Carnosine suppresses human glioma cells under normoxic and hypoxic conditions partly via inhibiting glutamine metabolism.

L-Carnosine (ß-alanyl-L-histidine) is a naturally occurring dipeptide, which has shown broad-spectrum anticancer activity. But the anticancer mechanisms and regulators remain unknown. In this study, we investigated the effects of carnosine on human glioma U87 and U251 cell lines under normoxia (21% O2) and hypoxia (1% O2). We showed that carnosine (25-75 mM) dose-dependently inhibited the proliferation of the glioma cells; carnosine (50 mM) inhibited their colony formation, migration, and invasion capacity. But there was no significant difference in the inhibitory effects of carnosine under normoxia and hypoxia. Treatment with carnosine (50 mM) significantly decreased the expression of glutamine synthetase (GS) at the translation level rather than the transcription level in U87 and U251 cells, both under normoxia and hypoxia. Furthermore, the silencing of GS gene with shRNA and glutamine (Gln) deprivation significantly suppressed the growth, migratory, and invasive potential of the glioma cells. The inhibitory effect of carnosine on U87 and U251 cells was partly achieved by inhibiting the Gln metabolism pathway. Carnosine reduced the expression of GS in U87 and U251 cells by promoting the degradation of GS through the proteasome pathway, shortening the protein half-life, and reducing its stability. Given that targeting tumor metabolism is a proven efficient therapeutic tactic, our results may present new treatment strategies and drugs for improving the prognosis of gliomas.

Synergistic Antitumor Effects on Drug-Resistant Breast Cancer of Paclitaxel/Lapatinib Composite Nanocrystals.

Drug resistance presents serious difficulties for cancer treatment. A combination of paclitaxel (PTX) and lapatinib (LAPA) shows potentials in multiple drug resistant cancers in the clinic, but it is almost impossible to deliver these two drugs to the tumor at the same time with the best proportion by simple co-administration of the respective current formualtions for their different pharmacokinetic profiles. Here composite nanocrystals of PTX and LAPA (cNC) were designed with a ratio of 2:1 (w/w), which was their intracellular ratio at the best synergistic efficacy on a drug-resistant cancer cell line (MCF-7/ADR). Such cNC were prepared using a bottom-up method to achieve a nearly spherical appearance and a narrow size distribution of 95.1 ± 2.1 nm. For nanocrystal stabilization, Polyethylene glycol (PEG) coating was introduced into the cNC via polydopamine (PDA) coating in order to get a PEGylated composite nanocrystal (cNC@PDA-PEG) with nanoscale size (170.5 ± 1.4 nm), considerable drug loading (PTX: 21.33 ± 1.48%, LAPA: 10.95 ± 1.24%) and good stability for at least 4 days in plasma-containing buffers. Differential scanning calorimeter (DSC) and XRD data both indicated the different crystalline states of the cNC as well as the cNC@PDA-PEG in comparison with bulk drugs. In vitro release data showed that PTX and LAPA were gradually and completely released from cNC@PDA-PEG in 3 days, while drug release from bulk drugs or cNC was only 30%. cNC@PDA-PEG also showed negligible hemolysis in vitro. Cellular uptake experiments in the MCF-7/ADR cell line showed that the nanocrystals entered the cells in a complete form through endocytosis and then released the drug in the cell. cNC@PDA-PEG inhibits the growth of this drug-resistant cell more effectively than the unmodified version (cNC). In summary, PEGylated PTX and LAPA composite nanocrystals showed the potential for treament of drug-resistant tumors by simultaneously delivering two drugs to tumor cells with the best proportion.

Kaposiform hemangioendothelioma complicated by Kasabach-Merritt phenomenon: ultrastructural observation and immunohistochemistry staining reveal the trapping of blood components.

