Surface d-Band Modulation via Biodirected Mineralization Enables Nanoenzymes to Inhibit Radiation-Induced T-Cell Exhaustion and Potentiate Immunoradiotherapy.

IBS23.01 Radiotherapy of OMD in Daily Clinical Practice

Objective To investigate the mechanism of cyclic adenosine monophosphate / protein kinase A signal transduction pathway in severe acute pancreatitis(SAP)-associated lung injury.Methods Seventy-two male healthy SD rats were completely randomized into three groups:sham operation (SO) group(n =8),SAP group and SAP plus H89 (cAMP inhibitor) group,then the latter two groups were divided into four sub-groups with eight rats in each sub-group according to the sampling time of 3,6,12 and 24 h,and the total numbers of groups were nine.The content change of TNF-α and IL-1β in serum,protein levels of cAMP-dependent protein kinase catalytic subunit (PKA C) and phosphorylated vasodilator-stimulated phosphoprotein(p-VASP) and the expression of VSAP mRNA in lung tissue were detected by enzyme-linked immunosorbent assay (ELISA),immunohistochemistry and quantitative real time PCR,respectively.Pathological changes of the pancreas and lung tissues were also observed.Results Compared with the SO group,the serum levels of TNF-α and IL-1β in the SAP group were obviously increased at different time points(P ＜0.05).Pathological changes of the pancreas and lung tissues were aggravated significantly.The protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were increased significantly (P ＜0.05)which peaked at 12 h in the SAP group [TNF-α was (266.07 ± 17.14) pg/mL,IL-1β(169.17 ±25.92) pg/mL,PKA C(210.69 ±6.32) × 103,p-VASP (56.62 ±0.57) × 103,VASP mRNA(2.06 ±0.21)],which had positive correlation with the serum level of TNF-α and IL-1β.Compared with the SAP group,pathological changes of the pancreas and lung tissues were alleviated significantly,the protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were decreased significantly in the SAP plus H89 group at different time points(P ＜ 0.05).Conclusion The cyclic adenosine monophosphate / protein kinase A signal transduction pathway is found to participate in the pathological process of SAP-associated lung injury through the up-regulations of TNF-α,IL-1 β and phospho-VASP. Key words: Pancreatitis; Cyclic AMP-dependent protein kinases; Lung injury; Vasodilator-stimulated phosphoprotein

MP64-16 HYPOFRACTIONATED STEREOTACTIC RADIOTHERAPY INCREASES BOTH TUMOR-INFILTRATING LYMPHOCYTES AND SUPPRESSIVE IMMUNE CELLS IN PROSTATE CANCER

Cyclic adenosine monophosphate (cAMP) is the firstly discoveried second messenger.cAMP controls a range of diverse physiological processes,including metabolicevents,calcium handling,learning and memory,cellgrowth and differentiation,apoptosis,and inflammation.Protein kinase A (PKA) had been considered as the sole downstream target of cAMP.However,recent studies have demonstrated thatEpac,a novel cAMPmediator,regulates manyphysiological processes either alone and (or) in concert with PKA.In this review,we will discuss the roles and probable mechanisms of Epac in the management of asthma in order to provide beneficial clue to find new therapy targets. Key words: Bronchial asthma; Epac; G protein couple receptor; Cyclic adenosine monophosphate; Protein kinase A

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) enhances the action of neuregulin, the most potent known mitogen for SCs, by synergistically increasing the activation of two crucial signaling pathways: ERK and Akt. However, the underlying mechanism of cross-talk between neuregulin and cAMP signaling remains mostly undefined. Here, we report that the activation of protein kinase A (PKA), but not that of exchange protein activated by cAMP (EPAC), enhances S-phase entry of SCs by synergistically enhancing the ligand-dependent tyrosine phosphorylation/activation of the neuregulin co-receptor, ErbB2-ErbB3. The role of PKA in neuregulin-ErbB signaling was confirmed using PKA inhibitors, pathway-selective cAMP analogs, and natural ligands stimulating PKA activity in SCs, such as adenosine and epinephrine. Two basic observations defined the synergistic action of PKA as "gating" for neuregulin-ErbB signaling: 1) the activation of PKA was not sufficient to induce S-phase entry or the activation of either ErbB2 or ErbB3; and 2) the presence of neuregulin was strictly required to ignite ErbB activation and thereby ERK and Akt signaling. However, PKA directly phosphorylated ErbB2 on Thr-686, a highly conserved intracellular regulatory site that was required for the PKA-mediated synergistic enhancement of neuregulin-induced ErbB2-ErbB3 activation and proliferation in SCs. The gating action of PKA on neuregulin-induced ErbB2-ErbB3 activation has important biological significance, because it insures signal amplification into the ERK and Akt pathways without compromising either the neuregulin dependence or the high specificity of ErbB signaling pathways.

