Prokaryotic expression of soluble IFN-λ1 recombinant protein with cold-shock system.

Interferon (IFN)-λ1, a member of type III IFN, possesses unique antiviral, anti-tumor, and immune modulation properties. IFN-λ alone or combined with other drugs is considered an essential therapeutic regimen in the clinic. Obtaining high-quality, biologically-active recombinant human IFN-λ1 (rhIFN-λ1) is of great practical significance. In this study, pCold-II-IFN-λ1 expression plasmid was correctly constructed, the rhIFN-λ1 was expressed in BL21(DE3) E.coli and reached the highest level under the optimal condition of 15 °C culture temperature and at 1 µg/L IPTG induction for 12 h. The soluble rhIFN-λ1 was purified by Ni-NTA affinity chromatography. The purified rhIFN-λ1 can effectively activate the JAK1-STAT1 signaling pathway and induce the expression of a large number of interferon-stimulated genes (ISG) including ISG15, ISG54, ISG56, TRAIL, OAS1, MX1, IRF7 and IRF9. In addition, rhIFN-λ1 can effectively inhibit the growth/proliferation of cervical cancer HeLa cells in a dose-dependent pattern. Collectively, the soluble rhIFN-λ1 was successfully expressed in BL21(DE3) E.coli with the cold-shock system, and the purified rhIFN-λ1 demonstrated excellent biological activity. This study lays a solid basis for acquiring high-quality rhIFN-λ1 and its clinical application.

Interferon gamma regulates a complex pro-survival signal network in chronic lymphocytic leukemia.

BACKGROUND: It is known that the microenvironmental cytokine interferon gamma (IFN-γ) provides a survival advantage for chronic lymphocytic leukemia (CLL) cells. However, the mechanisms involved in this effect have not been properly investigated. METHODS: Herein, we conducted a comprehensive screening of the effects of IFN-γ on signaling pathways and gene expression profiles in CLL cells by using western blotting, real-time quantitative reverse transcription (RT-qPCR) and high-throughput RNA sequencing (RNA-seq). RESULTS: We found that IFN-γ not only activated the pro-survival signal transducer and activator of transcription 3 (STAT3), but also activated the protein kinase B and extracellular signal-regulated kinase signaling pathways. RNA-seq analysis showed that IFN-γ stimulation changed the expression profiles of more than 500 genes, with 391 being up-regulated and 123 down-regulated. These genes are involved in numerous biological processes, including anti-apoptosis, cell migration, and proliferation. IFN-γ significantly up-regulated the expression of CD38, BCL6, CXCL9, BCL2A1, SCOS3, IL-10, HGF, EGFR, THBS-1, FN1, and MUC1, which encode proteins potentially associated with disease progression, worse prognosis or poor response to treatment. Blocking janus kinases1/2 (JAK1/2) or STAT3 signal by specific inhibitors affected the expression of most genes, suggesting a pivotal role of the JAK1/2-STAT3 pathway in IFN-γ pro-survival effects in CLL. CONCLUSIONS: Our data demonstrate that IFN-γ regulates a complex pro-survival signal network in CLL through JAK1/2-STAT3, which provides a rational explanation for IFN-γ promoting CLL cells survival and drug resistance.

PML-II regulates ERK and AKT signal activation and IFNα-induced cell death.

BACKGROUND: The requirement of promyelocytic leukaemia protein (PML) in interferon (IFN)-induced cell apoptosis is well-established. However, the exact mechanisms by which the multiple isoforms of PML protein participate in this process remain not well-understood. We previously demonstrated that PML isoform II (PML-II) positively regulates induced gene expression during a type I IFN response and evaluate here how PML-II contributes to IFNα-induced cell death. METHODS: HeLa cells were transiently depleted of PML-II by siRNA treatment and the response of these cells to treatment with IFNα assessed by molecular assays of mRNA and proteins associated with IFN and apoptosis responses. RESULTS: In HeLa cells, death during IFNα stimulation was reduced by prior PML-II depletion. PML-II removal also considerably decreased the induced expression of pro-apoptotic ISGs such as ISG54 (IFIT2), and substantially impaired or prevented expression of PUMA and TRAIL, proteins that are associated with the intrinsic and extrinsic apoptotic pathways respectively. Thirdly, PML-II depletion enhanced ERK and AKT pro-survival signaling activation suggesting that PML-II normally suppresses signaling via these pathways, and that lack of PML-II hence led to greater than normal activation of AKT signaling upon IFNα stimulation and consequently increased resistance to IFNα-induced apoptosis. CONCLUSIONS: The positive contribution of PML-II to the expression of various IFNα-induced pro-apoptotic proteins and its inhibition of pro-survival signaling together provide a mechanistic explanation for reduced apoptosis under conditions of PML deficiency and may account for at least part of the role of PML as a tumor suppressor gene. Video Abstract.

IFN-γ enhances CLL cell resistance to ABT-199 by regulating MCL-1 and BCL-2 expression via the JAK-STAT3 signaling pathway.

