Interacting with α7 nAChR is a new mechanism for AChE to enhance the inflammatory response in macrophages

Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses.     

Purinergic signaling pathway in severe COVID-19

Substantial efforts have been made to understand the immune response during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, in order to identify and characterize risk factors, immune mechanisms responsible for the induction of tissue injury and potential therapeutic targets. Purinergic signaling pathway has shown to modulate the inflammatory processes in the course of several infectious diseases, but its role in the coronavirus disease 2019 (COVID-19) has not been clearly defined. Inflammation is usually associated to the release of ATP from different cell types, starting a cascade of events through the activation of a set of different purinergic receptors. This review summarizes the evidence showing the involvement of the purinergic system in the inflammatory condition that characterizes severe COVID-19.     

Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Salvia miltiorrhiza Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out GSDME switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC via GSDME-dependent pyroptosis.     

ASIC1a promotes synovial invasion of rheumatoid arthritis via Ca2+/Rac1 pathway

Acid-sensitive ion channels (ASICs) as Ca²⁺ and Na⁺ cation channels are activated by changing in extracellular pH, which expressed in various diseases and participated in underlying pathogenesis. ASIC1a is involved in migration and invasion of various tumor cells. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) located at the edge of the synovium were identified as key players in the pathophysiological process of rheumatoid arthritis and reported to have many similar properties to tumor cells. Here, we investigated the roles of ASIC1a in synovial invasion in vivo and the migration and invasion of RA-FLSs in vitro.Our results showed ASIC1a highly expressed in RA synovial tissues and RA-FLSs. Inhibition of ASIC1a by PCTX-1 reduces synovial invasion and the expressions of MMP2, MMP9, p-FAK to protect articular cartilage in AA rats. Moreover, the acidity-promoted invasion and migration as well as the expressions of MMP2, MMP9, p-FAK of RA-FLSs were down-regulated by ASIC1a-RNAi and PCTX-1 while they were increased by overexpression-ASIC1a. ASIC1a mediated Ca²⁺ influx and the activation of Ras-related C3 botulinum toxin substrate 1(Rac1), which was decreased by the intracellular calcium chelating agent BAPTA-AM. Meanwhile, the migration and invasion as well as the expressions of MMP2, MMP9, p-FAK of RA-FLSs were decreased by Rac1 specific blocker NSC23766. In conclusion, this study indicated that ASIC1a may be a master regulator of synovial invasion via Ca²⁺/Rac1 pathway.     

Crosstalk among colon cancer-derived exosomes,fibroblast-derived exosomes,and macrophage phenotypes in colon cancer metastasis

Cellular crosstalk is an important mechanism in the pathogenesis of inflammatory disorders and cancers. One significant means by which cells communicate with each other is through the release of exosomes. Exosomes are extracellular vesicles formed by the outward budding of plasma membranes, which are then released from cells into the extracellular space. Many studies have suggested that microvesicles released by colon cancer cells initiate crosstalk and modulate the fibroblast activities and macrophage phenotypes. Interestingly, crosstalk among colon cancer cells, macrophages and cancer-associated fibroblasts maximizes the mechanical composition of the stromal extracellular matrix (ECM). Exosomes contribute to cancer cell migration and invasion, which are critical for colon cancer progression to metastasis. The majority of the studies on colorectal cancers (CRCs) have focused on developing exosomal biomarkers for the early detection and prediction of CRC prognosis. This study highlights the crosstalk among colon cancer-derived exosomes, macrophage phenotypes and fibroblasts during colon cancer metastasis.     

A hybrid bacterium with tumor-associated macrophage polarization for enhanced photothermal-immunotherapy

Remodeling the tumor microenvironment through reprogramming tumor-associated macrophages (TAMs) and increasing the immunogenicity of tumors via immunogenic cell death (ICD) have been emerging as promising anticancer immunotherapy strategies. However, the heterogeneous distribution of TAMs in tumor tissues and the heterogeneity of the tumor cells make the immune activation challenging. To overcome these dilemmas, a hybrid bacterium with tumor targeting and penetration, TAM polarization, and photothermal conversion capabilities is developed for improving antitumor immunotherapy in vivo. The hybrid bacteria (B.b@QDs) are prepared by loading Ag₂S quantum dots (QDs) on the Bifidobacterium bifidum (B.b) through electrostatic interactions. The hybrid bacteria with hypoxia targeting ability can effectively accumulate and penetrate the tumor tissues, enabling the B.b to fully contact with the TAMs and mediate their polarization toward M1 phenotype to reverse the immunosuppressive tumor microenvironment. It also enables to overcome the intratumoral heterogeneity and obtain abundant tumor-associated antigens by coupling tumor penetration of the B.b with photothermal effect of the QDs, resulting in an enhanced immune effect. This strategy that combines B.b-triggered TAM polarization and QD-induced ICD achieved a remarkable inhibition of tumor growth in orthotopic breast cancer.     

