were subjected to SCI induced by the application of vascular clips (force of 70 g) to the dura. Experimental groups (E1, E2, and E3 group) were given administration of etanercept immediately, 1 h, and 8 h after SCI. The control groups (C1, C2, and C3 group) were given administration of saline at the same time as experimental groups. Six rats of each group were killed 24 h after SCI in order to collect the samples for testing the expression of Bax and Bcl-2 by Western blot. The rest were killed 14 d after SCI for observing the pathological alteration using light microscopy. The recovery of motor function was graded using the modified murine Basso, Beattle, and Bresnahan (BBB). RESULTS: (1) The results of the expressions of Bax and Bcl-2 by Western blot: the gray value of the expression of Bax of E1 group was 165.423 ± 2.946, of E2 group 135.391 ± 3.045, of E3 group 108.543 ± 6.999, and of the control group 69.054 ± 0.774, and the gray value of the expression of Bcl-2 of E1 group was 58.854 ± 3.592, of E2 group 84.315 ± 2.138, of E3 group 125.091 ± 2.699, and of the control group 156.304 ± 2.490. (2) The results of BBB score: etanercept given immediately or 1 h after SCI could improve the recovery of the rats. There were significant differences in BBB score 14 d after SCI between E1 group (13.000 ± 1.095) and C1 group (7.167±0.753), E2 group (9.833 ± 1.472) and C2 group (7.000 ± 0.632) while there were no significant difference between E3 group (7.333 ± 0.516) and C3 group (6.833±0.753). (3) The result of histological alteration: histological alterations, such as necrosis, infiltration of lymphocytes and fibroblast and loss of nerve cells, were found attenuated in E1 and E2 groups, compared with C1 and C2 groups. There was no obvious difference between E3 and C3 groups. CONCLUSION: Administration of etanercept may inhibit the apoptosis after SCI, but this kind of effect may be too weak to improve the BBB score and histological alterations obviously when administration of etanercept is delayed 8 h after SCI. The clinical value of etanercept to SCI needs to be further validated.

[Effects of intrathecal injection of methylprednisolone sodium succinate in acute spinal cord injury rabbits].

OBJECTIVE: To investigate the effects and apoptosis of intrathecal injection of Methylprednisolone Sodium Succinate (MPss) for acute spinal cord injury (SCI) in New Zealand rabbits. METHODS: Seventy-two healthy New Zealand rabbits were used for the procedure and were randomly divided into two groups: SCI group and SHAM group, which was both divided into 6 subgroups, such as the vehicle group, the MPss intrathecal injection groups (1.5 mg/kg, 3.0 mg/kg, 6.0 mg/kg group), the MPss intravenous injection group and the combined injection group. TARLOV score was tested daily to evaluate the motor function. The rabbits were sacrificed 7 days after the surgery and the thoracic spinal cord sections and the sacral sections where MPss was injected were harvested for HE and TUNEL staining. Two-Factors Repeated Measures analysis of variance for TARLOV scores tested at various times and One-Way ANOVA analysis of variance for data between groups were used. RESULT: Seven days after surgery in SCI group, there was no statistical difference between the TARLOV scores of intrathecal injection of MPss 3.0 mg/kg group, 6.0 mg/kg group and MPss intravenous injection group (P > 0.05), which were all better than the vehicle group (F = 4.762, P < 0.05). Referring to the lymphocyte infiltration at the injury site in SCI group, there was statistical difference between MPss intrathecal injection 6.0 mg/kg group (1.33 ± 0.21) and the vehicle group (2.67 ± 0.21) (F = 5.793, P < 0.05) and no statistical difference between intrathecal injection of MPss 6.0 mg/kg group and MPss intravenous injection group (P > 0.05). As for the lymphocyte infiltration at the intrathecal injection site in SHAM group, there was statistical difference between MPss intrathecal injection 6.0 mg/kg group (2.50 ± 0.55) and the vehicle group (0.50 ± 0.55) (F = 17.333, P < 0.05). TUNEL staining in SCI group showed statistical difference between MPss intrathecal injection 6.0 mg/kg group (6.3 ± 1.5) and the vehicle group (20.3 ± 2.2) (F = 71.279, P < 0.05). CONCLUSIONS: Intrathecal injection of MPss can improve the functional recovery of lower limb and decrease apoptosis of neuron cells,which can provide same effects as the traditional intravenous injection of MPss in New Zealand rabbits.

Tamoxifen-resistant breast cancer cells possess cancer stem-like cell properties.

