Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

Clostridium perfringens α toxin damages the immune function, antioxidant capacity and intestinal health and induces PLCγ1/AMPK/mTOR pathway-mediated autophagy in broiler chickens.

Clostridium perfringens α toxin is generated by all types of C. perfringens and is closely related to necrotic enteritis in poultry. This study was conducted to investigate the effects of α toxin on immune function, antioxidant capacity, intestinal health and the underlying mechanisms in broiler chickens. A total of 144 twenty-day-old broiler chickens were randomly assigned to four treatments. On d 21, the birds were intraperitoneally injected with PBS (control group) or α toxin at 0.025, 0.1 or 0.4 U/kg of body weight. Samples were collected at 3 h and 24 h post injection (p.i.). Results showed that α toxin challenge linearly decreased the average daily gain during the 3 days after infection and decreased plasma IgA and IgM levels 3 h p.i. Plasma diamine oxidase and d-lactate levels were linearly elevated by α toxin challenge at 3 h p.i. and 24 h p.i. Alpha toxin challenge linearly decreased plasma and jejunal mucosal catalase, glutathione peroxidase and total superoxide dismutase activities at 3 h p.i. and linearly decreased glutathione peroxidase and total superoxide dismutase activities at 24 h p.i. The ileal villus height to crypt depth ratio decreased linearly with increasing α toxin levels at 3 h p.i. and 24 h p.i. Alpha toxin challenge linearly elevated jejunal IL-1ß, IL-6, IL-8 and tumor necrosis factor α mRNA expression at 3 h p.i. Additionally, α toxin challenge linearly reduced the jejunal claudin-1, claudin-3 and zonula occludens 1 mRNA expression at 3 h p.i. and the claudin-3, occludin and zonula occludens 1 mRNA expression at 24 h p.i. What's more, α toxin linearly increased the jejunal PLCγ1, AMPKα1 and ATG5 mRNA expression and linearly decreased the mTOR mRNA expression. In conclusion, C. perfringens α toxin challenge decreased body weight gain, impaired immune function, antioxidant capacity and intestinal health, and induced PLCγ1/AMPK/mTOR pathway-mediated autophagy. The recommended intraperitoneal injection dose for moderate injury was 0.1 U/kg of body weight and the recommended sampling time was 3 h p.i. in broiler chickens.

IL-8 from CD248-expressing cancer-associated fibroblasts generates cisplatin resistance in non-small cell lung cancer.

Chemotherapy-resistant non-small cell lung cancer (NSCLC) presents a substantial barrier to effective care. It is still unclear how cancer-associated fibroblasts (CAFs) contribute to NSCLC resistance to chemotherapy. Here, we found that CD248+ CAFs released IL-8 in NSCLC, which, in turn, enhanced the cisplatin (CDDP) IC50 in A549 and NCI-H460 while decreasing the apoptotic percentage of A549 and NCI-H460 in vitro. The CD248+ CAFs-based IL-8 secretion induced NSCLC chemoresistance by stimulating nuclear factor kappa B (NF-κB) and elevating ATP-binding cassette transporter B1 (ABCB1). We also revealed that the CD248+ CAFs-based IL-8 release enhanced cisplatin chemoresistance in NSCLC mouse models in vivo. Relative to wild-type control mice, the CD248 conditional knockout mice exhibited significant reduction of IL-8 secretion, which, in turn, enhanced the therapeutic efficacy of cisplatin in vivo. In summary, our study identified CD248 activates the NF-κB axis, which, consecutively induces the CAFs-based secretion of IL-8, which promotes NSCLC chemoresistance. This report highlights a potential new approach to enhancing the chemotherapeutic potential of NSCLC-treating cisplatin.

Complete genome sequence of a novel chrysovirus infecting Talaromyces neofusisporus.

