Ze-Qi-Tang formula inhibits MDSCs glycolysis through the down-regulation of p21/Hif1α/Glut1 signal in psoriatic-like mice.

BACKGROUND: Psoriasis is a chronic immune-mediated inflammatory skin disease that affects the quality of life and mental health of approximately 150 million people worldwide. Ze-Qi-Tang (ZQT) is a classic compound used in China for lung disease; however, its mechanism of action in psoriasis remains unclear. This study aimed to investigate the therapeutic effect of the ZQT formula on psoriasis and explore the underlying molecular mechanisms. METHODS: Peripheral blood samples were collected from patients with psoriasis and healthy individuals. Flow cytometry was used to detect changes in the proportions of myeloid-derived suppressor cells (MDSCs) and other immune cells. Psoriasis was induced in mice by the daily application of imiquimod. ZQT was administered separately or in combination with anti-Gr1 antibody to deplete MDSC. The glycolysis levels of the MDSCs were detected using a Seahorse analyzer. The p21/Hif1α/Glut1 pathway was identified and validated by mRNA sequence, RT-qPCR, WB, IF, and the application of p21 inhibitor UC2288. RESULTS: The number of MDSCs was significantly increased in patients with psoriasis, with the increased expression of p21, Hif1α, and Glut1 in MDSCs. ZQT significantly alleviated psoriasis-like skin lesions in mice. ZQT formula significantly reduced the number of MDSCs in psoriatic-like mice and enhanced their suppressive capacity for T cells. The efficacy of ZQT in alleviating psoriatic dermatitis is compromised by MDSC depletion. ZQT decreased the expressions of p21, Hif1α, and Glut1-induced glycolysis in MDSCs, thereby inhibiting Th17 cell differentiation. CONCLUSION: These suggest that ZQT alleviates IMQ-induced psoriatic dermatitis, by inhibiting p21/Hif1α/Glut1-induced glycolysis in MDSCs.

New monoterpenoid indole alkaloids from the stems of Tabernaemontana bovina Lour (Apocynaceae).

Two new monoterpenoid indole alkaloids, named taberibogines E and F (1 and 2), together with three known ones (3-5) were isolated from the stems of Tabernaemontana bovina Lour (Apocynaceae). Their structures including absolute configurations were elucidated from a combination of NMR and HRESIMS data and NMR calculations as well as DP4+ probability analyses. Compounds 1 and 2 exhibited inhibitory effects on LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages.

Experimental and Simulation Investigation of Nd Additions on As-Cast Microstructure and Precipitate Development in Mg-Nd System Alloys.

The microstructure and precipitate evolution of as-cast Mg-Nd alloys with different contents of Nd was investigated via experimental and simulation methods. The research showed that the as-cast microstructure of Mg-Nd alloy consisted of α-Mg dendrites and the intermetallic phases. A metastable ß phase precipitated, followed by α-Mg dendrites that could be confirmed as Mg12Nd by X-ray diffraction (XRD) analysis. The amount of ß-Mg12Nd presented a rising trend with increasing Nd additions. In addition, the tertiary phase was also observed in as-cast Mg-Nd alloy when Nd content was greater than 3 wt.%, which precipitated from the oversaturated α-Mg matrix. The tertiary phase should be ß1-Mg3Nd, which is also a metastable phase with a face-centered cubic lattice. However, it is a pity that the tertiary phase was not detected by the XRD technique. Moreover, an effective cellular automaton (CA) model was explored and applied to simulate the time-dependent α-Mg/ß1-Mg3Nd eutectic growth. The simulated results of α-Mg/ß1-Mg3Nd eutectic growth in Mg-3Nd presented that the growth of α-Mg dendrites was accompanied by the nucleation and growth of ß1-Mg3Nd precipitates and eventually formed a eutectic structure. The eutectic morphologies for Mg-Nd system alloys with different Nd contents were also simulated using the proposed model, and the results revealed that α-Mg dendrite was a refinement, and the amount of α-Mg/ß1-Mg3Nd eutectic was promoted, with increasing Nd content.

Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer.

Background: Natural killer (NK) cell-based immunotherapy is clinically limited due to insufficient tumor infiltration in solid tumors. We have previously found that the natural product rocaglamide (RocA) can enhance NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells by inhibiting autophagy, and autophagic inhibition has been shown to increase NK cell tumor infiltration in melanoma. Therefore, we hypothesized that RocA could increase NK cell infiltration in NSCLC by autophagy inhibition. Methods: Flow cytometry, RNA-sequencing, real-time PCR, Western blotting analysis, and xenograft tumor model were utilized to assess the infiltration of NK cells and the underlying mechanism. Results: RocA significantly increased the infiltration of NK cells and the expressions of CCL5 and CXCL10 in NSCLC cells, which could not be reversed by the inhibitions of autophagy/ULK1, JNK and NF-κB. However, such up-regulation could be suppressed by the inhibitions of TKB1 and STING. Furthermore, RocA dramatically activated the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway, and the inhibition/depletion of STING ablated the up-regulation of CCL5 and CXCL10, NK cell infiltration, and tumor regression induced by RocA. Besides, RocA damaged mitochondrial DNA (mtDNA) and promoted the cytoplasmic release of mtDNA. The mPTP inhibitor cyclosporin A could reverse RocA-induced cytoplasmic release of mtDNA. Conclusions: RocA could promote NK cell infiltration by activating cGAS-STING signaling via targeting mtDNA, but not by inhibiting autophagy. Taken together, our current findings suggested that RocA was a potent cGAS-STING agonist and had a promising potential in cancer immunotherapy, especially in NK cell-based immunotherapy.

Tanshinone IIA enhances susceptibility of non-small cell lung cancer cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5.

Natural killer (NK) cells have a great potential in cancer immunotherapy. However, their therapeutic efficacy is clinically limited owing to cancer cell immune escape. Therefore, it is urgently necessary to develop novel method to improve the antitumor immunity of NK cells. In the present study, it was found that the natural product tanshinone IIA (TIIA) enhanced NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells. TIIA in combination with adoptive transfer of NK cells synergistically suppressed the tumor growth of NSCLC cells in an immune-incompetent mouse model. Furthermore, TIIA significantly inhibited the tumor growth of Lewis lung cancer (LLC) in an immune-competent syngeneic mouse model, and such inhibitory effect was reversed by the depletion of NK cells. Moreover, TIIA increased expressions of ULBP1 and DR5 in NSCLC cells, and inhibition of DR5 and ULBP1 reduced the enhancement of NK cell-mediated lysis by TIIA. Besides, TIIA increased the levels of p-PERK, ATF4 and CHOP. Knockdown of ATF4 completely reversed the up-regulation of ULBP1 and DR5 by TIIA in all detected NSCLC cells, while knockdown of CHOP only partly reduced these enhanced expressions in small parts of NSCLC cells. These results demonstrated that TIIA could increase the susceptibility of NSCLC cells to NK cell-mediated lysis by up-regulating ULBP1 and DR5, suggesting that TIIA had a promising potential in cancer immunotherapy, especially in NK cell-based cancer immunotherapy.

Targeting CD155 by rediocide-A overcomes tumour immuno-resistance to natural killer cells.