BACKGROUND: Cancer stem cells (CSCs) are the cause of cancer recurrence because they are resistant to conventional therapy and contribute to cancer growth and metastasis. Endocrinotherapy is the most common breast cancer therapy and acquired tamoxifen (TAM) resistance is the main reason for endocrinotherapy failure during such therapy. Although acquired resistance to endocrine treatment has been extensively studied, the underlying mechanisms are unclear. We hypothesized that breast CSCs played an important role in TAM-induced resistance during breast cancer therapy. Therefore, we investigated the biological characteristics of TAM-resistant (TAM-R) breast cancer cells. METHODS: Mammosphere formation and tumorigenicity of wild-type (WT) and TAM-R MCF7 cells were tested by a mammosphere assay and mouse tumor xenografts respectively. Stem-cell markers (SOX-2, OCT-4, and CD133) and epithelial-mesenchymal transition (EMT) markers were tested by quantitative real-time (qRT)-PCR. Morphological observation was performed to characterize EMT. RESULTS: After induction of TAM resistance, TAM-R MCF7 cells exhibited increased proliferation in the presence of TAM compared to that of WT MCF7 cells (P < 0.05), indicating enhanced TAM resistance of TAM-R MCF7 cells compared to that of WT MCF7 cells. TAM-R MCF7 cells showed enhanced mammosphere formation and tumorigenicity in nude mice compared to that of WT MCF7 cells (P < 0.01), demonstrating the elevated CSC properties of TAM-R MCF7 cells. Consistently, qRT-PCR revealed that TAM-R MCF7 cells expressed increased mRNA levels of stem cell markers including SOX-2, OCT-4, and CD133, compared to those of WT MCF7 cells (P < 0.05). Morphologically, TAM-R MCF7 cells showed a fibroblastic phenotype, but WT MCF7 cells were epithelial-like. After induction of TAM resistance, qRT-PCR indicated that MCF7 cells expressed increased mRNA levels of Snail, vimentin, and N-cadherin and decreased levels of E-cadherin, which are considered as EMT characteristics (P < 0.05). CONCLUSION: TAM-R MCF7 cells possess CSC characteristics and may be responsible for TAM resistance during breast cancer therapy.

Bioinformatics analysis of breast cancer bone metastasis related gene-CXCR4.

OBJECTIVE: To analyze breast cancer bone metastasis related gene-CXCR4. METHODS: This research screened breast cancer bone metastasis related genes by high-flux gene chip. RESULTS: It was found that the expressions of 396 genes were different including 165 up-regulations and 231 down-regulations. The expression of chemokine receptor CXCR4 was obviously up-regulated in the tissue with breast cancer bone metastasis. Compared with the tissue without bone metastasis, there was significant difference, which indicated that CXCR4 played a vital role in breast cancer bone metastasis. CONCLUSIONS: The bioinformatics analysis of CXCR4 can provide a certain basis for the occurrence and diagnosis of breast cancer bone metastasis, target gene therapy and evaluation of prognosis.

[Effects of high glucose on in vitro invasiveness of human breast cancer cell line MDA-MB-435].

OBJECTIVE: To explore the effects and underlying mechanisms of high glucose on in vitro invasiveness of human breast cancer cell line MDA-MB-435. METHODS: The invasiveness of MDA-MB-435 was determined by Matrigel-coated transwell chambers. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were employed to analyze the cellular expression of matrix metalloproteinase-9/matrix metalloproteinase-2/E-cadherin (MMP-9/MMP-2/E-cadherin) gene/protein. RESULTS: The invasive breast cancer cell numbers of each group (Glu 5.5, 11 and 25 mmol/L) were 50 ± 5, 65 ± 6 and 77 ± 3 respectively. Cellular invasion was dramatically enhanced in the Glu 11 and 25 mmol/L group compared with the 5.5 mmol/L group. The MMP-9/MMP-2 protein expression increased significantly in the Glu 11 and 25 mmol/L groups compared with 5.5 mmol/L group while high glucose (Glu 11 and 25 mmol/L group) down-regulated significantly the E-cadherin mRNA/protein expression. CONCLUSION: High glucose can promote the in vitro invasiveness of human breast cancer cells through the altered expression of MMP-9/MMP-2/E-cadherin.

[Predictive value of (99)Tc(m)-MIBI scintimammography in evaluation of the efficacy of neoadjuvant chemotherapy in patients with operable breast cancer].