Kaposiform hemangioendothelioma (KHE), a borderline tumor of endothelial origin, is associated with Kasabach-Merritt phenomenon, characterized by profound thrombocytopenia and consumptive coagulopathy resulting from the localized intravascular coagulation (LIC) in the tumor. Previous studies have suggested that the trapping of blood components, including platelets, may underlie the LIC in KHE. However, more evidence is needed to support this hypothesis. In this study, one case of a Chinese infant with a KHE in the left arm was complicated by Kasabach-Merritt phenomenon. The tumor was partially resected and the sample was used for ultrastructural observation and immunohistochemistry staining of Glut-1. Ultrastructural observation found the trapping of erythrocytes, platelets, macrophages, and lymphocytes in the slit-like channels of the tumor nodules, and phagocytic vesicles in the cytoplasm of neoplastic cells. Immunohistochemistry staining further showed numerous Glut-1(+) erythrocytes in the channels. In conclusion, our results provided compelling morphological evidence of the trapping of blood components in KHE, which may interpret the LIC in the tumor and subsequent consumptive coagulopathy.

The adipogenesis in infantile hemangioma and the expression of adipogenic-related genes.

Infantile hemangioma, a common benign tumor of infancy, grows quickly in six months to one year after birth, then slowly involutes into fibrofatty tissue childhood. In this study, we observed the adipogenesis in hemangioma and investigated the expression of adipogenic differentiation-related genes. 33 fresh resected hemangioma samples were collected, including 18 proliferating cases (less than one year old), 9 involuting cases (from one to five years old), and 6 involuted cases (more than five years old). The pathological evolution of hemangioma was observed by H-E staining. The expression of Perilipin A was showed by immunohistochemistry staining. The expression and location of PPAR-γ (a key transcription factor in adipogenesis) was displayed by Immunofluorescence staining, with the co-staining of α-SMA and CD31. The expression of adipose differentiation-related genes including PPAR-γ2, LPL, CEBPA, and Perilipin A was detected by Quantitative real time PCR. The results of H-E and Immunohistochemical staining showed the increase of adipose cells as hemangioma developed from the proliferative phase to involuting phase and later to involuted phase. Immunofluorescence staining showed that PPAR-γ wa expressed in the perivascular cells in hemangioma. Quantitative PCR analysis showed a significant increase of PPAR-γ2, LPL, CEBPA and Perilipin A genes' expression in the involuting and involuted heangioma. In conclusion, the PPAR-γ(+) perivascular cells (specific mesenchymal stem cells or pericytes) contribute to the adipogenesis in hemangioma. The siginificantly increased expression of adipogenic differentiation-related genes in the involuting and involuted phase suggested that they played a role in the adipogenesis in hemangioma.

Over-expression of PPAR-γ2 gene enhances the adipogenic differentiation of hemangioma-derived mesenchymal stem cells in vitro and in vivo.

BACKGROUND: Most of infantile hemangiomas involute into fibrofatty tissue in childhood, which indicates adipogenesis during this period. Mesenchymal stem cells (MSCs) contribute to the adipogenesis in IH. In this study, we investigated the effects of overexpression of PPAR-γ2 gene on the adipogenic differentiation of Hemangioma-derived MSCs (Hem-MSCs), and discussed the possibility of targeted therapy via PPAR-γ pathway. METHODS: MSCs were isolated from proliferating hemangioma by their selective adhesion to plastic culture dishes. Recombinant lentivirus with PPAR-γ2 gene were prepared, and used to transfect Hem-MSCs. Transfected cells were cultured in adipogenic medium to observe the differentiation in vitro. And the cells were mixed with Matrigel, then subcutaneously injected into the back of nude mice to observe the differentiation in vivo. RESULTS: In the in vitro tests, Hem-MSCs with overexpression of PPAR-γ2 gene showed enhanced adipogenic differentiation with increased expression of adipogenic-related genes, including PPAR-γ2, ADD1, LPL, and CEBPA genes. In the in vivo tests, Hem-MSCs/Matrigel plugs with overexpression of PPAR-γ2 gene also showed accelerated adipogenesis and time-phased changes of above genes. CONCLUSIONS: Overexpression of PPAR-γ2 gene enhances and accelerates the adipogenic differentiation of Hem-MSCs in vitro and in vivo. The results may provide the preliminary evidences for the targeted therapy of IH via PPAR-γ signal pathway.

Kasabach-Merritt phenomenon in Chinese children: Report of 19 cases and brief review of literature.