A double-stranded RNA (dsRNA) mycovirus was isolated from Talaromyces neofusisporus isolate HJ1-6 and named "Talaromyces neofusisporus chrysovirus 1" (TnCV1). It was found to consist of four dsRNA segments (TnCV1-1, TnCV1-2, TnCV1-3, and TnCV1-4) with lengths of 3595 bp, 3063 bp, 3054 bp, and 2876 bp, respectively. Sequence analysis showed that TnCV1-1 contains an open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) of 1136 amino acids (aa), TnCV1-2 contains an ORF encoding a hypothetical protein of 906 aa, TnCV1-3 contains an ORF encoding a putative capsid protein (CP) of 938 aa, and TnCV1-4 contains an ORF encoding a hypothetical protein of 849 aa. The 5' and 3' untranslated regions (UTRs) of TnCV1-1, TnCV1-2, TnCV1-3, and TnCV1-4 showed a high degree of sequence similarity to each other. Phylogenetic analysis based on RdRp sequences suggested that TnCV1 is a new member of the genus Alphachrysovirus in the family Chrysoviridae. This is the first chrysovirus isolated from T. neofusisporus.

Circular RNA circ_0040823 inhibits the proliferation of acute myeloid leukemia cells and induces apoptosis by regulating miR-516b/PTEN.

OBJECTIVE: Endogenous circular RNAs (circRNAs) and microRNAs (miRNAs) have been shown to regulate the pathogenesis of acute myeloid leukemia (AML). The current study aimed to identify the role of circRNA 0040823 (circ_0040823) in AML. METHODS: Microarray datasets were analyzed to identify differentially expressed circRNAs in AML patients. Peripheral blood samples were obtained from healthy volunteers and AML patients for the measurement of circ_0040823 and miR-516b levels. The overexpression or knockdown of a target gene in AML cells was achieved by the transfection with lentiviral vectors or small interfering RNAs. BALB/c nude mice were inoculated with AML cells and monitored for tumor growth. Dual-luciferase reporter assay, RNA immunoprecipitation, and RNA pull-down assay were used to determine the binding relationship between circRNA and miRNA. RESULTS: circ_0040823 was significantly downregulated in AML patients and leukemia cells. Overexpression of circ_0040823 inhibited AML cell proliferation, and induced apoptosis and cell cycle arrest. Upregulation of circ_0040823 also repressed the growth of xenograft tumors in vivo. circ_0040823 acted as a miR-516b sponge and regulated key cellular events in leukemia cells via downregulating miR-516b. Moreover, tumor suppressor phosphatase and tensin homolog (PTEN) was a downstream target of miR-516b. The inhibition of miR-516b impaired the proliferation capacity of leukemia cells and induced apoptosis, while PTEN deficiency attenuated these effects. CONCLUSION: This study showed that circ_0040823 inhibited proliferation and induced apoptosis of AML cells by sponging miR-516b, thereby diminishing the regulatory effect of miR-516b on PTEN. These findings identified circ_0040823/miR-516b/PTEN as a new therapeutic target for AML.

Oncolytic reovirus induces ovarian cancer cell apoptosis in a TLR3-dependent manner.

Globally, ovarian cancer is the seventh most common cancer and the eighth-most common cause of cancer death among women with a five-year survival rate of less than 45%. Although reovirus is known to be effective for treating ovarian cancer, some types of tumor cells still exhibit resistance to reovirus. In order to solve this resistance problem in the treatment of ovarian cancer, we selected the reovirus-resistant OV-90 ovarian cancer cells to study reovirus oncolytic effects. We found that the viability of OV-90 cells decreased after reovirus double-stranded RNA (dsRNA) genome transfection. Interestingly, we observed that chemical blockage of the Toll-like receptor 3 (TLR3)-dsRNA binding complex in OV-90 cells and the inhibition of downstream TLR3 signaling disrupted OV-90 apoptosis triggered by reovirus dsRNA. Together, these results demonstrate that reovirus dsRNA induces reovirus-resistant tumor cell apoptosis through the TLR3 signaling pathway.

Reovirus enhances cytotoxicity of natural killer cells against colorectal cancer via TLR3 pathway.

