Donghua He

Immune evasion of mantle cell lymphoma: expression of B7-H1 leads to inhibited T-cell response to and killing of tumor cells.

Clinical trials of immunotherapy in mantle cell lymphoma have not yet delivered desirable results, partly because of the inhibitory machinery of the tumor and its microenvironment. Here we investigated the role of B7-H1, a member of the B7 family of co-stimulatory/co-inhibitory ligands, in mantle cell lymphoma-mediated immunosuppression. Allogeneic CD3+, CD4+ and CD8+ T cells were purified and co-cultured with irradiated mantle cell lymphoma cells. Mantle cell lymphoma-reactive T-cell lines from HLA-A*0201+ healthy blood donors were generated after in vitro restimulation, and were subjected to functional tests. We found that B7-H1 expressed on mantle cell lymphoma cells was able to inhibit T-cell proliferation induced by the tumor cells, impair the generation of antigen-specific T-cell responses, and render mantle cell lymphoma cells resistant to T-cell-mediated cytolysis. Blocking or knocking down B7-H1 on mantle cell lymphoma cells enhanced T-cell responses and restored tumor-cell sensitivity to T-cell-mediated killing in vitro and in vivo. Knocking down B7-H1 on mantle cell lymphoma cells primed more CD4+ or CD8+ memory effector T cells. Our study demonstrates for the first time that lymphoma cell-expressed B7-H1 may lead to the suppression of host anti-tumor immune responses in mantle cell lymphoma and targeting tumor cell B7-H1 may represent a novel approach to improve the efficacy of immunotherapy in patients with mantle cell lymphoma.

TLR4 inactivation protects from graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation. To clarify the role of Toll-like receptor 4 (TLR4), which is a major receptor for bacterial lipopolysaccharides (LPS), in the development of acute GVHD, we used a TLR4-knockout (TLR4−/−) mouse GVHD model and analyzed the underlying immunological mechanisms. When TLR4−/− mice were used as bone marrow and splenocyte cell graft donors or recipients, GVHD symptom occurrence and mortality were delayed compared to wild-type (TLR4+/+) mice. In addition, histopathological analyses revealed that in TLR4−/−→BALB/c chimeras, liver and small intestine tissue damage was reduced with minimal lymphocytic infiltration. In contrast to TLR4+/+, TLR4−/− mice dendritic cells did not express CD80, CD86, CD40, MHC-II or IL-12 during LPS induction and remained in an immature state. Furthermore, the ability of TLR4−/− mice spleen dendritic cells to promote allogeneic T-cell proliferation and, in particular, T-helper cell 1 (Th1) development was obviously attenuated compared with TLR4+/+ mice dendritic cells, and the levels of interferon-γ (IFN-γ) and IL-10, Th2-cell specific cytokines, were significantly higher in the serum of TLR4−/−→BALB/c than in TLR4+/+→BALB/c chimeric mice. Overall, our data revealed that TLR4 may play a role in the pathogenesis of GVHD and that targeted TLR4 gene therapy might provide a new treatment approach to reduce the risk of GVHD.

Triggering of toll-like receptor-4 in human multiple myeloma cells promotes proliferation and alters cell responses to immune and chemotherapy drug attack

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in the bone marrow and by recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections. The aim of this study was to investigate TLR initiated responses in MM cells including proliferation, anti-apoptosis and immune escape. Myeloma cell lines gene transcription, cell cycle and protein expression were detected by RT-PCR, real-time PCR, western blot, ELISA and flow cytometry analysis. 3H-thymidine was used for measuring cell proliferation, and Annexin V-PI flow cytometry for the detection of cell apoptosis. We show that human myeloma cell lines expressed TLRs，and LPS induced the proliferation and partially protected MM.1S and ARP-1 cells from adriamycin-induced apoptosis. LPS appears to induce proliferation via MyD88 and MAPKs signaling. In addition, LPS treatment upregulated myeloma cell secretion of cytokine IL-18 and expression of immunoregulatory factors B7-H1, B7-H2 and CD40 mRNA and helped myeloma cells to escape immune surveillance. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. See commentary: The TOLL of inflammation in multiple myeloma

Fibroblast activation protein α in tumor microenvironment: Recent progression and implications (Review)

Accumulated evidence has demonstrated that the microenvironment of a given tumor is important in determining its drug resistance, tumorigenesis, progression and metastasis. These microenvironments, like tumor cells, are vital targets for cancer therapy. The cross-talk between tumor cells and cancer-associated fibroblasts (CAFs, alternatively termed activated fibroblasts) is crucial in regulating the drug resistance, tumorigenesis, neoplastic progression, angiogenesis, invasion and metastasis of a tumor. Fibroblast activation protein α (FAPα) is a transmembrane serine protease and is highly expressed on CAFs present in >90% of human epithelial neoplasms. FAPα activity, alongside that of gelatinase and type I collagenase, has become increasingly important in cancer therapy due to its effectiveness in modulating tumor behavior. In this review, recent progression in the knowledge of the role of FAPα in tumor microenvironments is discussed.

