Hepatocyte Medium

Background CAR-T therapy has revolutionized treatments for many hematologic malignancies, but it has shown far less efficacy against solid tumors. One reason for this lo... More

Background CAR-T therapy has revolutionized treatments for many hematologic malignancies, but it has shown far less efficacy against solid tumors. One reason for this lower efficacy in solid tumors is increased antigen heterogeneity. This study utilizes a ligand-based CAR-T approach, which allows targeting of multiple receptors by a single ligand. A high expression of the ligand oncostatin M’s (OSM) receptors, oncostatin M receptor (OSMR), and/or leukemic inhibitory factor receptor (LIFR) were noted in osteosarcoma cell lines and patient samples. Osteosarcoma is a bone cancer where treatment options have been stagnant for close to 40 years. Thus, this study explores the therapeutic potential of OSM ligand-based CAR-T cells against osteosarcoma. Methods Third-generation CAR-T cells expressing human OSM on their surface were created, with the surface expression of OSM confirmed by flow cytometry. Co-incubation of OSM CARs and osteosarcoma in vitro was performed, with cell death assessed via Incucyte and PI detection by flow cytometry. CAR-Ts were injected i.v. into mice with osteosarcoma cell line xenografts, and metastatic osteosarcoma. New patient-derived samples were tested for OSMR and LIFR expression and vulnerability to OSM CAR T cells. A new PDX model (named KKOS) from a patient with metastatic treatment-resistant osteosarcoma was characterized and tested for its susceptibility to OSM CAR T cells. All cytotoxic in vivo experiments were performed with n=3–6 mice per group per experiment. Results OSM-CAR-T cells displayed cytotoxicity against osteosarcoma cell lines and patient samples expressing either one of OSM’s receptors in vitro and in vivo. Large increases in cytokine release, specifically IFNγ, were noted in a target-specific manner. One injection of OSM-CAR-T cells intravenously reduced tumor burden in two different mouse xenograft models. A similar anti-tumor effect was also noted in a metastatic model and a mouse model with multiple implanted KKOS tumors. Conclusions Human ligand-based OSM CAR-T cells displayed anti-tumor effects against multiple osteosarcoma cell lines and patient samples. These effects were demonstrated in vitro, in xenograft models, and against a model simulating metastatic disease. Overall, this data supports the continued study of OSM-CAR-T cells as a new therapeutic avenue for osteosarcoma. Less

For healthspan and lifespan, ERK, AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1,2,3,4,5,6,7. Here we examined whether IL... More

For healthspan and lifespan, ERK, AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1,2,3,4,5,6,7. Here we examined whether IL-11, a pro-inflammatory cytokine of the IL-6 family, has a negative effect on age-associated disease and lifespan. As mice age, IL-11 is upregulated across cell types and tissues to regulate an ERK–AMPK–mTORC1 axis to modulate cellular, tissue- and organismal-level ageing pathologies. Deletion of Il11 or Il11ra1 protects against metabolic decline, multi-morbidity and frailty in old age. Administration of anti-IL-11 to 75-week-old mice for 25 weeks improves metabolism and muscle function, and reduces ageing biomarkers and frailty across sexes. In lifespan studies, genetic deletion of Il11 extended the lives of mice of both sexes, by 24.9% on average. Treatment with anti-IL-11 from 75 weeks of age until death extends the median lifespan of male mice by 22.5% and of female mice by 25%. Together, these results demonstrate a role for the pro-inflammatory factor IL-11 in mammalian healthspan and lifespan. We suggest that anti-IL-11 therapy, which is currently in early-stage clinical trials for fibrotic lung disease, may provide a translational opportunity to determine the effects of IL-11 inhibition on ageing pathologies in older people. Less

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host... More

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. Less

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer deaths worldwide. However, current chemotherapeutic drugs for HCC are either poorly effective or exp... More

