Context-Dependent Modulation of Macrophage Plasticity by Cordycepin Drives Tumor Regression in Melanoma.

BackgroundCurrent ulcerative colitis (UC) therapies often cause adverse effects, and novel treatments are urgently needed. Cordycepin (COR), a bioactive compound from Cordyceps militaris, shows anti-inflammatory and intestinal protective potential, its precise role and mechanisms in UC remain unclear.PurposeThis study aimed to elucidate the effects and underlying mechanisms of COR on UC.MethodsUsing dextran sulfate sodium (DSS)-induced acute colitis mice and DSS-damaged human colonic epithelial cells as models, we evaluated and analyzed the effects and mechanisms of COR on UC by combining molecular docking, molecular dynamics simulations, in vivo/in vitro interventions with selective pharmacological antagonists, transcriptome sequencing and Western blotting verification.ResultsIn vitro experiments confirmed that COR exhibits protective effects on DSS-damaged colonic epithelial cells. Mechanistic studies revealed that COR elevates intracellular cAMP levels, and the selective adenosine A2A receptor (A2AAR) antagonist SCH58261 can block the protective effect of COR. Molecular docking and dynamics simulation analyses also demonstrated an interaction between COR and A2AAR at the molecular level. In vivo experiments further verified that oral administration of COR (5 mg/kg, 10 mg/kg) significantly ameliorated DSS-induced colitis in mice, manifested by reduced disease activity index, attenuated weight loss, improved colon shortening, decreased serum pro-inflammatory cytokines, alleviated colonic inflammation, and restored intestinal barrier function. Moreover, the therapeutic effect of COR on colitis could be blocked by SCH58261. Further investigations indicated that COR inhibits IL-6/IL-6R signaling in colonic tissues and suppresses phosphorylation-mediated activation of p38 MAPK and NF-κB p65 through A2AAR activation.ConclusionCOR ameliorates DSS-induced colitis by activating A2AAR to upregulate cAMP levels, inhibiting IL-6/IL-6R-mediated p38 MAPK and NF-κB activation. This study confirms A2AAR as a key therapeutic target, providing data support for the potential application of COR in UC treatment.

Cordycepin inhibits human extravillous trophoblast invasion by suppressing snail-mediated MMP2 expression.

A disintegrin and metalloproteinase domain 10 (ADAM10), a member of the ADAM family, is a cellular surface protein with potential adhesion and protease/convertase functions. The expression regulations in cancers by natural products [adenosine (AD) and its analogs, cordycepin (CD), and N6, N6-dimethyladenosine (m62A)], and immune regulation are unclear. As results, AD, CD, and m62A inhibited ADAM10 expression in various cancer cell lines, indicating their roles in anti-cancer agents. Further molecular docking with ADAM10 protein found the binding energies of all docking groups were <-7 kcal/mol for all small-molecules (AD, CD and m62A), suggesting very good binding activities. In addition, analysis of the immunomodulatory roles in cancer showed that ADAM10 was negatively correlated with immunomodulatory genes such as CCL27, CCL14, CCL25, CXCR5, HLA-B, HLA-DOB1, LAG3, TNFRSF18, and TNFRSF4 in bladder urothelial carcinoma, thymoma, breast invasive carcinoma, TGCT, kidney renal papillary cell carcinoma, SKCM and thyroid carcinoma, indicating the immune-promoting roles for ADAM10. LAG3 mRNA levels were reduced by both AD and CD in vivo. ADAM10 is also negatively associated with tumor immunosuppression and interrelated with the immune infiltration of tumors. Overall, the present study determined ADAM10 expression by AD, CD and m62A, and in AD or CD/ADAM10/LAG3 signaling in cancers, and suggested a potential method for immunotherapy of cancers by targeting ADAM10 using the small molecules AD, CD and m62A.

Efficient production of cordycepin in engineered Saccharomyces cerevisiae.

