Structure-Activity Relationship Analysis of Fluoxetine for Suppression of Inflammatory Cytokine Production.

There is accumulating evidence that selective serotonin reuptake inhibitors (SSRIs), clinically used as antidepressants, have a beneficial effect on inflammatory diseases such as coronavirus disease 2019 (COVID-19). We previously compared the inhibitory effects of five U.S. Food and Drug Administration (FDA)-approved SSRIs on the production of an inflammatory cytokine, interleukin-6 (IL-6), and concluded that fluoxetine (FLX) showed the most potent anti-inflammatory activity. Here, we investigated the structure-activity relationship of FLX for anti-inflammatory activity towards J774.1 murine macrophages. FLX suppressed IL-6 production induced by the TLR3 agonist polyinosinic-polycytidylic acid (poly(I : C)) with an IC50 of 4.76 µM. A derivative of FLX containing chlorine instead of the methylamino group lacked activity, suggesting that the methylamino group is important for the anti-inflammatory activity. FLX derivatives bearing an N-propyl or N-(pyridin-3-yl)methyl group in place of the N-methyl group exhibited almost the same activity as FLX. Other derivatives showed weaker activity, and the N-phenyl and N-(4-trifluoromethyl)benzyl derivatives were inactive. The chlorine-containing derivative also lacked inhibitory activity against TLR9- or TLR4-mediated IL-6 production. These derivatives showed similar structure-activity relationships for TLR3- and TLR9-mediated inflammatory responses. However, the activities of all amino group-containing derivatives against the TLR4-mediated inflammatory response were equal to or higher than the activity of FLX. These results indicate that the substituent at the nitrogen atom in FLX strongly influences the anti-inflammatory effect.

Transcriptome analysis of cultured human vascular endothelial cells after γ-ray irradiation and correlation analysis with ATP, ADP, and adenosine as secondary soluble factors.

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.

Extracellular Guanosine and Guanine Nucleotides Decrease Viability of Human Breast Cancer SKBR-3 Cells.

Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.

Involvement of Cannabinoid Receptors and Adenosine A2B Receptor in Enhanced Migration of Lung Cancer A549 Cells Induced by γ-Ray Irradiation.

Residual cancer cells after radiation therapy may acquire malignant phenotypes such as enhanced motility and migration ability, and therefore it is important to identify targets for preventing radiation-induced malignancy in order to increase the effectiveness of radiotherapy. G-Protein-coupled receptors (GPCRs) such as adenosine A2B receptor and cannabinoid receptors (CB1, CB2, and GPR55) may be involved, as they are known to have roles in proliferation, invasion, migration and tumor growth. In this study, we investigated the involvement of A2B and cannabinoid receptors in γ-radiation-induced enhancement of cell migration and actin remodeling, as well as the involvement of cannabinoid receptors in cell migration enhancement via activation of A2B receptor in human lung cancer A549 cells. Antagonists or knockdown of A2B, CB1, CB2, or GPR55 receptor suppressed γ-radiation-induced cell migration and actin remodeling. Furthermore, BAY60-6583 (an A2B receptor-specific agonist) enhanced cell migration and actin remodeling in A549 cells, and this enhancement was suppressed by antagonists or knockdown of CB2 or GPR55, though not CB1 receptor. Our results indicate that A2B receptors and cannabinoid CB1, CB2, and GPR55 receptors all contribute to γ-radiation-induced acquisition of malignant phenotypes, and in particular that interactions of A2B receptor and cannabinoid CB2 and GPR55 receptors play a role in promoting cell migration and actin remodeling. A2B receptor-cannabinoid receptor pathways may be promising targets for blocking the appearance of malignant phenotypes during radiotherapy of lung cancer.

Luminal B Breast Cancer Coexpressing p62 and ALDH1A3 Is Less Susceptible to Radiotherapy.

