In vitro culture with gemcitabine augments death receptor and NKG2D ligand expression on tumour cells

Much effort has been made to try to understand the relationship between chemotherapeutic treatment of cancer and the immune system. Whereas much of that focus has been on the direct effect of chemotherapy drugs on immune cells and the release of antigens and danger signals by malignant cells killed by chemotherapy, the effect of chemotherapy on cells surviving treatment has often been overlooked. In the present study, tumour cell lines: A549 (lung), HCT116 (colon) and MCF-7 (breast), were treated with various concentrations of the chemotherapeutic drugs cyclophosphamide, gemcitabine (GEM) and oxaliplatin (OXP) for 24 hours in vitro. In line with other reports, GEM and OXP upregulated expression of the death receptor CD95 (fas) on live cells even at sub-cytotoxic concentrations. Further investigation revealed that the increase in CD95 in response to GEM sensitised the cells to fas ligand treatment, was associated with increased phosphorylation of stress activated protein kinase/c-Jun N-terminal kinase and that other death receptors and activatory immune receptors were co-ordinately upregulated with CD95 in certain cell lines. The upregulation of death receptors and NKG2D ligands together on cells after chemotherapy suggest that although the cells have survived preliminary treatment with chemotherapy they may now be more susceptible to immune cell-mediated challenge. This re-enforces the idea that chemotherapy-immunotherapy combinations may be useful clinically and has implications for the make-up and scheduling of such treatments

S-100 protein positive cells in nasopharyngeal carcinoma (NPC): absence of prognostic significance. A clinicopathological and immunohistochemical study of 40 cases

An immunohistochemical study of S-100 protein in 43 nasopharyngeal carcinomas (NPC) of known clinical evolution (33 primary and 10 metastatic) is presented. Sixty per cent of primary site cases as well as all metastatic forms showed S-100 protein positive cells intermingled with tumour cells. These S-100 positive elements were identified as Langerhans cells. No significant differences were found when correlating S-100 protein positivity and histological NPC variants, neither in age nor in sex of patients. Statistical analysis failed to demonstrate any positive correlation between S-100 protein reactivity and clinical survival

Generation and physiological roles of linear ubiquitin chains

Ubiquitination now ranks with phosphorylation as one of the best-studied post-translational modifications of proteins with broad regulatory roles across all of biology. Ubiquitination usually involves the addition of ubiquitin chains to target protein molecules, and these may be of eight different types, seven of which involve the linkage of one of the seven internal lysine (K) residues in one ubiquitin molecule to the carboxy-terminal diglycine of the next. In the eighth, the so-called linear ubiquitin chains, the linkage is between the amino-terminal amino group of methionine on a ubiquitin that is conjugated with a target protein and the carboxy-terminal carboxy group of the incoming ubiquitin. Physiological roles are well established for K48-linked chains, which are essential for signaling proteasomal degradation of proteins, and for K63-linked chains, which play a part in recruitment of DNA repair enzymes, cell signaling and endocytosis. We focus here on linear ubiquitin chains, how they are assembled, and how three different avenues of research have indicated physiological roles for linear ubiquitination in innate and adaptive immunity and suppression of inflammation

RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord

ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned \u3e50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG’s). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNFα-induced protein 2 (TNFAIP2) as a major network “hub” gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF’s involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNFα signaling may be helpful in ALS patients

Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1

Peer reviewedPublisher PD

SMG1 and NIK regulate apoptosis induced by Smac mimetic compounds

Smac mimetic compounds (SMCs) are experimental small molecules that induce tumour necrosis factor alpha (TNFα)-dependent cancer cell death by targeting the inhibitor of apoptosis proteins. However, many cancer cell lines are resistant to SMC-mediated apoptosis despite the presence of TNFα. To add insight into the mechanism of SMC-resistance, we used functional siRNA-based kinomic and focused chemical screens and identified suppressor of morphogenesis in genitalia-1 (SMG1) and NF-κB-inducing kinase (NIK) as novel protective factors. Both SMG1 and NIK prevent SMC-mediated apoptosis likely by maintaining FLICE inhibitory protein (c-FLIP) levels to suppress caspase-8 activation. In SMC-resistant cells, the accumulation of NIK upon SMC treatment enhanced the activity of both the classical and alternative nuclear factor-κB pathways, and increased c-FLIP mRNA levels. In parallel, persistent SMG1 expression in SMC-resistant cells repressed SMC-mediated TNFα-induced JNK activation and c-FLIP levels were sustained. Importantly, SMC-resistance is overcome by depleting NIK and SMG1, which appear to facilitate the downregulation of c-FLIP in response to SMC and TNFα treatment, leading to caspase-8-dependent apoptosis. Collectively, these data show that SMG1 and NIK function as critical repressors of SMC-mediated apoptosis by potentially converging on the regulation of c-FLIP metabolism

Suppression of MMP-2 Attenuates TNF-α Induced NF-κB Activation and Leads to JNK Mediated Cell Death in Glioma

BACKGROUND: Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines. METHODOLOGY/PRINCIPAL FINDINGS: MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-α with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKKβ and pIκBα expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-α, TRADD, IKKβ and pIκBα expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-κB activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections. CONCLUSION/SIGNIFICANCE: In summary, our study implies a role of MMP-2 in the regulation of TNF-α mediated constitutive NF-κB activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo

LUBAC prevents lethal dermatitis by inhibiting cell death induced by TNF, TRAIL and CD95L

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone

Ligand Bound β1 Integrins Inhibit Procaspase-8 for Mediating Cell Adhesion-Mediated Drug and Radiation Resistance in Human Leukemia Cells

BACKGROUND: Chemo- and radiotherapeutic responses of leukemia cells are modified by integrin-mediated adhesion to extracellular matrix. To further characterize the molecular mechanisms by which β1 integrins confer radiation and chemoresistance, HL60 human acute promyelocytic leukemia cells stably transfected with β1 integrin and A3 Jurkat T-lymphoma cells deficient for Fas-associated death domain protein or procaspase-8 were examined. METHODOLOGY/PRINCIPAL FINDINGS: Upon exposure to X-rays, Ara-C or FasL, suspension and adhesion (fibronectin (FN), laminin, collagen-1; 5–100 µg/cm(2) coating concentration) cultures were processed for measurement of apoptosis, mitochondrial transmembrane potential (MTP), caspase activation, and protein analysis. Overexpression of β1 integrins enhanced the cellular sensitivity to X-rays and Ara-C, which was counteracted by increasing concentrations of matrix proteins in association with reduced caspase-3 and -8 activation and MTP breakdown. Usage of stimulatory or inhibitory anti β1 integrin antibodies, pharmacological caspase or phosphatidylinositol-3 kinase (PI3K) inhibitors, coprecipitation experiments and siRNA-mediated β1 integrin silencing provided further data showing an interaction between FN-ligated β1 integrin and PI3K/Akt for inhibiting procaspase-8 cleavage. CONCLUSIONS/SIGNIFICANCE: The presented data suggest that the ligand status of β1 integrins is critical for their antiapoptotic effect in leukemia cells treated with Ara-C, FasL or ionizing radiation. The antiapoptotic actions involve formation of a β1 integrin/Akt complex, which signals to prevent procaspase-8-mediated induction of apoptosis in a PI3K-dependent manner. Antagonizing agents targeting β1 integrin and PI3K/Akt signaling in conjunction with conventional therapies might effectively reduce radiation- and drug-resistant tumor populations and treatment failure in hematological malignancies

The Role of MMP7 and Its Cross-Talk with the FAS/FASL System during the Acquisition of Chemoresistance to Oxaliplatin

Background: The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells. Principal Findings: For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53+/+, RHCT116 p53−/−) from the parental HT29, HCT116 p53+/+ and HCT116 p53−/− colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53−/− but not in the RHCT116 p53+/+. Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53−/− but not in the RHCT116 p53+/+. Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells. Conclusions: Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals