Effect of scoparone on neurite outgrowth in PC12 cells

The neurite outgrowth-promoting effects of scoparone isolated from the stem bark of Liriodendron tulipifera were investigated in PC12 cells. At a concentration of 200 microM, scoparone markedly induced neurite outgrowth from PC12 cells. Scoparone at 200 microM also enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentration of Ca2+ were also increased by 200 microM scoparone. In addition, scoparone at 200 microM increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+/calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparone-induced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells.     

Inhibition of NGF-induced neurite outgrowth of rat pheochromocytoma cells (PC12) following administration of dioxyamphetamine

Amphetamine analogs are known to induce not only neurotoxicity at serotonergic axon terminals but also neocortical neuronal degeneration. However, a much less studied aspect involves the impact of amphetamine exposure on neuronal development. The present study investigated whether pretreatment of PC12 cells with dioxyamphetamine (DA) alters differentiation of PC12 cells by NGF and, if so, which components of the Ras/Raf/MEK/ERK pathway known to be involved in the differentiation response to NGF are particularly affected. Though exposure of PC12 cells to DA 1 h prior to NGF treatment resulted in apopotosis, several PC12 cells survived. However, neurite outgrowth of these NGF-responsive cells was repressed. Immunoblots of whole cell extracts revealed a strong induction rather than inhibition of ERK phosphorylation up to 48 h after DA/NGF treatment. Our results indicate that NGF-mediated neurite outgrowth was inhibited by pretreatment with DA, and this blockage of NGF-induced neuritogenesis was not due to an inhibition of ERK phosphorylation.     

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis,inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer''s disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aβ1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.     

The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells

The nervous system is highly sensitive to various environmental stresses, such as ischemia. Stress response mechanisms that result in neuroprotection, including the induction of heat shock proteins (HSP), are not well understood. We examined the effect of KNK437, a compound that inhibits the synthesis of inducible heat shock proteins, on neuronal differentiation in rat pheochromocytoma PC12 cells. KNK437 decreased the expression of HSP70, and induced the neurite outgrowth of PC12 cells in the absence of stress stimulation, although with lower efficacy than nerve growth factor (NGF). Neurite outgrowth stimulated by KNK437 and NGF was blocked by inhibitors of ERK mitogen-activated protein (MAP) kinase, p38 MAP kinase, and glycogen synthase kinase 3β signaling pathways. NGF, and not KNK437, induced acetylcholine esterase (AChE) activity, a functional differentiation marker, indicating that KNK437 utilizes a mechanism distinct from that of NGF. KNK437 enhanced the activity of low dose NGF treatment on neurite outgrowth induction and ERK phosphorylation in PC12 cells, a finding that identifies KNK437 as a possible nerve regeneration agent. This compound may be a useful tool for the investigation of neuronal differentiation and neuroprotection against environmental stress.     

Neurite outgrowth of PC12 mutant cells induced by orange oil and d-limonene via the p38 MAPK pathway

We studied the effects of natural essential oil on neurite outgrowth in PC12m3 neuronal cells to elucidate the mechanism underlying the action of the oils used in aromatherapy. Neurite outgrowth can be induced by nerve growth factor (NGF), where ERK and p38 MAPK among MAPK pathways play important roles in activating intracellular signal transduction. In this study, we investigated whether d-limonene, the major component of essential oils from oranges, can promote neurite outgrowth in PC12m3 cells, in which neurite outgrowth can be induced by various physical stimulations. We also examined by which pathways, the ERK, p38 MAPK or JNK pathway, d-limonene acts on PC12m3 cells. Our results showed that neurite outgrowth can be induced when the cells are treated with d-limonene. After treatment with d-limonene, we observed that p38 MAPK is strongly activated in PC12m3 cells, while ERK is weakly activated. In contrast, JNK shows little activity. A study using an inhibitor of p38 MAPK revealed that neurite outgrowth in PC12m3 cells is induced via the activation of p38 MAPK by d-limonene. The results thus indicate that d-limonene may promote neural cell differentiation mainly via activation of the p38 MAPK pathway.     

