Anti-apoptotic Response Research Articles

Activity of distinct growth factor receptor network components in breast tumors uncovers two biologically relevant subtypes

BackgroundThe growth factor receptor network (GFRN) plays a significant role in driving key oncogenic processes. However, assessment of global GFRN activity is challenging due to complex crosstalk among GFRN components, or pathways, and the inability to study complex signaling networks in patient tumors. Here, pathway-specific genomic signatures were used to interrogate GFRN activity in breast tumors and the consequent phenotypic impact of GRFN activity patterns.MethodsNovel pathway signatures were generated in human primary mammary epithelial cells by overexpressing key genes from GFRN pathways (HER2, IGF1R, AKT1, EGFR, KRAS (G12V), RAF1, BAD). The pathway analysis toolkit Adaptive Signature Selection and InteGratioN (ASSIGN) was used to estimate pathway activity for GFRN components in 1119 breast tumors from The Cancer Genome Atlas (TCGA) and across 55 breast cancer cell lines from the Integrative Cancer Biology Program (ICBP43). These signatures were investigated for their relationship to pro- and anti-apoptotic protein expression and drug response in breast cancer cell lines.ResultsApplication of these signatures to breast tumor gene expression data identified two novel discrete phenotypes characterized by concordant, aberrant activation of either the HER2, IGF1R, and AKT pathways (“the survival phenotype”) or the EGFR, KRAS (G12V), RAF1, and BAD pathways (“the growth phenotype”). These phenotypes described a significant amount of the variability in the total expression data across breast cancer tumors and characterized distinctive patterns in apoptosis evasion and drug response. The growth phenotype expressed lower levels of BIM and higher levels of MCL-1 proteins. Further, the growth phenotype was more sensitive to common chemotherapies and targeted therapies directed at EGFR and MEK. Alternatively, the survival phenotype was more sensitive to drugs inhibiting HER2, PI3K, AKT, and mTOR, but more resistant to chemotherapies.ConclusionsGene expression profiling revealed a bifurcation pattern in GFRN activity represented by two discrete phenotypes. These phenotypes correlate to unique mechanisms of apoptosis and drug response and have the potential of pinpointing targetable aberration(s) for more effective breast cancer treatments.

HCMV activation of ERK-MAPK drives a multi-factorial response promoting the survival of infected myeloid progenitors.

Viral binding and entry provides the first trigger of a cell death response and thus how human cytomegalovirus (HCMV) evades this - particularly during latent infection where a very limited pattern of gene expression is observed - is less well understood. It has been demonstrated that the activation of cellular signalling pathways upon virus binding promotes the survival of latently infected cells by the activation of cell encoded anti-apoptotic responses. In CD34+ cells, a major site of HCMV latency, ERK signalling is important for survival and we now show that the activation of this pathway impacts on multiple aspects of cell death pathways. The data illustrate that HCMV infection triggers activation of pro-apoptotic Bak which is then countered through multiple ERK-dependent functions. Specifically, ERK promotes ELK1 mediated transcription of the key survival molecule MCL-1, along with a concomitant decrease of the pro-apoptotic BIM and PUMA proteins. Finally, we show that the elimination of ELK-1 from CD34+ cells results in elevated Bak activation in response to viral infection, resulting in cell death. Taken together, these data begin to shed light on the poly-functional response elicited by HCMV via ERK-MAPK to promote cell survival.

Transient activation of the PI3K/Akt pathway promotes Newcastle disease virus replication and enhances anti-apoptotic signaling responses.