Metformin mainly gives play to the hypoglycemic effect by reducing hepatic gluconeogenesis and activating glucose utilization of peripheral tissues. It is especially important that inhibiting hepatic gluconeogenesis in the hypoglycemic effect of metformin, of which molecular mechanisms are complicated and diverse, and are related to drug concentrations. Metformin, at pharmacologic concentrations, may directly inhibit mitochondria glycerophosphate dehydrogenase, resulting in reduced nicotinamide adenine dinucleotide(NADH)accumulation in cytosol and reducing pyruvate/lactate ratio, thus inhibiting gluconeogenesis. This effect is dependent on neither AMP nor adenosine monophosphate-activated protein kinase(AMPK). Metformin at pharmacologic concentrations may also promote subunit assembly of AMPK to activate AMPK directly, thereby inhibiting hepatic gluconeogenesis. This effect is independent on AMP while dependent on AMPK. Metformin, at supra-pharmacologic concentrations, may inhibit mitochondrial complex Ⅰ, thus decrease ATP/AMP ratio, thereby activating AMPK and inhibiting hepatic gluconeogenesis. The reduction of ATP/AMP ratio also inhibits gluconeogenesis directly by energy chargeing mechanism; the accumulation of AMP inhibits adenylate cyclase, reducing levels of cyclic AMP and protein kinase A(PKA)activity, abrogating phosphorylation of critical protein targets of PKA, and blocking hepatic glucagon signaling. Metformin at supra-pharmacologic concentrations also inhibits AMP deaminase, and bypasses mitochondrial respiratory chain to increase AMP level, which activates AMPK and inhibits gluconeogenesis. Metformin at supra-pharmacologic concentrations also inhibits hepatic gluconeogenesis by protein kinase Cζ(PKCζ)-liver kinase B1(LKB1)-AMPK phosphorylation cascade. In addition, before metformin enters the blood, it activates intestinal mucosa(probablly L-cells)AMPK to increase glucagon-like peptide 1(GLP-1)secretion. GLP-1 acts on afferent neuron of intestines vagus to inhibit hepatic gluconeogenesis by vagus efferent neuron of nucleus tractus solitarii(intestines-brain-liver axis route). (Chin J Endocrinol Metab, 2016, 32: 716-722) Key words: Metformin; Diabetes mellitus; Adenosine monophosphate; AMP-activated protein kinase; Mitochondria; Gluconeogenesis