Kasabach-Merritt phenomenon (KMP) is life-threatening, charactered by the profound thrombocytopenia and consumptive coagulopathy associated with vascular tumors. The therapy of KMP still remains challenging. In this study, we retrospectively analyzed the clinical data of KMP treated in Nanjing Children's Hospital and Jinling Hospital, China, and brief reviewed the literature on KMP. From Jan. 2005 to Dec. 2014, a total of 19 cases of KMP were enrolled into this study. Laboratory results showed that seven patients had typical disseminated intravascular coagulation (DIC), and others were atypical DIC. CT scanning showed the low-density tumor with obvious intensification in enhanced scanning, and the large distorted arteries in association with the tumor. After the admission, the patients received the infusion of platelets and the applying of dipyridamole, steroids, and other necessary drugs. Eight patients underwent complete surgical removal of the tumor, or partial removal with subsequent chemotherapy of vincristine. Three patients underwent only the chemotherapy of vincristine. Eight patients underwent the intralesional injection of absolute ethanol. Pathological examination showed eighteen samples were kaposiform hemangioendothelioma, and one tufted agioma. In our cases, six patients died from extensive hemorrhage and subsequent multiple organ failure. The others survived. In conclusion, KMP in Chinese children has typical symptoms. Kaposiform hemangioendothelioma is the most frequent vascular tumor associated with KMP. The individual treatments with surgical management, chemotherapy with vincristine, and intralesional injection of absolute ethanol can achieve good results in most of the patients with KMP.

PDGFR-ß (+) perivascular cells from infantile hemangioma display the features of mesenchymal stem cells and show stronger adipogenic potential in vitro and in vivo.

Infantile hemangioma, a common benign tumor of infancy, grows quickly in the first year of life, and then regresses slowly to fibrofatty tissue in childhood. The accumulation of fibrofatty tissue in hemangioma involution indicates adipogenesis during this period. Perivascular cells (PCs) from multiple organs display multi-lineage differentiation, including adipogenesis. So we supposed that PCs in hemangioma may contribute to the adipogenesis in the involution. In this study, PDGFR-ß (+) PCs was isolated from hemangioma tissue (hemangioma-derived perivascular cells, Hem-PCs) by fluorescence-activated cell sorter. In vitro, Hem-PCs showed fibroblast-like morphology. Immunofluorescence staining and flow cytometry showed Hem-PCs expressed MSCs markers CD105, CD90, CD29 and vimentin, pericyte markers α-SMA and PDGFR-ß, stem cell marker CD133, and the adipogenic transcription factor PPAR-γ, but not hematopoietic/endothelial markers CD45, CD34, CD31, and flt-1. In vitro inductions confirmed multi-lineage differentiation of Hem-PCs, especially strong adipogenic potential. Then a murine model was established to observe in vivo differentiation of Hem-PCs by subcutaneous injection of cells/Matrigel compound into nude mice. The results showed Hem-PCs differentiated into adipocytes in vivo. To the best of our knowledge, this is the first study reporting the isolation of multipotential PDGFR-ß (+) PCs from hemangioma, and observing their adipogenic differentiation in vivo. PCs may be the cellular basis of adipogenesis in hemangioma involution, and may be the target cells of adipogenic induction to promote hemangioma involution.

Rosiglitazone accentuates the adipogenesis of hemangioma-derived mesenchymal stem cells induced by adipogenic media.

Hemangioma-derived mesenchymal stem cells (Hem-MSCs) expressed PPAR-γ, the key transcription factor in adipogenesis. We supposed that rosiglitazone, the agonist of PPAR-γ, may promote the adipogenesis of Hem-MSCs. In this study, MSCs were isolated from proliferating hemangioma. Four groups were set up, which were Group A (DMEM-LG/10% FBS), Group B (1 µM rosiglitazone + DMEM-LG/10% FBS), Group C (adipogenic media), and Group D (1 µM rosiglitazone + adipogenic media). Cells were cultured in the medium above. On the day 7 and 14, Oil Red "O" staining and Western blot were performed to detect the cytoplasmic lipid and perilipin A in the cells. The results showed that cytoplasmic lipid appeared in Group C and D, and no cytoplasmic lipid in Group A and B on the day 7 and 14. Analysis of Oil Red "O" staining showed the area of staining in Group D was significantly larger than that in Group C. Analysis of western blot showed no expression of perilipin A in Group A and B, and upregulated expression in Groups C and D, with the greater upregulation in Group D. In conclusion, our study demonstrated that rosiglitazone promoted the adipogenesis of Hem-MSCs initiated by adipogenic media via the activation of PPAR-γ pathway. The results may put forward the possibility of treating hemangioma via PPAR-γ pathway.

