SC Cells Research Articles

Generalized Topology of a Hybrid Switched- Capacitor Multilevel Inverter for High- Frequency AC Power Distribution

The generalized topology of a hybrid switched-capacitor multilevel inverter (SCMLI) for high-frequency ac (HFAC) power distribution system is presented. By employing the series–parallel conversion technique, the proposed SCMLI can achieve self-balancing of capacitor voltages and voltage step-up with arbitrary numbers of SC cells. The output power quality is improved by the intermediate voltage levels provided by the bidirectional switched converter connected to the dc source string. Besides, several active switches can be replaced with diodes if the large phase difference between output current and voltage is not required. Since the voltage ripple magnitude of the SC units is inversely proportional to the fundamental output frequency, the proposed SCMLI is more preferable for HFac applications. This paper elucidates the working principle of the proposed inverter with the detailed circuit and switching states’ description under staircase modulation. The mathematical analyses associated with the capacitor voltage ripples, sizing, and power loss are also provided. A 19-level MLI with a voltage gain of three was realized by cooperating two SC units with a voltage source string consisting of three symmetric dc sources. The operation and performance of the proposed inverter have been validated by the simulation and experiment on the inverter. The results showed that the inverter performed better at HFac condition and the power quality was satisfactory with the 19-level staircase output modulated at the fundamental frequency.

Broad-spectrum receptor tyrosine kinase inhibitors overcome de novo and acquired modes of resistance to EGFR-targeted therapies in colorectal cancer.

It is increasingly appreciated that 3D cultures are more predictive of in vivo therapeutic efficacy than 2D cultures. Using in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR, we previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab. The de novo mode of cetuximab resistance in SC cells could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. We now show that crizotinib also overcomes acquired cetuximab resistance in CC-CR cells. Phospho-RTK array analysis showed increased phosphorylation of several RTKs, including MET and RON, in SC and CC-CR cells compared to cetuximab-sensitive CC counterparts. Furthermore, other multi-RTK inhibitors cabozantinib and BMS-777607 helped overcome cetuximab resistance, as measured by 3D colony growth and activation state of key signaling molecules. Conversely, addition of RTK ligands HGF and NRG1 induced cetuximab resistance in CC cells, which could be blocked by addition of crizotinib. We further determined the mechanism of the cooperative effect of cetuximab and crizotinib by FACS analysis and observed increased cell cycle arrest in G1 phase in cetuximab-resistant CRC 3D cultures. Finally, we show that crizotinib overcomes cetuximab resistance in vivo in SC nude mice xenografts. Thus, our work shows that multi-RTK inhibition strategy is a potent, broadly applicable strategy to overcome resistance to EGFR-targeted therapeutics in CRC and highlights the relevance of 3D cultures in these studies.Statement of implication: Using in vitro 3D CRC cultures and in vivo CRC xenografts, we show that parallel inhibition of multiple RTKs with small molecule inhibitors overcomes de novo and acquired resistance to EGFR-directed therapies in CRC.

Testosterone augments FSH signaling by upregulating the expression and activity of FSH-Receptor in Pubertal Primate Sertoli cells

The synergistic actions of Testosterone (T) and FSH via testicular Sertoli cells (Sc) regulate male fertility. We have previously reported that the actions of these hormones (T and FSH) in infant monkey testes are restricted only to the expansion of Sc and spermatogonial cells. The robust differentiation of male Germ cells (Gc) occurs after pubertal maturation of testis. The present study was aimed to investigate the molecular basis of the synergy between T and FSH action in pubertal primate (Macaca mulatta) Sc. Using primary Sc culture, we here have demonstrated that T (but not FSH) downregulated AMH and Inhibin-β-B (INHBB) mRNAs in pubertal Sc. We also found that, prolonged stimulation of T in pubertal Sc significantly elevated the expression of genes involved in FSH signaling pathway like FSH-Receptor (FSHR), GNAS and RIC8B, and this was associated with a rise in cAMP production. T also augmented FSH induced expression of genes like SCF, GDNF, ABP and Transferrin (TF) in pubertal Sc. We therefore conclude that T acts in synergy with FSH signaling in pubertal Sc. Such a coordinated network of hormonal signaling in Sc may facilitate the timely onset of the first spermatogenic wave in pubertal primates and is responsible for quantitatively and qualitatively normal spermatogenesis.