OBJECTIVE: To investigate the value of technetium-99m methoxyisobutylisonitrile ((99)Tc(m)-MIBI) imaging in predicting the efficacy of neoadjuvant chemotherapy (NCT) and prognosis in patients with operable breast cancer. METHODS: Sixty five patients with breast cancer underwent (99)Tc(m)-MIBI scintimammography before NCT, and static planar images were taken at 10 min and 180 min after scintimammography. The clearance rate was calculated in each patient, correlation between the clearance rate and efficacy of NCT, and the disease free survival rate were analyzed. RESULTS: The mean clearance rate of 65 patients was (17.4 ± 6.8)%. The efficacy of NCT was 86.2% (CR 4 cases, PR 52 cases, SD 8 cases, and PD 1 case), and the mean clearance rate of patients with good response or poor response of chemotherapy were (15.5 ± 5.0)% and (29.2 ± 3.2)%, respectively. There was a significant difference between the two groups. The average disease free survival rate in the group with low clearance rate was (75.8%, P = 0.046), significantly higher than that in the group with high clearance rate (53.1%). CONCLUSION: Scintimammography of (99)Tc(m)-MIBI may be used to evaluate the efficacy and prognosis of NCT for patients with operable breast cancer.

[Predicative value of 99mTc-MIBI scintimammography in neoadjuvant chemotherapeutic effect of patients with operable breast cancer].

OBJECTIVE: To evaluate the relationship between the clearance rate of technetium-99m methoxyisobutylisonitrile ((99m)Tc-MIBI) in scintimammography and the efficacy of neoadjuvant chemotherapy (NCT) of patients with operable breast cancer. METHODS: Seventy-eight patients with breast cancer underwent (99m)Tc-MIBI scintimammography at pre-NCT. And static planar images were taken at 10 min and 180 min post-scintimammography. The clearance rate was calculated in each patient. And the efficacy of NCT was evaluated after 2 cycles. RESULTS: The clearance rates of patients with a poor or good efficacy of chemotherapy were 24.21% ± 6.38% (n = 14) and 14.13% ± 5.98% (n = 64) respectively. There was significant difference between two groups. And a significant correlation was observed between the efficacy of chemotherapy and the clearance rate of (99m)Tc-MIBI (r = -0.539, P < 0.001). CONCLUSION: The scintimammography of (99m)Tc-MIBI may be employed to evaluate the efficacy of NCT.

Defects in mesenchymal stem cell self-renewal and cell fate determination lead to an osteopenic phenotype in Bmi-1 null mice.

In parathyroid hormone-related protein 1-84 [PTHrP(1-84)] knockin mice, expression of the polycomb protein Bmi-1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi-1(-/-) mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM-MSCs) from Bmi-1(-/-) mice ex vivo in culture. Neonatal Bmi-1(-/-) mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis. Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar-gamma expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1-84) knockin mouse. The efficiency of colony-forming unit fibroblast (CFU-F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase-positive CFU-F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar-gamma expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM-MSCs were associated with upregulation of senescence-associated tumor-suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi-1(-/-) mice suggests that Bmi-1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi-1 maintains self-renewal of BM-MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM-MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi-1 therefore plays a critical role in promoting osteogenesis.

[Correlation of expression of heparanase to angiogenesis and prognosis of breast cancer].

BACKGROUND & OBJECTIVE: Heparanase is a heparan sulfate proteoglycan cleaving enzyme. It helps to degrade extracellular matrix and basement membrane, promote angiogenesis, and accelerate tumor metastasis. This study was to investigate correlation of heparanase expression to angiogenesis and prognosis of breast cancer. METHODS: Immunohistochemistry was used to detect heparanase and microvessel density (MVD) in 120 specimens of infiltrative ductal breast cancer, and 10 specimens of normal breast tissue. Correlation of heparanase expression to clinicopathologic factors and prognosis of breast cancer were analyzed using Chi-square test, t test, Kaplan-Meier method, and log-rank test. RESULTS: Positive rate of heparanase in breast cancer was 65% (78/120), significantly higher than that in normal breast tissue (0, 0/10) (P< 0.05). MVD in breast cancer was 53.84+/-13.45, significantly higher than that in control group (33.32+/-8.55) (P< 0.01). Expression of heparanase positively correlated with tumor size, histological grade, lymph node metastasis, and clinical stage (P< 0.05) of breast cancer, and negatively correlated with 5-year survival rate (P< 0.05). MVD in heparanase positive group was much higher than that in heparanase negative group (P< 0.05), MVD positively correlated with heparanase expression (r=0.358,P< 0.01). CONCLUSION: Heparanase may promote angiogenesis, and may closely correlate with prognosis of breast cancer.