Differences in neuritogenic response to nitric oxide in PC12 and PC12h cells

We have demonstrated that a natural iridoid compound, genipin, induces neurite outgrowth through the nitric oxide (NO)-cGMP-protein kinase G signaling pathway in PC12h cells. PC12 cells, the parental cell line of PC12h cells, have been shown to carry out neurite extension that accompanies NO production in response to nerve growth factor (NGF). This neurite outgrowth was significantly inhibited by NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, in both PC12 and PC12h cells, suggesting that the neuritogenesis is NO-dependent in both cells. In this report, we investigated whether genipin also induces neurite outgrowth in PC12 cells in order to determine the NO-dependent neurotrophic action of genipin in more than just one cell type. Genipin induced marked neurite outgrowth in PC12h cells but not in PC12 cells. The genipin-induced neurite outgrowth was significantly inhibited by L-NAME in PC12h cells. An NO donor, NOR4, also significantly induced neurite outgrowth in a concentration-dependent manner in PC12h cells but not in PC12 cells. On the other hand, NGF-primed PC12 cells exhibited significant neurite extension, which was inhibited by L-NAME, in response to genipin. Interestingly, NGF-primed PC12 cells responded to NOR4 extending neurites and expressed detectable neuronal NO synthase protein which is not detected in naive PC12 cells. These results suggest that genipin exerts a neuritogenic action on neuronal cells which are responsive to NO itself. Furthermore, the results also suggest that PC12h cells are more suitable for the study of NO-dependent neuronal function than PC12 cells which were not responsive to NO.     

Nerve growth factor expression by PLG-mediated lipofection

Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2+/-8.3% of NIH3T3 cells with luciferase activity of 3.97x10(7)RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.     

Functional role of JNK in neuritogenesis of PC12-N1 cells

JNKs, also known as SAPKs, are activated in response to a wide variety of factors including growth factors, cytokines, UV radiation, and heat shock. In the rat pheochromocytoma PC12 cells, the JNK signaling pathway mediates diverse functions such as differentiation and apoptosis. We have previously shown that activated JNK is required for later stages of neuritogeneis induced by NGF in a variant PC12 cell line (N1). Here, the functional role of JNK in N1 cells is further investigated. We show that NGF treatment, which induces extensive neurite branching and cell soma enlargement in the N1 cells, stimulates a biphasic activation of JNK. The first phase of activation is rapid and transient, beginning at 15 min after NGF exposure and lasting approximately 45 min. The second phase of activation is sustained, beginning at 9-12 h of NGF treatment and lasting for at least 24 h. Similar biphasic pattern of JNK activation is also observed in the parental PC12 cells. Using the specific JNK inhibitor SP600125, we show that the biphasic activation is necessary for neurite outgrowth and branching, and that inhibition of either phase suppresses neuritogenesis in the N1 cells. These results suggest that dynamic JNK activation may play a key role in neurite outgrowth during neuronal development.     

ERK在NGF诱导PC12 细胞分化中的作用

目的探讨细胞外信号调节激酶(ERK)在NGF。诱导的PC12细胞分化中的作用机制。方法以NGF处理PC12细胞建立分化模型,运用免疫印迹检测不同浓度不同作用时间时NGF对ERK1／2蛋白和磷酸化ERK1／2蛋白水平的影响,并观察MAPK／ERK激酶(MEK)抑制剂U0126对NGF诱导的细胞形态学改变的影响。结果ERK1／2蛋白的磷酸化呈现NGF剂量和时间依赖性。NGF作用细胞5min即可观察到明显的ERK1／2蛋白磷酸化,持续1h左右,2h时降低到初始水平,而细胞形态的改变出现在NCF作用12h以后。倒置相差显微镜观察可见PC12细胞分化的程度与ERK1／2活化持续的时间正相关,U0126可完全即时抑制ERK1／2的活化,而ERK1／2活化的抑制可完全阻断。NGF诱导的PC12细胞分化。结论ERK1／2的活化是PC12细胞发生分化的必需事件,其活化时间的长短对分化具有决定作用。     

Induction of neurite outgrowth in PC12 cells by the medium-chain fatty acid octanoic acid