BACKGROUND: Cetuximab has been approved for use for first-line treatment of patients with wild-type KRAS metastatic colorectal cancer (CRC). However, treatment with cetuximab has shown limited efficacy as a CRC monotherapy. In addition, natural killer (NK) cell function is known to be severely attenuated in cancer patients. The goal of this study was to develop a new strategy to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells, in combination with cetuximab against CRC cells. METHODS: Ex vivo expanded NK cells were stimulated with reovirus, and reovirus-activated NK cells mediated ADCC assay were performed on CRC cells in combination with cetuximab. The synergistic antitumor effects of reovirus-activated NK cells and cetuximab were tested on DLD-1 tumor-bearing mice. Finally, Toll-like receptor 3 (TLR3) knockdown in NK cells, along with chemical blockade of TLR3/dsRNA complex, and inhibition of the TLR3 downstream signaling pathway, were performed to explore the mechanisms by which reovirus enhances NK cell cytotoxicity. RESULTS: We first confirmed that exposure of NK cells to reovirus enhanced their cytotoxicity in a dose-dependent manner.We then investigated whether reovirus-activated NK cells exposed to cetuximab-bound CRC cells exhibited greater anti-tumor efficacy than either monotherapy. Co-culture of CRC cell lines with reovirus-activated NK cells indicated that NK cytotoxicity was significantly higher in combination with cetuximab, regardless of KRAS mutation status or EGFR expression level. We also found that reovirus activation of NK cells, in conjunction with cetuximab, resulted in significantly stronger anti-tumor efficacy.Finally, TLR3 knockdown, inhibition of TLR3/dsRNA complex or TBK1/IKKε demonstrated that activation of NK cells by reovirus was dependent on TLR3 and its downstream signaling pathway. CONCLUSIONS: This study demonstrated that combination treatment of reovirus-activated NK cells with cetuximab synergistically enhances their anti-tumor cytotoxicity, suggesting a strong candidate strategy for clinical treatment of CRC.

Upregulation of TRIB2 by Wnt/ß-catenin activation in BRAFV600E papillary thyroid carcinoma cells confers resistance to BRAF inhibitor vemurafenib.

PURPOSE: The BRAFV600E mutation is an oncogenic driver associated with aggressive tumor behaviors and increased mortality among patients with papillary thyroid cancer (PTC). Although the BRAF inhibitor vemurafenib gave promising results in BRAFV600E-mutant PTC, resistance development remains a major clinical challenge. This study aimed to explore the mechanisms underlying drug resistance in PTC. METHODS: Two vemurafenib-resistant PTC cell lines (KTC1 and BCPAP) were established by continuous treatment with vemurafenib for 5 months. The knockdown and upregulation of Tribbles homolog 2 (TRIB2) in PTC cells were achieved by the transfection with short hairpin RNA against TRIB2 or recombinant lentiviral vector carrying TRIB2, respectively. The ß-catenin inhibitor, ICG-001, was used for the inhibition of the Wnt/ß-catenin signaling in PTC cells. RESULTS: Vemurafenib-resistant PTC cells showed higher TRIB2 expression, upregulated ERK and AKT activation, enhanced invasive capacity, and increased epithelial-mesenchymal transition compared to the drug-sensitive groups. TRIB2 knockdown repressed the activation of ERK and AKT, inhibited invasion and EMT, and induced apoptosis of PTC cells. TRIB2 deficiency also enhanced the sensitivity of both PTC cells to vemurafenib. Vemurafenib-resistant PTC cells showed elevated expression of ß-catenin in both cytoplasm and nucleus. The pre-incubation of cells with ß-catenin inhibitor significantly inhibited TRIB2 expression, suppressed EMT, and repressed the activation of ERK and AKT in vemurafenib-resistant cells. CONCLUSION: Our study showed that the upregulation of TRIB2 by the Wnt/ß-catenin activation confers resistance to vemurafenib in PTC with BRAFV600 mutation. These findings support the potential use of TRIB2 as a therapeutic target for resistant PTC.