[The role of the expression of PPAR-gamma gene in the adipogenesis in hemangioma evolution].

OBJECTIVE: To investigate the role of the expression of PPAR-gamma gene in the adipogenesis in hemangioma evolution. METHODS: Routine immunohistochemistry staining of Perilipin A, the marker antigen of adipocytes, was performed to observe the adipogenesis in hemangioma. Immunofluorescence staining of PPAR-gamma, the important transcription factor in promoting adipogenesis, was carried out to observe its location in hemangioma tissue, with the co-staining of alpha-SMA and CD31. And RT-PCR was used to examine the expression of PPAR-gamma gene in hemangioma in different stages. RESULTS: In the evolution of hemangioma, the number of adipocytes increased continuously. And the tumor was replaced by fibrofatty tissue finally. PPAR-gamma was located in the nuclei of perivascular cell in hemangioma tissue. The expression of PPAR-gamma gene in hemangioma increased in the evolution of hemangioma, but still was lower than that in normal fat tissue from children. CONCLUSION: The expression of PPAR-gamma in the perivascular cells suggests that they may contribute to the adipogenesis in hemangioma involution.

[Orbicular oris muscle flap wrapped by acellular dermis for the treatment of upper lip atrophy in children].

OBJECTIVE: To investigate the treatment of upper lip atrophy resulted by previous therapy. METHODS: From Mar. 2008 to Mar. 2012, 4 cases with upper lip atrophy resulted by radiotherapy and sclerosing agent injection were treated with lower orbicularis oris muscle flap wrapped by acellular dermis. The thickness and height of upper lip were increased to improve the lip atrophy. RESULTS: Primary wound healing was achieved in all the 4 cases. All the patients were followed up for 3 years with obvious improvement and inconspicuous scar. CONCLUSION: The volume of lower lip in children is not sufficient as donor site. The lower orbicularis oris muscle flap wrapped by acellular dermis can effectively improve the lip thickness and vermilion portion of upper lip.

Mesenchymal stem cells in infantile hemangioma reside in the perivascular region.

Infantile hemangioma grows quickly in the first year of life and regresses slowly to fibrofatty tissue during childhood; mesenchymal stem cells (MSCs) have been reported to contribute to this adipogenesis. Recent studies have shown the perivascular origin of MSCs in multiple organs. We hypothesized that MSCs in hemangioma might also reside in the perivascular region. We isolated MSCs from proliferating hemangioma by their selective adhesion to plastic culture dishes. Mesenchymal stem cells from bone marrow (BM-MSCs) and foreskin-derived fibroblasts were used as controls. Flow cytometry and immunofluorescence staining were used to examine their antigen profiles; in vitro induction of multi-lineage differentiation was performed to test their pluripotency. Platelet-derived growth factor R-ß (PDGFR-ß), CD133, and peroxisome-proliferator-activated receptor gamma (PPAR-γ) were selected as the markers to observe MSCs in hemangioma by immunohistochemistry staining, with costaining of CD31 and alpha-smooth muscle actin (α-SMA). Hemangioma-derived MSCs (Hem-MSCs) had fibroblast-like morphology; they expressed the MSC markers CD105, CD90, CD29, and vimentin and did not express the hematopoietic/endothelial markers CD45, CD34, CD31, and flt-1; Hem-MSCs also expressed CD133 and PPAR-γ. Most Hem-MSCs expressed PDGFR-ß and α-SMA; in contrast, the expression of PDGFR-ß and α-SMA in BM-MSCs was very weak. The Hem-MSCs differentiated into adipocytes, osteoblasts, and chondroblasts in vitro. This confirmed their pluripotency. Immunohistochemistry showed the colocalization of PDGFR-ß/α-SMA, CD133/α-SMA, and PPAR-γ/α-SMA in the perivascular region. MSCs were successfully obtained from proliferating hemangioma, revealing the perivascular origin of MSCs in hemangioma.

