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Glioblastoma (GBM), as the immunologically cold tumor, respond poorly to programmed cell death 1 (PD-1) immune checkpoint inhibitors because of insufficient immune infiltration. Herein, through the analysis of The Cancer Genome Atlas data and clinical glioma samples, we found Wnt/β-catenin signal was activated in GBM and inversely related to the degree of immune cell (CD8+) infiltration and programmed cell death ligand 1 (PD-L1) expression. Blockade of Wnt/β-catenin signal could inhibit GBM U118 cells' growth and migration, and upregulate their PD-L1 expression which indicated the possible better response to anti-PD-1 immunotherapy. Besides, in a co-culture system comprising U118 cells and Jurkat cells, Wnt inhibition alleviated Jurkat cell's apoptosis and enhanced its cytotoxic function as evidenced by obviously increased effector cytokine IFNγ secretion and lactate dehydrogenase release. Moreover, the enhanced anti-GBM effect of PD-1 antibody triggered by Wnt inhibition was observed in GL261 homograft mouse model, and the upregulation of immune cell (CD4+/CD8+) infiltration and IFNγ secretion in tumor tissues suggested that Wnt/β-catenin inhibition could inflame cold tumor and then sensitize GBM to PD-1 blockade therapy. Taken together, our study verified the blockade of Wnt/β-catenin signal could augment the efficacy of PD-1 blockade therapy on GBM through directly inhibiting tumor proliferation and migration, as well as facilitating T-cell infiltration and PD-L1 expression in tumor microenvironment.

Supplementary data file including supplementary figure 1-4 and supplementary table 1-4.

This research aims to investigate the effect of gemcitabine (GEM) on various activities and functions of macrophages. Phagocytosis, cell autophagy and reactive oxygen species (ROS) were analysed by laser scanning confocal microscope. The cell cycle status and major histocompatibility complex II (MHC-II) expression were examined by flow cytometry. Inflammatory cytokine secretion such as tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) was detected by Elisa assay. The expression of proteins was analysed by western blot method. The results revealed that GEM-induced immune inhibition of M1-type RAW264.7 macrophages activated by interferon-γ (IFN-γ) and lipopolysaccharide (LPS). We also found that GEM inhibited autophagy, as evidenced by the reduced formation of autophagosome-like vacuoles and autophagosomes. Further study showed that incubation of activated macrophages with the autophagy inhibitor 3-MA induced immune suppression. In contrast, treatment with the autophagy inducer trehalose (Tre) restored phagocytosis, TNF-α and IL-6 secretion, and MHC-II expression in GEM-induced immune-inhibited macrophages. GEM reduced immune effect of M1-type RAW264.7 macrophages via inhibiting TNF-α, IL-6 and MHC-II expression. Furthermore, activation of autophagy by Tre reversed GEM-induced immune inhibition of RAW264.7 macrophages. Supplementary Information The online version contains supplementary material available at 10.1007/s12026-021-09210-7.

Glioblastoma (GBM), as the immunologically cold tumor, respond poorly to programmed cell death 1 (PD-1) immune checkpoint inhibitors because of insufficient immune infiltration. Herein, through the analysis of The Cancer Genome Atlas data and clinical glioma samples, we found Wnt/β-catenin signal was activated in GBM and inversely related to the degree of immune cell (CD8+) infiltration and programmed cell death ligand 1 (PD-L1) expression. Blockade of Wnt/β-catenin signal could inhibit GBM U118 cells' growth and migration, and upregulate their PD-L1 expression which indicated the possible better response to anti-PD-1 immunotherapy. Besides, in a co-culture system comprising U118 cells and Jurkat cells, Wnt inhibition alleviated Jurkat cell's apoptosis and enhanced its cytotoxic function as evidenced by obviously increased effector cytokine IFNγ secretion and lactate dehydrogenase release. Moreover, the enhanced anti-GBM effect of PD-1 antibody triggered by Wnt inhibition was observed in GL261 homograft mouse model, and the upregulation of immune cell (CD4+/CD8+) infiltration and IFNγ secretion in tumor tissues suggested that Wnt/β-catenin inhibition could inflame cold tumor and then sensitize GBM to PD-1 blockade therapy. Taken together, our study verified the blockade of Wnt/β-catenin signal could augment the efficacy of PD-1 blockade therapy on GBM through directly inhibiting tumor proliferation and migration, as well as facilitating T-cell infiltration and PD-L1 expression in tumor microenvironment.

