Design, synthesis, and biological evaluation of novel 8-substituted quercetin derivatives targeting the ß­catenin/B-cell lymphoma 9 interaction.

The ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential target for aberrantly active Wnt/ß-catenin signaling which actively participates in initiating and progressing of many cancers. Herein, we discovered novel 8-substituted quercetin derivatives with potential inhibitory activities targeting ß-catenin/BCL9 PPI. Among all the derivatives, compound B4 displayed the most promising PPI inhibitory activity with an IC50 value of 2.25 µM in a competitive fluorescence polarization assay and a KD value of 1.44 µM for the ß-catenin protein. Furthermore, B4 selectively inhibited the growth of colorectal cancer (CRC) cells, suppressed the transactivation of Wnt signaling, and downregulated the expression of oncogenic Wnt target gene. Especially, B4 showed potent anti-CRC activity in vivo with the tumor growth inhibition (TGI) of 75.99 % and regulated the tumor immune microenvironment.

The antitumor effect of mycelia extract of the medicinal macrofungus Inonotus hispidus on HeLa cells via the mitochondrial-mediated pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Inonotus hispidus (I. hispidus), known as shaggy bracket, has been used extensively in China and some East Asian countries as a traditional medicinal macrofungus to treat difficult diseases, such as diabetes, gout, and arthritis. Modern pharmacological research has shown that I. hispidus has an important application value in antitumor treatment. However, the main anti-cervical cancer activity substances from its mycelia and its mechanisms are still not clear. AIMS OF THE STUDY: To enrich the germplasm resources of I. hispidus, to reveal the antitumor activity of the extract from the mycelium of I. hispidus against cervical cancer, and to preliminarily analyze its action mechanism. MATERIALS AND METHODS: The SH3 strain was isolated from wild fruiting bodies and identified by morphology and molecular biology. The antitumor active component from the mycelium of I. hispidus was isolated and identified with liquid chromatography-tandem mass spectrometry. The cell viability was assessed by MTT assay. The cell cycle distribution, apoptotic cell detection, and mitochondrial membrane potential were detected by flow cytometer. The expression of apoptosis-related proteins was assessed by Western blotting. The inhibition of tumor growth in vivo was assessed by a mouse xenograft model. RESULTS: The SH3 strain was isolated and identified as a new strain of I. hispidus. The antitumor active component containing cyclic peptides from the mycelium of I. hispidus (CCM) was isolated for the first time. In addition, we found that CCM had a strong inhibitory effect on HeLa proliferation in vitro and in vivo. Mechanically, the CCM blocked the cell cycle at the G0/G1 phase, decreased the mitochondrial membrane potential, and eventually promoted apoptosis of HeLa cells through the mitochondria-mediated pathway by upregulating the expression levels of Bax, cytochrome C, cleaved caspase-9, and cleaved caspase-3 and downregulating the expression level of Bcl-2. CONCLUSIONS: Our study not only enriches the strain resources of I. hispidus but also confirms that the mycelium of this strain has active components that can inhibit cervical cancer. This is highly significant for the development of active drugs and drug lead molecules for treating cervical cancer.

[The In Vivo Intervention and Relative Mechanism of Genistein on the Inflammation and Thrombophilia in Lymphoma-Bearing Mice].

OBJECTIVE: To investigate the in vivo intervention and relative mechanism of Genistein (GEN) on tumor-associated inflammatory and tumor thrombophilia in lymphoma-bearing mice. METHODS: Forty female Balb/c mice aged 5-6 weeks were injected with murine-derived Pro B-cell lymphoma cell line 38B9 to establish a lymphoma mouse model, which was randomly divided into control group, tumor-bearing group, GEN drug intervention group and cyclophosphamide (CTX）drug intervention group. Histopathologic was used to evaluate the tumorigenesis. Tumor formation was observed, and tumor tissues were collected of HE and immunohistochemical staining. ELISA and flow cytometry were used to detect the expression of inflammatory factors and the changes of thrombus indices in plasma after intervention of GEN and Cyclophosphamide (CTX) respectively. Immunohistochemistry method was used to detect the expression of CD19 in tomor tissues of tummor bearing mice. RESULTS: After 14 days of tumor bearing, the mice were tumorigenic. The lymphoma cells were diffusely distributed in the tumor tissue and the expression of CD19 in the tumor tissue was positive. The inflammatory factors such as IL-6, NETs and CLEC-2, and thrombotic indices such as TF, FIB and D-D in lymphoma-bearing mice were significantly higher than those before tumor-injection and lower than those after drug-intervention (all P<0.05). The levels of CLEC-2 and D-D in GEN group were significantly lower than those in CTX group (P<0.05). CONCLUSION: Tumor-associated inflammation and thrombophilia exist in lymphoma-bearing mice. GEN shows better anti-inflammatory and anti-thrombotic effects compared with CTX by interfering with tumor inflammatory factors.