It has been shown that polyunsaturated fatty acids such as arachinonic and docosahexanoic acids but not monounsaturated and saturated long-chain fatty acids promote basal and nerve growth factor (NGF)-induced neurite extension of PC12 cells, a line derived from a rat pheochromocytoma. On the other hand, short-chain fatty acids and valproic acid (2-propylpentanoic acid) enhance the growth of neurite processes of the cells only in the presence of inducers. In this study, we demonstrated that straight medium-chain fatty acids (MCFAs) at millimolar concentrations alone potently induced neuronal differentiation of PC12 cells. Hexanoic, heptanoic and octanoic acids dose-dependently induced neurite outgrowth of the cells: their maximal effects determined 2 days after addition to the culture medium were more marked than the effect of NGF. PC12 cells exposed to octanoic acid expressed increased levels of the neuronal marker beta-tubulin isotype III. Nonanoic, decanoic, and dodecanoic acids also induced growth of neurite processes, but their maximal effects were less marked than that of octanoic acid. In contrast, the polyunsaturated fatty acid linoleic acid and short-chain fatty acids had only slight or almost no effects on neurite formation in the absence of NGF. The effect of octanoic acid was synergistic with or additive to the effects of NGF and dibutyryl cyclic AMP. Octanoic acid upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), critical signaling molecules in neuronal differentiation, but not phosphorylation of Akt, a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K). Moreover, growth of neurites induced by octanoic acid was potently inhibited by treatment of cells with the p38 MAPK inhibitor SB203580 and the ERK kinase inhibitor PD98059 but not inhibited and only slightly inhibited by the JNK inhibitor SP600125 and the PI3K inhibitor wortmannin, respectively. Taken together, our results indicate that MCFAs, including octanoic acid, induced neurite outgrowth of PC12 cells in the absence of NGF and suggest that the activation of p38 MAPK and ERK pathways is involved in this process.     

Enhanced gene delivery to PC12 cells by a cationic polypeptide

Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders. We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors. The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli. It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum. The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors. The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide. These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons.     

Syntaxin 6 regulates nerve growth factor-dependent neurite outgrowth

Neurite outgrowth is crucial for neural circuit formation. Intracellular membrane trafficking is involved in the cell surface expansion that is necessary for neurite outgrowth. It is known that syntaxin 6 is predominantly located in the Golgi region in undifferentiated PC12 cells and that it regulates trans-Golgi network trafficking and the secretory pathway via its coiled-coil domains. However, whether it also regulates neurite outgrowth remains unknown. In this paper, we found that syntaxin 6 was located both in the Golgi apparatus and the distal tips of the neurites of nerve growth factor (NGF)-treated PC12 cells. We also showed that the overexpression of the first coiled-coil domain of syntaxin 6 inhibited NGF-dependent neurite outgrowth. However, the coiled-coil domain-disrupting mutant had little effect on neurite outgrowth. These results suggest that the first coiled-coil domain of syntaxin 6 plays a crucial role in NGF-dependent neurite outgrowth.     

Human mast cells express functional TrkA and are a source of nerve growth factor

Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions. Despite reports that rodent mast cells proliferate in the presence of nerve growth factor (NGF), human mast cells reportedly do not respond to this factor. To determine if human mast cells express the NGF receptors, TrkA tyrosine receptor and the low affinity NGF receptor (LNGFR), we first analyzed the mRNA expression by RT-PCR of TrkA and LNGFR in a human mast cell line (HMC-1) and in human mast cells cultured in the presence of stem cell factor. Both HMC-1 and cultured human mast cells were found to express TrkA but not LNGFR. TrkA protein was demonstrated by Western blot analysis of HMC-1 lysates. Using flow cytometric analysis and mast cell tryptase as a mast cell marker, both HMC-1 cells and cultured human mast cells were shown to co-express tryptase and TrkA. Treatment of mast cells with NGF resulted in phosphorylation of TrkA on tyrosine residues as detected by immunoblotting with an antiphosphotyrosine antibody. Furthermore, NGF induced the immediate early gene c-fos in HMC-1 cells. HMC-1 cells and cultured human mast cells were also found to express NGF mRNA, and conditioned medium from HMC-1 cells stimulated neurite outgrowth from chicken embryonic sensory ganglia in culture. This effect was blocked by anti-NGF. Thus, mast cells express functional TrkA and synthesize NGF, suggesting a mechanism by which NGF may act as an autocrine factor for human mast cells, and by which mast cells and nerves may interact.     