BACKGROUND/AIM: We have reported that p62 (also known as sequestosome 1) is needed for survival/proliferation and tumor formation by aldehyde dehydrogenase 1 (ALDH1) -positive cancer stem cells (CSCs) and that p62high ALDH1A3high expression is associated with a poor prognosis in luminal B breast cancer. However, the association between p62high ALDH1A3high and the benefit from radiotherapy in patients with luminal B breast cancer remains unclear. MATERIALS AND METHODS: Datasets from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) were downloaded, and data from p62high ALDH1A3high luminal B patients treated without or with radiotherapy were analyzed by Kaplan-Meier and multivariate Cox regression analyses. We also performed an in vitro tumor sphere formation assay after X-ray irradiation using p62-knockdown ALDH1high luminal B BT-474 cells. RESULTS: p62high ALDH1A3high patients had poorer clinical outcomes than other luminal B breast cancer patients treated with radiotherapy. The combination of p62 DsiRNA KD and X-ray irradiation suppressed in vitro tumor sphere formation by ALDH1high BT-474 cells. These results suggest that p62 is involved in the reduced effect of X-ray irradiation on ALDH1-positive luminal B breast CSCs. CONCLUSION: p62 and ALDH1A3 may serve as prognostic biomarkers for luminal B breast cancer patients treated with radiotherapy. Additionally, the combination of p62 inhibition and radiotherapy could be useful for targeted strategies against ALDH1-positive luminal B breast CSCs.

Profiling Differential Effects of 5 Selective Serotonin Reuptake Inhibitors on TLRs-Dependent and -Independent IL-6 Production in Immune Cells Identifies Fluoxetine as Preferred Anti-Inflammatory Drug Candidate.

Excessive proinflammatory cytokine production induced by abnormal activation of Toll-like receptor (TLR) signaling, for example, by SARS-CoV-2 infection, can cause a fatal cytokine storm. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine and fluvoxamine, used to treat depression, were recently reported to reduce the risk of severe disease in patients with coronavirus disease 2019 (COVID-19), but the mechanisms of the anti-inflammatory effects of SSRIs, and which SSRI would be most suitable as an anti-inflammatory drug, remain unclear. Here, we examined the inhibitory effects of 5 FDA-approved SSRIs, paroxetine, fluoxetine, fluvoxamine, sertraline and escitalopram, on the production of interleukin-6 (IL-6) induced by stimulation with multiple TLR agonists in murine macrophages and dendritic cells, and on the production of cytokines induced by concanavalin A in murine lymphocytes. In J774.1 murine macrophage cells, pretreatment with SSRIs significantly suppressed IL-6 release induced by TLR3 agonist poly(I:C), TLR4 agonist LPS or TLR9 agonist CpG ODN, but did not affect IL-6 release induced by TLR7 agonists imiquimod or resiquimod. In accordance with the results obtained in J774.1 cells, pretreatment with SSRIs also suppressed IL-6 release induced by a TLR3, TLR4 or TLR9 agonist in bone marrow-derived dendritic cells and peritoneal cells of C57BL/6 mice. On the other hand, interestingly, sertraline alone among the SSRIs amplified IL-6 production induced by TLR7 agonists in murine dendritic cells, though not in macrophages. Concanavalin A-induced production of IL-6 or IL-2 in murine lymphocytes was suppressed by SSRIs, suggesting that SSRIs also inhibit TLRs-independent IL-6 production. Since SSRIs suppressed both IL-6 production induced by multiple TLR agonists in macrophages or dendritic cells and TLR-independent IL-6 production in lymphocytes, they are promising candidates for treatment of patients with cytokine storm, which is mediated by overactivation of multiple TLRs in a complex manner, leading to the so-called IL-6 amplifier, an IL-6 overproduction loop. However, the 5 SSRIs examined here all showed different effects. Overall, our results suggest that fluoxetine may be the most promising candidate as an anti-inflammatory drug. An examination of the structural requirements indicated that the N-methyl group of fluoxetine has a critical role in the inhibition of IL-6 production.

Involvement of CD73 and A2B Receptor in Radiation-Induced DNA Damage Response and Cell Migration in Human Glioblastoma A172 Cells.