Discovery of Novel 3-Phenylpiperidine Derivatives Targeting the ß-Catenin/B-Cell Lymphoma 9 Interaction as a Single Agent and in Combination with the Anti-PD-1 Antibody for the Treatment of Colorectal Cancer.

Direct disruption of the ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential strategy for colorectal cancer (CRC) treatment through inhibiting oncogenic Wnt activity. Herein, a series of 3-phenylpiperidine derivatives were synthesized and evaluated as ß-catenin/BCL9 PPI inhibitors. Among them, compound 41 showed the best IC50 (0.72 µM) in a competitive fluorescence polarization assay and a KD value of 0.26 µM for the ß-catenin protein. This compound selectively inhibited the growth of CRC cells, suppressed Wnt signaling transactivation, and downregulated oncogenic Wnt target gene expression. In vivo, 41 showed potent anti-CRC activity and promoted the infiltration and function of cytotoxic T lymphocytes while decreasing the infiltration of regulatory T-cells (Tregs). Furthermore, the combination of 41 and the anti-PD-1 antibody (Ab) efficiently enhanced anti-CRC efficacy, first verifying the in vivo efficacy of the small-molecule ß-catenin/BCL9 PPI inhibitor and anti-PD-1 Ab in combination.

Design, synthesis and biological evaluation of quercetin derivatives as novel ß-catenin/B-cell lymphoma 9 protein-protein interaction inhibitors.

The ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential target for the suppression of hyperactive Wnt/ß-catenin signaling that is vigorously involved in cancer initiation and development. Herein, we first described quercetin and its derivatives had potential inhibitory effects on ß-catenin/BCL9 PPI. The most potent compound, quercetin-3'-O-(4-methylpiperazine-1-yl) propyl (C1), directly binded with ß-catenin and disrupted the ß-catenin/BCL9 interaction in both the protein level and the cellular context. C1 also effectively inhibited colorectal cancer in vitro and showed better selectivity in inhibiting hyperactive Wnt/ß-catenin signaling cells like CT26 and HCT116. And we further confirmed that C1 could inhibit CT26 tumor growth in vivo and regulate the tumor immune microenvironment. This study provides a good chemical probe to explore ß-catenin-related biology and a drug-like quercetin derivative as novel ß-catenin/BCL9 PPI inhibitors for further drug development.

Tumor-infiltrated activated B cells suppress liver metastasis of colorectal cancers.

More than 40% of patients with late-stage colorectal cancer (CRC) develop liver metastasis (LM). Which immune cells play important roles in CRC-LM and contribute to the difference between left-sided CRC (LCC) and right-sided CRC (RCC) remain unclear. By single-cell RNA sequencing (scRNA-seq), we not only find that activated B cells are significantly depleted in CRC with LM, but also find a subtype of B cells developed from activated B cells, namely immature plasma cell population alpha (iMPA), highly correlated with metastasis. Mechanistically, inhibition of the Wnt and transforming growth factor ß (TGF-ß) pathways in cancer cell promotes activated B cell migration via the SDF-1-CXCR4 axis. This study reveals that B cell subpopulations in the tumor immune microenvironment (TIME) play a key role in CRC-LM as well as in LCC and RCC. The preventive effects of modulating B cell subpopulations in CRC may provide a rationale for subsequent drug development and CRC-LM management.

RORγt agonist enhances anti-PD-1 therapy by promoting monocyte-derived dendritic cells through CXCL10 in cancers.

