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Chondrosarcoma is the second most common primary malignancy of bone, and one of the most difficult bone tumors to diagnose and treat. It is well known that increased levels of vascular endothelial growth factor-C (VEGF-C) promote active tumor lymphangiogenesis and lymphatic tumor spread to regional lymph nodes. Brain-derived neurotrophic factor (BDNF) is known to promote metastasis in human chondrosarcoma cells. Knowing more about the mechanism of BDNF in VEGF-C expression and lymphangiogenesis in human chondrosarcoma would improve our understanding as how to prevent chondrosarcoma angiogenesis and metastasis, which currently lacks effective adjuvant treatment. Here, we found that BDNF expression was at least 2.5-fold higher in the highly migratory JJ012(S10) cell line as compared with the primordial cell line (JJ012). In addition, VEGF-C expression and secretion was markedly increased in JJ012(S10) cells. Conditioned medium from JJ012(S10) cells significantly promoted migration and tube formation of human lymphatic endothelial cells (LECs), whereas knockdown of BDNF attenuated LEC migration and tube formation by suppressing VEGF-C production in JJ012(S10) cells. Mechanistic investigations indicated that BDNF facilitated VEGF-C-dependent lymphangiogenesis through the MEK/ERK/mTOR signaling pathway. We also showed that microRNA (miR)-624-3p expression was negatively regulated by BDNF via the MEK/ERK/mTOR cascade. Importantly, BDNF knockdown profoundly inhibited tumor-associated lymphangiogenesis in vivo. Further analyses identified that BDNF promoted tumor lymphangiogenesis by downregulating miR-624-3p in human chondrosarcoma tissues. In conclusion, this study is the first to reveal the mechanism underlying BDNF-induced lymphangiogenesis. We suggest that BDNF may serve as a promising therapeutic target for the restriction of VEGF-C-mediated tumor lymphangiogenesis and lymphatic metastasis.

Mitochondrial DNA synthesis requires the supply of thymidine triphosphate (dTTP) independent of nuclear DNA replication. In resting and differentiating cells that withdraw from the cell cycle, mitochondrial thymidine kinase 2 (TK2) mediates thymidine monophosphate (dTMP) formation for the dTTP biosynthesis in mitochondria. However, a thymidine monophosphate kinase (TMPK) that phosphorylates dTMP to form thymidine diphosphate (dTDP) in mitochondria remains undefined. Here, we identified an expressed sequence tag cDNA, which encodes a TMPK with a mitochondrial import sequence at its N‐terminus designated as TMPK2. HeLa cells expressing TMPK2 fused to green fluorescent protein (GFP) displayed green fluorescence in mitochondria. Over‐expression of TMPK2 increased the steady‐state level of cellular dTTP and promoted the conversion of radioactive labeled‐thymidine and ‐dTMP to dTDP and dTTP in mitochondria. TMPK2 RNA was detected in several tissues and erythroblastoma cell lines. We also generated TMPK2 antibody and used it for immunofluorescence staining to demonstrate endogenous expression of TMPK2 in mitochondria of erythroblastoma cells. Finally, we showed that TMPK2 protein expression was upregulated in monocyte/macrophage differentiating cells, suggesting the coordinated regulation of TMPK2 expression with the terminal differentiation program.

We establish a triple-stacking capillary electrophoresis (CE) separation method to monitor methotrexate (MTX) and its eight metabolites in cerebrospinal fluid (CSF). Three stacking methods with different mechanisms were combined and incorporated into CE separation. Complete stacking and sharp peaks were achieved. Firstly, the optimized buffer (60 mM phosphate containing 15% THF and 100 mM SDS) was filled into the capillary, which was followed by the higher conductivity buffer (100 mM phosphate, 2 psi for 45 s). The analytes extracted from CSF were injected at 2 psi for 99.9 s, which provided long sample zones and pH junction for focusing. Finally, the stacking step was performed by sweeping, and separation was achieved by micellar electrokinetic chromatography. The results of the linear regression equations indicated high linearity (r ≥ 0.9981) over the range of 0.5-7 μM. In intra- and inter-batch results, all data of RSD and RE were below 11%, indicating good precision and accuracy of this method. The LODs (S/N = 3) were 0.1 μM for MTX, 7-hydroxymethotrexate (7-OHMTX) and MTX-polyglutamates (MTX-(Glu)(n, n = 2-5)), 0.2 μM for MTX-(Glu)(6), and 0.3 μM for 2,4-diamino-N(10)-methylpteroic acid (DAMPA) and MTX-(Glu)(7). Our method was implemented for analysis of MTX and its metabolites in the CSF, and could be used for evaluation of its curative effects of acute lymphoblastic leukemia patients. The data were also confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results showed good coincidence.