Glucocorticoids-Resistant Leukemic B-Cells Undergo a Phenotypic Change That Increases Sensitivity to SRC/ABL Inhibition

▪Glucocorticoids (GCs) have a crucial role in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is considered a strong prognostic marker of relapse, which occurs in about 20% of pediatric B-ALL patients. GCs exert therapeutic effects by inducing cell cycle arrest and eventually, cell death. However, their precise mechanisms of action and resistance are not fully understood. We have previously demonstrated the importance of developmental state in treatment failure in B-ALL (Good Z Nat Med 2018 and Bendall SC Cell 2014), specifically at the developmental transition between pro-B to pre-B cells. Using this developmental framework, we examined the effect of GCs on healthy and malignant early B cells progenitors to better understand mediators of GC resistance.To understand the transcriptional programs mediating early B-cell differentiation, we sorted three B-cell developmental populations (pre-pro-B, pro-B/pre-B I and pre-B II) and performed RNA-seq analysis. Ingenuity pathway analysis revealed a coordinated upregulation of B cell developmental genes and Glucocorticoid Receptor (GCR) pathway genes in healthy pre-B I cells, suggesting a role of GCs in healthy B-cell development. We confirmed expression of the GCR in healthy pre-B I cells by mass cytometry (CyTOF). Further, in vitro treatment of healthy B-cells with GCs (dexamethasone, dex 1 uM) demonstrated pre-B I cycling cells to be most sensitive to GC-induced cell death.Given the importance of GCs in B-ALL treatment, we investigated the GCR pathway and its relationship with B-cell development in 18 B-ALL primary samples (14 diagnosis, 4 relapse; including Ph+=1; MLL rearranged=2; TEL-AML translocated=3). Samples were profiled by CyTOF with a 40-antibody panel including surface markers, signaling molecules, transcription factors, apoptosis and cell cycle molecules. To better comprehend the relation of GCR pathway with BCR signaling, primary cells were analyzed after 48 hours of treatment with dex (1uM), SRC/ABL inhibitor dasatinib (das, 100 nM) or their combination.Similar to results obtained in healthy B-cells, we found the highest expression of GCR in the pre B-I cells in all patients (p<0.001). Interestingly, samples from relapse or diagnostic high-risk groups demonstrated the highest expression of GCR in this cell population. As expected, dex induced cell cycle arrest and cell death in almost all the samples (88%), while two samples were completely unresponsive. Moreover, combined treatment resulted in increased cell death in 29% of patients, compared to dex or das alone.Interestingly, we also observed consistent patterns of phenotypic modulation in dex-resistant cells and to understand how these were related to B-cell development we performed developmental classification (Good Z Nat Med 2018) of leukemic cells in dex-treated versus untreated conditions. This analysis revealed a large decrease of early B-cell progenitors and corresponding increase of more mature subsets of either B and non-B cells (false discovery rate, FDR<0.001), suggesting a differentiating effect of GCs. This phenomenon was even more prominent when we performed, for some primary samples (n=4), longer culture up to six days. Moreover, dex-resistant cells showed aberrant activation of prpS6 and pCREB that was blunted by the combination with das resulting in a reversion of the phenotype and killing of the majority of the cells.To determine if this effect was directly related to GCR expression, we compared results between a GCR+ cell line (NALM6) and GCR- cell line (REH). Consistent with the primary samples, the combination of dex with das more effectively killed NALM6 cells yet the REH were unresponsive to either treatment. However, overexpressing GCR in REH cells reversed dex-resistance, also becoming more sensitive to the combined treatment with das. Similar to primary cells, NALM6 and REH GCR+ cells showed a phenotypic change when treated with dex, that will be further investigated to clarify the relationship between the dex-induced phenotype and its apoptotic effect.In summary, these data suggest a dual role of glucocorticoids on early B-cells; mediating both apoptosis and differentiation in relationship to cell cycle status and GCR level. These findings may carry therapeutic implications and suggest cell types vulnerable to BCR signaling inhibition as a therapeutic intervention to overcome GCs resistance. DisclosuresNolan:Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

IL-11 antagonist suppresses Th17 cell-mediated neuroinflammation and demyelination in a mouse model of relapsing-remitting multiple sclerosis

IL-11 induced differentiation and expansion of Th17 cells in patients with early relapsing-remitting multiple sclerosis (RRMS). In mice with relapsing-remitting experimental autoimmune encephalomyelitis (RREAE), IL-11 exacerbated disease, induced demyelination in the central nervous system (CNS), increased the percentage of IL-17A+CD4+ Th17 cells in the CNS in the early acute phase, and up-regulated serum IL-17A levels and the percentage of IL-17A+CD4+ Th17 cells in lymph nodes, and IFN-γ+CD4+ T cells in spinal cord in the RR phase. IL-11 antagonist suppressed RREAE disease activities, inhibited IL-17A+CD4+ cell infiltration and demyelination in the CNS, and decreased the percentage of IL-17A+CD4+ T cells in peripheral blood mononuclear cells and ICAM1+CD4+ T cells in brain and SC. Diffusion Tensor Imaging indicated that IL-11 antagonist inhibited demyelination in several brain regions. We conclude that by suppressing Th17 cell-mediated neuroinflammation and demyelination, IL-11 antagonist can be further studied as a potential selective and early therapy for RRMS.