[Segmental monobloc osteotomy and bi-directional distraction for the treatment of Crouzon syndrome in an infant].

OBJECTIVE: To discuss the segmental monobloc osteotomy and bi-directional distraction for the treatment of Crouzon syndrome in an infant. METHODS: A 9-month-old female infant underwent monobloc osteotomy through combined intra- and extra-cranial way. The facial skull was divided into frontal, orbital and maxillary segments. The external distractor was used to move the frontal segment, orbital segment and midface forward. The internal distractor was used to move the maxilla downward. The consolidation lasted for 3 months after distraction. RESULTS: The osteotomy and distraction were successfully completed. The distraction distance reached 18 mm, showing by distractor. The real distraction distance of facial bone was 12 mm, documented by 3-D image. The skull deformity and severe depression of midface improved a lot. The exophthalmos and underbite were corrected. The obstructive sleep apnea also improved markedly. However, the downward movement of maxilla was limited. CONCLUSIONS: Monobloc osteotomy with external distractor, as well as maxillary distraction, could be used for Crouzon syndrome in infant. It is safe and effective method which can be performed in an early age for Crouzon syndrome with obstructive sleep apnea.

[Diagnosis and treatment of children sinus pericranii].

OBJECTIVE: To discuss the diagnosis and treatment of children sinus pericranii (SP). METHODS: From Jan. 2000 to Dec. 2008, 12 cases of SP were treated. The clinical data and CT results were studied. If the SP had no communication with the intracranial vessels, DSA was performed to know its venous drainage. The operation procedures included excision of bump, followed by hemostasis. Then the local flap was used to cover the cranial defect. RESULTS: 12 cases all had scalp bump whose size was changed with body position. Three-dimensional CT showed a crater-like depression and multiple honeycomb diploic holes in the skull. 6 cases with venous malformation received DSA. 12 patients were operated. CONCLUSIONS: SP can be diagnosed with typical CT results and symptoms. DSA should be performed if the communication with the intracranial vessels is existed. The main methods include operation, embolization and reserved observation. Medicine injection is not recommended due to the high risk.

[Management of congenital scalp defects in infants].

OBJECTIVE: To investigate the management of congenital scalp defects in infants. METHODS: From 1996 to 2008, 6 infants with congenital scalp defects were treated with dressing change, flap transposition, or tissue expansion combined with skull defect reconstruction. RESULTS: Parietal scalp defects in 6 cases were healed after treatment. 5 cases were followed up for 3 months to one year. 2 cases had scalp alopecia in some areas. The scar was inconspicuous in the other 3 cases. CONCLUSIONS: Dressing change is suitable for small scalp defect, while flap transposition should be used for medium defect. For large full-thickness cranial defect, dressing change and tissue expansion should be performed at the first stage, followed by skull defect reconstruction and expanded flap transposition.

[Treatment of airway obstruction with mandibular distraction osteogenesis in Pierre Robin syndrome].

OBJECTIVE: To investigate the feasibility of mandibular distraction osteogenesis (MDO) in the treatment of airway obstruction in Pierre Robin syndrome (PRS). METHODS: From 2007 to 2009, 8 newborns with PRS were treated with MDO. The mandibular distractors were fixed after bilateral oblique mandibular osteotomy. The distraction was started one day after operation, three times a day. The distraction distance was 1.2 mm per day until it reached about 12 -20 mm (mean 15 mm). RESULTS: The distraction was successfully completed in 8 cases with no complication. The distraction distance reached 15 mm, as we expected. The discontinuity cyanosis, inspiratory dyspnea, dystithia were improved after distraction. CONCLUSIONS: MDO is feasible and safe for the treatment of airway obstruction in PRS.