Cordyceps sinensisis, a kind of precious natural crude drugs and edible mushrooms, were used as tonic food in East Asia area and enjoyed an extensive praise for its medicinal functions. Cordyceps militaris, as a substitute for C. sinensis, is a widely distributed species, which can be cultivated in various medium. In this study, the contents of major bioactive components, cordycepin and adenosine in fruiting bodies and mycelia from the nature C. sinensis and artificial cultural C. militaris were investigated using improved HPLC method. The results showed the mean contents of cordycepin and adenosine in the fruiting bodies of C. militaris were 2.654 ± 0.02 and 2.45 ± 0.03 mg/g, those in C. sinensis were 0.9801 ± 0.01 and 1.643 ± 0.03 mg/g, while those in the mycelium of C. militaris were 0.9040 ± 0.02 and 1.592 ± 0.03 mg/g, respectively. The concentration of cordycepin and adenosine in the fruiting bodies of C. militaris were higher than that in natural C. sinensis, while the fermented mycelium of C. militaris were similar with natural C. sinensis.

There is increasing evidence that adenosine (ADO) and dopamine (DA) interact directly in the basal ganglia via actions at ADO A2a and DA D2 receptors, respectively. The purpose of this study was to determine 1) the extent to which these receptors modulate endogenous GABA release in discrete regions of the rat basal ganglia and 2) whether GABA release is modulated by a direct and opposing interaction between ADO A2a and DA D2 receptors. Tissue slices of striatum (STR) containing globus pallidus (GP; STR/GP) and micropunches of STR, GP, and substantia nigra pars reticulata (SNr) were studied. Radioligand binding demonstrated that ADO A1, ADO A2a, and DA D2 receptors were present in each of the tissue preparations with the exception of SNr, in which ADO A2a receptors were not detected. Stimulation of ADO A2a receptors with CGS 21680 (1-10 nM) increased electrically stimulated GABA release in STR/GP slices and GP micropunches. Consistent with the lack of A2a receptors in SNr, CGS 21680 had no effect on GABA release from this region. In contrast, stimulation of DA D2 receptors with N-0437 (1-100 nM) inhibited evoked GABA release from STR/GP slices and both GP and SNr micropunches. The D2-mediated inhibition of GABA release in GP was abolished in the presence of CGS 21680 (10 nM). These experiments demonstrate that stimulation of ADO A2a and DA D2 receptors has opposing effects on endogenous GABA release in STR and GP. These opposing actions may explain the antagonistic interactions between ADO and DA that have been observed in behavioral studies and support the hypothesis that the striatopallidal efferent system is an important anatomical substrate for the A2a/D2 receptor interaction.

As a cellular protease, transmembrane serine protease 2 (TMPRSS2) plays roles in various physiological and pathological processes, including cancer and viral entry, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, we conducted expression, mutation, and prognostic analyses for the TMPRSS2 gene in pan-cancers as well as in COVID-19-infected lung tissues. The results indicate that TMPRSS2 expression was highest in prostate cancer. A high expression of TMPRSS2 was significantly associated with a short overall survival in breast invasive carcinoma (BRCA), sarcoma (SARC), and uveal melanoma (UVM), while a low expression of TMPRSS2 was significantly associated with a short overall survival in lung adenocarcinoma (LUAD), demonstrating TMPRSS2 roles in cancer patient susceptibility and severity. Additionally, TMPRSS2 expression in COVID-19-infected lung tissues was significantly reduced compared to healthy lung tissues, indicating that a low TMPRSS2 expression may result in COVID-19 severity and death. Importantly, TMPRSS2 mutation frequency was significantly higher in prostate adenocarcinoma (PRAD), and the mutant TMPRSS2 pan-cancer group was significantly associated with long overall, progression-free, disease-specific, and disease-free survival rates compared to the wild-type (WT) TMPRSS2 pan-cancer group, demonstrating loss of functional roles due to mutation. Cancer cell lines were treated with small molecules, including cordycepin (CD), adenosine (AD), thymoquinone (TQ), and TQFL12, to mediate TMPRSS2 expression. Notably, CD, AD, TQ, and TQFL12 inhibited TMPRSS2 expression in cancer cell lines, including the PC3 prostate cancer cell line, implying a therapeutic role for preventing COVID-19 in cancer patients. Together, these findings are the first to demonstrate that small molecules, such as CD, AD, TQ, and TQFL12, inhibit TMPRSS2 expression, providing novel therapeutic strategies for preventing COVID-19 and cancers.