Mesenchymal stem cell carriers enhance antitumor efficacy induced by oncolytic reovirus in acute myeloid leukemia.

Chemotherapy is the main treatment for acute myeloid leukemia (AML), but the therapeutic efficacy is modest, and most commonly manifests as relapse from remission. Thus, improving long-term AML survival is a crucial clinical challenge. In recent years, oncolytic virotherapy has provided an alternative approach for AML treatment. The use of oncolytic reoviruses has been explored in more than 30 clinical trials for safety and feasibility issues. However, like other oncolytic viruses, neutralizing antibodies (NAbs) reduce therapeutic efficacy. To tackle this problem, human umbilical cord mesenchymal stem cells (hUC-MSCs) were used to deliver reovirus using in vitro and in vivo models. Human UC-MSCs were successfully loaded with reovirus, without impairing biological function.We also observed in vitro protective effects of hUC-MSCs on reovirus in the presence of NAbs. In the immunocompromised AML mouse model, hUC-MSCs effectively carried reoviruses to tumor lesions and significantly prolonged the survival of AML xenografts in mice in the presence of a high titer anti-reovirus antibody (p = 0.001). However, reovirus-induced activation of AKT, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and NF-κB signaling led to the maintenance of intrinsic migratory properties and secretion of pro-inflammatory cytokines from hUC-MSCs, particularly CXCL10. In immuno-competent AML mice, MSCs carrying reovirus triggered immune responses, and eventually inhibited tumor growth. Therefore, these results suggest that MSCs as carriers of oncolytic reoviruses can enhance the antitumor efficacy of virotherapy.

A Comparative Analysis of Methods for Titering Reovirus.

BACKGROUND: A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers. OBJECTIVE: Real-time unlabeled cell analysis (RTCA) was used to establish a standard curve of correlation between half-cell index time (CIT50) and virus titer. At the same time, the virus titer from tunable resistance pulse detection (TRPS) technology was compared with the traditional median tissue culture infectious dose (TCID50) method to evaluate the feasibility and application value of the RTCA technique and TRPS technology. METHODS: Cell index (CI) values for L929 cells under different culture conditions were detected, and the appropriate initial cell inoculation density was screened. The half-cell index (CI50) values of reovirus infected L929 cells with TCID50 titers were analyzed by RTCA, the CI50-TCID50 standard curve was created, and a regression equation was developed. RTCA, TCID50, and TRPS methods were used to detect the reovirus titer obtained by the amplification, and the sensitivity and feasibility of the CIT50-TCID50 standard curve method were analyzed. The virus titer was detected by TRPS technology and the TCID50 method. RESULTS: L929 cells were best propagated at an initial density of 6 × 103 cells/well. After infecting L929 cells with different titers of reference reovirus, the linear correlation of CIT50 and TCID50 was y = -2.1806x + 71.023 (R2 = 0.9742). The titer resulting from the RTCA assay was 7×109.6821 pfu/mL, from the TRPS assay was 4.52×1010 pfu/mL, and from the TCID50 assay was 7×109.467 pfu/mL. CONCLUSION: The CIT50-TCID50 standard curve method established by the RTCA technique can be used to quantitatively detect reovirus titer with L929 cells. Compared with the TCID50 method, it takes a relatively short time and has high sensitivity and accuracy. The TRPS technology requires even less time to quantify the virus, but its precision is lower than that of the TCID50 method and RTCA technology. This study provides new technical methods for assessing the virulence of infectious live reovirus particles. Lay Summary: After amplification of the virus, we need to detect the virus titers (the virulence of the virus). The traditional method is to use the virus to infect cells, and then the virus titers can be calculated by 50% of the cells infected. However, this traditional method is time consuming. The ways of RTCA (a real-time cell analysis technique) and TRPS (a nano-bioparticle analysis technique) help us to detect viral titers. The consistency of these three methods determines their feasibility and accuracy. If they are feasible, then these two simple technologies will provide new ideas for detecting viral titers.