To investigate the production and mechanism of chemokine (C-C motif) ligand 5 (CCL5) by macrophages in U14 cervical cancer-bearing mice during infection. The U14 cervical cancer cells were injected in C57BL/6 mice to induce tumor-bearing condition. Lipopolysaccharide (LPS) was injected into C57BL/6 mice to induce infection. The protein expression of CCL5 in the serum and the CCL5 mRNA expression in inflammatory cells were measured by ELISA and fluorescence quantitative-PCR in four groups. Macrophages were induced in the tumor conditioned medium (TCM) which extracted from mice serum. The protein expression levels of CCL5, prostaglandin E2 (PGE2) and cyclic adenosine monophosphate (cAMP) in the medium and CCL5, PGE2 and cAMP mRNA expression in the macrophages were detected in different groups. In order to determine whether the inhibition was related to PGE2, selective cyclooxygenase 2(COX-2) inhibitor NS398 was used to reverse this phenomenon and protein kinase A (PKA) inhibitor H89 demonstrated the mechanism through blocking cAMP/PKA signaling pathway. (1) The protein and mRNA level of CCL5 in tumor-bearing mice were respectively (151 ± 35) pg/ml and 1.0, which were lower than those in the tumor-free mice (691 ± 85) pg/ml and 4.5 ± 0.8, there were significant difference between them (all P < 0.05). The protein and mRNA level of PGE2 in tumor-bearing mice were (1 198 ± 83) pg/ml and 5.8 ± 0.8, which were higher than those in the tumor-free mice (187 ± 25) pg/ml and 1.0, the difference were significant (all P < 0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice were (4 049 ± 141) pg/ml and 31.5 ± 2.0, which were higher than those in the tumor-bearing + LPS mice (1 951 ± 71) pg/ml and 12.1 ± 2.8, the difference were also significant (P < 0.05). The protein and mRNA level of PGE2 in tumor-free + LPS mice were (676 ± 70) pg/ml and 3.4 ± 0.4, which were lower than those in tumor-bearing + LPS mice (2 550 ± 382) pg/ml and 11.6 ± 0.9, the difference were also significant (all P < 0.05). (2) Macrophages were cultured in vitro using TCM derived from mice. The protein and mRNA level of CCL5 in tumor-bearing mice TCM were respectively (1 626 ± 177) pg/ml and 28.6 ± 1.2, which were higher than those in the tumor-free mice TCM [(27 ± 3) pg/ml and 1.0], there were significant difference (P < 0.05). The protein and mRNA level of PGE2 in tumor-bearing mice TCM were (790 ± 156) pg/ml and 1.7 ± 0.3, which were higher than those in the tumor-free mice TCM [(448 ± 115) pg/ml, 1.0], the difference were significant (all P < 0.05). The protein and mRNA level of cAMP in tumor-bearing mice TCM were (164 ± 30) pg/ml and 1.6 ± 0.3, which weres higher than those in the tumor-free mice TCM [(118 ± 25) pg/ml,1.0], the difference were significant (all P < 0.05). The protein and mRNA level of CCL5 in tumor-free + LPS mice TCM were (10 475 ± 742) pg/ml and 212.0 ± 5.7, which were higher than those in the tumor-bearing + LPS mice TCM [(6 375 ± 530) pg/ml, 142.3 ± 2.5], the difference were significant (all P < 0.05). The protein and mRNA level of PGE2 in tumor-free + LPS mice TCM were (2 438 ± 95) pg/ml and 4.3 ± 0.7, which weres lower than those in the tumor-bearing + LPS mice TCM [(3 441 ± 163) pg/ml, 5.9 ± 0.3], the difference were significant (all P < 0.05). The protein and mRNA level of cAMP in tumor-free + LPS mice TCM were (340 ± 13) pg/ml and 4.1 ± 0.4, which were lower than those in the tumor-bearing + LPS mice TCM [(542 ± 42) pg/ml, 5.4 ± 0.5], the difference were significant (all P < 0.05). (3) Using COX-2 inhibitor NS398 in the tumor-bearing + LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (7 691 ± 269) pg/ml and 159.0 ± 8.9, (2 820 ± 152) pg/ml and 4.9 ± 0.3, (465 ± 8) pg/ml and 4.3 ± 0.4, respectively, and there were significant difference (all P < 0.05), compared to before treatment. Using PKA inhibitor H89 in the tumor-bearing + LPS mice, the protein and mRNA level of CCL5, PGE2 and cAMP were (8 375 ± 520) pg/ml and 177.0 ± 8.8, (2 650 ± 35) pg/ml and 4.7 ± 0.4, (368 ± 13) pg/ml and 3.1 ± 0.7, respectively, and there were significant difference (all P < 0.05), compared to before treatment. TCM of U14 cells activated macrophages to release PGE2 could inhibit the expression of CCL5 levels by cAMP/PKA signaling pathway.

Kinesins are motor proteins that transport their cargos along microtubules in an ATP-dependent manner. The testis-specific kinesin KIF17b was shown to directly regulate cAMP-response element modulator (CREM)-dependent transcription by determining the subcellular localization of the activator of CREM in testis (ACT), the testis-specific coactivator of CREM in postmeiotic male germ cells. CREM is a crucial transcriptional regulator of many important genes required for spermatid maturation, as demonstrated by the complete block of sperm development at the first steps of spermiogenesis in crem-null mice. To better understand the complex regulation of postmeiotic germ cell differentiation, we further characterized the ACT-KIF17b interaction, the function of KIF17b, and the signaling pathways governing its action. In this study, we demonstrated that the abilities of KIF17b to shuttle between the nuclear and the cytoplasmic compartments and to transport ACT are neither dependent on its motor domain nor on microtubules, thus revealing a novel microtubule-independent function for kinesins. We also showed that the cyclic AMP-dependent protein kinase A mediates the phosphorylation of KIF17b, and this modification is important for its subcellular localization. These results indicate that cyclic AMP signaling controls CREM-mediated transcription in male germ cells through modification of KIF17b function.

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14-18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.