Viral infection activates a host's cellular phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which is involved in cell differentiation, growth, survival, and apoptosis. To elucidate molecular mechanisms in the pathogenesis of Newcastle disease virus (NDV), we demonstrated that NDV transiently activates the PI3K/Akt pathway in chicken cells at an early phase of infection. Its activation was observed as early as 15 min post-infection and gradually weakened after 24 h. Incubating cells with a PI3K inhibitor, LY294002 or wortmannin, prior to NDV infection decreased NDV progeny yields and suppressed Akt phosphorylation at early times post-infection. Akt activation is triggered by NDV-GM or NDV-F48E9 and is abolished by methyl β-cyclodextrin and chlorpromazine. Treatment following NDV-La Sota infection had no obvious effect. However, inhibiting PI3K activation promoted apoptotic responses during an early stage of NDV infection. The pan caspase inhibitor ZVAD-FMK mitigated the reduction in Akt phosphorylation by inhibiting PI3K activation, which indicates the signaling pathway promotes cell survival and, in turn, facilitates viral replication. By suppressing premature apoptosis upon NDV infection, the PI3K/Akt pathway enhances the anti-apoptotic response.

Preexposure of MCF-7 breast cancer cell line to dexamethasone alters the cytotoxic effect of paclitaxel but not 5-fluorouracil or epirubicin chemotherapy.

PurposeGlucocorticoids (GCs) are often administered prior to any chemotherapeutics to prevent the secondary effects of anticancer agents. Glucocorticoid receptors (GRs) are expressed in several types of cancer cells, particularly in several histological types of breast cancer. Activation of GRs is not associated with any specific cellular response. Both proapoptotic and antiapoptotic responses have been observed, depending on the study or the type of breast cancer cells. Therefore, it is of relevance to investigate the possible modulation of apoptotic effect of chemotherapeutic agents when cancerous cells have previously been exposed to GCs.MethodsIn vitro cell growth was assayed by counting MCF-7 cells upon exposure to epirubicin (25 nM), 5-fluorouracil (5-FU) (15 µM), and paclitaxel (15 nM), either with or without prior exposure to the GC dexamethasone (Dex) (100 nM).ResultsFollowing preexposure to Dex, the antiapoptotic activity of paclitaxel was significantly reduced by 8.5% (p<0.05), but the activities of epirubicin and 5-FU remained unaltered.ConclusionIn light of the finding that the response of MCF-7 cells pretreated with Dex was significantly reduced, we recommend that the function of GCs should be defined more precisely if they are to be used in conjunction with chemotherapy.

The flashlights on a distinct role of protein kinase C δ: Phosphorylation of regulatory and catalytic domain upon oxidative stress in glioma cells

Glioblastoma multiforme are considered to be aggressive high-grade tumors with poor prognosis for patient survival. Photodynamic therapy is one of the adjuvant therapies which has been used for glioblastoma multiforme during last decade. Hypericin, a photosensitizer, can be employed in this treatment. We have studied the effect of hypericin on PKCδ phosphorylation in U87 MG cells before and after light application. Hypericin increased PKCδ phosphorylation at tyrosine 155 in the regulatory domain and serine 645 in the catalytic domain. However, use of the light resulted in apoptosis, decreased phosphorylation of tyrosine 155 and enhanced serine 645. The PKCδ localization and phosphorylation of regulatory and catalytic domains were shown to play a distinct role in the anti-apoptotic response of glioma cells. We hypothesized that PKCδ phosphorylated at the regulatory domain is primarily present in the cytoplasm and in mitochondria before irradiation, and it may participate in Bcl-2 phosphorylation. After hypericin and light application, PKCδ phosphorylated at a regulatory domain which is in the nucleus. In contrast, PKCδ phosphorylated at the catalytic domain may be mostly active in the nucleus before irradiation, but active in the cytoplasm after the irradiation. In summary, light-induced oxidative stress significantly regulates PKCδ pro-survival and pro-apoptotic activity in glioma cells by its phosphorylation at serine 645 and tyrosine 155.

IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study.

BackgroundPediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment.Methods and FindingsWe performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome.To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor’s ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL.ConclusionsUsing an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome.