BACKGROUND: The overall response rate to checkpoint blockade remains unsatisfactory, partially due to the immune-suppressive tumor microenvironment. A retinoic acid-related orphan receptor γt (RORγt) agonist (LYC-55716) is currently used in clinical trials combined with anti-PD-1, but how the Th17 cell transcription factor RORγt enhances antitumor immunity of PD-1 in the tumor microenvironment remains elusive. METHODS: The expression of mRNA was analyzed using qPCR assays. Flow cytometry was used to sort and profile cells. Cell migration was analyzed using Transwell assays. Biacore was used to determine the binding affinity to the RORγt protein. The RORγt GAL4 cell-based reporter gene assay was used to measure activity in the RORγt driven luciferase reporter gene expression. RESULTS: We designed a potent and selective small-molecule RORγt agonist (8-074) that shows robust antitumor efficacy in syngeneic tumor models and improves the efficacy of anti­PD­1 in a murine lung cancer model. RORγt agonist treatment increased intratumoral CD8+ T cells, which were correlated with CXCL10 and monocyte-derived dendritic cells (MoDCs). In addition, the RORγt agonist promoted Type 17 T cell migration by upregulating CCL20 and CCR6 expression, and Type 17 T cell tumor infiltration. CCL20 induces MoDCs migration, and CXCL10 derived from MoDCs promotes CD8+ T cell migration. CONCLUSION: Our results revealed that the RORγt agonist improved the efficacy of anti-PD-1. The RORγt agonist increased the migration of MoDCs, which increased the local levels of CXCL10, thus promoting CD8+ T cell tumor infiltration. Our findings provide the mechanistic insights implicating the RORγt agonist in immunotherapy and offer a strategy for targeting the RORγt agonist to improve PD-1 antibody efficacy in cancers.

[Establishment of an Animal Model of Vesicoureteral Reflux Renal Injury through Partial Bladder Outlet Obstruction].

OBJECTIVE: To establish an animal model of reflux renal damage through bladder outlet obstruction. METHODS: Sixty male C57BL/6 mice aged 6-8 weeks were randomly assigned to a control group, a sham operation group, and a partial bladder outlet obstruction (PBOO) group, with 20 mice in each group. Laparotomy were performed on the PBOO mice under anesthesia in order to separate the bladder necks and to perform guided partial ligation of the bladder neck with a metal rod of 0.3 mm diameter. Mice in the sham operation group had laparotomy and had their bladder necks separated without ligation. The control group did not receive any treatment. 7 days after the surgery, 12 surviving mice were randomly selected from each group to observe the general changes of the bladder, ureter, renal pelvis and kidney. Retrograde urography was performed through the bladder. Kidney tissues were extracted for histopathological analysis. The expression levels of Vimentin, proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were examined with Western blot, immunohistochemistry and immunofluorescence staining tests, respectively. RESULTS: Compared with the control and sham operation group, the bladder, ureter, and renal pelvis of the mice in the PBOO group were significantly enlarged, vesicoureteral reflux was more obvious, the kidney volume and mass increased ( P<0.001), and renal parenchyma became thinner ( P<0.000 1). Histopathological staining showed glomerular atrophy, renal tubule expansion, tubulointerstitial inflammatory cell infiltration, glomerular basement membrane hyperplasia and obvious interstitial fibrosis. Western blot, immunofluorescence and immunohistochemistry staining showed that the expression levels of Vimentin, PCNA and α-SMA in kidney tissue were elevated ( P<0.000 1). CONCLUSION: After PBOO, the bladder, ureter, and kidney of the mice showed obvious morphological alteration and presented reflux renal fibrosis-like damage. This can be used as an animal model to study the pathological alteration mechanism and therapeutic measures of renal fibrosis caused by bladder outlet obstruction.

Radiotherapy Combined with PD-1 Inhibition Increases NK Cell Cytotoxicity towards Nasopharyngeal Carcinoma Cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) in endemic regions and younger patients is characterized by a prominent lymphomononuclear infiltration. Radiation is the principal therapeutic modality for patients with NPC. Recent data suggest that the efficacy of radiotherapy in various cancers can be augmented when combined with immune checkpoint blockade. Here, we investigate the effect of radiotherapy on the killing of NPC cells by Natural Killer (NK) cells. METHODS: NPC cell lines and a patient-derived xenograft were exposed to NK cells in the context of radiotherapy. Cytotoxicity was measured using the calcein-release assay. The contribution of the PD-L1/PD-1 checkpoint and signaling pathways to killing were analyzed using specific inhibitors. RESULTS: Radiotherapy sensitized NPC cells to NK cell killing and upregulated expression of PD-1 ligand (PD-L1) in NPC cells and PD-1 receptor (PD-1) in NK cells. Blocking of the PD-L1/PD-1 checkpoint further increased the killing of NPC cells by NK cells in the context of radiotherapy. CONCLUSION: Radiation boosts the killing of NPC cells by NK cells. Killing can be further augmented by blockade of the PD-L1/PD-1 checkpoint. The combination of radiotherapy with PD-L1/PD-1 checkpoint blockade could therefore increase the efficacy of radiotherapy in NPC tumors.