Ischemia-reperfusion injury is a common clinical challenge in surgery, which has a significant impact on the prognosis of patients. It is a complex, multi-factor, multi-cell intertwined pathological process. Many scholars had explored its mechanism, but the drugs used in clinical practice still need to be further developed. Cordycepin (3’-deoxyadenosine), a natural derivative of adenosine, is one of the important active ingredients in Cordyceps militaris. It has many pharmacological activities such as anti-inflammatory, anti-aging, anti-tumor, immune regulation and metabolism. In this review, we summarized the protective effects and mechanism of cordycepin in ischemia-reperfusion injury in recent years. Key words: Ischemia-reperfusion injury; Cordycepin; Anti-inflammatory; Anti-oxidation

Objective To explore the effects of antipsychotics on peripheric platelet mRNA level of adenosine A2a receptor (ADA2aR) in un-medicated patients with schizophrenia.Methods The mRNA level of ADA2aR in 30 un-medicated patients with schizophrenia was evaluated with real-time polymerase chain reaction (RT-PCR) at baseline and after six-week antipsychotic treatment,and 30 healthy volunteers were examined as controls.Results There were no significant difference ( P ＞ 0.05 ) in the mRNA level of ADA2aR between schizophrenics (747.6 ±282.3) and controls (692.7 ± 286.7) at baseline.There were also no significant correlation between mRNA level of ADA2aR and scores of Positive and Negative Symptoms Scales (PANSS) (P ＞ 0.05 ) at baseline. After six-week antipsychotic treatment,the mRNA level of ADA2aR in schizophrenics ( 873.2 ± 206.2) were significantly higher than the baseline (747.6 ± 282.3 )and in controls ( 635.4 ± 263.2 ) ( all P ＜ 0.05 ).No significant correlation between the mRNA level of ADA2aR and PANSS scores were found in schizophrenics and controls at baseline and endpoint (P ＞ 0.05 ).There were also no significant correlation between changes in the mRNA level of ADA2aR and PANSS scores ( P ＞ 0.05 ) in schizophrenics and controls.Conclusions The results suggested that antipsychotics treatment have effect on the gene expression level of adenosine A2a receptor on platelet and changes in gene expression level of adenosine A2a receptor could have no relationship with clinical symptom of schizophrenics. Key words: Schizophrenia; Adenosine; Antipsychotic agents; Blood platelets; Gene expression

Inflammaging in male reproductive organs covers a wide variety of problems, including sexual dysfunction and infertility. In this study, the beneficial effects of cordycepin (COR), isolated from potential medicinal fungi Cordyceps militaris, in aging-associated testicular inflammation and serum biochemical changes in naturally aged rats were investigated. Male Sprague Dawley rats were divided into young control (YC), aged control (AC), and COR (5, 10, and 20 mg/kg) treated aged rat groups. Aging-associated serum biochemical changes and inflammatory parameters were analyzed by biochemical assay kits, Western blotting, and real-time RT-PCR. Results showed a significant (p < 0.05) alteration in the total blood cell count, lipid metabolism, and liver functional parameters in AC group when compared with YC group. However, COR-treated aged rats ameliorated the altered biochemical parameters significantly (p < 0.05 and p < 0.01 at 5, 10, and 20 mg/kg, respectively). Furthermore, the increase in the expression of inflammatory mediators (COX-2, interleukin (IL)-6, IL-1β, and tissue necrosis factor-alpha) in aged rat testis was significant (p < 0.05) when compared with YC group. Treatment with COR at 20 mg/kg to aged rats attenuated the increased expression of inflammatory mediators significantly (p < 0.05). Mechanistic studies revealed that the potential attenuating effects exhibited by COR in aged rats was mediated by regulation of NF-κB activation and MAPKs (c-Jun N-terminal kinase, extracellular signal-regulated kinase 1/2, and p38) signaling. In conclusion, COR restored the altered serum biochemical parameters in aged rats and ameliorated the aging-associated testicular inflammation proving the therapeutic benefits of COR targeting inflammaging-associated male sexual dysfunctions.