CONTEXT: Therapeutic benefits of immunotherapy are restricted by cancer immune-resistance mechanisms. Rediocide-A (Red-A), a natural product extracted from Traditional Chinese Medicine, is a promising agent to battle against cancer which acts as an immune checkpoint inhibitor. OBJECTIVE: To investigate the effect of Red-A on NK-cell tumouricidal activity. MATERIALS AND METHODS: NK cells were co-cultured with A549 or H1299 cells and treated with 10 or 100 nM Red-A for 24 h. Cells treated with 0.1% dimethyl sulphoxide (DMSO) was employed as vehicle control. NK cell-mediated cytotoxicity was detected by biophotonic cytotoxicity and impedance assay. Degranulation, granzyme B, NK cell-tumour cell conjugates and ligands profiling were detected by flow cytometry. Interferon-γ (IFN- γ) production was assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Red-A increased NK cell-mediated lysis of A549 cells by 3.58-fold (21.86% vs. 78.27%) and H1299 cells by 1.26-fold (59.18% vs. 74.78%), compared to vehicle control. Granzyme B level was increased by 48.01% (A549 cells) and 53.26% (H1299 cells) after 100 nM Red-A treatment. INF-γ level was increased by 3.23-fold (A549 cells) and 6.77-fold (H1299 cells) after 100 nM Red-A treatment. Red-A treatment down-regulated the expression level of CD155 by 14.41% and 11.66% in A549 cells and H1299 cells, respectively, leading to the blockade of tumour immuno-resistance to NK cells. CONCLUSIONS: Red-A overcomes immuno-resistance of NSCLCs to NK cells by down-regulating CD155 expression, which shows the possibility of developing checkpoint inhibitors targeting TIGIT/CD155 signalling to overcome immuno-resistance of cancer cells.

A high-throughput assay for screening natural products that boost NK cell-mediated killing of cancer cells.

Context: Natural killer (NK) cells can eliminate malignant cells and play a vital role in immunosurveillance. Administration of natural compounds represents a promising approach for antitumor immunotherapy, which may enhance the NK cell activity via multiple mechanisms.Objective: Establishing approaches to evaluate the effect of select natural products on NK cell-mediated cytotoxicity.Materials and methods: We selected a natural product library containing 2880 pure compounds, which was provided by the National Centre for Drug Screening of China. 0.1% DMSO was employed as a negative control, and 100 U/mL human recombinant IL-2 was employed as a positive control. To evaluate the % of tumour cells which were killed by NK cells, expanded NK cells were co-cultured with tumour cells and then treated with natural products at the concentration of 10 µM. After 24-h co-incubation, luminescent signal was detected and percent lysis was calculated.Results: We report on the results of a three-round high-throughput screening effort that identified 20-deoxyingenol 3-angelate (DI3A) and its analogue ingenol 3-angelate (I3A) as immuno enhancers which boosts NK cell-mediated killing of non-small cell lung cancer cells (NSCLCs). Biophotonic cytotoxicity assay and calcein release assay were used as two well-established NK cell cytotoxicity detection assays to validate the immuno-enhancing effects of DI3A and I3A, which was achieved by increasing degranulation and interferon-gamma secretion of NK cells.Conclusions: Our newly established ATP-based method was a valuable and information-rich screening tool to investigate the biological effects of natural products on both NK cells and tumour cells.

The Ancient Chinese Decoction Yu-Ping-Feng Suppresses Orthotopic Lewis Lung Cancer Tumor Growth Through Increasing M1 Macrophage Polarization and CD4+ T Cell Cytotoxicity.