Small Rab GTPase Rab7b promotes megakaryocytic differentiation by enhancing IL-6 production and STAT3-GATA-1 association

Induction of the differentiation of human leukemia cells is a useful strategy in treatment of human leukemia. However, the molecular mechanisms involved in leukemia cell differentiation have not been fully elucidated. Interleukin 6 (IL-6) is a pleiotropic cytokine acting on a variety of cell types, and plays important roles in hematopoiesis. GATA binding protein 1 (GATA-1) is an important transcription factor involved in either megakaryocytic or erythrocytic differentiation. Herein we report that Rab7b, a late endosome/lysosome-localized myeloid small GTPase, promotes phorbol-12-myristate-13-acetate (PMA)-induced megakaryocytic differentiation by increasing nuclear factor κB (NF-κB)-dependent IL-6 production and subsequently enhancing the association of activated signal transducer and activator of transcription 3 (STAT3) with GATA-1. By using PMA-induced megakaryocytic differentiation of leukemia cells as a model, we investigated the roles of Rab7b in megakaryocytic differentiation. We find that Rab7b can potentiate PMA-induced upregulation of megakaryocytic markers, production of IL-6, and activation of NF-κB. Inhibitor of NF-κB and neutralizing antibodies for IL-6 or the IL-6 signaling receptor gp130 can block the effects of Rab7b in megakaryocytic differentiation. In Rab7b-silenced cells, PMA-induced activation of NF-κB, IL-6 production, and megakaryocytic differentiation are impaired. Furthermore, we demonstrate that IL-6-induced activation of STAT3 and the subsequent association of STAT3 with GATA-1 may contribute to PMA-induced and Rab7b-mediated transcriptional upregulation of megakaryocytic differentiation markers. Therefore, our data suggest that Rab7b may play important roles in megakaryopoiesis by activating NF-κB and promoting IL-6 production. Our study also indicates that the IL-6-induced association of STAT3 with GATA-1 may regulate megakaryocytic differentiation.

Vernodalol enhances TRAIL-induced apoptosis in diffuse large B-cell lymphoma cells

Tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) is a potent anti‐tumor agent that triggers apoptosis in cells from multiple types of carcinoma but not in normal cells. However, diverse mechanisms are associated with insensitivity to TRAIL in various cancers. TRAIL efficacy may be enhanced by combining TRAIL with a sensitizer. In this study, vernodalol, a sesquiterpene lactone, sensitized diffuse large B‐cell lymphoma (DLBCL) cells to TRAIL‐induced apoptosis. Vernodalol increased the expression of death receptor (DR) 5, and silencing of DR5 with a small interfering RNA (siRNA) reduced the effect of vernodalol on TRAIL‐mediated apoptosis. Additionally, vernodalol up‐regulated the expression of CCAAT/enhancer‐binding protein (C/EBP) homologous protein (CHOP), a transcription factor. Inhibition of CHOP with a siRNA diminished DR5 expression and vernodalol‐induced sensitization to the TRAIL treatment. In addition, a c‐Jun N‐terminal kinase (JNK) inhibitor blocked the vernodalol‐induced up‐regulation of DR5, indicating that the effect depended on JNK activation. Furthermore, the down‐regulation of induced myeloid leukaemia cell differentiation protein (Mcl‐1) played an important role in vernodalol/TRAIL‐induced apoptosis, as Mcl‐1 overexpression prevented this apoptotic effect. Moreover, the vernodalol/TRAIL combination inhibited tumor growth in a xenograft model. Based on our results, vernodalol enhanced TRAIL‐induced apoptosis by down‐regulating Mcl‐1 and up‐regulating DR5, and the effects of DR5 depended on JNK activation and CHOP induction. Therefore, combining TRAIL with vernodalol, a naturally occurring agent, may represent a promising therapeutic approach for DLBCL.