Although clinical outcomes of CLL have improved with the use of BCL-2 inhibitor, ABT-199, acquired resistance eventually occurs in many cases, which leads to CLL disease progression. Thus, understanding the mechanisms that mediate this relapse is important to design improved therapies. Herein, we report that cytokine IFN-γ, secreted by dysfunctional T cells, enhanced CLL cells resistance to ABT-199. IFN-γ stimulation significantly increased the expression of BCL-2, MCL-1 and BCL-xL. Blocking JAK1/2-STAT3 signaling pathway impaired the expression of these anti-apoptotic proteins after IFN-γ stimulation. The combination of ABT-199 with JAK1/2 inhibitor Ruxolitinib or STAT3 inhibitors Stattic and C188-9 increased malignant B cell death. In summary, we show that IFN-γ enhanced CLL cells resistance to ABT-199 at least in part by up-regulating BCL-2, MCL-1 and BCL-xL expression via JAK1/2-STAT3 pathway, and thus blocking this pathway with inhibitors increased ABT-199 efficiency to induce CLL cell apoptosis, suggesting a potential therapeutically relevant combination to overcome ABT-199 resistance.

poly(I:C) synergizes with proteasome inhibitors to induce apoptosis in cervical cancer cells.

Cervical cancer is one of the most common malignancies in women, with a poor survival rate. Thus, there is a need to define effective combination strategies to improve therapy. In this study, we report that dsRNA poly(I:C) up-regulated the expression of IFNß and apoptosis-associated genes in cervical cancer cells, activating both intrinsic and extrinsic apoptotic pathways, and eventually inducing cell death. Similarly, proteasome inhibitors also effectively induced cervical cancer cell apoptosis, probably through prevention of p53 degradation, inhibiting NF-κB signal activation and decreasing BCL-2 expression. Importantly, the combination of poly(I:C) with proteasome inhibitors enhanced caspase-8 and caspase-9 activation, and synergistically induced cervical cancer cell apoptosis. Both activated p38 signals and increased ROS levels, and their combination extended these effects. Collectively, we show that the activation of multiple pro-apoptotic pathways by poly(I:C) and proteasome inhibitors underpin a synergistic effect on inducing cervical cancer cell death, suggesting a potential therapeutic combination with clinical relevance.

Icotinib derivatives as tyrosine kinase inhibitors with anti-esophageal squamous carcinoma activity.

Previous report showed that a variety of icotinib derivatives bearing different 1,2,3-triazole moieties, which could be readily prepared via copper (I)-catalyzed cycloaddition (CuAAC) reaction between icotinib and different azides, exhibited interesting activity against different lung cancer cell lines such as H460, H1975, H1299, A549 or PC-9. To further expand the application scope of the compounds and to validate the function of triazole groups in drug design, the anti-cancer activity of these compounds against esophageal squamous carcinoma (ESCC) cells was tested herein. Preliminary MTT experiments suggested that these compounds were active against different ESCC cell lines such as KYSE70, KYSE410, or KYSE450 as well as their drug-resistant ones. Especially, compound 3l showed interesting anticancer activity against these cell lines. The mode of action was studied via molecular docking, SPR experiments and other biochemical studies, and 3l exhibited higher binding potential to wild-type EGFR than icotinib did. In vivo anticancer study showed that 3l could inhibit tumor growth of cell-line-derived xenografts in ESCC. Study also suggested that 3l was a potent inhibitor for EGFR-TK pathway. Combining these results, 3l represents a promising lead compound for the design of anti-cancer drugs against ESCC.

[Phylogenetic analysis of human/swine/avian gene reassortant H1N2 influenza A virus isolated from a pig in China].

OBJECTIVE: Our aim in this study was to determine the genetic characterization and probable origin of the H1N2 swine influenza virus (A/Swine/Guangxi/13/2006) (Sw/GX/13/06) from lung tissue of a pig in Guangxi province, China. METHODS: Eight genes of Sw/GX/13/06 were cloned and genetically analyzed. RESULTS: The hemagglutinin (HA), nucleoprotein (NP), matrix (M) and non-structural (NS) genes of Sw/GX/13/06 were most closely related to genes from the classical swine H1N1 influenza virus lineage. The neuraminidase (NA) and PB1 genes were most closely related to the corresponding genes from the human influenza H3N2 virus lineage. The remaining two genes PA and PB2 polymerase genes were most closely related to the genes from avian influenza virus lineage. CONCLUSION: Phylogenetic analyses revealed that Sw/GX/13/06 was a human/swine/avian H1N2 virus, and closely related to H1N2 viruses isolated from pigs in United States (1999-2001) and Korea (2002). To our knowledge, Sw/GX/13/06 was the first triple-reassortant H1N2 influenza A virus isolated from a pig in China. Whether the Sw/GX/13/06 has a potential threat to breeding farm and human health remains to be further investigated.

Promyelocytic Leukemia Protein Isoform II Promotes Transcription Factor Recruitment To Activate Interferon Beta and Interferon-Responsive Gene Expression.

To trigger type I interferon (IFN) responses, pattern recognition receptors activate signaling cascades that lead to transcription of IFN and IFN-stimulated genes (ISGs). The promyelocytic leukemia (PML) protein has been implicated in these responses, although its role has not been defined. Here, we show that PML isoform II (PML-II) is specifically required for efficient induction of IFN-ß transcription and of numerous ISGs, acting at the point of transcriptional complex assembly on target gene promoters. PML-II associated with specific transcription factors NF-κB and STAT1, as well as the coactivator CREB-binding protein (CBP), to facilitate transcriptional complex formation. The absence of PML-II substantially reduced binding of these factors and IFN regulatory factor 3 (IRF3) to IFN-ß or ISGs promoters and sharply reduced gene activation. The unique C-terminal domain of PML-II was essential for its activity, while the N-terminal RBCC motif common to all PML isoforms was dispensable. We propose a model in which PML-II contributes to the transcription of multiple genes via the association of its C-terminal domain with relevant transcription complexes, which promotes the stable assembly of these complexes at promoters/enhancers of target genes, and that in this way PML-II plays a significant role in the development of type I IFN responses.