Downregulation of Mir-146a-5p, Mir-99b-5p, Mir-143-3p, Mir-10a-5p and Mir-151a-3p Associated with PI3K/AKT, p53, NF-Kb, and Fanconi Anemia/BRCA Signaling Pathways Are Observed in Imatinib-Resistant Chronic Myeloid Leukemia Patients without Detectable BCR-ABL kinase Domain Mutations

IntroductionChronic myeloid leukemia (CML) patients who do not achieve landmark responses are considered imatinib (IM)-resistant. IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, although many IM-resistant patients do not harbor KD mutations. The pathogenesis of this phenomenon is unclear. Aberrations in BCR-ABL independent downstream signaling pathways, namely PI3K/AKT, p53, NF-kB and Fanconi anaemia (FA)/BRCA, have been implicated. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and are notoriously promiscuous, with one miRNA regulating many mRNAs. In recognition of this, we sought to identify dysregulated miRNAs associated with the above pathways in patients with IM-resistant CML.MethodsEight patients with chronic phase CML who demonstrated primary resistance to IM and tested negative for KD mutations via Sanger sequencing were enrolled. Two healthy volunteers constituted the control group. Peripheral blood samples were taken and miRNA extracted from the white cells. MiRNA profiling was performed using miRNA sequencing (miRNA-seq) of the human microRNAome. MiRNA expression analyses (miEA) were performed on 4 sets of miRNAs associated with 4 different signaling pathways derived from publicly available databases: 237 miRNAs for PI3K/AKT, 223 miRNAs for p53, 221 miRNAs for NF-kB, and 126 miRNAs for FA/BRCA. MiRNA expression levels of the patients were compared with normal controls to obtain the differential expression of the miRNAs. MiRNAs that occur in all 4 pathways were further investigated in silico for their putative gene targets (Tarbase v7.0). Gene targets which were commonly linked with 3 different miRNAs were identified.ResultsMiRNA-seq yielded a total of 790 miRNAs. MiEA showed downregulation of 5 miRNAs in all 4 signaling pathways above: miR-146a-5p, miR-99b-5p, miR-143-3p and miR-10a-5p, miR-151a-3p. MiR-146a-5p and miR-99b-5p were the first and second most downregulated miRNAs in all 4 pathways. Putative gene targets that were commonly modulated by 3 miRNAs were: CDK6 (miR-143-3p, miR-10a-5p, miR-151a-3p), MDM2 (miR-146a-5p, miR-143-3p, miR-10a-5p), PTGS2 (miR-146a-5p, miR-143-3p, miR-10a-5p) and REV3L (miR-146a-5p, miR-143-3p, miR-151a-3p), as shown in Table 1.ConclusionsThe PI3K/AKT pathway is activated in response to IM-exposure to IM-naïve cells, implying a role in IM resistance. As cell cycle modulators, both p53 and NF-kB pathways are regulated within the PI3K/AKT pathway via AKT. FANCD2 is a central FA/BRCA pathway regulator and its transcription is maintained by AKT/mTOR via enhancement of NF-kB activity. The complexity of the interactions above has impeded efforts in identifying a unifying target in counteracting IM resistance. Thus, it is of significance that our results showed 5 differentially expressed miRNAs that consistently featured in the above signaling pathways, 4 of which remarkably influence PI3K/AKT and p53.In our study, PI3K/AKT and p53 pathways are inextricably linked with the same 4 miRNAs (miR-146a-5p, miR-143-3p, miR-10a-5p and miR-151a-3p) targeting the same 2 genes (MDM2 and CDK6) in these 2 pathways. MDM2 is an ubiquitin ligase that regulates the stability of p53a. AKT facilitates the functions of MDM2 to promote p53 ubiquitination. Conversely, inhibition of MDM2 leads to activation of p53 in response to cellular stresses. CDK6 is a cell cycle regulator and its overexpression influences the accumulation of pro-apoptotic p53 proteins, postulating a role in DNA damage response. Both PTGS2 and REV3L genes were commonly targeted by 3 miRNAs, within the NF-kB and FA/BRCA pathways respectively. PTGS2 overexpression has been shown in gastrointestinal adenocarcinomas; it induces carcinoma cell migration and invasion via activation of PI3K/AKT signaling. REV3L is the catalytic subunit of DNA polymerase ζ and plays a central role in DNA damage tolerance and chemoresistance towards DNA damaging agents. Notably, the NF-kB and FA/BRCA pathways were juxtaposed to the PI3K/AKT/p53 system via shared miRNA regulators.This provokes interesting questions regarding IM-resistance: are anti-apoptotic signaling and DNA damage response mechanisms driven simultaneously by these miRNAs? Can these miRNAs be potential biomarkers and therapeutic targets in IM-resistant CML? More studies are needed to further validate our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Regulatory-Associated Protein of Mechanistic Target of Rapamycin Prevents Hyperoxia-Induced Lung Injury by Enhancing Autophagy and Reducing Apoptosis in Neonatal Mice.