Sickle cell dehydration: Pathophysiology and therapeutic applications.

Cell dehydration is a distinguishing characteristic of sickle cell disease and an important contributor to disease pathophysiology. Due to the unique dependence of Hb S polymerization on cellular Hb S concentration, cell dehydration promotes polymerization and sickling. In double heterozygosis for Hb S and C (SC disease) dehydration is the determining factor in disease pathophysiology. Three major ion transport pathways are involved in sickle cell dehydration: the K-Cl cotransport (KCC), the Gardos channel (KCNN4) and Psickle, the polymerization induced membrane permeability, most likely mediated by the mechano-sensitive ion channel PIEZO1. Each of these pathways exhibit unique characteristics in regulation by oxygen tension, intracellular and extracellular environment, and functional expression in reticulocytes and mature red cells. The unique dependence of K-Cl cotransport on intracellular Mg and the abnormal reduction of erythrocyte Mg content in SS and SC cells had led to clinical studies assessing the effect of oral Mg supplementation. Inhibition of Gardos channel by clotrimazole and senicapoc has led to Phase 1,2,3 trials in patients with sickle cell disease. While none of these studies has resulted in the approval of a novel therapy for SS disease, they have highlighted the key role played by these pathways in disease pathophysiology.

Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3β-hydroxysteroid dehydrogenase (3β-HSD) in pigs

Recent studies have suggested that apelin has a role in controlling female reproduction. The aims of the present study were, firstly, to investigate the gene expression (mRNA and protein) and immunolocalization of apelin and its receptor APJ in corpora lutea (CL) of pigs collected during the early (CL1), middle (CL2) and late (CL3) luteal phase. Using real time PCR and immunoblotting techniques, it was observed that apelin gene expression was similar in CL1 and CL2, and less in CL3, while relative abundance APJ mRNA and abundance of the protein were similar in CL1 and CL3 and greater in CL2. There was apelin staining in the cytoplasm of both small (SC) and large (LC) luteal cells with the greatest intensity in CL2. In the cytoplasm of CL1, only a few SC cells stained for APJ; in CL2, APJ was located in the cell membrane of LC and in the cytoplasm of SC; and in CL3 was located in the membrane with moderate cytoplasmic APJ staining. Intense APJ staining was noted in epithelium of blood vessels of CL2–3. Secondly, there was an effect of apelin on progesterone (P4) secretion in CL2 and on the molecular mechanisms of these cells. Stimulatory effects of apelin on P4 secretion, 3β–hydroxysteroid dehydrogenase (HSD) activity and protein abundance were observed and this was inhibited in response to APJ and adenosine 5′-monophosphate-activated protein kinase (AMPKα) kinase blockers. In conclusion, the presence of apelin/APJ in the CL of pigs and stimulatory effects of apelin on P4 secretion and 3β-HSD levels suggest potential auto/paracrine regulation by apelin in the luteal phase of the estrous cycle. Moreover, the involvement of APJ and AMPKα kinase in apelin activity in CL was confirmed.

Modulation of Schlemm's canal endothelial cell stiffness via latrunculin loaded block copolymer micelles.

Increased stiffness of Schlemm's canal endothelial cells (SC cells) is a major contributing factor to the increased pressure characteristic of primary open-angle glaucoma. New treatments for glaucoma are being developed using actin depolymerizers and rho kinase inhibitors to address this increased stiffness. However, these agents have off-target effects and are not as potent as had been hoped. We have developed a micellar nanocarrier assembled from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers capable of encapsulating latrunculin A (Lat A) with the goal of modulating SC cell stiffness. Lat A-loaded nanocarriers were similar in size and morphology to unloaded poly (ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) micelles, loaded Lat A at 62% encapsulation efficiency, and retained loaded Lat A for at least 22 days. The continued functional activity of Lat A following encapsulation within micelles was verified in murine macrophages, which are known to display decreased endocytosis in response to Lat A-dependent cytoskeletal disruption. Endocytic inhibition remained unchanged when comparing equal concentrations of micelle-loaded versus free form Lat A. Uptake of Lat A-loaded micelles by human SC cells was verified in vitro with no sign of cytotoxicity, and modulation of SC cell stiffness was measured by atomic force microscopy. Lat A-loaded micelles significantly decreased SC cell stiffness, which resulted in visible changes in cell morphology as observed by confocal microscopy. Our results demonstrate that PEG-bl-PPS micelles represent a tunable platform for the controlled intracellular delivery of latrunculin. These self-assembled polymeric nanobiomaterials may support the rational design and engineering of delivery systems for the treatment of glaucoma. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1771-1779, 2018.

