Grifolic acid causes osteosarcoma cell death in vitro and in tumor-bearing mice.

Grifolic acid is a natural compound isolated from the fungus Albatrellus confluens. In the present study, we assessed the effects of grifolic acid on human osteosarcoma cells. We found that grifolic acid dose- and time-dependently induced cell death in the U-2 OS, MG-63, Saos-2, and 143B human osteosarcoma cell lines. Grifolic acid decreased osteosarcoma cell mitochondrial membrane potential, ATP production, and cellular NADH levels, but did not impact mitochondrial membrane potential in isolated mitochondria from human osteosarcoma cells. Intratumoral injection of grifolic acid also promoted tumor cell death and prolonged survival in nude mice bearing human osteosarcoma xenografts. Grifolic acid had no obvious toxicity in mice, with no histological changes in liver, kidney, lung, or heart, and no changes in blood cell counts or levels of plasma total protein, alanine aminotransferase, or aspartate aminotransferase. These results show that grifolic acid induces osteosarcoma cell death by inhibiting NADH generation and ATP production without obvious toxicity. Intratumoral injection of grifolic acid may be a promising anti-osteosarcoma therapeutic option in patients.

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.

Int7G24A variant of transforming growth factor-beta receptor 1 is associated with osteosarcoma susceptibility in a Chinese population.

The TGF-beta signaling pathway is important in the development and invasion of cancers. Int7G24A is an intronic variant of TGF-beta receptor type 1 and has been shown to be associated with the occurrence of some kinds of cancers. Nevertheless, the association of this polymorphism with osteosarcoma is unknown. In this study, we evaluated Int7G24A variant frequencies in osteosarcoma cases. The case-control study involved 168 osteosarcoma patients and 168 age- and gender-matched controls. The blood samples were obtained, and Int7G24A variant was determined by PCR amplification and DNA sequencing. The odds ratio (OR) and 95% confidence interval (95% CI) for the Int7G24A polymorphism were calculated using unconditional logistic regression adjusted for age and gender. Three analysis models, which are the dominant model, additive model and recessive model, were used to analyze the contribution of Int7G24A variant to osteosarcoma susceptibility. Heterozygotic and homozygotic Int7G24A variants were 33.93 and 6.55% in total 168 cases, while they were 28.57 and 2.98%, respectively, in total 168 controls. The ORs for homozygosity and heterozygosity of Int7G24A allele were 1.56 [95% CI, 0.98-1.83] and 2.89 [95% CI, 1.46-4.92] in additive model. The ORs of Int7G24A genotypes in dominant model and in recessive model were 1.75 [95% CI, 1.21-2.68] and 2.21 [95% CI, 1.34-4.72], respectively. There were significant increases in Int7G24A variants in osteosarcoma cases when compared to control in every three models. Further analysis showed that Int7G24A genotypes were not associated with gender and osteosarcoma location of the cases. However, Int7G24A was significantly increased in the cases less than 20 years old. Moreover, Int7G24A was significantly associated with increased distant metastasis of osteosarcoma. It is concluded that Int7G24A is a polymorphism of TGFBR1 that is associated with the susceptibility and distant metastasis of osteosarcoma.

Association between TGFBR1*6A and osteosarcoma: a Chinese case-control study.

BACKGROUND: TGFBR1*6A is a common hypomorphic variant of transforming growth factor beta receptor 1 (TGFBR1). TGFBR1*6A is associated with an increased cancer risk, but the association of this polymorphism with osteosarcoma remains unknown. We have measured the frequency of TGFBR1*6A variants in osteosarcoma cases and controls. METHODS: Our case-control study is based on 168 osteosarcoma patients and 168 age- and gender-matched controls. Blood samples were obtained and the TGFBR1*6A variant determined by PCR amplification and DNA sequencing. The odds ratio (OR) and 95% confidence interval (95% CI) for the TGFBR1*6A polymorphism were calculated by unconditional logistic regression, adjusted for both age and gender. Three models - dominant, additive and recessive - were used to analyze the contribution of the TGFBR1*6A variant to osteosarcoma susceptibility. RESULTS: Heterozygotic and homozygotic TGFBR1*6A variants represented 50.4% and 6.0% of the 168 cases, whereas the controls had 18. 5% and 1.3%, respectively. ORs for homozygosity and heterozygosity of the TGFBR1*6A allele were 4.6 [95% CI, 2.33-7.97] and 2.9 [95% CI, 1.59-5.34] in the additive model. There were significant increases in the TGFBR1*6A variants in osteosarcoma cases compared to control in all 3 models. Further analysis showed that TGFBR1*6A genotypes were not associated with gender, age, or tumor location. However, TGFBR1*6A was significantly associated with less metastasis. CONCLUSIONS: TGFBR1*6A, a dominant polymorphism of TGFBR1, is associated with increased susceptibility and metastasis spread of osteosarcoma.