More efficient drug delivery system and formulation with less adverse effects are needed for the clinical application of broad-spectrum antineoplastic agent doxorubicin (DOX). Here we obtained outer-membrane vesicles (OMVs), a nano-sized proteoliposomes naturally released by Gram-negative bacteria, from attenuated Klebsiella pneumonia and prepared doxorubicin-loaded O0MVs (DOX-OMV). Confocal microscopy and in vivo distribution study observed that DOX encapsulated in OMVs was efficiently transported into NSCLC A549 cells. DOX-OMV resulted in intensive cytotoxic effects and cell apoptosis in vitro as evident from MTT assay, Western blotting and flow cytometry due to the rapid cellular uptake of DOX. In A549 tumor-bearing BALB/c nude mice, DOX-OMV presented a substantial tumor growth inhibition with favorable tolerability and pharmacokinetic profile, and TUNEL assay and H&E staining displayed extensive apoptotic cells and necrosis in tumor tissues. More importantly, OMVs’ appropriate immunogenicity enabled the recruitment of macrophages in tumor microenvironment which might synergize with their cargo DOX in vivo. Our results suggest that OMVs can not only function as biological nanocarriers for chemotherapeutic agents but also elicit suitable immune responses, thus having a great potential for the tumor chemoimmunotherapy., Graphical abstract The attenuated Klebsiella pneumonia derived outer-membrane vesicles (OMVs), as a kind of biological drug-delivery carriers, are highly effective in transporting the chemotherapy drug doxorubicin (DOX) into non-small-cell lung cancer (NSCLC) A549 cells. Moreover, they can elicit appropriate immune responses, thereby enhancing the anti-NSCLC effect of DOX with no obvious toxicity in vivo.Image 1

Diosgenin, a natural steroidal saponin, can exert antitumor effect by regulating immune function and improving intestinal microbiota. The response to anti-PD-1 immunotherapy is associated with intestinal microbiota and effector T cells in tumor microenvironment. We hypothesize that the modulation of diosgenin on intestinal microbiota can facilitate antitumor immunity and the therapeutic efficacy of PD-1 antibody. In melanoma-bearing C57BL/6 mice, we observed that the anti-melanoma effect of diosgenin relied more on antitumor immunity than direct tumor inhibition activity evidenced by obvious CD4+/CD8+ T-cell infiltration and IFN-γ expression in tumor tissues, and it could improve the compositions of intestinal microbiota. Antibiotics impaired the therapeutic efficacy and immunity responses of diosgenin through disturbing intestinal microbiota, indicating the importance of intestinal microbiota in diosgenin’s in vivo antitumor activity. More importantly, the combined administration of PD-1 antibody with diosgenin aggravated the tumor necrosis and apoptosis by eliciting augmented T-cell responses. Taken together, diosgenin can be used as a microecological regulator to induce antitumor immunity and improve the efficacy of immune checkpoint antibody, making it more suitable for the treatment of malignant tumors.

