Immunosuppressive effect of bone marrow-derived mesenchymal stem cells in inflammatory microenvironment favours the growth of B16 melanoma cells

Mesenchymal stem cells (MSCs) are studied for their potential clinical use in regenerative medicine, tissue engineering and tumour therapy. However, the therapeutic application of MSCs in tumour therapy still remains limited unless the immunosuppressive role of MSCs for tumour growth in vivo is better understood. In this study, we investigated the mechanism of MSCs favouring tumour escape from immunologic surveillance in inflammatory microenvironment. We first compared the promotive capacity of bone marrow-derived MSCs on B16 melanoma cells growth in vivo, pre-incubated or not with the inflammatory cytokines interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We showed that the development of B16 melanoma cells is faster when co-injected with MSCs pre-incubated with IFN-γ and TNF-α compared with control groups. Moreover, tumour incidence increases obviously in allogeneic recipients when B16 melanoma cells were co-injected with MSCs pre-incubated with IFN-γ and TNF-α. We then demonstrated that the immunosuppressive function of MSCs was elicited by IFN-γ and TNF-α. These cytokine combinations provoke the expression of inducible nitric oxide synthase (iNOS) by MSCs. The impulsive effect of MSCs treated with inflammatory cytokines on B16 melanoma cells in vivo can be reversed by inhibitor or short interfering RNA of iNOS. Our results suggest that the MSCs in tumour inflammatory microenvironment may be elicited of immunosuppressive function, which will help tumour to escape from the immunity surveillance.     

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.     

Effect of tumor necrosis factor-α and interferon-γ on the growth of a human salivary gland cell line

Interferon-γ (IFN-γ) is a product of activated T-lymphocytes, and tumor necrosis factor-α (TNF-α) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF-α and IFN-γ on a human submandibular gland epithelial cell line (HSG). IFN-γ caused a concentration-dependent decrease in HSG cell growth (～70% in 6 days). Conversely, TNF-α alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF-α and 1,000 units/ml of IFN-γ), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine-induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3–6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN-γ alone and that of IFN-γ and TNF-α in combination were blocked completely using an antibody to the IFN-γ receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up-regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM-1) and histocompatibility antigen HLA-DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN-γ, with or without TNF-α, resulted in increased levels of ICAM-1 and the acquisition of HLA-DR expression. These aggregate data suggest that IFN-γ alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF-α. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Autophagy inhibition via Becn1 downregulation improves the mesenchymal stem cells antifibrotic potential in experimental liver fibrosis

Liver fibrosis (LF) is the result of a vicious cycle between inflammation-induced chronic hepatocyte injury and persistent activation of hepatic stellate cells (HSCs). Mesenchymal stem cell (MSC)-based therapy may represent a potential remedy for treatment of LF. However, the fate of transplanted MSCs in LF remains largely unknown. In the present study, the fate and antifibrotic effect of MSCs were explored in a LF model induced by CCl₄ in mouse. Additionally, MSCs were stimulated in vitro with LF-associated factors, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β1 (TGF-β1), to mimic the LF microenvironment. We unveiled that MSCs exhibited autophagy in response to the LF microenvironment through Becn1 upregulation both in vivo and in vitro. However, autophagy suppression induced by Becn1 knockdown in MSCs resulted in enhanced antifibrotic effects on LF. The improved antifibrotic potential of MSCs may be attributable to their inhibitory effects on T lymphocyte infiltration, HSCs proliferation, as well as production of TNF-α, IFN-γ, and TGF-β1, which may be partially mediated by elevated paracrine secretion of PTGS2/PGE₂. Thus, autophagy manipulation in MSCs may be a novel strategy to promote their antifibrotic efficacy.     

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.     

Modulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) by interferon-γ in a human salivary gland cell line