Silencing of long isoforms of nuclear factor erythroid 2 like 1 primes macrophages towards M1 polarization.

Macrophages are a major component of the immune system and play an important role in regulating the magnitude, duration, and quality of the inflammatory response. Dissecting the functions of transcription factors regulating macrophage activation is important for understanding the inflammatory responses. Nuclear factor erythroid 2 like 1 (NFE2L1, also known as Nrf1) is a CNC-bZIP protein, which has multiple isoforms. While the exact physiological functions of various isoforms of NFE2L1 are still under investigation, accumulating evidence indicate that long isoforms of NFE2L1 (NFE2L1(L)) are important regulators in the antioxidant response, proteasome homeostasis and inflammation. In this study, we found that NFE2L1(L) was upregulated in response to LPS stimulation in RAW264.7 macrophages. Stable knockdown of Nfe2l1(L) (Nfe2l1(L)-KD) in RAW264.7 cells resulted in increased expression of multiple genes indicative of M1 polarization, including Il6, Il1β, Cox2, and Ccl2, under both resting and LPS-challenged conditions. In addition, lentiviral shRNA-mediated silencing of NFE2L1(L) in human monocytic SC and THP1 cells also significantly increased mRNA expression of IL6, IL1β, and TNFα. Furthermore, transient silence of NFE2L1(L) in primary human monocytes isolated from peripheral blood by nucleofection with small interfering RNA resulted in increased expression of IL6 and TNFα. Analysis of the key transcription factors involved in M1 polarization revealed that Nfe2l1(L)-KD RAW264.7 cells have increased mRNA and protein expression and phosphorylation of STAT1 and STAT3 under both resting and M1 polarized conditions. Activation of the NFκB, ERK1/2 and p38 pathways in response to LPS was not affected by the reduction of NFE2L1(L). Moreover, Nfe2l1(L)-KD cells were found to have elevated levels of intracellular ROS, but macrophage M1 polarization induced by Nfe2l1(L) silence was independent of ROS accumulation. Collectively, our results show that knockdown of Nfe2l1(L) leads macrophages to M1 polarization by disinhibition of STAT1/3, and not through the NFκB, ERK1/2 and/or p38 signaling pathways. These findings indicate that NFE2L1(L) functions as a negative regulator of M1 polarization and pro-inflammatory response in macrophages.

Inhibition of IL-6-JAK/Stat3 signaling in castration-resistant prostate cancer cells enhances the NK cell-mediated cytotoxicity via alteration of PD-L1/NKG2D ligand levels.

To investigate whether IL‐6 signaling affects the susceptibility of castration‐resistant prostate cancer (CRPC) cells to cytotoxic action of natural killer (NK) cells, CRPC cell lines (having different IL‐6 levels) were developed by lentiviral transduction. While observing no secreted IL‐6 level in parental C4‐2 and CWR22Rv1 cells, we found the IL‐6 expression/secretion in these cells was induced after the transduction process and the IL‐6 level difference in C4‐2siIL‐6/sc and CWR22siIL‐6/sc cell CRPC cell sets could be detected. We then found that IL‐6‐knockdown cells were more susceptible to NK cell cytotoxicity than control cells due to lowered programmed death receptor ligand 1 (PD‐L1) and increased NK group 2D (NKG2D) ligand levels. In animal studies, to concur with the in vitro results, we found that IL‐6‐expressing cell‐derived tumors were more resistant to NK cell action than the tumors of IL‐6‐knockdown cells. Further, we discovered that JAK‐Stat3 is the most critical IL‐6 downstream signaling that modulates PD‐L1/NKG2D ligand levels in CRPC cells. Furthermore, inhibition of the JAK or Stat3 signaling effectively increased the susceptibility of C4‐2sc and CWRsc cells to NK cell cytotoxicity. We observed the most effective cytotoxicity when the PD‐L1 Ab and JAK inhibitor (or Stat 3 inhibitor) were used together. These results suggest that the strategy of targeting IL‐6 signaling (or its downstream signaling) may enhance the NK cell‐mediated immune action to CRPC tumors, thus yielding clinical implications in developing future immunotherapeutics of exploiting this strategy to treat patients with CRPC.

