Lactate dehydrogenase A promotes nasopharyngeal carcinoma progression through the TAK1/NF-κB Axis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor that originates in the nasopharyngeal mucosa and is common in China and Southeast Asian countries. Cancer cells reprogram glycolytic metabolism to promote their growth, survival and metastasis. Glycolysis plays an important role in NPC development, but the underlying mechanisms remain incompletely elucidated. Lactate dehydrogenase A (LDHA) is a crucial glycolytic enzyme, catalyzing the last step of glycolysis. This study aims to investigate the exact role of LDHA, which catalyzes the conversion of pyruvate into lactate, in NPC development. METHODS AND RESULTS: The western blot and immunohistochemical (IHC) results indicated that LDHA was significantly upregulated in NPC cells and clinical samples. LDHA knockdown by shRNA significantly inhibited NPC cell proliferation and invasion. Further knockdown of LDHA dramatically weakened the tumorigenicity of NPC cells in vivo. Mechanistic studies showed that LDHA activated TGF-ß-activated kinase 1 (TAK1) and subsequent nuclear factor κB (NF-κB) signaling to promote NPC cell proliferation and invasion. Exogenous lactate supplementation restored NPC cell proliferation and invasion inhibited by LDHA knockdown, and this restorative effect was reversed by NF-κB inhibitor (BAY 11-7082) or TAK1 inhibitor (5Z-7-oxozeaenol) treatment. Moreover, clinical sample analyses showed that LDHA expression was positively correlated with TAK1 Thr187 phosphorylation and poor prognosis. CONCLUSIONS: Our results suggest that LDHA and its major metabolite lactate drive NPC progression by regulating TAK1 and its downstream NF-κB signaling, which could become a therapeutic target in NPC.

GFAT1-linked TAB1 glutamylation sustains p38 MAPK activation and promotes lung cancer cell survival under glucose starvation.

Reprogrammed cell metabolism is deemed as one of the hallmarks of cancer. Hexosamine biosynthesis pathway (HBP) acts as an "energy sensor" in cells to regulate metabolic fluxes. Glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of HBP, is broadly found with elevated expression in human cancers though its exact and concrete role in tumorigenesis still remains unknown and needs further investigation. P38 mitogen-activated protein kinase (MAPK) is an important component of stress-signaling pathway and plays a critical role in cell fate decision, whereas the underlying mechanism of its activation under nutrient stress also remains elusive. In this study, we show that glucose deprivation induces the interaction of GFAT1 with transforming growth factor ß-activated kinase 1 binding protein 1 (TAB1) in a TAB1 S438 phosphorylation-dependent manner. Subsequently, the binding of GFAT1 to TAB1 facilitates TTLL5-GFAT1-TAB1 complex formation, and the metabolic activity of GFAT1 for glutamate production further contributes to TTLL5-mediated TAB1 glutamylation. In consequence, TAB1 glutamylation promotes the recruitment of p38α MAPK and thus drives p38 MAPK activation. Physiologically, GFAT1-TAB1-p38 signaling promotes autophagy occurrence and thus protects tumor cell survival under glucose deficiency. Clinical analysis indicates that both GFAT1 and TAB1 S438 phosphorylation levels correlate with the poor prognosis of lung adenocarcinoma patients. These findings altogether uncover an unidentified mechanism underlying p38 MAPK signaling regulation by metabolic enzyme upon nutrient stress and provide theoretical rationality of targeting GFAT1 for cancer treatment.

Effects of Salvia miltorrhiza in neural differentiation of rat mesenchymal stem cells with optimized protocol.

AIM OF THE STUDY: The present study was aimed to explore the effects of Salvia miltorrhiza in inducing rMSCs to differentiate into functional neurons. MATERIALS AND METHODS: rMSCs were isolated and cultured in vitro, then Salvia miltorrhiza was added to induce rMSCs to differentiate repeatedly for 5 times with an optimized protocol, and neurophysiological functions such as action potential, endocytosis and exocytosis of the induced cells were investigated. RESULTS: About 98% of rMSCs expressed markers related to neural stem cells after treatment with preinduction medium, but they remained fibroform, the classical morphological state of MSCs, after exposure to induction medium for 2h, and the induced cells showed a neural shape. Next, fetal bovine serum (FBS) was added into the induction medium, transforming the neuron-like cells into fibroform cells. Finally, after exposure to induction medium, the cells could be transformed into neuron-like cells again. After the procedure was repeated 5 times, the induced cells displayed a classical neural shape and more than 95% of them expressed neural markers, including TUJ-1, NF and synaptophysin. Furthermore, the induced cells displayed neurophysiological functions, as characterized by action potential, endocytosis and exocytosis in response to a solution with a high concentration of potassium (K(+)). CONCLUSION: Salvia miltorrhiza can induce rMSCs to differentiate into neurons with neurophysiological functions efficiently by an optimized protocol.

