SphK2 confers 5-fluorouracil resistance to colorectal cancer via upregulating H3K56ac-mediated DPD expression.

Aberrant sphingolipid metabolism has been implicated in chemoresistance, but the underlying mechanisms are still poorly understood. Herein we revealed a previously unrecognized mechanism of 5-fluorouracil (5-FU) resistance contributed by high SphK2-upregulated dihydropyrimidine dehydrogenase (DPD) in colorectal cancer (CRC), which is evidenced from human CRC specimens, animal models, and cancer cell lines. TMA samples from randomly selected 60 CRC specimens firstly identified the clinical correlation between high SphK2 and increased DPD (p < 0.001). Then the regulatory mechanism was explored in CRC models of villin-SphK2 Tg mice, SphK2-/-mice, and human CRC cells xenografted nude mice. Assays of ChIP-Seq and luciferase reporter gene demonstrated that high SphK2 upregulated DPD through promoting the HDAC1-mediated H3K56ac, leading to the degradation of intracellular 5-FU into inactive α-fluoro-ß-alanine (FBAL). Lastly, inhibition of SphK2 by SLR080811 exhibited excellent inhibition on DPD expression and potently reversed 5-FU resistance in colorectal tumors of villin-SphK2 Tg mice. Overall, this study manifests that SphK2high conferred 5-FU resistance through upregulating tumoral DPD, which highlights the strategies of blocking SphK2 to overcome 5-FU resistance in CRC.

Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function.

BACKGROUND/AIMS: Calcium-activated chloride channels (CaCCs) regulate numerous physiological processes including cell proliferation, migration, and extracellular matrix secretion. T16Ainh-A01 and CaCCinh-A01 are selective inhibitors of CaCCs. But it is unknown whether these two compounds have functional effects on cardiac fibroblasts (CFs). METHODS: Primary CFs were obtained by enzymatic dissociation of cardiomyocytes from neonatal rat hearts. Intracellular Ca2+ ([Ca2+]i) and Cl- ([Cl-]i) were measured using the fluorescent calcium indicators (Fluo-4 AM) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide respectively. The expression of anoctamin-1 (ANO1) and α-smooth muscle actin (α-SMA) was detected by quantitative RT-PCR, immunofluorescence, and western blotting. A hydroxyproline assay was used to examine collagen secretion. Cell proliferation, cell cycle distribution, and cell migration were assessed by Cell Counting Kit-8, flow cytometry, and Transwell assays, respectively. RESULTS: ANO1 was preferentially expressed on the nuclear membrane and partially within intracellular compartments around the nucleus. T16Ainh-A01 and CaCCinh-A01 displayed different inhibitory effects on [Cl-]i in CFs. T16Ainh-A01 considerably decreased [Cl-]i in the nucleus, whereas CaCCinh-A01 reduced [Cl-]i in intracellular compartments around the nucleus, and both inhibitors exhibited a minimal effect on [Ca2+]i in CFs. ANO1 and α-SMA expression levels were significantly repressed by CaCCinh-A01. T16Ainh-A01 showed a marked inhibitory effect on the mRNA levels of ANO1 and α-SMA, but had a negligible effect on ANO1 at the protein level. T16Ainh-A01 and CaCCinh-A01 led to the significant repression of cell proliferation, cell migration, and collagen secretion in CFs. CONCLUSION: Our findings indicate that T16Ainh-A01 and CaCCinh-A01 have the potential to inhibit the proliferation and collagen secretion of CFs and may serve as novel anti-fibrotic therapeutic drugs in the future.

Effects of linagliptin and liraglutide on glucose- and angiotensin II-induced collagen formation and cytoskeleton degradation in cardiac fibroblasts in vitro.