Objective To explore the molecular mechanism of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway in γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma QBC939 cell line. Methods QBC939 cells were cultured in different groups and treated withγ-aminobutyric acid (GABA), GABA+ 8 Br (cAMP agonists), GABA+ H89 (PKA antagonist) for 48 hours. (4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the proliferation of QBC939 cells. Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) binding assay was used to detect apoptosis in the QBC939 cells. Enzymelinkedimmunosorbentassay (ELISA) assay was detected to the expression of cAMP and PKA. Western blotting was applied to check the expression of PKAI and PKAII and extracellular regulated protein kinases (ERK) proteins in different groups of QBC939 cells. Animal models of cholangiocarcinoma bearing nude mice were established by subcutaneous injection of QBC939 cells and randomized into 2 groups: control and GABA-treated groups. The effect of GABA was evaluated after 5 weeks, including tumor volume. The expression of PKA Ⅰ and PKA Ⅱ and ERK was detected by Western blotting in xenograft tumors. Results MTT and flow cytometry (FCM) assays all showed that the effect of GABA inhibitory on the proliferation and induced apoptosis of QBC939 cells could be antagonized by H89, but not 8 Br. GABA significantly increased the content of cAMP and PKA, the content of cAMP was enhanced by 8 Br, the content of PKA was antagonized by H89. Western blotting analysis showed that GABA significantly down-regulated the expression of PAK I protein (0.087 8±0.003 0 vs. 0.152 1±0.003 0, t=29.687, P<0.05), up-regulated the expression of PKAⅡ, and also decreased the expression of ERK protein protein (0.368 3±0.007 0 vs. 0.468 7±0.010 0, t=13.647, P<0.05). Xenograft tumor volume [ (0.500±0.020 vs. 0.320±0.030) cm3,t=15.354, P<0.05]. The expression of PKAI and ERK were significantly decreased, and PKA II was increased in GABA-treated group as compared with control group. Conclusion GABA may inhibit the growth of cholangiocarcinoma cells QBC939 through cAMP/PKA, down-regulated PAK I, up-regulated PKA II, and decreased the expression of the ERK perhaps is one of its anti-tumor mechanisms. Key words: Bile duct neoplasms; Gamma-aminobutyric acid; Cyclic adenosine monophosphate; Protein kinase A; Extracellular regulated protein kinases

To study the effect of cyclic adenosine monophosphate on aquaporin 8 (AQP8) expression and distribution in human amnion-derived WISH cells. Human amnion-derived WISH cells were cultured. The cells were divided into control group and study group at random. The study group was established by exposure to various concentrations of 8-Br-cAMP. Western analysis was used to quantify AQP8 expression levels. RT-PCR was used to quantify AQP8 mRNA expression levels. Immunofluorescence was used to determine the localization of AQP8 in WISH cells. With increase of cAMP, AQP8 mRNA and protein expressions significantly increased in WISH cells in vitro. When the concentration of cAMP reached 200 micromol/L, AQP8 mRNA and protein expressions were highest. Incubation with cAMP (200 micromol/L) for 2 hours resulted in a 2-fold increase in AQP8 mRNA level, and incubation for 8 hours resulted in a peak. After incubation for 16 hours AQP8 mRNA level began to descend, and after 24 hours it decreased to baseline. Incubation with cAMP for 8 hours AQP8 protein level began to increase. Incubation for 24 hours resulted in a peak in AQP8 protein level, and after incubation for 48 hours it began to decline. By immunofluorescence microscopy after incubation with cAMP (200 micromol/L) cells, AQP8 labeling in plasma membrane was enhanced and intracellular AQP8 labeling was decreased. The cAMP triggers translocation of AQP8 from cytosol to the plasma membrane via vesicle-transporting related protein instead of AQP8 itself. cAMP may upregulate the transcription of target gene protein kinase A. The cAMP may be the critical regulatory medium of AQP8 in WISH cells.

(P46) Toxicity Analysis of Concurrent Stereotactic Body Radiotherapy and Immunotherapy for Primary and Oligometastatic Cancer

The utilisation of neoadjuvant immunotherapy has demonstrated promising preliminary clinical outcomes for early-stage resectable non-small-cell lung cancer (NSCLC). Nevertheless, it is imperative to develop novel neoadjuvant combination therapy regimens incorporating immunotherapy to further enhance the proportion of patients who derive benefit. Recent studies have revealed that stereotactic body radiotherapy (SBRT) not only induces direct tumour cell death but also stimulates local and systemic antitumour immune responses. Numerous clinical trials have incorporated SBRT into immunotherapy for advanced NSCLC, revealing that this combination therapy effectively inhibits local tumour growth while simultaneously activating systemic antitumour immune responses. Consequently, the integration of SBRT with neoadjuvant immunotherapy has emerged as a promising strategy for treating resectable NSCLC, as it can enhance the systemic immune response to eradicate micrometastases and recurrent foci post-resection. This review aims to elucidate the potential mechanism of combination of SBRT and immunotherapy followed by surgery and identify optimal clinical treatment strategies. Initially, we delineate the interplay between SBRT and the local tumour immune microenvironment, as well as the systemic antitumour immune response. We subsequently introduce the preclinical foundation and preliminary clinical trials of neoadjuvant SBRT combined with immunotherapy for treating resectable NSCLC. Finally, we discussed the optimal dosage, schedule, and biomarkers for neoadjuvant combination therapy in its clinical application. In conclusion, the elucidation of potential mechanism of neoadjuvant SBRT combined immunotherapy not only offers a theoretical basis for ongoing clinical trials but also contributes to determining the most efficacious therapy scheme for future clinical application.

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.