In vivo MR imaging of magnetically labeled mesenchymal stem cells transplanted into rat liver through hepatic arterial injection.

OBJECTIVE: The aim of this study was to evaluate in vivo magnetic resonance imaging (MRI) for tracking the magnetically labeled mesenchymal stem cells (MSCs) transplanted into rat liver through hepatic arterial injection. MATERIALS AND METHODS: MSCs, harvested from the bone marrow of Wistar rats and expanded by the adhesion method, were labeled with both Feridex and 4',6-diamidino-2-phenylindole (DAPI). Cell transplantation was performed by injection of 1 x 10(6) labeled cells (n = 20) or unlabeled cells (n = 10) via hepatic artery into rat livers treated with 2% carbon tetrachloride to induce acute liver necrosis. MR imaging was performed on a clinical 1.5 T MR scanner with a T(2)*-weighted gradient-echo sequence immediately before and at 1 h, 3 days, 7 days and 14 days after transplantation, and the signal-to-noise ratios (SNRs) were measured in liver, spleen, kidney and muscle. After MR examination, the animals were sacrificed, and the liver, kidney, lung and muscle were prepared for fluorescence observation and Prussian Blue staining. RESULTS: In the group treated with labeled cells, the SNR of the liver after cell transplantation was 3.12 +/- 0.43 at 1 h, 7.98 +/- 1.05 at 3 days and 11.46 +/- 1.41 at 7 days. These values were significantly lower than the pre-transplantation SNR (14.40 +/- 0.37). In the group treated with unlabeled cells, no significant difference could be found between after and before transplantation liver SNRs. Prussian Blue staining showed iron particles, contained within the cytoplasm and distributed in the liver parenchyma, which corresponded to the DAPI-stained fluorescent nuclei under the fluorescence microscope. CONCLUSION: The magnetically labeled MSCs transplanted into rat liver through hepatic arterial injection can be detected and monitored in vivo with a 1.5 T clinical MR scanner for up to 7 days after cell transplantation.

[Tracing magnetically labeled mesenchymal stem cells transplanted into rat livers by MRI].

OBJECTIVE: To trace magnetically labeled MSCs transplanted into the rat livers by magnetic resonance imaging (MRI). METHODS: Feridex and DAPI labeled rat mesenchymal stem cells (MSCs) were injected via portal veins into carbon tetrachloride treated rats. MRI was performed with a clinical 1.5 T MRI machine immediately before the MSCs injection and at h 1, d 3, d 7, and d 14 after the injection, and then the signal-to-noise ratio (SNR) was measured. MRI findings were compared with the liver histopathologies after the slides were stained with fluorescence dye and Prussian blue. RESULTS: The SNR for liver was 1.10+/-0.26 at hour 1, 8.18+/-1.55 at day 3, 11.08+/-1.30 at day 7, and 14.15+/-1.02 at day 14 respectively. Within 7 days after the MSCs transplantation, the SNRs of the livers were significantly lower than those before the transplantation (P less than 0.05). Histologically, the blue fluorescent particles under the fluorescence microscopy matched in distribution with the iron particles on the Prussian blue stained slides. CONCLUSION: The magnetically labeled MSCs transplanted into livers give rise to an obvious signal decrease, and can be tracked with a 1.5 T clinical MRI machine for up to 7 days after MSCs transplantation.

[Comparison of the effect of ambroxol and dexamethasone on the expression of pulmonary surfactant proteins in the fetal rat lungs].