Cytotoxic effect of in vitro expanded NK cell-carrying oncolytic reovirus on colorectal cancer cells / 中国肿瘤生物治疗杂志

@# Objective: To evaluate whether human NK cells expanded in vitro can be used as carrier cells of reovirus and to investigate its clinical application value. Methods: Expansion of human NK cells in vitro, and flow cytometry was used to analyse the purity of CD3-CD56+ cells. Expanded NK cells were loaded with reovirus and observed by confocal microscopy, to determining the location of reovirus on NK cells. CCK-8 assay was used to detect reovirus-induced oncolysis of expanded NK cells carrying reovirus (Reo-NK) to tumor cells in the presence of neutralizing antibodies; Real-time fluorescence quantitative PCR was used to assess the relative expression of viral RNA in tumor cells. Cytotoxicity assay were performed to detect Reo-NK cells against KRAS mutant (DLD-1) and KRAS wild type (CaCo-2, HT29) colorectal cancer cell lines, ELISA matched paired antibodies assay was performed to measure the perforin level released by NK cells. Results: Confocal microscopy demonstrated that NK cells retained reovirus on the surface. Expanded NK cells could delivery reovirus to tumor cells in the presence of neutralizing antibodies, and the reovirus after delivery still had significant oncolytic activity (P<0.01); Corresponding qPCR result displayed that the expression of viral RNA in tumor cells significantly increased over time (P<0.01). Compared with NK group, Reo-NK group evidently enhanced the cytotoxicity on colorectal cancer cell lines with both KRAS gene mutant and wild (all P<0.05), and significantly increased the release of perforin (all P<0.05). Conclusion: In vitro expanded NK cells provide a convincing cell carrier for reovirus, while reovirus enhances the cytotoxicity of NK cells, and the combination of the two show a stronger killing effect on colorectal cancer cells，that has important clinical application value.

Roles of genetic and microenvironmental factors in cancer epithelial-to-mesenchymal transition and therapeutic implication.

Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells lose their cell-cell contacts resulting in the formation of mesenchymal cells with migratory properties. Increasing evidence indicate EMT plays a key role in the invasion, metastasis and therapeutic resistance of cancer and maintenance of the phenotype of cancer stem cells (CSCs), which makes the prognosis of patients worse. The progression of cancer from epithelial tissue towards a malignant phenotype is driven by multiple factors that remodel the tissue architecture. This review summarizes and analyzes current studies of genetic and microenvironmental factors in inducing and maintaining cancer EMT and therapeutic implications. This will enable a better understanding of the contribution of EMT-associated factors to cancer progression and highlights that genetic factors and tumor microenvironment responsible for EMT could be used as attractive targets for therapeutic intervention.

Correction: Cytokine-induced killer cell delivery enhances the antitumor activity of oncolytic reovirus.

[This corrects the article DOI: 10.1371/journal.pone.0184816.].

Cytokine-induced killer cell delivery enhances the antitumor activity of oncolytic reovirus.

Oncolytic viruses (OV) have recently emerged as a promising therapeutic modality in cancer treatment. OV selectively infect and kill tumor cells, while sparing untransformed cells. The direct cytotoxic effects combined with the capacity to trigger an immune response make OV an appealing combination partner in the burgeoning field of cancer immunotherapy. One of the leading OV therapeutic candidates is the double-stranded RNA virus reovirus. In order to improve the oncolytic activity of reovirus and allow for systemic administration despite the prevalence of neutralizing antibodies, cytokine-induced killer (CIK) cells were explored as cell carriers for reovirus delivery. In this study, CIK cells were successfully loaded with reovirus ex vivo, and viral replication was limited in CIK cells. Confocal microscopy and flow cytometry demonstrated that CIK cells retained reovirus on the surface. Moreover, CIK cells could promote reovirus infection of tumor cells in the presence of neutralizing antibodies; meanwhile, cytotoxicity of CIK cells was increased after loading with reovirus. These findings support further investigation of reovirus and CIK combination for antitumor therapy.