Discovery of the anti-angiogenesis effect of eltrombopag in breast cancer through targeting of HuR protein

HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited in vitro cell proliferation of multiple cancer cell lines and macrophages, and the in vivo anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The in vivo data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the in vitro anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and in vitro Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.     

Bioresponsive immune-booster-based prodrug nanogel for cancer immunotherapy

The combination of chemotherapy and immunotherapy motivates a potent immune system by triggering immunogenic cell death (ICD), showing great potential in inhibiting tumor growth and improving the immunosuppressive tumor microenvironment (ITM). However, the therapeutic effectiveness has been restricted by inferior drug bioavailability. Herein, we reported a universal bioresponsive doxorubicin (DOX)-based nanogel to achieve tumor-specific co-delivery of drugs. DOX-based mannose nanogels (DM NGs) was designed and choosed as an example to elucidate the mechanism of combined chemo-immunotherapy. As expected, the DM NGs exhibited prominent micellar stability, selective drug release and prolonged survival time, benefited from the enhanced tumor permeability and prolonged blood circulation. We discovered that the DOX delivered by DM NGs could induce powerful anti-tumor immune response facilitated by promoting ICD. Meanwhile, the released mannose from DM NGs was proved as a powerful and synergetic treatment for breast cancer in vitro and in vivo, via damaging the glucose metabolism in glycolysis and the tricarboxylic acid cycle. Overall, the regulation of tumor microenvironment with DOX-based nanogel is expected to be an effectual candidate strategy to overcome the current limitations of ICD-based immunotherapy, offering a paradigm for the exploitation of immunomodulatory nanomedicines.     

Proposed mechanisms for the extracellular release of PD-L1 by the anticancer saponin platycodin D

Platycodin D (PTD) is an oleanane-type terpenoid saponin, isolated from the plant Platycodon grandiflorus. PTD displays multiple pharmacological effects, notably significant anticancer activities in vitro and in vivo. Recently, PTD was shown to trigger the extracellular release of the immunologic checkpoint glycoprotein PD-L1. The reduction of PD-L1 expression at the surface of cancer cells leads to interleukin-2 secretion and T cells activation. In the present review, we have analyzed the potential origin of this atypical PTD-induced PD-L1 release to propose a mechanistic explanation. For that, we considered all published scientific information, as well as the physicochemical characteristics of the natural product (a modeling analysis of PTD and the related saponin β -escin is provided). On this basis, we raise the hypothesis that the capacity of PTD to induce PD-L1 extracellular release derives from two main mechanisms: (i) a drug-promoted shedding of membrane PD-L1 by metalloproteases or more likely, (ii) a cholesterol binding-related effect, that would lead to perturbation of membrane raft domains, limiting the recruitment of proteins like TLR4. The drug-induced membrane effects (frequently observed with saponin drugs), associated with a production of interferon-γ,can favor the release of proteins like PD-L1 into membrane vesicles. Our analysis supports the hypothesis that PTD is a cholesterol-dependent lipid raft-modulating agent able to promote the formation of PD-L1 containing extracellular vesicles. The anticancer potential of PTD and its capacity to modulate the functioning of the PD-1/PD-L1 checkpoint should be further considered.     

Suppression of TRIM8 by microRNA-182-5p restricts tumor necrosis factor-α-induced proliferation and migration of airway smooth muscle cells through inactivation of NF-Κb

MicroRNAs (miRNAs) have emerged as critical modulators involved in the regulation of airway remodeling in asthma. MicroRNA-182-5p (miR-182-5p) has been reported as a key miRNA in regulating the proliferation and migration of various cell types, and its dysfunction contributes is implicated in a wide range of pathological processes. Yet, it remains unknown whether miR-182-5p modulates the proliferation and migration of airway smooth muscle (ASM) cells during asthma. In the present study, we aimed to determine the potential role of miR-182-5p in regulating the proliferation and migration of ASM cells induced by tumor necrosis factor (TNF)-α in vitro. We found that TNF-α stimulation markedly reduced miR-182-5p expression in ASM cells. Gain-of-function experiments showed that miR-182-5p upregulation suppressed the proliferation and migration of ASM cells induced by TNF-α. By contrast, miR-182-5p inhibition had the opposite effect. Notably, tripartite motif 8 (TRIM8) was identified as a target gene of miR-182-5p. TRIM8 expression was induced by TNF-α stimulation, and TRIM8 knockdown markedly impeded TNF-α-induced ASM cell proliferation and migration. Moreover, miR-182-5p overexpression or TRIM8 knockdown significantly downregulated the activation of nuclear factor-κB (NF-κB) induced by TNF-α. However, TRIM8 restoration partially reversed the miR-182-5p-mediated inhibitory effect on TNF-α-induced ASM cell proliferation and migration. In conclusion, our study indicates that miR-182-5p restricts TNF-α-induced ASM cell proliferation and migration through downregulation of NF-κB activation via targeting TRIM8. The results of our study highlight the potential importance of the miR-182-5p/TRIM8/NF-κB axis in the airway remodeling of asthma.     