Administration of supplemental oxygen remains a critical clinical intervention for survival of preterm infants with respiratory failure. However, prolonged exposure to hyperoxia can augment pulmonary damage, resulting in developmental lung diseases embodied as hyperoxia-induced acute lung injury and bronchopulmonary dysplasia (BPD). We sought to investigate the role of autophagy in hyperoxia-induced apoptotic cell death in developing lungs. We identified increased autophagy signaling in hyperoxia-exposed mouse lung epithelial-12 cells, freshly isolated fetal type II alveolar epithelial cells, lungs of newborn wild-type mice, and human newborns with respiratory distress syndrome and evolving and established BPD. We found that hyperoxia exposure induces autophagy in a Trp53-dependent manner in mouse lung epithelial-12 cells and in neonatal mouse lungs. Using pharmacological inhibitors and gene silencing techniques, we found that the activation of autophagy, upon hyperoxia exposure, demonstrated a protective role with an antiapoptotic response. Specifically, inhibiting regulatory-associated protein of mechanistic target of rapamycin (RPTOR) in hyperoxia settings, as evidenced by wild-type mice treated with torin2 or mice administered (Rptor) silencing RNA via intranasal delivery or Rptor+/-, limited lung injury by increased autophagy, decreased apoptosis, improved lung architecture, and increased survival. Furthermore, we identified increased protein expression of phospho-beclin1, light chain-3-II and lysosomal-associated membrane protein 1, suggesting altered autophagic flux in the lungs of human neonates with established BPD. Collectively, our study unveils a novel demonstration of enhancing autophagy and antiapoptotic effects, specifically through the inhibition of RPTOR as a potentially useful therapeutic target for the treatment of hyperoxia-induced acute lung injury and BPD in developing lungs.

Hydrogen Sulfide Therapy in Diabetes-Accelerated Atherosclerosis: A Whiff of Success.

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in diabetes (1). Atherosclerosis occurs earlier and with greater severity in the population with diabetes, leading to a much higher risk of myocardial infarction, stroke, and limb ischemia and amputation. Although numerous factors contribute to the etiology of atherosclerosis, oxidative stress and inflammation play a fundamental role and both processes are exacerbated in diabetes. Given the rapidly growing worldwide incidence of diabetes, there is a critical need for new therapies that target atherogenesis and its clinical manifestations in patients with diabetes. The gasotransmitters nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have emerged as crucial regulators of vascular disease in diabetes (2). Although NO and CO have been extensively studied, less is known regarding the role of H2S in diabetes. H2S is a colorless, water-soluble gas with the characteristic smell of rotten eggs. It is generated by the metabolism of cysteine by the enzymes cystathionine β-synthase and cystathionine γ-lyase or by the concerted action of cysteine amino transferase and 3-mercaptopyruvate sulfurtransferase (Fig. 1). H2S is also produced nonenzymatically from glucose, glutathione, thiosulfate, and sulfur-containing proteins and by the bacterial reduction of sulfur in the intestinal tract (3,4). Although long considered a toxic gas, studies in the past decade have revealed important physiological roles for H2S. H2S promotes blood flow by dilating blood vessels and inhibiting platelet aggregation (3,5). It also exerts potent antioxidant, antiapoptotic, anti-inflammatory, and angiogenic responses. H2S …

Remote preconditioning as a novel "conditioning" approach to repair the broken heart: potential mechanisms and clinical applications.