Release characteristics and bioactivity of gelatin-tricalcium phosphate membranes covalently immobilized with nerve growth factors

The gelatin-tricalcium phosphate membranes were cross-linking with low concentration glutaraldehyde solution (GTG). This material has good mechanical property, biocompatibility, and is feasible for surgical manipulation. For axonal regeneration, nerve growth factors (NGF) were immobilized onto the composite (GTG) with carbodiimide. The purpose of this study was to evaluate the release characteristics and bioactivity of NGF after covalent immobilization onto the GTG membranes (GEN). NGF immobilized onto and released from the composite was quantified using ELISA method. PC 12 cells were cultured on the GTG and GEN composites. Cell survival, cytotoxicity, and cellular activity were evaluated by total protein content, LDH activity, and MTT assay respectively. Neurite outgrowth assay was used to evaluate the biological activity of NGF released from GEN composite. From ELISA measurement, the releasing curve for NGF showing two distinctive parts with different slopes indicated that NGF were released from the composite in diffusion-controlled mechanism and degradation-controlled mechanism respectively. While culturing with PC 12 cells, LDH leakage results implied that whether GTG composite cross-linked with NGF or not showed little cytotoxicity. The total protein content and cellular activity of PC 12 cells were lower on GTG and GEN membranes than control group. However, 56%+/-3.98 of PC 12 cells showed significant neurite outgrowth on GEN membranes which was statistically higher than GTG without NGF immobilization. In addition, sustained release of bioactive NGF for two months had been demonstrated by neurite outgrowth assay. From these experiments, it can be concluded that the technique used in the present study is capable of immobilizing NGF onto GTG membranes covalently and remaining the bioactivity of NGF. Therefore, GEN composite can be materials for sustained release of bioactive NGF and a candidate for future therapeutic application in nerve repair.     

Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells

We previously found that the methanol extract of a marine brown alga, Sargassum macrocarpum showed marked nerve growth factor (NGF)-dependent neurite outgrowth promoting activity to PC12D cells. The active substance purified was elucidated to be sargachromenol. The median effective dose (ED50) was 9 microM against PC12D cells in the presence of 10 ng/ml NGF, although it showed no neurotrophic effect on its own. Pretreatment of cells with protein kinase A (PKA) inhibitor or U0126 substantially suppressed the sargachromenol-enhanced neurite outgrowth from PC12D cells, suggesting that the activation of cyclic AMP-mediated protein kinase and mitogen-activated protein (MAP) kinase 1/2 was apparently required for the action of sargachromenol. On the other hand, sargachromenol significantly promoted the survival of neuronal PC12D cells at 0-50 ng/ml NGF in serum-free medium. Neither PKA inhibitor nor U0126 could inhibit the survival supporting effect of sargachromenol, whereas wortmannin significantly blocked the sargachromenol-induced survival supporting effect on neuronal PC12D cells, suggesting that sargachromenol rescued neuronal PC12D cells by activating phosphatidylinositol-3 kinase. These results demonstrate that sargachromenol promotes neuronal differentiation of PC12D cells and supports the survival of neuronal PC12D cells via two distinct signaling pathways.     

Purinergic stimulation of cell division and differentiation: mechanisms and pharmacological implications.

Extracellular purine nucleosides and nucleotides in micromolar concentrations stimulate proliferation of a variety of cell types in vitro and in vivo. As well they act synergistically with NGF to stimulate neurite outgrowth from PC12 cells. A variety of purine nucleosides and deoxyribonucleosides promote cell proliferation and increase intracellular cAMP. Their activities are inhibited by adenosine A2 receptor antagonists. Only adenosine interacts with the A2 receptor. We propose that the other nucleosides and deoxyribonucleosides inhibit extracellular adenosine deaminase, thereby increasing the extracellular concentration of adenosine. The nucleotides apparently act by stimulating P2y receptors coupled to inositol phosphate metabolism. We propose that the nucleosides and nucleotides act synergistically with other growth factors because each has distinct but complementary second messenger systems. If our hypotheses are correct, it should prove possible to modulate the growth and morphogenesis in several cell types using drugs that inhibit or stimulate adenosine A2 or purine P2y receptor agonists or the second messenger systems coupled to these receptors.     