AIM: Glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors can not only lower blood glucose levels, but also alleviate cardiac remodeling after myocardial ischemia and hypertension. In the present study, we investigated the effects of a DPP-4 inhibitor (linagliptin) and a GLP-1 activator (liraglutide) on glucose- and angiotensin II (Ang II)-induced collagen formation and cytoskeleton reorganization in cardiac fibroblasts in vitro, and elucidated the related mechanisms. METHODS: Cardiac fibroblasts were isolated from the hearts of 6-week-old C57BL/6 mice, and then exposed to different concentrations of glucose or Ang II for 24 h. The expression of fibrotic signals (fibronectin, collagen-1, -3 and -4), as well as ERK1/2 and NF-κB-p65 in the fibroblasts was examined using Western blotting assays. F-actin degradation was detected under inverted laser confocal microscope in fibroblasts stained with Rhodamine phalloidin. RESULTS: Glucose (1-40 mmol/L) and Ang II (10-8-10-5 mol/L) dose-dependently increased the expression of fibronectin, collagens, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. High concentrations of glucose (≥40 mmol/L) and Ang II (≥10-6 mol/L) caused a significant degradation of F-actin (less assembly F-actin fibers and more disassembly fibers). ERK1/2 inhibitor U0126 (10 µmol/L) and NF-κB inhibitor JSH-23 (10 µmol/L) both markedly suppressed glucose- and angiotensin II-induced fibronectin and collagen expressions in cardiac fibroblasts. Furthermore, pretreatment with liraglutide (10-100 nmol/L) or linagliptin (3 and 30 nmol/L) significantly decreased glucose- and Ang II-induced expression of fibrotic signals, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. Moreover, pretreatment with liraglutide (30 nmol/L) or liraglutide (100 nmol/L) markedly inhibited glucose-induced F-actin degradation, however, only liraglutide inhibited Ang II-induced F-actin degradation. CONCLUSION: Linagliptin and liraglutide inhibit glucose- and Ang II-induced collagen formation in cardiac fibroblasts via activation of the ERK/NF-κB/pathway. Linagliptin and liraglutide also markedly inhibit glucose-induced F-actin degradation in cardiac fibroblasts, but only liraglutide inhibits Ang II-induced F-actin degradation.

Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples.

AIM: To establish an easily-handled method to isolate mesenchymal stem cells (MSCs) from coagulated human bone marrow samples. METHODS: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated, treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 °C for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast (CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents. RESULTS: The average CFU-F number of urokinase-treated samples (16.85 ± 11.77/10(6)) was comparable to that of un-coagulated control samples (20.22 ± 10.65/10(6), P = 0.293), which was significantly higher than those of mechanically-cut clots (6.5 ± 5.32/10(6), P < 0.01) and untreated clots (1.95 ± 1.86/10(6), P < 0.01). The CFU-F numbers decreased after samples were stored, but those of control and urokinase-treated clots remained higher than the other two groups. Consistently, the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots. The attached cells were fibroblast-like in morphology and homogenously positive for CD44, CD73 and CD90, and negative for CD31 and CD45. Also, they could be induced to differentiate into osteoblasts and adipocytes in vitro. CONCLUSION: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.

Gata4, Tbx5 and Baf60c induce differentiation of adipose tissue-derived mesenchymal stem cells into beating cardiomyocytes.

The adipose tissue-derived mesenchymal stem cells (ADMSCs) are extensively utilized in tissue engineering, regenerative medicine and cell therapy. ADMSCs can differentiate into cardiomyocytes, and it has been shown that over-expression of a cocktail of factors can induce ectopic heart formation and program cardiogenesis in ESCs. However, which genes are responsible for differentiation of ADMSCs into beating cardiomyocyte-like cells remains unknown. In this study we have shown that the combination of Gata4, Tbx5 and Baf60c is sufficient for inducing ADMSCs to form cardiomyocytes. It also appears that, while Gata4 and Baf60c are key inducers of myocardial differentiation, Tbx5 is essential for the ability of cardiac cells to contract. These findings provide additional experimental references for myocardial tissue engineering in the emerging field of cell-based therapy of heart diseases.