Background: The tumor microenvironment (TME) has a deep influence on cancer progression and has become into a new target for cancer treatment. In our previous study, we found that Yu-Ping-Feng (YPF), an ancient Chinese herbal decoction, significantly inhibited the Lewis lung cancer (LLC) tumor growth in a subcutaneous xenograft tumor model, and prolonged the survival of tumor-bearing mice. But the regulation of Yu-Ping-Feng on tumor microenvironment is unknown. Methods: To access the effect of Yu-Ping-Feng on non-small cell lung cancer, an orthotopic luciferase stably expressed Lewis lung cancer tumor model was established on C57BL/6 mice, and then the survival and the tumor growth were evaluated. To address the tumor microenvironment immune regulation, the percentages of CD4+ T cells, CD8+ T cells, natural killer cells (NK), regulatory T cells (Treg), macrophages, and myeloid-derived suppressor cells (MDSC) in spleens and tumor tissues, the macrophage polarization and CD4+ T cell cytotocixity were analyzed by flow cytometry, biophotonic cell killing activity assay, real-time PCR and western-blot. Results: Yu-Ping-Feng significantly prolonged orthotopic lung tumor-bearing mouse survival, and increased the percentages of CD4+ T cell and M1 macrophages and the cytotoxicity of CD4+ T cells. Yu-Ping-Feng significantly enhanced macrophage-mediated lysis of LLC in a concentration-dependent manner, and had no effect on CD4+ T cell-mediated lysis of LLC, but significantly increased CD4+ T cell-mediated lysis after co-incubated with macrophages. In addition, Yu-Ping-Feng induced M1 macrophage polarization through promoting the phosphorylation of STAT1. Conclusion: Yu-Ping-Feng induced M1 macrophages polarization, and then activated CD4+ T lymphocytes, resulting in killing of LLC cells. Yu-Ping-Feng was a potent regulator of M1 macrophage polarization and might have a promising application in tumor immunotherapy.

The histone deacetylase inhibitor valproic acid inhibits NKG2D expression in natural killer cells through suppression of STAT3 and HDAC3.

NKG2D is a major activating receptor of NK cells and plays a critical role in tumor immunosurveillance. NKG2D expression in NK cells is inhibited by the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and enhanced by the narrow-spectrum HDAC inhibitor entinostat. We previously demonstrated that entinostat enhanced NKG2D transcription by increasing acetylation of Histones H3 and H4. However, the mechanism by which VPA reduces NKG2D expression in NK cells is not known. We have also shown that NKG2D transcription is regulated by STAT3 phosphorylation. In this study, we investigated regulation of NKG2D expression in NK cells by VPA and entinostat by assessing protein expression, phosphorylation, and interaction of HDACs and STAT3. We find that VPA selectively inhibits STAT3 tyrosine705 phosphorylation, but entinostat does not. STAT3 complexes with HDAC3, and HDAC3 inhibition represses STAT3 phosphorylation and therefore NKG2D expression. NK cells from STAT3 wild-type mice downregulate NKG2D in response to VPA, but not NK cells from STAT3 knockout mice. These results show that VPA is a potent inhibitor of STAT3 phosphorylation and demonstrate that histone acetylation and STAT3 tyrosine705 phosphorylation cooperate in regulating NKG2D expression in NK cells.

Enhancement of NK cell-mediated lysis of non-small lung cancer cells by nPKC activator, ingenol 3,20 dibenzoate.

The IFN-γ production is crucial for NK cell-mediated lysis of cancer cells. Thus increasing the IFN-γ production by NK cells may be an ideal strategy to improve their tumoricidal effect. Since the focus on new drug development has shifted towards natural products, limited information is out there about natural products that enhance the IFN-γ production by NK cells. In this study, through a high-throughput screening, we have identified a natural product ingenol 3,20 dibenzoate (IDB), an activator of tumor suppressor protein kinase C (PKC) isozymes, could increase the IFN-γ production and degranulation by NK cells, especially when NK cells were stimulated by non-small lung cancer (NSCLC) cells. IDB also significantly enhanced the NK cell-mediated lysis of NSCLC cells. Furthermore, PKC inhibitor, sotrastaurin abrogated IDB-induced IFN-γ production, degranulation and cytotoxicity, but did not affect IFN-γ production by NK cells without IDB treatment and NSCLC cell stimulation. The IFN-γ neutralization reversed the IDB-induced enhancement of NK cell mediated killing. In conclusion, our study indicated that IDB enhanced NK cell-mediated lysis of NSCLC cells is dependent on specific PKC mediated IFN-γ production and degranulation. Thus, IDB may have a promising application in clinic for NK cell-based cancer immunotherapy.

Comparative Evaluation of the Chemical Composition, Antioxidant and Antimicrobial Activities of the Volatile Oils of Hawk Tea from Six Botanical Origins.

In this study, volatile oils of six Hawk tea varieties were studied for their chemical composition, antioxidant and antimicrobial activities to screen the most suitable botanical origins of Hawk tea. A total of 72 components were separated and identified from the six oils. The major constituents of the volatile oils were: α-pinene, camphene, limonene, 1,8-cineole, linalool, cis-nerolidol, and germacrene B. Moreover, the volatile oils were evaluated for antioxidant potential and antimicrobial activities. The results showed that all volatile oils exhibited acceptable antioxidant and antimicrobial activities, which suggested that these volatile oils may serve as natural alternatives to synthetic antioxidants and preservatives to be applied in food and pharmaceutical industries. Principal component analysis results denoted that some major compounds may be closely related to the antioxidant and antimicrobial activities. It also showed that the volatile oils from Litsea coreana var. lanuginosa and Litsea pungens Hemsl. were characterized by positive values of first two principal components, indicating higher active chemical compounds and antioxidant and antimicrobial activities compared with other species. Thus, they were temporarily considered as good sources of Hawk tea.

Near-Infrared Light-Responsive Hydrogel for Specific Recognition and Photothermal Site-Release of Circulating Tumor Cells.

Isolation of single circulating tumor cells (CTCs) from patients is a very challenging technique that may promote the process of individualized antitumor therapies. However, there exist few systems capable of highly efficient capture and release of single CTCs with high viability for downstream analysis and culture. Herein, we designed a near-infrared (NIR) light-responsive substrate for highly efficient immunocapture and biocompatible site-release of CTCs by a combination of the photothermal effect of gold nanorods (GNRs) and a thermoresponsive hydrogel. The substrate was fabricated by imprinting target cancer cells on a GNR-pre-embedded gelatin hydrogel. Micro/nanostructures generated by cell imprinting produce artificial receptors for cancer cells to improve capture efficiency. Temperature-responsive gelatin dissolves rapidly at 37 °C; this allows bulk recovery of captured CTCs at physiological temperature or site-specific release of single CTCs by NIR-mediated photothermal activation of embedded GNRs. Furthermore, the system has been applied to capture, individually release, and genetically analyze CTCs from the whole blood of cancer patients. The multifunctional NIR-responsive platform demonstrates excellent performance in capture and site-release of CTCs with high viability, which provides a robust and versatile means toward individualized antitumor therapies and also shows promising potential for dynamically manipulating cell-substrate interactions in vitro.

Photoresponsive immunomagnetic nanocarrier for capture and release of rare circulating tumor cells.

Isolation, release and culture of rare circulating tumor cells (CTCs) may, if implemented, promote the progress of individualized anti-tumor therapies. To realize the release of CTCs without disruption of their viability for further culture and analysis, we designed an effective photocontrolled CTC capture/release system by combination of photochemistry and immunomagnetic separation. 7-Aminocoumarin was synthesized as the phototrigger to bridge the connection between the anti-EpCAM antibody and the magnetic beads. The coumarin moieties produced cleavage of a C-O bond under both ultraviolet (UV) and near-infrared (NIR) light illumination, breaking the bridge and releasing CTCs from the immunomagnetic beads. Compared with conventional immunomagnetic separation systems, the negative influence of absorbed immunomagnetic beads on further CTCs culture and analysis was effectively eliminated. The system can specifically recognize 102 MCF-7 cells in 1 mL of human whole blood samples with 90% efficiency and 85% purity. Under the irradiation of UV and NIR light, 73 ± 4% and 52 ± 6% of captured cells were released with a viability of 90% and 97%, respectively. Furthermore, this technique has been used to detect CTCs from whole blood of cancer patients with high purity. This study demonstrates that the photochemical-based immunomagnetic separation method for isolating, releasing and culturing CTCs from clinic patients may provide new opportunities for cancer diagnosis and personalized therapy.