TMEM16A inhibits angiotensin II-induced basilar artery smooth muscle cell migration in a WNK1-dependent manner

Vascular smooth muscle cell (VSMC) migration plays a critical role in the pathogenesis of many cardiovascular diseases. We recently showed that TMEM16A is involved in hypertension-induced cerebrovascular remodeling. However, it is unclear whether this effect is related to the regulation of VSMC migration. Here, we investigated whether and how TMEM16A contributes to migration in basilar artery smooth muscle cells (BASMCs). We observed that AngII increased the migration of cultured BASMCs, which was markedly inhibited by overexpression of TMEM16A. TMEM16A overexpression inhibited AngII-induced RhoA/ROCK2 activation, and myosin light chain phosphatase (MLCP) and myosin light chain (MLC20) phosphorylation. But AngII-induced myosin light chain kinase (MLCK) activation was not affected by TMEM16A. Furthermore, a suppressed activation of integrinβ3/FAK pathway, determined by reduced integrinβ3 expression, FAK phosphorylation and F-actin rearrangement, was observed in TMEM16A-overexpressing BASMCs upon AngII stimulation. Contrary to the results of TMEM16A overexpression, silencing of TMEM16A showed the opposite effects. These in vitro results were further demonstrated in vivo in basilar arteries from VSMC-specific TMEM16A transgenic mice during AngII-induced hypertension. Moreover, we observed that the inhibitory effect of TMEM16A on BASMC migration was mediated by decreasing the activation of WNK1, a Cl⁻-sensitive serine/threonine kinase. In conclusion, this study demonstrated that TMEM16A suppressed AngII-induced BASMC migration, thus contributing to the protection against cerebrovascular remodeling during AngII-infused hypertension. TMEM16A may exert this effect by suppressing the RhoA/ROCK2/MLCP/MLC20 and integrinβ3/FAK signaling pathways via inhibiting WNK1. Our results suggest that TMEM16A may serve as a novel therapeutic target for VSMC migration-related diseases, such as vascular remodeling.     

Scutellarin ameliorates cartilage degeneration in osteoarthritis by inhibiting the Wnt/β-catenin and MAPK signaling pathways

Osteoarthritis (OA) is a chronic inflammatory disease that is the basis of cartilage extracellular matrix degeneration and joint inflammation. Scutellarin is an herbal flavonoid glucuronide, isolated from the Chinese traditional herb Erigeron breviscapus, has been reported to have anti-inflammatory effect. Here, we showed that Scutellarin could inhibit inflammation and protects cartilage from degeneration in vitro and in vivo. Scutellarin downregulate the mRNA and protein expression of MMP1, MMP13, and ADAMTS-5, Wnt3a, Frizzled7 and promote the expression of Collagen II and Aggrecan. Moreover, scutellarin inhibit the migration of β-catenin and phosphorylation of p38 into the nucleus, which may relate to the mediation of the Wnt/β-catenin and MAPK signaling pathway. Furthermore, scutellarin significantly inhibit the cartilage degradation of DMM-induced OA mice by safranin-O and fast green staining. In conclusion, our study indicates that scutellarin may be a potential drug for the treatment of OA.     

Chemo-photothermal immunotherapy for eradication of orthotopic tumors and inhibition of metastasis by intratumoral injection of polydopamine versatile hydrogels

Cancer remains one of the leading causes of death globally and metastasis always leads to treatment failure. Here, we develop a versatile hydrogel loading photothermal agents, chemotherapeutics, and immune-adjuvants to eradicate orthotopic tumors and inhibit metastasis by combinational therapy. Hydrogel networks were synthesized via the thiol-Michael addition of polydopamine (PDA) with thiolated hyaluronic acid. PDA acted as a cross-linking agent and endowed the hydrogel with excellent photothermal property. Meanwhile, a chemotherapeutic agent, doxorubicin (DOX), was loaded in the hydrogel via π?π stacking with PDA and an immune-adjuvant, CpG-ODN, was loaded via electrostatic interaction. The release of DOX from the hydrogel was initially slow but accelerated due to near infrared light irradiation. The hydrogels showed remarkably synergistic effect against 4T1 cancer cells and stimulated plenty of cytokines secreting from RAW264.7 cells. Moreover, the hydrogels eradicated orthotopic murine breast cancer xenografts and strongly inhibited metastasis after intratumoral injection and light irradiation. The high anticancer efficiency of this chemo-photothermal immunotherapy resulted from the strong synergistic effect of the versatile hydrogels, including the evoked host immune response. The combinational strategy of chemo-photothermal immunotherapy is promising for highly effective treatment of breast cancer.     

Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.     

Magnetic systems for cancer immunotherapy

Immunotherapy is a rapidly developing area of cancer treatment due to its higher specificity and potential for greater efficacy than traditional therapies. Immune cell modulation through the administration of drugs, proteins, and cells can enhance antitumoral responses through pathways that may be otherwise inhibited in the presence of immunosuppressive tumors. Magnetic systems offer several advantages for improving the performance of immunotherapies, including increased spatiotemporal control over transport, release, and dosing of immunomodulatory drugs within the body, resulting in reduced off-target effects and improved efficacy. Compared to alternative methods for stimulating drug release such as light and pH, magnetic systems enable several distinct methods for programming immune responses. First, we discuss how magnetic hyperthermia can stimulate immune cells and trigger thermoresponsive drug release. Second, we summarize how magnetically targeted delivery of drug carriers can increase the accumulation of drugs in target sites. Third, we review how biomaterials can undergo magnetically driven structural changes to enable remote release of encapsulated drugs. Fourth, we describe the use of magnetic particles for targeted interactions with cellular receptors for promoting antitumor activity. Finally, we discuss translational considerations of these systems, such as toxicity, clinical compatibility, and future opportunities for improving cancer treatment.     

Notch1 contributes to TNF-α-induced proliferation and migration of airway smooth muscle cells through regulation of the Hes1/PTEN axis

Notch1 has been implicated in asthma pathogenesis. However, the function of Notch1 in regulating airway smooth muscle (ASM) cell proliferation and migration during airway remodeling of asthma remains unknown. Using an in vitro model induced by tumor necrosis factor (TNF)-α, we reported in this study that Notch1 participated in TNF-α-induced proliferation and migration of ASM cells. Our results demonstrated that Notch1 expression was significantly upregulated in ASM cells exposed to TNF-α. Notch1 inhibition significantly repressed TNF-α-induced ASM cell proliferation and migration, while Notch1 overexpression promoted the opposite effect. Moreover, Notch1 inhibition downregulated the expression of Notch-1 intracellular domain (NICD) and Hes1, while upregulated PTEN expression in TNF-α-exposed cells. Notably, Hes1 overexpression partially reversed the Notch1-inhibition-mediated inhibitory effect on TNF-α-induced ASM cell proliferation and migration. In addition, the promoting effect of Notch1 inhibition on PTEN expression was markedly abrogated by Hes1 overexpression. Overall, these findings demonstrated that Notch1 inhibition repressed TNF-α-induced ASM cell proliferation and migration by modulating the Hes1/PTEN signaling axis, a finding that highlights the involvement of Notch1/Hes1/PTEN in regulating airway remodeling of asthma.     

PEP06 polypeptide 30 is a novel cluster-dissociating agent inhibiting αv integrin/FAK/Src signaling in oral squamous cell carcinoma cells

Collectively migrating tumor cells have been recently implicated in enhanced metastasis of epithelial malignancies. In oral squamous cell carcinoma (OSCC), αv integrin is a crucial mediator of multicellular clustering and collective movement in vitro; however, its contribution to metastatic spread remains to be addressed. According to the emerging therapeutic concept, dissociation of tumor clusters into single cells could significantly suppress metastasis-seeding ability of carcinomas. This study aimed to investigate the anti-OSCC potential of novel endostatin-derived polypeptide PEP06 as a cluster-dissociating therapeutic agent in vitro. Firstly, we found marked enrichment of αv integrin in collectively invading multicellular clusters in human OSCCs. Our study revealed that metastatic progression of OSCC was associated with augmented immunostaining of αv integrin in cancerous lesions. Following PEP06 treatment, cell clustering on fibronectin, migration, multicellular aggregation, anchorage-independent survival and colony formation of OSCC were significantly inhibited. Moreover, PEP06 suppressed αv integrin/FAK/Src signaling in OSCC cells. PEP06-induced loss of active Src and E-cadherin from cell–cell contacts contributed to diminished collective migration of OSCC in vitro. Overall, these results suggest that PEP06 polypeptide 30 inhibiting αv integrin/FAK/Src signaling and disrupting E-cadherin-based intercellular junctions possesses anti-metastatic potential in OSCC by acting as a cluster-dissociating therapeutic agent.