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer deaths worldwide. However, current chemotherapeutic drugs for HCC are either poorly effective or expensive, and treatment with these drugs has not led to satisfactory outcomes. In a 2012 case report, we described our breakthrough finding in two advanced HCC patients, of whom one achieved complete remission of liver tumors and the other a normalized α-fetoprotein level, along with complete remission of their lung metastases, after the concomitant use of thalidomide and cyproheptadine. We assumed the key factor in our effective therapy to be cyproheptadine. In this study, we investigated the antiproliferative effects and molecular mechanisms of cyproheptadine. Methods: The effect of cyproheptadine on cell proliferation was examined in human HCC cell lines HepG2 and Huh-7. Cell viability was assayed with Cell Counting Kit-8; cell cycle distribution was analyzed by flow cytometry. Mechanisms underlying cyproheptadine-induced cell cycle arrest were probed by western blot analysis. Results: Cyproheptadine had a potent inhibitory effect on the proliferation of HepG2 and Huh-7 cells but minimal toxicity in normal hepatocytes. Cyproheptadine induced cell cycle arrest in HepG2 cells in the G1 phase and in Huh-7 cells at the G1/S transition. The cyproheptadine-induced G1 arrest in HepG2 cells was associated with an increased expression of HBP1 and p16, whereas the G1/S arrest in Huh-7 cells was associated with an increase in p21 and p27 expression and a dramatic decrease in the phosphorylation of the retinoblastoma protein. Additionally, cyproheptadine elevated the percentage of Huh-7 cells in the sub-G1 population, increased annexin V staining for cell death, and raised the levels of PARP and its cleaved form, indicating induction of apoptosis. Finally, cyproheptadine-mediated cell cycle arrest was dependent upon the activation of p38 MAP kinase in HepG2 cells and the activation of both p38 MAP kinase and CHK2 in Huh-7 cells. Conclusions: Our results demonstrate that a non-classical p38 MAP kinase function, regulation of cell cycle checkpoints, is one of the underlying mechanisms promoted by cyproheptadine to suppress the proliferation of HCC cells. These results provide evidence for the drug's potential as a treatment option for liver cancer. Less

Hepatitis C virus (HCV) replication is associated with endoplasmic reticulum (ER) and its infection triggers ER stress. In response to ER stress, ER overload response (EO... More

Hepatitis C virus (HCV) replication is associated with endoplasmic reticulum (ER) and its infection triggers ER stress. In response to ER stress, ER overload response (EOR) can be activated, which involves the release of Ca2+ from ER, production of reactive oxygen species (ROS) and activation of nuclear factor κB (NF-κB). We have previously reported that HCV NS4B expression activates NF-κB via EOR-Ca2+-ROS pathway. Here, we showed that NS4B expression and HCV infection activated cancer-related NF-κB signaling pathway and induced the expression of cancer-related NF-κB target genes via EOR-Ca2+-ROS pathway. Moreover, we found that HCV-activated EOR-Ca2+-ROS pathway had profound effects on host cell viability and HCV replication. HCV infection induced human hepatocyte death by EOR-Ca2+-ROS pathway, whereas activation of EOR-Ca2+-ROS-NF-κB pathway increased the cell viability. Meanwhile, EOR-Ca2+-ROS-NF-κB pathway inhibited acute HCV replication, which could alleviate the detrimental effect of HCV on cell viability and enhance chronic HCV infection. Together, our findings provide new insights into the functions of EOR-Ca2+-ROS-NF-κB pathway in natural HCV replication and pathogenesis. Less

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have g... More

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury. Less

Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studie... More

Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studies have demonstrated that inhibition of the persistent STAT3 signaling pathway results in decreased proliferation and increased apoptosis in human cancer cells, indicating that STAT3 is a viable molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable small molecule, named LY5, to target STAT3 in medulloblastoma cells. LY5 inhibited persistent STAT3 phosphorylation and induced apoptosis in human medulloblastoma cell lines expressing constitutive STAT3 phosphorylation. The inhibition of STAT3 signaling by LY5 was confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, bcl-XL, survivin, and micro-RNA-21. LY5 also inhibited the induction of STAT3 phosphorylation by interleukin-6 (IL-6), insulin-like growth factor (IGF)-1, IGF-2, and leukemia inhibitory factor in medulloblastoma cells, but did not inhibit STAT1 and STAT5 phosphorylation stimulated by interferon-γ (IFN-γ) and EGF, respectively. In addition, LY5 blocked the STAT3 nuclear localization induced by IL-6, but did not block STAT1 and STAT5 nuclear translocation mediated by IFN-γ and EGF, respectively. A combination of LY5 with cisplatin or x-ray radiation also showed more potent effects than single treatment alone in the inhibition of cell viability in human medulloblastoma cells. Furthermore, LY5 demonstrated a potent inhibitory activity on cell migration and angiogenesis. Taken together, these findings indicate LY5 inhibits persistent and inducible STAT3 phosphorylation and suggest that LY5 is a promising therapeutic drug candidate for medulloblastoma by inhibiting persistent STAT3 signaling. Less