Interleukin-32α promotes the proliferation of multiple myeloma cells by inducing production of IL-6 in bone marrow stromal cells

Multiple myeloma (MM) is a malignant plasma disease closely associated with inflammation. In MM bone marrow microenvironment, bone marrow stromal cells (BMSCs) are the primary source of interleukin-6 (IL-6) secretion, which promotes the proliferation and progression of MM cells. However, it is still unknown how the microenvironment stimulates BMSCs to secrete IL-6. Interleukin-32 (IL-32) is a newly identified pro-inflammatory factor. It was reported that in solid tumors, IL-32 induces changes in other inflammatory factors including IL-6, IL-10, and TNF-α. The aim of this study was to investigate the expression of IL-32 and the role of IL-32 in the MM bone marrow microenvironment. Our data illustrate that MM patients have higher expression of IL-32 than healthy individuals in both bone marrow and peripheral blood. We used ELISA and qRT-PCR to find that malignant plasma cells are the primary source of IL-32 production in MM bone marrow. ELISA and Western blot analysis revealed that recombinant IL-32α induces production of IL-6 in BMSCs by activating NF-κB and STAT3 signaling pathways, konckdown of IL-32 receptor PR3 inhibit this process. Knockdown of IL-32 by shRNA decreased the proliferation in MM cells that induced by BMSCs. In conclusion, IL-32 secreted from MM cells has paracrine effect to induce production of IL-6 in BMSCs, thus feedback to promote MM cells growth.

BAFF is involved in macrophage-induced bortezomib resistance in myeloma

We aimed to characterize the role of B-cell activating factor (BAFF) in macrophage-mediated resistance of multiple myeloma (MM) cells to bortezomib (bort), and to further understand the molecular mechanisms involved in the process. First, we detected BAFF and its three receptors on myeloma cells and macrophages using the quantitative reverse transcriptase-polymerase chain reaction and flow cytometry. The secretion of BAFF was tested in patients with MM, MM cell lines, and macrophages. The ability of macrophages to protect MM cells from bort-induced apoptosis was significantly attenuated using BAFF-neutralizing antibody in the co-culture system or knocking down the expression of BAFF in macrophages with small interfering RNA. We also showed that the MM–macrophage interaction through BAFF and its receptors was primarily mediated by the activation of Src, Erk1/2, Akt, and nuclear factor kappa B signaling and the suppression of caspase activation induced by bort. Our data demonstrated that BAFF played a functional role in the macrophage-mediated resistance of MM cells to bort, suggesting that targeting BAFF may provide a basis for the molecular- and immune-targeted therapeutic approach.

Quercetin induces cell apoptosis of myeloma and displays a synergistic effect with dexamethasone in vitro and in vivo xenograft models

Quercetin, a kind of dietary flavonoid, has shown its anticancer activity in many kinds of cancers including hematological malignancies (acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, and MM) in vitro and in vivo. However, its effects on MM need further investigation. In this study, MM cell lines were treated with quercetin alone or in combination with dexamethasone. In order to observe the effects in vivo, a xenograft model of human myeloma was established. Quercetin inhibited proliferation of MM cells (RPMI8226, ARP-1, and MM.1R) by inducing cell cycle arrest in the G2/M phase and apoptosis. Western blot showed that quercetin downregulated c-myc expression and upregulated p21 expression. Quercetin also activated caspase-3, caspase-9, and poly(ADP-ribose)polymerase 1. Caspase inhibitors partially blocked apoptosis induced by quercetin. Furthermore, quercetin combined with dexamethasone significantly increased MM cell apoptosis. In vivo xenograft models, quercetin obviously inhibited tumor growth. Caspase-3 was activated to a greater extent when quercetin was combined with dexamethasone. In conclusion, quercetin alone or in combination with dexamethasone may be an effective therapy for MM.

Pirh2 mediates the sensitivity of myeloma cells to bortezomib via canonical NF-κB signaling pathway

Clinical success of the proteasome inhibitor established bortezomib as one of the most effective drugs in treatment of multiple myeloma (MM). While survival benefit of bortezomib generated new treatment strategies, the primary and secondary resistance of MM cells to bortezomib remains a clinical concern. This study aimed to highlight the role of p53-induced RING-H2 (Pirh2) in the acquisition of bortezomib resistance in MM and to clarify the function and mechanism of action of Pirh2 in MM cell growth and resistance, thereby providing the basis for new therapeutic targets for MM. The proteasome inhibitor bortezomib has been established as one of the most effective drugs for treating MM. We demonstrated that bortezomib resistance in MM cells resulted from a reduction in Pirh2 protein levels. Pirh2 overexpression overcame bortezomib resistance and restored the sensitivity of myeloma cells to bortezomib, while a reduction in Pirh2 levels was correlated with bortezomib resistance. The levels of nuclear factor-kappaB (NF-κB) p65, pp65, pIKBa, and IKKa were higher in bortezomib-resistant cells than those in parental cells. Pirh2 overexpression reduced the levels of pIKBa and IKKa, while the knockdown of Pirh2 via short hairpin RNAs increased the expression of NF-κB p65, pIKBa, and IKKa. Therefore, Pirh2 suppressed the canonical NF-κB signaling pathway by inhibiting the phosphorylation and subsequent degradation of IKBa to overcome acquired bortezomib resistance in MM cells.