Bacterial determination, emerging as a critical step in the understanding of increasingly serious bacterial contaminations, remains a major challenge. Herein, a novel chemiluminescence biosensor was exploited for the ultrasensitive determination of nuclease activity and bacteria, in which, hemin, the chemiluminescent (CL) tag molecule was encapsulated into ordered mesopores of mesoporous silica nanoparticles with a specific DNA gate. The capped DNA could be specifically switched upon exposure to the DNA nuclease or bacterial lysate and allowed for an increased release of the encapsulated hemin, which therefore resulted in an obviously enhanced CL signal for the luminol-H2O2 system. Attributed to this unique behavior with the linear or sigmoidal relationship between CL intensity and DNA nuclease or bacterial concentration, the as-prepared CL biosensor could detect S1 nuclease activity in the concentration range 0.01-10.0 U with a detection limit of 0.1 mU, and Escherichia coli O157:H7 (E. coli) or Staphylococcus aureus (S. aureus) in the concentration ranges 101 to 109 cfu mL-1. The detection limit of E. coli and S. aureus was calculated to be 3.0 and 2.5 cfu mL-1, respectively, which was comparable or even better than that of previous studies. Thus, this detection method could achieve detectable levels without cell enrichment overnight. Moreover, the proposed biosensing system could be conducted in the homogeneous solution without separation and washing, greatly improving the reaction efficiency and simplifying the procedure. As expected, the novel CL biosensor promised a great potential for simple and convenient detection of nuclease and bacteria in fields such as food bacterial contamination, pharmaceuticals, and clinical analysis.

Allicin can be used as fumigant to protect food and cultural relics from fungal contamination because of its strong antifungal activity and the characteristics of high volatility and no residues. However, the obvious disadvantages such as high minimal inhibitory concentration and instability prevent it from wide application. In this study, a stable derivative of allicin, S-ethyl ethanethiosulfinate (ALE), was synthesized. We further explored its antifungal activity and apoptosis-inducing effect, as well as the underlying mechanism. ALE had an excellent capability of inhibiting spore germination and mycelial growth of Penicillium chrysogenum observed by inverted microscope and scanning electron microscopy. XTT colorimetric assay indicated ALE could reduce the cell viability obviously and IC50 was 0.92 μg/ml, only 1/42 of allicin (38.68 μg/ml). DHR 123 ROS Assay Kit, flow cytometry assay and confocal immunofluorescence revealed intercellular ROS generation and metacaspase-dependent apoptosis triggered by ALE, while antioxidant tocopherol could reverse ALE-induced cytotoxicity effect and metacaspase activation. These results indicate that ALE induces metacaspase-dependent apoptosis through ROS generation, thus possesses an effective antifungal activity. This new derivative of allicin might be developed as a high efficient alternative to the conventional fungicides for food storage and cultural relic protection.

Slow transit constipation (STC) is a common disease characterized by markedly delayed colonic transit time as a result of colonic motility dysfunction. It is well established that STC is mostly caused by disorders of relevant nerves, especially the enteric nervous system (ENS). Colonic electrical stimulation (CES) has been regarded as a valuable alternative for the treatment of STC. However, little report focuses on the underlying nervous mechanism to normalize the delayed colonic emptying and relieve symptoms. In the present study, the therapeutic effect and the influence on ENS triggered by CES were investigated in STC beagles. The STC beagle model was established by oral administration of diphenoxylate/atropine and alosetron. Histopathology, electron microscopy, immunohistochemistry, Western blot analysis and immunofluorescence were used to evaluate the influence of pulse train CES on myenteric plexus neurons. After 5 weeks of treatment, CES could enhance the colonic electromyogram (EMG) signal to promote colonic motility, thereby improving the colonic content emptying of STC beagles. HE staining and transmission electron microscopy confirmed that CES could regenerate ganglia and synaptic vesicles in the myenteric plexus. Immunohistochemical staining showed that synaptophysin (SYP), protein gene product 9.5 (PGP9.5), cathepsin D (CAD) and S-100B in the colonic intramuscular layer were up-regulated by CES. Western blot analysis and immunofluorescence further proved that CES induced the protein expression of SYP and PGP9.5. Taken together, pulse train CES could induce the regeneration of myenteric plexus neurons, thereby promoting the colonic motility in STC beagles.