RORγt agonist synergizes with CTLA-4 antibody to inhibit tumor growth through inhibition of Treg cells via TGF-ß signaling in cancer.

To date, the overall response rate to checkpoint blockade remains unsatisfactory, partially due to the limited understanding of the tumor immune microenvironment. The retinoic acid-related orphan receptor γt (RORγt) is the key transcription factor of T helper cell 17 (Th17) cells and plays an essential role in tumor immunity. In this study, we used JG-1, a potent and selective small-molecule RORγt agonist to evaluate the therapeutic potential and mechanism of action of targeting RORγt in tumor immunity. JG-1 promotes Th17 cells differentiation and inhibition of regulatory T (Treg) cells differentiation. JG-1 demonstrates robust tumor growth inhibition in multiple syngeneic models and shows a synergic effect with the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) antibody. In tumors, JG-1 not only promotes Th17 cells differentiation and increases C-C Motif Chemokine Receptor 6 (CCR6)- Chemokine (C-C motif) ligand 20 (CCL20) expression, but also inhibits both the expression of transforming growth factor-ß1 (TGF-ß1) and the differentiation and infiltration of Treg cells. In summary, JG-1 is a lead compound showing a potent activity in vitro and robust tumor growth inhibition in vivo with synergetic effects with anti-CTLA-4.

Discovery of a novel, potent and selective small-molecule inhibitor of PD-1/PD-L1 interaction with robust in vivo anti-tumour efficacy.

BACKGROUND AND PURPOSE: PD-1/PD-L1 antibodies have achieved great success in clinical treatment. However, monoclonal antibody drugs also have challenges, such as high manufacturing costs, poor diffusion, low oral bioavailability and limited penetration into tumour tissue. The development of small-molecule inhibitors of PD-1/PD-L1 interaction represents a promising perspective to overcome the above challenges in cancer immunotherapy. EXPERIMENTAL APPROACH: We explored structural activity relationships and used biochemical assays to generate a lead compound (ZE132). CD8+ T-cells killing assay and Ifng expression assay were used to verify the in vitro cellular activity of ZE132. Off-target study was performed to verify the selectivity. Syngeneic mouse models were used to verify the in vivo activity of ZE132 in tumour immune microenvironment (TIME). We also performed pharmacokinetics profiling in mice and The Cancer Genome Atlas database analysis. KEY RESULTS: ZE132 can effectively inhibit the PD-1/PD-L1 interactions in vitro, and it has a potent affinity to PD-L1. ZE132 shows robust anti-tumour effects in vivo, better than anti-PD-1 antibody. In the analysis of TIME, we found that ZE132 treatment promotes cytotoxic T-cell tumour infiltration and induces IL-2 expression. In addition, ZE132 elicits strong inhibitory effects on the mRNA expression of TGF-ß, which may serve as a potential biomarker to predict responsiveness to PD-1/PD-L1 immunotherapies. CONCLUSION AND IMPLICATIONS: We identified a new lead compound ZE132 targeting PD-1/PD-L1 interactions, not only showing favourable drug-like properties in vitro and in vivo but also showing the advantage of overcoming the barrier of TIME compared to anti-PD-1 antibody.

Resibufogenin inhibited colorectal cancer cell growth and tumorigenesis through triggering ferroptosis and ROS production mediated by GPX4 inactivation.

Resibufogenin (RB) has been used for cancer treatment, but the underlying mechanisms are still unclear. This study aimed to investigate the effects of RB treatment on colorectal cancer (CRC) cells, and to determine the underlying mechanisms. The cell counting kit-8 assay was used to determine cell viability. Cell morphology was observed under light microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was employed to detect cell apoptosis. Intracellular ferrous iron (Fe2+ ), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species levels were detected by using commercial iron assay kit, MDA assay kit, GSH assay kit, and 2,7-dichlorodihydrofluorescein diacetate probes, respectively. The protein expressions were determined by Western blot and immunohistochemistry. RB inhibited cell viability in the CRC cell lines (HT29 and SW480) in a dose- and time-dependent manner, and caused cytotoxicity to the normal colonic epithelial cell line (NCM460) at high dose. Similarly, RB induced morphological changes in CRC cells from normal to round shape, and promoted cell death. Of note, RB triggered oxidative stress and ferroptotic cell death in CRC cells, and only ferroptosis inhibitors (deferoxamine and ferrostatin-1), instead of inhibitors for other types of cell death (apoptosis, autophagy, and necroptosis), reversed the inhibitory effects of RB on CRC cell proliferation. Furthermore, glutathione peroxidase 4 (GPX4) was inactivated by RB treatment, and overexpression of GPX4 alleviated RB-induced oxidative cell death in CRC cells. Consistently, the in vivo experiments validated that RB also triggered oxidative stress, and inhibited CRC cells growth and tumorigenicity in mice models. RB can inhibit CRC cells growth and tumorigenesis by triggering ferroptotic cell death in a GPX4 inactivation-dependent manner.