Gelatinases have been shown to be regulated by many cytokines and growth factors, and have been implicated in the pathogenesis of certain autoimmune diseases via tissue destruction. High levels of several cytokines, including IFN-γ and TNF-α, have been demonstrated in the salivary gland microenvironment of patients with Sjo¨︁gren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of this disease is not well understood. We hypothesized that IFN-γ with or without (±) TNF-α could be playing a role in the pathogenesis of SS via the regulation of matrix metalloproteinase (MMP) levels. This study examined the role of IFN-γ and (+) TNF-α in the regulation of the matrix metalloproteinases, MMP-2 (72 kD gelatinase A) and MMP-9 (92 kD gelatinase B). A human salivary gland cell line (HSG) has been used as a possible in vitro model to study the role of IFN-γ + TNF-α in the pathogenesis of SS. The HSG cell line, in the presence of IFN ± TNF-α, displays increased MMP-2 and MMP-9 gelatinolytic activity, protein and RNA levels. The increase in MMP activity was partially blocked with an antibody against the IFN-γ receptor, and this was associated with a complete inhibition of the previously described IFN-γ ± TNF-α antiproliferative effect. However, incubation of IFN-γ treated HSG cells with the synthetic MMP inhibitor BB94 did not alleviate this antiproliferative effect. In addition, we demonstrate that there are very high levels of MMP-9 in the saliva of patients with SS when compared to healthy control subjects. These data suggest that cytokines could be regulating MMP production by salivary epithelial cells and thus indicate a potential role for these cells in the pathogenesis of SS. J. Cell. Physiol. 171:117–124, 1997. © 1997 Wiley-Liss, Inc.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

Pre-conditioning mesenchymal stromal cell spheroids for immunomodulatory paracrine factor secretion

Background aimsMesenchymal stromal cells (MSCs) exhibit the inherent potential to regulate multiple signaling pathways and cell types that contribute to the pathogenesis of inflammatory and immune diseases. However, more recent studies have suggested that the secretion of immunomodulatory factors by MSCs can be enhanced by three-dimensional aggregation or pro-inflammatory cytokine treatment.MethodsHuman MSC spheroids were formed by forced aggregation into agarose micro-wells and subsequently cultured in either minimal essential medium alpha supplemented with fetal bovine serum or serum-free, defined MesenCult-XF medium (STEMCELL Technologies, Vancouver, Canada). A subset of the spheroids were treated with pro-inflammatory cytokines interferon (IFN)-γ or tumor necrosis factor (TNF)-α or both for 4 days. Immunomodulatory factor (prostaglandin E₂, indoleamine 2,3-dioxygenase, transforming growth factor-β1 and interleukin-6) secretion was quantified after 4 days of culture, and the immunomodulatory activity of MSCs was assessed by quantifying activated macrophage expression of TNF-α after trans-well co-culture.ResultsCulturing human MSCs as three-dimensional aggregates increased secretion of immunomodulatory paracrine factors, which was enhanced further by treatment with IFN-γ and TNF-α, demonstrating that these parameters can synergistically enhance endogenous human MSC immunomodulatory properties. However, immunomodulatory factor secretion was found to be highly dependent on the composition of cell culture medium. Human MSCs cultured in MesenCult-XF medium displayed significantly less expression of prostaglandin E₂, indoleamine 2,3-dioxygenase, transforming growth factor-β1 and interleukin-6 compared with human MSCs cultured in medium supplemented with fetal bovine serum. Finally, pre-conditioning of human MSC spheroids with IFN-γ and TNF-α resulted in greater immunomodulatory activity in a macrophage co-culture assay.ConclusionsAltogether, engineering the environment of human MSCs to develop pre-conditioning strategies for enhancing human MSC immunomodulation may be a simple approach for improving MSC-based therapies for the treatment of inflammatory and immune diseases.     

Activated T-cells and pro-inflammatory cytokines differentially regulate prostaglandin E2 secretion by mesenchymal stem cells

In recent years it has become clear that mesenchymal stem or stromal cells (MSCs) are capable of modulating inflammatory and immune responses through interaction with a wide variety of cells. Whereas several studies indicated that PGE2 is one of the chief soluble mediators involved in these processes, here we investigated prostaglandin E2 (PGE2) production of murine bone marrow- (BM-) and adipose tissue- (Ad-) derived MSCs stimulated with pro-inflammatory cytokines TNF-α and IFN-γ, or co-cultured with ConA-induced T-cell blasts. We found that both MSC populations are able to produce high amounts of PGE2 in MSC/activated T-cell co-cultures. This effect was markedly attenuated when direct cell-cell contact was prevented in transwell system, indicating that the elicitation of the PGE2 secretion of MSCs is contact-dependent in this experimental setting. In contrast, when soluble recombinant pro-inflammatory cytokines were added to the MSC cultures, TNF-α and IFN-γ act synergistically to induce PGE2 production, whereas only high amount of TNF-α but not IFN-γ was able to do so alone. Although the PGE2 secretion by MSCs was completely abrogated by addition of indomethacin under all culture conditions tested, L-NMA, a NOS inhibitor could only partially inhibit it when the cells were elicited in the concomitant presence of TNF-α and IFN-γ. These results, combined with others, suggest that NO acts downstream of IFN-γ but upstream of COX2. Taken together, our findings demonstrate that the induction of PGE2 secretion by BM- and Ad-MSCs is not mediated by a single or unique, nonredundant molecular mechanism under different experimental conditions.     