OBJECTIVE: To investigate the effects of maternally administered dexamethasone and ambroxol on the mRNA levels of surfactant proteins (SP-A, SP-B and SP-C) expression in fetal rat lungs at gestational age day 19. METHODS: A 19-day fetal rat lung model was employed. In situ hybridization was used to detect the expression of SP-B mRNA in alveolar type II cell, and the levels of SP-A, SP-B and SP-C mRNAs were detected by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: (1) SP-B mRNA was detected in situ in alveolar type II cells in fetal rat lung of day 19 gestational age; (2) In the late developmental period of fetal rat lungs, alveolar type II cells were also found around bronchus; (3) Comparing to beta-actin mRNA, the relative values of SP-A, SP-B and SP-C mRNAs were 0.81 +/- 0.26, 0.97 +/- 0.20 and 0.88 +/- 0.11 in fetal lung in the control group. The relative values of mRNAs of SP-A, SP-B and SP-C to beta-actin were 1.04 +/- 0.16, 1.28 +/- 0.29, 1.09 +/- 0.25 in fetal lungs of the ambroxol injected rats, and were 1.08 +/- 0.25, 1.23 +/- 0.35, 1.21 +/- 0.25 in fetal lungs of the dexamethasone injected rats, respectively. Both ambroxol and dexamethasone-treated rats had significantly higher mRNA expression of surfactant proteins compared to the control saline injected animals (P < 0.05). (4) There were no significant differences between ambroxol and dexamethasone in the effects of increasing expressions of surfactant protein mRNAs (P > 0.05). CONCLUSION: Antepartum administration of both ambroxol and dexamethasone can significantly increase fetal lung SP-A, SP-B and SP-C mRNAs expression.

[Study on mesenchymal stem cells entering the brain through the blood-brain barrier].

OBJECTIVE: Neonatal hypoxic-ischemic encephalopathy (HIE) harms the lives and health of newborn infants and children severely. The prognosis is not satisfied, especially of the severe HIE. Mesenchymal stem cells (MSCs) can secrete a series of growth factors and neurotrophic factors. As well they have the potential ability to differentiate to the neural cells in vitro and in vivo. Therefore MSCs transplantation has been employed as a source of progenitor cells for cell therapy in patients with HIE in order to promote recovery of brain function and reduce the sequelae. Studies have shown that MSCs could enter the cerebral parenchyma and differentiate to neural cells through systemic infusion, but most of the researches applied adult stroke animal models. This study used neonatal HIE models to test the hypothesis that MSCs could enter the brain of newborn Wistar rats through the blood-brain barrier (BBB) by intraperitoneal infusion followed by observing the characteristics of the distribution and differentiation of MSCs in brain tissues, and exploring the effects of hypoxic-ischemic brain damage to the penetration and differentiation of MSCs. METHODS: Isolation and purification of MSCs were established from the whole bone marrow of juvenile Wistar rats by removing the nonadherent cells in primary and passage cultures. For cellular identification, MSCs of three to five passages were continuously pre-labeled with 5-bromo-2-deoxyuridine (BrdU) for 72 hours before transplantation. Animal models of HIE were built in 7-day-postnatal Wistar rats according to the method described by Rice. Two hours after hypoxia-ischemia, rats in HIE group (n = 8) were intraperitoneally infused with MSCs (4 x 10(6), 0.5 ml). In control group (n = 8), 7-day-postnatal normal Wistar rats were intraperitoneally infused with the same amount of MSCs. All rats were sacrificed and their cerebra were sectioned by cryomicrotome 14 days after transplantation. Immunohistochemical staining with chromogen diaminobenzidine (DAB) was used to detect and measure the cells derived from MSCs, and study the characteristics of distribution. To determine the differentiation of the BrdU positive cells entering the brains, immunofluorescence double labeling for BrdU and neural cells specific antigens was performed. RESULTS: MSCs were distributed throughout the cerebra in both groups at the 14th day after transplantation. The number of MSCs detected was 2415 +/- 226 in the control group, and 3626 +/- 461 in HIE group, respectively (t = 6.68, P < 0.05). More BrdU reactive cells were observed in the right ischemic hemisphere (1904 +/- 267) than in the contralateral hemisphere (1723 +/- 204), (t = 4.47, P < 0.05). No significant difference was found while comparing both cerebral hemispheres of the control group (t = 0.31, P > 0.05). In the HIE group, MSCs distributed more extensively, and some focal aggregations of MSCs were noticed. A few MSCs expressed Nestin-protein marker of neural progenitor cells, and almost none of the MSCs which expressed proteins characteristic of neuron (e.g. NSE) and astrocyte (e.g. GFAP) was detected at the 14th day after transplantation. CONCLUSION: 1. MSCs could enter the cerebral parenchyma through BBB and migrate throughout the brain by intraperitoneal infusion. 2. More MSCs injected intraperitoneally were localized and directed to the sites of hypoxic-ischemic brain damage. 3. Transplanted MSCs could not differentiate to neuron and astrocyte without other interventions during 14 days after transplantation.