Remote ischemic preconditioning (RIPC) is a novel strategy of protection against ischemia-reperfusion (IR) injury in the heart (and/or other organs) by brief episodes of non-lethal IR in a distant organ/tissue. Importantly, RIPC can be induced noninvasively by limitation of blood flow in the extremity implying the applicability of this method in clinical situations. RIPC (and its delayed phase) is a form of relatively short-term adaptation to ischemia, similar to ischemic PC, and likely they both share triggering mechanisms, whereas mediators and end-effectors may differ. It is hypothesized that communication between the signals triggered in the remote organs and protection in the target organ may be mediated through substances released from the preconditioned organ and transported via the circulation (humoral pathways), by neural pathways and/or via systemic anti-inflammatory and antiapoptotic response to short ischemic bouts. Identification of molecules involved in RIPC cascades may have therapeutic and diagnostic implications in the management of myocardial ischemia. Elucidation of the mechanisms of endogenous cardioprotection triggered in the remote organ could lead to the development of diverse pharmacological RIPC mimetics. In the present article, the authors provide a short overview of RIPC-induced protection, proposed underlying mechanisms and factors modulating RIPC as a promising cardioprotective strategy.

The NF-κB pathway participates in the response to sulfide stress in Urechis unicinctus

The NF-κB pathway is known to be involved in regulating apoptosis, inflammation and immunity in organisms. In this study, we first identified full-length cDNA sequences of two key molecules in the NF-κB pathway, namely, NEMO and p65, and characterized their responses in the hindgut of Urechis unicinctus (Echiura, Urechidae) exposed to sulfide. The full-length of cDNA was 2491 bp for U. unicinctus NEMO (UuNEMO) and 1971 bp for U. unicinctus p65 (Uup65), and both polyclonal antibodies were prepared using UuNEMO or Uup65 expressed prokaryotically with the sequence of their whole open reading frame. Immunoprecipitation and Western blotting showed that the NF-κB pathway was activated in U. unicinctus exposed to sulfide, in which the content of UuNEMO ubiquitination and nuclear Uup65 increased significantly (p < 0.05) in hindgut tissue of U. unicinctus exposed to sulfide. Furthermore, the mRNA level of UuBcl-xL, a downstream anti-apoptosis gene of the NF-κB pathway, increased significantly (p < 0.05) from 48 h to 72 h and the mRNA level of UuBax, a Bcl-xL antagonist gene, decreased significantly (p < 0.05) at 48 h in the hindgut of U. unicinctus exposed to 50 μM sulfide. During the 150 μM sulfide exposure, the level of UuBcl-xL showed no obvious change, whereas the UuBax mRNA level increased significantly (p < 0.05) at 72 h post-exposure to 150 μM sulfide. We suggested that the activated NF-κB pathway up-regulates UuBcl-xL expression, and evokes an anti-apoptotic response to resist sulfide damage at 50 μM in U. unicinctus. Meanwhile, a Bax-mediated pro-apoptotic response occurs when U. unicinctus is exposed to 150 μM sulfide.