The expression analysis of nanog in the developing rat myocardial tissues.

OBJECTIVES: To investigate the expression dynamic of nanog gene in the development of rat myocardial tissues. METHODS: SD rats were studied at 5 time points before and after birth. The techniques of immunohistochemistry, immunofluorescence, western blotting and RT-PCR were used to investigate the expression of nanog gene in the rat myocardial tissues at different embryonic (E) and postnatal (P) stages, and image analysis system was used for the quantitative analysis. RESULTS: The immunohistochemistry, immunofluorescence and western blotting analyses have shown that expression of nanog protein was highest in the rat myocardial tissues at E18, then it gradually declined at postnatal stages (P<0.05), and became nearly undetectable in most myocardial tissues at P30 with very few remaining nanog-positive cells. RT-PCR result indicated that the expression of nanog gene was strong at E18, but gradually decreased from E18 to P30. CONCLUSION: The mRNA transcription and protein translation of nanog gene in the rat heart gradually decreased with every consecutive growth stage. This indicates that nanog gene has potential regulatory functions in the differentiation of myocardial cells during rat development.

CD73+ adipose-derived mesenchymal stem cells possess higher potential to differentiate into cardiomyocytes in vitro.

Adipose-derived mesenchymal stem cells (ADMSCs) are an attractive adult-derived stem cell population for cardiovascular repair. ADMSCs are heterogeneous cell populations with pluripotent capacity to differentiate into different types of cells. In the present study, we investigated the biological characteristics and differentiation potential of CD73-positive (CD73(+)) and CD73-negative (CD73(-)) ADMSCs. Our results show that in terms of morphological shape, CD73(+)-ADMSCs are mainly small-sized cells, whereas CD73(-)-ADMSCs are big-sized cells; both subpopulations can equally differentiate into adipocytes and osteoblasts in vitro. However, the CD73(+)-ADMSCs possess a higher potential to differentiate into cardiomyocytes than the CD73(-)-ADMSCs. The expression of the cardiac-specific genes, cTnT, Gata4, and Nkx2.5, is much higher in the CD73(+)-ADMSCs than in the CD73(-)-ADMSCs. Furthermore, Nanog expression at both the mRNA and protein levels is significantly higher in CD73(+)-ADMSCs than in CD73(-)-ADMSCs, suggesting that CD73(+)-ADMSCs are an undifferentiated subpopulation that can differentiate into cardiomyocytes in vitro more efficiently. Therefore, this study facilitates a better understanding of the differentiation of the ADMSCs subgroups and attempts to identify if CD73 is a useful marker for sorting and purifying the subpopulation of ADMSCs with a higher capacity for differentiation into cardiomyocytes.

Distribution of dopamine receptors D1- and D2-immunoreactive neurons in the dorsal motor nucleus of vagus in rats.

The dorsal motor nucleus of vagus (DMV) plays an important role in the regulation of gastrointestinal function. Dopamine (DA) exerts potent neuromodulatory effects on the motoneurons in the DMV via dopamine receptors (DRs). However, the distribution of DRs and their neurochemical phenotypes in the DMV are unclear. In the present study, the distribution of DRs D1- and D2-immunoreactive (IR) neurons and their neurochemical phenotypes in the DMV of rats were investigated using a double-labeling immunofluorescence technique combined with confocal microscopy. The results indicated that a considerable quantity of D1 and D2 was expressed throughout the DMV. A large amount of choline acetyltransferase (ChAT)-IR and a few tyrosine hydroxylase (TH)-IR neurons were observed in the DMV. Nearly all of the neurons were also D1-IR and D2-IR. In conclusion, the present study demonstrates the wide distribution of D1 and D2 in the cholinergic and catecholaminergic neurons in the DMV of rats. The DRs might play an important role in the regulation of DA on the activity of cholinergic and catecholaminergic neurons in the DMV.