[Comparison between Gd-DTPA-enhanced MRI and USPIO-enhanced MRI in diagnosing benign and metastatic cervical lymph nodes in rabbit models].

OBJECTIVE: To compare the diagnostic value of gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA)-enhanced MRI with ultrasmall superparamagnetic iron oxide (USPIO)-enhanced MRI in differentiating reactive hyperplastic lymph nodes from metastatic lymph nodes in rabbit models. METHODS: Reactive hyperplastic cervical lymph node model was established in 18 rabbits as hyperplasia group, and tumor-bearing lymph node model was established in another 18 rabbits as tumor group. For Gd-DTPA-enhanced MRI, T1WI and T2WI were performed on 9 animals of each model, and T1WI was acquired 80 seconds after administration of Gd-DTPA. For USPIO-enhanced MRI, T1WI, T2WI and T2*WI were performed on another 9 animals of each model before and 24 hours after administration of USPIO. MRI images were analyzed and correlated with surgical specimens and pathological results. RESULTS: In the tumor group, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of Gd-DTPA-enhanced MRI were 62.5%, 91.3%, 88.2%, 70.0% and 76.6%, respectively. The corresponding values of USPIO-enhanced MRI were 95.0%, 90.9%, 90.5%, 95.2% and 92.9%, respectively. The sensitivity and accuracy of USPIO-enhanced MRI differ significantly from those of Gd-DTPA-enhanced MRI. In the hyperplasia group, the accuracy of Gd-DTPA-enhanced MRI was 74.2%, while 87.1% for USPIO-enhanced MRI. CONCLUSION: USPIO-enhanced MRI has higher accuracy in diagnosing metastatic lymph nodes than Gd-DTPA-enhanced MRI.

Ghrelin stimulates proliferation of human osteoblastic TE85 cells via NO/cGMP signaling pathway.

Ghrelin regulates bone formation and osteoblast proliferation, but the detailed signaling pathway for its action on osteoblasts remains unclear. In human osteoblastic TE85 cells, we observed the effects and intracellular signaling pathway of ghrelin on cell proliferation using BrdU incorporation method. Ghrelin, at 10(-10)-10(-8) M concentration, significantly increased BrdU incorporation into TE85 cells. The action of ghrelin was inhibited by D: -Lys3-GHRP-6, a selective antagonist of GHS-R. Nitric oxide (NO) scavenger hemoglobin and the NO synthase inhibitor NAME eliminated the stimulatory action of ghrelin on proliferation, while NO donor SNAP and NO synthase substrate L-AME stimulated proliferation of osteoblastic TE85 cells. The cGMP analogue, 8-Br-cGMP, stimulated TE85 cell proliferation, and ghrelin did not enhance proliferation in the presence of 8-Br-cGMP. Inhibition of cGMP production by the guanylate cyclase inhibitor prevented ghrelin-induced osteoblastic TE85 cell proliferation. In conclusion, ghrelin stimulates proliferation of human osteoblastic TE85 cells via intracellular NO/cGMP signaling pathway.

[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.

[Biomechanical study on the composite of allogenic decalcified bone matrix gelatin and bone cement].

OBJECTIVE: To evaluate the biomechanical properties and structural characteristics of various composites of partially decalcified allogenic bone matrix gelatin and bone cement at different ratios. METHODS: According to Urist method, partially decalcified allogenic bone matrix gelatin was prepared and mixed with bone cement at different ratios of 0, 400, 500, and 600 mg/g. Then the comparisons of these composites were performed in microstructure, ultimate compression strength and ultimate bending strength properties. RESULTS: The electronic microscope showed that the bone particles and bone cement were distributed evenly in the composite, irregularly connecting by multiple points; with the increase of bone particles and decrease of bone cement in the composite, there were more and more natural crevices, varying from 100 microns to 400 microns in width, in the biomaterials. Of all the composites with the ratios of 0, 400, 500, and 600 mg/g, the measurements of ultimate compression strength were (71.7 +/- 2.0) MPa, (46.9 +/- 3.3) MPa, (39.8 +/- 4.1) MPa, and (32.2 +/- 3.4) MPa, respectively; and the measurements of ultimate bending strength were (65.0 +/- 3.4) MPa, (38.2 +/- 4.0) MPa, (33.1 +/- 4.3) MPa and (25.3 +/- 4.6) MPa, respectively. CONCLUSION: The composite of partially decalcified allogenic bone matrix gelatin and bone cement has a good biomechanical property and could be easily fabricated and re-shaped, which make it available to be used clinically as an idea bone graft biomaterial.