HIF-2α诱导MSCs向内皮细胞分化的机制

缺氧诱导因子-2（hypoxia—inducible factor-2,HIF-2）是一种转录因子。其主要活性作用部位是HIF-2α亚基,与HIF-10L有高度同源性,但二者在基因调节中有不同的作用,HIF-2α能优先表达某些靶基因如EPO、IGF、VEGFR等,是近年来研究较热门的一种促血管新生因子。骨髓间充质干细胞（mesenchy—real stem cells,MSCs）向内皮细胞分化、促进血管新生及管腔形成在很大程度上取决于HIF-2α的调节作用,此调节过程主要依赖于VEGF／VEGFR信号途径。     

Cytokine physiognomies of MSCs from varied sources confirm the regenerative commitment post-coculture with activated neutrophils

The interaction of mesenchymal stromal cells (MSCs) with paracrine signals and immunological cells, and their responses and regenerative commitment thereafter, is understudied. In the current investigation, we compared MSCs from the umbilical cord blood (UCB), dental pulp (DP), and liposuction material (LS) on their ability to respond to activated neutrophils. Cytokine profiling (interleukin-1α [IL-1α], IL-2, IL-4, IL-6, IL-8, tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], transforming growth factor-β [TGF-β]), cellular proliferation and osteogenic differentiation patterns were assessed. The results showed largely comparable cytokine profiles with higher TNF-α and IFN-γ levels in LSMSCs owing to their mature cellular phenotype. The viability and proliferation between LS/DP/UCB MSCs were comparable in the coculture group, while direct activation of MSCs with lipopolysaccharide (LPS) showed comparable proliferation with significant cell death in UCB MSCs and slightly higher cell death in the other two types of MSC. Furthermore, when MSCs post-neutrophil exposure were induced for osteogenic differentiation, though all the MSCs devoid of the sources differentiated, we observed rapid and significant turnover of DPMSCs positive of osteogenic markers rather than LS and UCB MSCs. We further observed a significant turnover of IL-1α and TGF-β at mRNA and cytokine levels, indicating the commitment of MSCs to differentiate through interacting with immunological cells or bacterial products like neutrophils or LPS, respectively. Taken together, these results suggest that MSCs have more or less similar cytokine responses devoid of their anatomical niche. They readily switch over from the cytokine responsive cell phenotype at the immunological microenvironment to differentiate and regenerate tissue in response to cellular signals.     

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation

Background aimsMesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α–pretreated human bone marrow–derived MSCs on resting or activated T cells.MethodsMSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed.ResultsUnprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions.ConclusionsUnprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.     

Stabilization of HIF-2α Induces sVEGFR-1 Production from Tumor-Associated Macrophages and Decreases Tumor Growth in a Murine Melanoma Model

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.     

Benzyl isothiocyanate suppresses pancreatic tumor angiogenesis and invasion by inhibiting HIF-α/VEGF/Rho-GTPases: pivotal role of STAT-3