Objective To explore the expression of the A2A adenosine receptor and the inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in human degenerative nucleus pulposus (NP) cells after they have been treated with a pulsed electromagnetic field (PEMF). Methods Human degenerative NP cells were cultured in vitro and treated using an 0.8mT PEMF with a pulse frequency of 50Hz. The pulse width was 150μs and the exposure time was 30min, repeated 5 times at 12 hour intervals. The expression of the A2A adenosine receptor in NP cells was determined using western blotting and reverse transcription polymerase chain reactions. The expression of the inflammatory cytokines IL-1β and TNF-α were detected using enzyme-linked immunosorbent assays (ELISA). The human degenerative NP cells were also treated with an antagonist and agonist of the A2A adenosine receptor, and the expression of IL-1β and TNF-α were also determined using ELISA. Results After the PEMF treatment the expression of the A2A adenosine receptor increased significantly, while the expression of IL-1β and TNF-α decreased significantly. However, the A2A adenosine receptor antagonist reversed the inhibitory effect of the PEMF on the expression of IL-1β and TNF-α, while the agonist played an opposite role. Conclusion A PEMF can significantly inhibit the expression of IL-1β and TNF-α in human degenerative NP cells, which could be related to up-regulation of the expression of the A2A adenosine receptor in those cells. Key words: Pulsed electromagnetic fields; Adenosine receptors; Nucleus pulposus cells; Inflammatory cytokines

Adenosine A2A receptors mediate hypoxic inhibition of fetal breathing in sheep

Adenosine is a multi-functional physiological molecule found abundantly in the body. It is one of the important components of ATP cellular energy metabolism. Adenosine has diverse actions as a ligand on many different types of cells and tissues acting via specific receptors. Currently, four subtypes of adenosine receptors are described, namely, the A1, A2A, A2B and A3 receptors. Neuroblastoma, mostly found in young children, is a malignant tumor derived from peripheral neurons in the body. Several different types of neuroblastoma cell lines of human origin have been established and contributed to the studies of neuroblastoma itself, neuronal differentiation, neurotransmitters, alcoholism, Alzheimer's disease and other neuronal diseases and disorders. In 1987, it was shown by Abbracchio et al. that a human neuroblastoma cell line, IMR32, could be induced to differentiate into cells that have a more neuronal morphology, with long neurites, by an adenosine receptor agonist 5'-N-ethylcarboxamideadenosine (NECA) 2. 'Neuronal differentiation' is expected to be a new alternative to the conventional clinical therapies, such as surgery, chemotherapy and radiotherapy. Unlike IMR32, PC12 cells, a rat adrenal pheochromocytoma cell line, resembling human neuroblastoma cell lines and also expressing the A2 subtype of adenosine receptors, was shown not to differentiate under stimulation of the A2A subtype of adenosine receptors 3. Moreover, adenosine inhibited neuronal differentiation in mouse dorsal root ganglion cells presumably via the A1 subtype 4. The mechanism(s) of these confusing effects of adenosine on neuronal differentiation require examination. First, a detection method for each of the adenosine receptor subtypes was developed using reverse transcriptase polymerase chain reaction (RT-PCR). This provided a sensitive, non-radioactive, analytical tool. Subtype-specific, four pairs of PCR primers, corresponding to the A1, A2A, A2B and A3 receptors, were designed and synthesized. The RT-PCR study revealed the presence of adenosine A1, A2A and A2B receptor mRNAs in untreated SH-SY5Y cells. These PCR primers were also designed so that they would allow multiplex PCR. Optimization of conditions for multiplex PCR was conducted, allowing it to detect several adenosine receptor subtypes simultaneously, and it was proven to be partially successful. In the study of differentiation, the use of the designed PCR primers was not quantitative to measure the levels of adenosine receptors due to variations of the expressions levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, a house-keeping gene commonly used as the internal control in PCR or northern blot analysis. An adequate neuronal differentiation model system was established in order to study the possible role(s) of adenosine in neuronal differentiation. Nerve growth factor (NGF), a well-known inducer of differentiation of rat PC12 cells, did not show any apparent differentiation effects on human neuroblastoma SH-SY5Y cells. All-trans retinoic acid (50 µM) induced distinct neuronal differentiation in SH-SY5Y cells, however ethanol, used as a vehicle for retinoic acid, was also shown to have effects on this cell line causing morphological changes. Adenosine (100 µM) alone also did not induce marked differentiation in this cell line probably due to the presence of adenosine in serum. Adenosine deaminase-resistant, synthetic adenosine analogues were used and demonstrated enhancement of differentiation. A serum deprivation-induced differentiation in SH-SY5Y was found to be a consistent and useful model to evaluate the effects of other factors on differentiation in this cell line. This serum deprivation-induced differentiation was also found to accompany a substantial rise in the expression of neurofilament-H (NF-H), one of the marker proteins for neuronal differentiation, at the protein level. Using this model, the possible involvement of adenosine signaling via its receptors was investigated. Treatment of cells with selective adenosine analogues for the A1 and A2A subtypes, 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) and 2-[4-(2-carboxylethyl)phenylamino]-5'-N-ethylcarboxamido (CGS21680, 30 and 100 nM), respectively, enhanced the differentiation induced by serum deprivation at day 7 by approximately 60% and 70%, respectively. These enhancing effects of agonists were blocked by selective antagonists, 8-cyclophenyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinalzolin-5-amine (CGS15943), respectively. Simultaneous co-stimulation of the A1 and A2A subtypes with these agonists gave no further effects compared to the enhancing effects exerted by CCPA or CGS21680 alone. Signal transduction pathways were examined using various protein kinase inhibitors. A selective protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 100 nM) alone greatly enhanced the differentiation induced by serum deprivation in this cell line. No additive or synergistic effects of 10 nM H-89 with either the A1 or A2A receptor agonist were seen. A selective mitogen-activated protein kinase kinase (MAPKK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098,059) showed a similar pattern to H-89: 100 nM PD098,059 alone caused enhanced differentiation in serum deprivation-induced SH-SY5Y cells. The combination of PD098,059 and adenosine agonists did not show any further enhancement of differentiation. On the contrary, a selective protein kinase C (PKC) inhibitor, chelerythrine, suppressed the differentiation (by 51%) by serum deprivation at 1 uM, and at 100 nM, chelerythrine suppressed the enhancement of differentiation caused by CCPA and CGS21680 with no effect on the basic level of differentiation, indicating the possible involvement of PKC both in the differentiation induced by serum deprivation and the adenosine receptor-induced potentiation. Surprisingly, contrary to the assumption that the stimulation of PKA induces or assists neuronal differentiation, H-89 (20 uM) alone exerted a prompt differentiation (44% at day 2) in SH-SY5Y cells in the presence of the normal serum concentration (10%). This data suggests that the previously assumed role of PKA in differentiation must be re-evaluated. This H-89-induced differentiation model was shown to have a different differentiation mechanism to the previous serum deprivation-induced differentiation. Establishment of these new differentiation study models will add further options to explore neuronal differentiation, especially, of human type.