The protective effect of regucalcin against radiation-induced damage in testicular cells

AimsRegucalcin (RGN), a protein broadly expressed in the male reproductive tract, has shown to have beneficial effects on spermatogenesis suppressing chemical-induced apoptosis. This study aimed to evaluate whether RGN overexpression ameliorates the spermatogenic phenotype after radiation treatment. Main methodsTransgenic rats overexpressing RGN (Tg-RGN) and their wild-type (Wt) counterparts were exposed to a single dose of X-rays (6Gy), and at ten weeks after irradiation, the testicular status and the epididymal sperm parameters were evaluated. The expression of RGN and several cell cycle and apoptosis regulators, the enzymatic activity of caspase-3, and RGN immunostaining were also assessed. Key findingsTg-RGN animals displayed higher gonadosomatic index, and augmented sperm viability and motility relatively to their Wt counterparts after irradiation, as well as higher frequency of normal sperm morphology and a diminished incidence of head-defects. The differences in reproductive parameters were underpinned by a lower rate of apoptosis, as evidenced by the reduced activity of caspase-3, lower levels of caspase-8, and increased Bcl-2/Bax ratio in the testis of Tg-RGN animals. Supporting the involvement of RGN in the anti-apoptotic response, an enhanced expression of RGN was observed in irradiated rats. SignificanceTransgenic-overexpression of RGN protected against radiation-induced testicular damage, which strengthens the role of this protein protecting cells from the damage of external agents. These findings also indicated that the modulation of RGN testicular levels would be a mechanism for fertility preservation in men undergoing oncological treatment.

Myeloma Drug Resistance Induced by Binding of Myeloma B7-H1 (PD-L1) to PD-1.

B7 homolog 1 (B7-H1)-expressing myeloma cells not only inhibit myeloma-specific cytotoxic T lymphocytes (CTL), but also confer a proliferative advantage: resistance to antimyeloma chemotherapy. However, it remains unknown whether B7-H1 expressed on myeloma cells induces cellular responses associated with aggressive myeloma behaviors. To address this question, we analyzed the proliferation and drug sensitivity of B7-H1-expressing myeloma cells transfected with B7-H1-specific short-hairpin RNA or treated with programmed cell death (PD)-1-Fc-coupled beads. Knockdown of B7-H1 expression in myeloma cells significantly inhibited cell proliferation and increased apoptosis induced by the chemotherapeutic alkylating agent melphalan, with downregulation of the expression of cell cycle-related genes (CCND3 and CDK6) and antiapoptotic genes (BCL2 and MCL1). B7-H1 molecules thus contributed to myeloma cell-cycle progression and suppression of drug-induced apoptosis. B7-H1-expressing myeloma cells had a higher affinity for PD-1 than for CD80. PD-1-Fc bead-treated myeloma cells also became resistant to apoptosis that was induced by melphalan and the proteasome inhibitor bortezomib. Apoptosis resistance was associated with the PI3K/AKT pathway. Both myeloma cell drug resistance and antiapoptotic responses occurred through the PI3K/AKT signaling pathway, initiated from "reverse" stimulation of B7-H1 by PD-1. Therefore, B7-H1 itself may function as an oncogenic protein in myeloma cells. The interaction between B7-H1 on myeloma cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces drug resistance in myeloma cells through the PI3K/AKT signaling pathway. These observations provide mechanistic insights into potential immunotherapeutic benefits of blocking the B7-H1-PD-1 pathway. Cancer Immunol Res; 4(9); 779-88. ©2016 AACR.

Protection from interferon-β-induced neuronal apoptosis through stimulation of muscarinic acetylcholine receptors coupled to ERK1/2 activation.

Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis. The effects of the ACh receptor agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using western blots, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and overexpression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression. In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 receptors, blocked by Gq/11 antagonist YM254890 and PKC inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by overexpression of ERK2 and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, Oxo-M reduced IFN-β-induced apoptosis; this effect was antagonized by blockade of M1 /M3 receptors and ERK1/2. Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.