Our previous studies have shown that benzyl isothiocyanate (BITC) suppresses pancreatic tumor growth by inhibiting STAT-3; however, the exact mechanism of tumor growth suppression was not clear. Here we evaluated the effects and mechanism of BITC on pancreatic tumor angiogenesis. Our results reveal that BITC significantly inhibits neovasularization on rat aorta and Chicken-Chorioallantoic membrane. Furthermore, BITC blocks the migration and invasion of BxPC-3 and PanC-1 pancreatic cancer cells in a dose dependant manner. Moreover, secretion of VEGF and MMP-2 in normoxic and hypoxic BxPC-3 and PanC-1 cells was significantly suppressed by BITC. Both VEGF and MMP-2 play a critical role in angiogenesis and metastasis. Our results reveal that BITC significantly suppresses the phosphorylation of VEGFR-2 (Tyr-1175), and expression of HIF-α. Rho-GTPases, which are regulated by VEGF play a crucial role in pancreatic cancer progression. BITC treatment reduced the expression of RhoC whereas up-regulated the expression of tumor suppressor RhoB. STAT-3 over-expression or IL-6 treatment significantly induced HIF-1α and VEGF expression; however, BITC substantially suppressed STAT-3 as well as STAT-3-induced HIF-1α and VEGF expression. Finally, in vivo tumor growth and matrigel-plug assay show reduced tumor growth and substantial reduction of hemoglobin content in the matrigel plugs and tumors of mice treated orally with 12 μmol BITC, indicating reduced tumor angiogenesis. Immunoblotting of BITC treated tumors show reduced expression of STAT-3 phosphorylation (Tyr-705), HIF-α, VEGFR-2, VEGF, MMP-2, CD31 and RhoC. Taken together, our results suggest that BITC suppresses pancreatic tumor growth by inhibiting tumor angiogenesis through STAT-3-dependant pathway.     

MicroRNA-22 regulates hypoxia signaling in colon cancer cells

MicroRNAs (MiRNAs) are short, non-coding RNA that regulate a variety of cellular functions by suppressing target protein expression. We hypothesized that a set of microRNA regulate tumor responses to hypoxia by inhibiting components of the hypoxia signaling pathway. We found that miR-22 expression in human colon cancer is lower than in normal colon tissue. We also found that miR-22 controls hypoxia inducible factor 1α (HIF-1α) expression in the HCT116 colon cancer cell line. Over-expression of miR-22 inhibits HIF-1α expression, repressing vascular endothelial growth factor (VEGF) production during hypoxia. Conversely, knockdown of endogenous miR-22 enhances hypoxia induced expression of HIF-1α and VEGF. The conditioned media from cells over-expressing miR-22 contain less VEGF protein than control cells, and also induce less endothelial cell growth and invasion, suggesting miR-22 in adjacent cells influences endothelial cell function. Taken together, our data suggest that miR-22 might have an anti-angiogenic effect in colon cancer.     

Decursin promotes HIF-1α proteasomal degradation and immune responses in hypoxic tumour microenvironment

BackgroundHypoxia and HIF-1α are important regulators of tumour growth and angiogenesis and could be attractive targets for cancer therapeutics. Decursin is an active compound extracted from the roots of Angelica gigas and has been shown to have potent anti-cancer and anti-angiogenic activities. However, whether decursin regulates HIF-1α activity and immune responses under hypoxic conditions is not yet understood.PurposeThe aim of this study was to identify whether decursin exhibits anti-cancer activity by targeting HIF-1α.Study designWe investigated whether decursin regulates HIF-1α protein stability and increases its degradation. In addition, we determined if decursin increases immune responses in tumour microenvironment to identify its hypoxia-associated anti-cancer activities.Materials and methodsWe performed the hypoxia-responsive element promoter–reporter assay, Western blot analysis, immune-fluorescence assay, semi-quantitative RT-PCR and ELISA for VEGF secretion, CCK-8 assay for cell proliferation, TUNEL assay for apoptosis and invasion assay in A549 human lung cancer or HCT116 human colon cancer cells. In vivo Lewis lung carcinoma (LLC) allograft mouse model was used to check tumour growth and immune responses in tumour microenvironment by immunohistochemistry analysis.ResultsWe observed that decursin inhibited HIF-1 activation under hypoxia by down-regulating the protein level of its subunit HIF-1α. It increased oxygen-dependant hydroxylation and ubiquitination of HIF-1α to promote HIF-1α degradation. Decursin also decreased mRNA expression of HIF-1α target genes. Decursin suppressed cancer cell proliferation, induced apoptosis and inhibited cancer cell invasion under hypoxia in cancer cells. In the allograft mouse tumour model, decursin reduced the hypoxic area and HIF-1α and PD-L1 expression. Infiltrating T cells (CD3+), helper T cells (CD4+) and cytotoxic (CD8+) T cells were accumulated, but regulatory T cells (Foxp3) and myeloid-derived suppressor cell-mediated immune suppressors (Arg1) were attenuated by decursin.ConclusionOur results suggest that decursin is a novel HIF-1α inhibitor that functions by promoting its proteasomal degradation and that it also helps improve T cell activation in tumour microenvironment; these findings provide new explanations about its anti-cancer and anti-angiogenic activity mechanisms.