Anti-PD-1 antibody increases NK cell cytotoxicity towards nasopharyngeal carcinoma cells in the context of chemotherapy-induced upregulation of PD-1 and PD-L1.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a highly malignant epithelial cancer linked to Epstein-Barr virus (EBV) infection. Tumors are characterized by a lymphomononuclear infiltrate and the number of natural killer (NK) cells in tumors appears to be of prognostic significance. Standard treatment for NPC in adolescents and young adults consists of induction chemotherapy followed by radiochemotherapy. Though survival rates are above 80%, the majority of patients suffer from long-term side-effects, mainly related to radiotherapy. The addition of immunotherapy to induction chemotherapy could improve tumor response. METHODS: We have investigated the killing of NPC cells by NK cells in the context of chemotherapy, using a panel of three nasopharyngeal carcinoma cell lines and a patient-derived xenograft. Cytotoxicity was measured using the calcein-release assay, while the contribution of different checkpoints and signaling pathways to killing was studied by siRNA-mediated gene silencing and chemical inhibitors. RESULTS: Chemotherapeutics cisplatin, 5-fluorouracil and gemcitabine sensitized NPC cells to killing by NK cells. Chemotherapeutics led to upregulation of PD-1 in NK cells and PD-L1 in NPC cells via NF-κB. Inhibition of the PD-L1/PD-1 checkpoint by an anti-PD-1 antibody or siRNA increased NK-cell cytotoxicity towards NPC cells. CONCLUSION: The addition of an anti-PD-1 antibody to chemotherapy in patients with NPC could increase the efficacy of induction chemotherapy. If confirmed in a clinical trial, more efficient induction therapy could allow the dose of radiotherapy to be reduced and thereby diminish severe late effects of such therapy.

The emerging role of the MiR-1272-ADAM9-CDCP1 signaling pathway in the progression of glioma.

Glioma is a primary, malignant, and aggressive brain tumor in adults. To develop new therapeutic strategies for glioma, we must determine its underlying mechanisms. In the present study, we aimed to investigate the potential role of miR-1272-ADAM9-CDCP1 signaling in the progression of glioma. We found that ectopic expression of miR-1272 produced significant inhibitory effects on cell proliferation and migration and was associated with cell cycle G0/G1 arrest in A172 and SHG44 glioma cells. Using the luciferase reporter assay, we identified ADAM9 as a target of miR-1272. The expression of ADAM9 was markedly decreased or increased after overexpression or inhibition, respectively, of miR-1272 in glioma cells. Moreover, overexpression of ADAM9 reversed the inhibitory effects of miR-1272 on glioma cell progression. Furthermore, CDCP1 served as a potential downstream molecule of miR-1272/ADAM9 signaling in glioma and promoted the proliferation and migration of glioma. Results derived from clinical samples and online databases confirmed correlations between the expression of ADAM9 and CDCP1 and both the severity and prognosis of glioma. In conclusion, these results suggest that miR-1272 and CDCP1 may act as novel regulators in glioma. The miR-1272/ADAM9/CDCP1 pathway may serve as a potential candidate pathway for the prevention of glioma.

Spiral Field Generation in Smith-Purcell Radiation by Helical Metagratings.

Moving electrons interacting with media can give rise to electromagnetic radiations and has been emerged as a promising platform for particle detection, spectroscopies, and free-electron lasers. In this letter, we investigate the Smith-Purcell radiation from helical metagratings, chiral structures similar to deoxyribonucleic acid (DNA), in order to understand the interplay between electrons, photons, and object chirality. Spiral field patterns can be generated while introducing a gradient azimuthal phase distribution to the induced electric dipole array at the cylindrical interface. Experimental measurements show efficient control over angular momentum of the radiated field at microwave regime, utilizing a phased electromagnetic dipole array to mimic moving charged particles. The angular momentum of the radiated wave is determined solely by the handedness of the helical structure, and it thus serves as a potential candidate for the detection of chiral objects. Our findings not only pave a way for design of orbital angular momentum free-electron lasers but also provide a platform to study the interplay between swift electrons with chiral objects.

Interferon beta increases NK cell cytotoxicity against tumor cells in patients with nasopharyngeal carcinoma via tumor necrosis factor apoptosis-inducing ligand.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an EBV-associated neoplasm occurring endemically in Southeast Asia and sporadically all over the world. In children and adolescents, high cure rates have been obtained using chemotherapy, radiochemotherapy and maintenance therapy with interferon beta (IFNß). The mechanism by which IFNß contributes to a low systemic relapse rate has not yet been fully revealed. PATIENTS AND METHODS: NK cells and serum samples from two patients with NPC were analyzed before and at different time points during IFNß therapy, for assessment of TRAIL expression and NK cell cytotoxicity. Cytotoxicity was measured using the calcein release assay and the contribution of different death effector pathways was analyzed using specific inhibitors. RESULTS: Treatment with IFNß induced TRAIL expression on patients' NK cells and increased their cytotoxicity against NPC targets in vitro. NK cell-mediated cytotoxicity was predominately mediated via TRAIL. IFNß also induced the production of soluble TRAIL (sTRAIL) by NK cells and its release upon contact with NPC cells. IFNß treatment increased serum levels of sTRAIL in patients. Moreover, sTRAIL concentrated from patients' serum samples induced apoptosis ex vivo in NPC cells from a patient-derived xenograft. CONCLUSION: Increased cytotoxicity of NK cells against NPC cells and increased serum levels of biologically active TRAIL in patients treated with IFNß could be a means to eliminate micrometastatic disease and explain the low systemic relapse rate in this patient group.

Interferon ß and Anti-PD-1/PD-L1 Checkpoint Blockade Cooperate in NK Cell-Mediated Killing of Nasopharyngeal Carcinoma Cells.

Nasopharyngeal carcinoma (NPC) is a highly malignant epithelial cancer linked to EBV infection. Addition of interferon-ß (IFNß) to chemo- and radiochemotherapy has led to survival rates >90% in children and adolescents. As NPC cells are sensitive to apoptosis via tumor necrosis factor-related apoptosis inducing ligand (TRAIL), we explored the role of TRAIL and IFNß in the killing of NPC cells by natural killer (NK) cells. NPC cells, including cells of a patient-derived xenograft were exposed to NK cells in the presence or absence of IFNß. NK cells killed NPC- but not nasoepithelial cells and killing was predominately mediated via TRAIL. Incubation of NK cells with IFNß increased cytotoxicity against NPC cells. Concomitant incubation of NK- and NPC cells with IFNß before coculture reduced cytotoxicity and could be overcome by blocking the PD-1/PD-L1 axis leading to the release of intracellular TRAIL from NK cells. In conclusion, combination of IFNß and anti-PD-1, augmenting cytotoxicity of NK cells against NPC cells, could be a strategy to improve NPC-directed therapy and warrants further evaluation in vivo.

Dexamethasone-induced Krüppel-like factor 9 expression promotes hepatic gluconeogenesis and hyperglycemia.

Chronic glucocorticoid therapy has serious side effects, including diabetes and fatty liver. However, the molecular mechanisms responsible for steroid-induced diabetes remain largely enigmatic. Here, we show that hepatic Krüppel-like factor 9 (Klf9) gene expression is induced by dexamethasone and fasting. The overexpression of Klf9 in primary hepatocytes strongly stimulated Pgc1a gene expression through direct binding to its promoter, thereby activating the gluconeogenic program. However, Klf9 mutation abolished the stimulatory effect of dexamethasone on cellular glucose output. Adenovirus-mediated overexpression of KLF9 in the mouse liver markedly increased blood glucose levels and impaired glucose tolerance. Conversely, both global Klf9-mutant mice and liver-specific Klf9-deleted mice displayed fasting hypoglycemia. Moreover, the knockdown of Klf9 in the liver in diabetic mouse models, including ob/ob and db/db mice, markedly lowered fasting blood glucose levels. Notably, hepatic Klf9 deficiency in mice alleviated hyperglycemia induced by chronic dexamethasone treatment. These results suggest a critical role for KLF9 in the regulation of hepatic glucose metabolism and identify hepatic induction of KLF9 as a mechanism underlying glucocorticoid therapy-induced diabetes.