Thaliporphine, an alkaloid from Neolitsea konishii, exerts antioxidant, anti-inflammatory, and anti-apoptotic responses in guinea pig during cardiovascular collapse in inflammatory disease

Excessive production of inflammatory cytokines and reactive oxygen species causes circulatory abnormalities leading to insufficient oxygen supply, which results in organ ischaemia and failure. Cardiovascular collapse enhances the risk of death in inflammatory disease. The present study examined the protective effects of thaliporphine, an alkaloid derived from Neolitsea konishii, during cardiovascular collapse in inflammatory disease, which was induced by LPS injection following coronary artery occlusion in guinea pig. Thaliporphine alleviated oxidative stress by preserving SOD activity and accompanied with the alleviation in inflammatory response, was observed to decrease NO, TNF-α secretion and hyperglycaemic response. In addition, thaliporphine inhibited cell death-related pathways activation, as seen by reduced JNK phosphorylation, bax expression, and capase-3 activity, leading to the reduction of myocardial infarct size and prolongation of QT interval in heart. The findings indicate that thaliporphine induces antioxidant, anti-inflammatory, and anti-apoptotic effects and may be developed for the prevention of inflammatory disease with cardiovascular system disorder.

Inducible HGF-secreting Human Umbilical Cord Blood-derived MSCs Produced via TALEN-mediated Genome Editing Promoted Angiogenesis.

Mesenchymal stem cells (MSCs) promote therapeutic angiogenesis to cure serious vascular disorders. However, their survival period and cytokine-secretory capacity are limited. Although hepatocyte growth factor (HGF) can accelerate the rate of angiogenesis, recombinant HGF is limited because of its very short half-life (<3–5 minutes). Thus, continuous treatment with HGF is required to obtain an effective therapeutic response. To overcome these limitations, we produced genome-edited MSCs that secreted HGF upon drug-specific induction. The inducible HGF expression cassette was integrated into a safe harbor site in an MSC chromosome using the TALEN system, resulting in the production of TetOn-HGF/human umbilical cord blood-derived (hUCB)-MSCs. Functional assessment of the TetOn-HGF/hUCB-MSCs showed that they had enhanced mobility upon the induction of HGF expression. Moreover, long-term exposure by doxycycline (Dox)-treated TetOn-HGF/hUCB-MSCs enhanced the anti-apoptotic responses of genome-edited MSCs subjected to oxidative stress and improved the tube-formation ability. Furthermore, TetOn-HGF/hUCB-MSCs encapsulated by arginine-glycine-aspartic acid (RGD)-alginate microgel induced to express HGF improved in vivo angiogenesis in a mouse hindlimb ischemia model. This study showed that the inducible HGF-expressing hUCB-MSCs are competent to continuously express and secrete HGF in a controlled manner. Thus, the MSCs that express HGF in an inducible manner are a useful therapeutic modality for the treatment of vascular diseases requiring angiogenesis.

Anisomycin-induced GATA-6 degradation accompanying a decrease of proliferation of colorectal cancer cell

Transcription factor GATA-6 plays a key role in normal cell differentiation of the mesoderm and endoderm. On the other hand, GATA-6 is abnormally overexpressed in many clinical gastrointestinal cancer tissue samples, and accelerates cell proliferation or an anti-apoptotic response in cancerous tissues. We previously showed that activation of the JNK signaling cascade causes proteolysis of GATA-6. In this study, we demonstrated that anisomycin, a JNK activator, stimulates nuclear export of GATA-6 in a colorectal cancer cell line, DLD-1. Concomitantly, anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest in a plate culture. However, it did not induce apoptosis under growth arrest conditions. Furthermore, the growth of DLD-1 cells in a spheroid culture was suppressed by anisomycin. Although 5-FU showed only a slight inhibitory effect on 3D spheroid cultures, the same concentration of 5-FU together with a low concentration of anisomycin exhibited strong growth inhibition. These results suggest that the induction of GATA-6 dysfunction may be more effective for chemotherapy for colorectal cancer, although the mechanism underlying the synergistic effect of 5-FU and anisomycin remains unknown.