Hydroxyapatite nanoparticles promote TLR4 agonist-mediated anti-tumor immunity through synergically enhanced macrophage polarization.

Objective To investigate cellular and behavioral effects of adenosine A2A receptor antagonist in a rat model of levodopa-induced motor complications.Methods The hemi-parkinsonian rat model was produced by stereotaxically injecting 6-OHDA to right medial forebmin bundle(MFB).Animals were intraperitoneally treated with levodopa 50 mg/kg plus benserazide 12.5 mg/kg twice a day for 22 days levodopa + vehicle.Rotational duration was estimated.After they were sacrificed,the expression of adenosine A2A receptor was observed by immunohistochemistry and Western blot.Results CSC,reversing the shortened rotational duration induced by levodopa,prolonged the rotational duration.This effect was maintained fil the end of the treatment.The chronic levedopa treatment induced an upregulation of adenosine A2A receptor expression in the lesioned striatum (IOD,(11.55±2.75)×104).The subsequent CSC treatment decreased the adenosine A2A receptor expression to the level of control (IOD,(6.02±1.29)× 104) and PD group (IOD,(5.60±1.83)×104>,F=33.31,P<0.05).Conclusion These results suggest that adenosine A2A receptor is probably involved in the development of levodopa induced motor complications and adenosine A2A receptor antagonist could be useful in the treatment of motor comphcations in parkinsonian patients. Key words: Parkinson disease; Movement disorders; Receptor,adenosine A2A; Caffeine; Levodopa