The multi-herbal medicine Gongjin-dan enhances memory and learning tasks via NGF regulation

Nerve growth factor (NGF) decreases degeneration of cholinergic neurons, improves memory loss, and increases long-term potentiation and learning tasks. Therefore, NGF mimetics or NGF inducers may be important targets for the treatment of various neurodegenerative disorders. Traditionally, Gongjin-dan (GJD) has been used clinically for the treatment of central nervous system disorders. In this study, we examined the effects of GJD on NGF mimetic activity in PC12 cells and the induction of NGF secretion in primary astrocytes. Moreover, we also measured neuron survival by MAP-2 staining in an immobilization stress rat model and induction of long-term potentiation by the MEA system in rat hippocampus slices treated with dexamethasone. The behavioral syndrome by novel object test was also performed in mice. GJD increased neurite outgrowth in PC12 cells and NGF secretion in primary astrocytes. Also, it reduced neuronal cell death and increased long-term potentiation in the rat hippocampus. Moreover, the number of entries, the time spent and the distance moved in the center area of the test region by the mice was increased by oral administration of GJD in comparison with the distance moved over the total area. These data suggest that administration of GJD may improve memory and learning tasks via NGF regulation, and that it may have a potential for multiple function neuroprotection via NGF regulation.     

Ganglioside GM1 binding toxins and human neuropathy-associated IgM antibodies differentially promote neuritogenesis in a PC12 assay

PC12 cells undergo neuritogenesis upon nerve growth factor (NGF) activation of the TrkA receptor, an effect mimicked by the ganglioside GM1 binding B-subunit of cholera toxin (CTB). Modulation of neuritogenesis by a GM1 ligand indicates a possible pathway for pathophysiological actions of neuropathy-associated anti-GM1 antibodies. Here we examine the ability of GM1 binding toxins and antibodies to induce neuritogenesis, using a PC12 neurite outgrowth assay. Cholera toxin (CT) and commercially prepared CTB (sCTB, contaminated with traces of the adenyl cyclase activating CT A-subunit) were highly neuritogenic. Recombinant cholera toxin B-subunit (rCTB, free from CTA) induced a much smaller effect, suggesting that the potent effects of sCTB are largely due to contaminating CTA. The recombinant GM1 binding B-subunit of Escherichia coli heat-labile enterotoxin (rETxB) exhibited no neuritogenic activity, whilst rETx holotoxin, which activates adenyl cyclase, was highly neuritogenic. Monoclonal anti-GM1 IgM antibodies from human neuropathy subjects induced small neuritogenic effects. These data indicate that GM1/ligand interaction does not necessarily lead to neuritogenesis and suggest that a specialisation of CTB, not shared by anti-GM1 antibodies or rETxB, is required to activate TrkA. Our data also indicate that antibodies are unlikely to exert major modulatory effects on TrkA activity in patients with anti-GM1 antibody-associated peripheral neuropathies.     

Hypoxia induces T-cell apoptosis by inhibiting chemokine C receptor 7 expression: the role of adenosine receptor A2

Hypoxia is a major characteristic of the tumor microenvironment, and its effects on immune cells are proposed to be important factors for the process of tumor immune escape. It has been reported that hypoxia affects the function of dendritic cells and the antitumor function of T cells. Here we discuss the effects of hypoxia on T-cell survival. Our results showed that hypoxia induced apoptosis of T cells. Adenosine and adenosine receptors (AR) are important to the hypoxia-related signaling pathway. Using AR agonists and antagonists, we demonstrated that hypoxia-induced apoptosis of T cells was mediated by A_2a and A_2b receptors. Furthermore, we are the first, to our knowledge, to report that hypoxia significantly inhibited the expression of chemokine C receptor 7 (CCR7) of T cells via the A₂R signal pathway, perhaps representing a mechanism of hypoxia-induced apoptosis of T cells. Collectively, our research demonstrated that hypoxia induces T-cell apoptosis by the A₂R signaling pathway partly by suppressing CCR7. Blocking the A₂R signaling pathway and/or activation of CCR7 can increase the anti-apoptosis function of T cells and may become a new strategy to improve antitumor potential.