Carbon monoxide promotes gastric wound healing in mice via the protein kinase C pathway

Previous studies have shown that carbon monoxide (CO) is involved in a variety of physiological and pathophysiological processes including anti-inflammatory, anti-apoptotic and anti-oxidant responses. However, it remains unclear whether CO promotes gastric ulcer healing. In the present study, we evaluated the efficacy of CO-saturated saline in the treatment of gastric ulcers and its underlying mechanism. Acute gastric ulcers were induced in C57BL/6 male mice using acetic acid. A CO-saturated solution was prepared by bubbling 50% CO gas into saline. To investigate the effect of CO on gastric mucosal healing, CO solution was orally administrated twice a day beginning on day 3 after the induction of gastric ulcer. Mice were sacrificed on day 7 after ulcer induction. The stomach was removed, and the ulcerated lesions were measured. In vitro wound healing assays were used to determine the mechanism of action of CO in the restoration of murine gastric epithelial cells. The oral administration of CO solution accelerated the gastric ulcer healing by promoting re-epithelialization. Furthermore, the wound healing assay performed using the murine gastric epithelial cells revealed that the CO-saturated medium enhanced cell migration through the activation of protein kinase C (PKC). Based on these data, CO may represent a novel therapeutic approach for the treatment of gastric mucosal injuries.

β3 integrin promotes chemoresistance to epirubicin in MDA-MB-231 through repression of the pro-apoptotic protein, BAD

Resistance to anthracycline based chemotherapy is a major limitation in the treatment of breast cancer, particularly of the triple negative sub-type that lacks targeted therapies. Resistance that arises from tumor-stromal interaction facilitated by integrins provides the possibility of targeted disruption. In the present study, we demonstrate that integrin β3 signaling inhibits apoptosis induced by a DNA-damaging chemotherapeutic agent, epirubicin, in MDA-MB-231 breast cancer cells. Drug efflux based mechanisms do not contribute to this effect. We show that integrin β3 employs the PI3K-Akt and the MAPK pathway for enabling cell survival and proliferation. Further, our results indicate that integrin β3 helps inhibit epirubicin induced cytotoxicity by repression of the pro-apoptotic protein BAD, thus promoting an anti-apoptotic response. Myristoylated RGT peptide and a monoclonal antibody against integrin β3 brought about a reversal of this effect and chemosensitized the cells. These results identify β3 integrin signaling via repression of BAD as an important survival pathway used by breast cancer cells to evade chemotherapy induced stress.

Neferine prevents NF-κB translocation and protects muscle cells from oxidative stress and apoptosis induced by hypoxia.

Neferine (Nef), a bisbenzylisoquinoline alkaloid from lotus seed embryo has a wide range of pharmacological activities. Possible molecular mechanism for the cytoprotective action of Nef during hypoxic stress has not been explored till now. Hence, this is an attempt to elucidate the molecular mechanism involved in the Nef mediated cytoprotection on hypoxia-induced cell injury. Cytoprotective dose of Nef in muscle cells (Human rhabdomyosarcoma cells) exposed to hypoxia was determined by MTT assay. Nef at 500 nM offered better cytoprotection and was used for all the experiments. ROS, intracellular calcium accumulation and mitochondrial membrane (ΔψM) potential were measured using fluorescent probes. Further, we evaluated the effect Nef on hypoxia induced inflammatory and apoptotic responses by FACS and analyzing the expression patterns of NF-κB, COX-2, HIF-1α, caspase-3, caspase-9, Bcl2, and Bax. The results of this study revealed that pretreatment of the cells with Nef significantly decreased the ΔψM and ROS in the cells subjected to hypoxia. Further, Nef inhibited NF-κB there by inhibiting the expression of its downstream regulator COX-2, while it induced the functional HIF-1α expression. The results also indicate that Nef significantly inhibited the ROS dependent mitochondrial mediated apoptosis induced during hypoxia. The cytoprotection elicited by Nef in a model of hypoxia induced cell death involves both anti-inflammatory and anti-apoptotic response. These results suggest that Nef may be used as prophylactic agent against the hypoxic challenge. © 2016 BioFactors, 42(4):407-417, 2016.