Background Compound 6, as a novel hybrid of 3-benzyl coumarin seco-B-ring derivative and nitric oxide (NO) donor phenylsulfonylfuroxan, has the potential to develop into an anticancer drug because it displays significant antiproliferation activitity for various solid cancer cell lines including non-small-cell lung cancer (NSCLC) cells. Purpose We attempt to uncover the capacities of compound 6 to induce apoptosis and autophagy in NSCLC cells, as well as the underlying mechanism involved in this process. Methods The effect of compound 6 on cell viability was evaluated in A549 cells by MTT assay. Apoptosis was mainly detected by flow cytometry. The induction of autophagy was observed by transmission electron microscopy (TEM), confocal microscopy as well as western-blotting technique. The expression of all related-proteins including PI3K/Akt/mTOR signaling pathway were also examined by western-blotting technique. Results Above all, distinct growth inhibition and caspase-dependent apoptosis were detected in A549 cells administered with compound 6. Then, we confirmed the induction of autophagy triggered by compound 6 in A549 cells. Noticeably, blocking autophagy using a series of inhibitors and ATG5 siRNA had little effect on the cytotoxicity of compound 6, elucidating nonprotective autophagy triggered in NSCLC cells. Further research illustrated that PI3K/Akt/mTOR signaling pathway was involved in compound 6-induced apoptosis, and 3-MA as well as LY294002 had synergistic inhibiting effect on proliferation of A549 cells through the pathway mentioned above. Conclusion These findings raise a rationale that this 3-benzyl coumarin seco-B-ring derivative and phenylsulfonylfuroxan hybrid could be a promising candidate for developing as a therapeutic agent toward NSCLC, and the combination therapy through PI3K/Akt/mTOR signaling pathway may result in optimized treatment outcomes.

Background Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Compound 8b, a novel coumarin derivative with phenylsulfonylfuroxan group, shows significant antiproliferation activity against lung adenocarcinoma cell with low toxicity. Purpose This study aims to uncover the potential of compound 8b in relation to apoptosis as well as autophagy induction in lung adenocarcinoma cells. Study design The cytotoxicity and apoptosis of A549 and H1299 cells induced by compound 8b were detected by MTT, microscope and western blot analysis. Autophagy was determined by TEM, confocal microscopy and western blot analysis. Akt/mTOR and Erk signaling pathway were also examined by western blot analysis. Results First, significant growth inhibition and caspase-dependent apoptosis were observed in compound 8b-treated A549 and H1299 cells. Then, we confirmed compound 8b-induced autophagy by autophagosomes formation, upregulated expression of autophagy-related protein LC3-II and autophagic flux. Importantly, abolishing autophagy using inhibitors and ATG5 siRNA enhanced the cytotoxicity of compound 8b, indicating the cytoprotective role of autophagy in lung adenocarcinoma. Further mechanistic investigations suggested that Akt/mTOR and Erk signaling pathways contributed to autophagy induction by compound 8b. Conclusion This results demonstrate that compound 8b induces caspase-dependent apoptosis as well as cytoprotective autophagy in lung adenocarcinoma cells, which may provide scientific evidence for developing this furoxan-based NO-releasing coumarin derivative as a potential anti-lung adenocarcinoma therapeutic agents.

Sorafenib as an effective multikinase inhibitor has been approved for the clinical treatment against advanced hepatocellular carcinoma (HCC). HCC treatment requires usually combined therapy because of its complex pathogenesis. Ceramide has been confirmed to induce remarkable apoptosis in human tumor cells and has attracted increasing attention in investigations on combination therapy. In this paper, the anti-HCC effect of sorafenib combined with C2-ceramide was investigated on cell vitality, apoptosis, and migration, and the underlying mechanism was examined using flow cytometry and western blot. Bel7402 cells coincubated with sorafenib and C2-ceramide exhibited lower cell vitality and more irregular cellular morphology and cell cycle arrest. Sorafenib plus C2-ceramide stimulated significantly the production of reactive oxygen species (ROS) and mitochondrial depolarization, which promoted caspases-dependent cell apoptosis as illustrated by related protein expression including caspase 3, caspase 9, Bax, Bcl-2, and cytochrome c. Combination treatment of sorafenib and C2-ceramide inhibited obviously cell growth and proliferation via PI3K/AKT/mTOR and Erk signaling pathways. Furthermore, the combination treatment was proved to inhibit cell migration and epithelial-mesenchymal transition (EMT). These findings indicated that the combination of C2-ceramide and sorafenib provided synergistic inhibitory effects on HCC cells.