Glioblastoma is the most common malignant tumor of the central nervous system and is treated with a combination of surgery, radiation and chemotherapy. However, the tumor often acquires radiation resistance, which is characterized by an increased DNA damage response (DDR). Here, we show that CD73, which generates extracellular adenosine from ATP, and A2B receptor, which is activated by adenosine, are involved in the γ-radiation-induced DDR and the enhanced migration ability of human glioblastoma cell line A172. To investigate DDR, we evaluated ataxia telangiectasia mutated (ATM) activation and focus formation of histone H2A isoform γ (γH2AX) and p53-binding protein 1 (53BP1) in the nucleus of A172 cells after γ-irradiation. Antagonists of A2B receptor and CD73, or knockdown with small interfering RNA (siRNA), suppressed γ-radiation-induced DDR and promoted γ-radiation-induced cell death, as well as suppressing γ-radiation-induced cell migration and actin remodeling. These results suggest that activation of A2B receptor by extracellular adenosine generated via CD73 promotes γ-radiation-induced DDR, leading to recovery from DNA damage, and also enhances cell migration and actin remodeling. The CD73-A2B receptor pathway may be a promising target for overcoming radiation resistance and the acquisition of malignant phenotypes during radiotherapy of glioblastoma.

ATP and ADP enhance DNA damage repair in γ-irradiated BEAS-2B human bronchial epithelial cells through activation of P2X7 and P2Y12 receptors.

Agents that promote DNA repair may be useful as radioprotectants to minimize side effects such as radiation pneumonia caused by damage to normal cells during radiation therapy to treat lung cancer. We have reported that extracellular nucleotides and nucleosides are involved in the P2 or P1 receptor-mediated DNA damage response (DDR) after γ-irradiation. Here, we investigated the effects of ATP, UTP, GTP, ITP and their metabolites on the γH2AX/53BP1 focus formation in nuclei (a measure of γ-irradiation-induced DDR) and the survival of γ-irradiated immortalized human bronchial epithelial (BEAS-2B) cells. Fluorescence immunostaining showed that ATP and ADP increase DDR and DNA repair, and exhibit radioprotective effects as evaluated by colony formation assay. These effects of ATP or ADP were blocked by inhibitors of P2X7 or P2Y12 receptor, respectively, and by ERK1/2 inhibitor. ATP and ADP enhanced phosphorylation of ERK1/2 by suppressing MKP-1 and MKP-3 expression after γ-irradiation. These results indicate that ATP and ADP exhibit radioprotective effects by phosphorylation of ERK1/2 via activation of P2X7 and P2Y12 receptors, respectively, to promote γ-irradiation-induced DDR and DNA repair. ATP and ADP appear to be candidates for radioprotectants to reduce damage to non-cancerous cells during lung cancer radiotherapy by promoting DDR and DNA repair.

Involvement of adenosine A2B receptor in radiation-induced translocation of epidermal growth factor receptor and DNA damage response leading to radioresistance in human lung cancer cells.

BACKGROUND: Adenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR. METHODS: We investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation. RESULTS: A2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression. CONCLUSION: Activation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage. GENERAL SIGNIFICANCE: Our results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.

Involvement of A2B Receptor in DNA Damage Response and Radiosensitizing Effect of A2B Receptor Antagonists on Mouse B16 Melanoma.

It is therapeutically important to elucidate the factors involved in the radiation resistance of tumors. We previously showed that ATP is released from mouse melanoma B16 cells in response to γ-irradiation, but the role of adenosine, a metabolite of ATP, is still unclear. Here, we show that the adenosine A2B receptor is involved in DNA damage repair and radioresistance in mouse melanoma B16 cells. The DNA damage response after γ-irradiation was attenuated by pretreatment with A2B receptor antagonists, such as PSB603, while it was enhanced by pretreatment with A2B receptor agonists, such as BAY60-6583. γ-Irradiation decreased the cell survival rate, and pretreatment with PSB603 further reduced the survival rate. On the other hand, pretreatment with BAY60-6583 increased the cell survival rate after irradiation. The DNA damage response and the cell survival rate after γ-irradiation were both decreased in A2B-knockdown cells. In vivo experiments in mice confirmed that tumor growth was suppressed and delayed in the irradiated group pretreated with PSB603, compared with the irradiation-alone group. Our results indicate that adenosine A2B receptor contributes to radioresistance, and could be a new target for the development of agents to increase the efficacy of radiotherapy.

Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells.

Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.