Leather Waste-Derived Biochar with High Performance for Supercapacitors

Activated biochars from solid residues have been very successful in many applications due to their high surface area, porosity properties, and surface chemistry. Our work, leather waste-derived biochar obtained from pyrolysis of footwear leather wastes at 700°C, followed by a chemical activation with KOH has been prepared, and characterized by SEM, physisorption, XPS and Raman. Results showed that activation with KOH increases the biochar BET specific surface area by almost 100 times. The biochars were tested as electrode materials for supercapacitors in four different aqueous electrolytes, where the KOH activated biochar (AL700) shows higher capacitances. A maximum specific capacitance of 268 Fg-1 (5 mVs-1) was obtained in 0.5M H2SO4 electrolyte for the AL700. A supercapacitor cell was assembled showing excellent performance, with a capacitance of 52 Fg-1 at 0.5 mA g-1 with only 8% of capacitance loss after 5000 charge/discharge cycles, reveling a high stability. The maximum specific energy and specific power determined were 12.8 Wh kg-1 and 1.8 kW kg-1, respectively. These results indicate that it is possible to use the footwear leather waste in energy storage devices, such as in supercapacitors showing a major improvement in SC cells performance, compared to commercial carbons.

Audiogenic Epilepsy and Structural Features of Superior Colliculus in KM Rats

Using immunoblotting, we showed that in rats of audiogenic epilepsy (AE) prone strain (Krushinsky- Molodkina, KM) the superior colliculus tissue (SC) contains significantly less quantity of glial neurotrophic factor (GDNF), beta-tubulin and actin in comparison to the same brain region in "0" rats, nonprone to AE. This fact led to the suggestion that the histological structure of the SC in KM rats could differ significantly from that of the "0" strain. Using neuromorphologу technique, we demonstrated that the total number of SC cells, as well as the number of neurons were significantly less in KM rats than in the "0" strain rats. Particularly strong differences were found in the deep layers of SC, the area of terminals from IC. Further studies of the midbrain structures, will help to identify the novel aspects of neural networks, involved in the genesis of AE in rats of KM strain.

Comparing the degradation effect of a ‘two-cell’ Supercapacitor-module with and without voltage equalization circuit(s) under experimental self-discharge and load cycling tests

The problem of over-voltage is common in SC modules under cycling conditions. To prevent this, the voltage equalization circuit is usually used as a tool to balance SC cells in a module to increase the life expectancy of the system. There is lack of detailed experimental analysis on its performance on degradation proximity testing. This paper therefore focuses on demonstrating the importance of voltage equalization circuits specifically on their performance in degradation proximity between two-cell modules experimentally. Three types of common active balancing equalization circuits were first tested and the best performing circuit was used to compare with a commercial Maxwell voltage equalization circuit under self-discharge and load cycling tests after 1115h of continuous cycles under high temperature. The periodic capacitance (C) and ESR parameters calculated from charge-discharge characterization test results over 1115h shows that the active balancing circuit produced a more even/balance parameter regression between each two-cells in a module over time.

IL-6 Mediates Macrophage Infiltration after Irradiation via Up-regulation of CCL2/CCL5 in Non-small Cell Lung Cancer.

Radiotherapy is effective in reducing primary tumors, however, it may enhance macrophage infiltration to tumor sites, accelerating tumor progression in several ways. We investigated whether radiation can increase macrophage infiltration into non-small cell lung carcinoma (NSCLC) cells. Analysis of in vitro macrophage (differentiated THP-1 cells) migration to either nonirradiated or irradiated tumor cells showed increased migration to the irradiated tumor cells. Because the IL-6 levels in A549 and H157 cells were significantly increased after irradiation, we then investigated whether this increased IL-6 level contributes to radiation-induced macrophage migration. Radiation-induced macrophage infiltration was reduced when IL-6 was knocked down in tumor cells, indicating a positive IL-6 role in this process. To validate this in vitro result, an orthotopic mouse model was developed using a luciferase-tagged H157siIL-6/scramble control (sc) cell set. After tumors developed, the lungs were irradiated, and infiltration of endogenous macrophages and tail-vein injected fluorescent macrophages to tumor sites was investigated. In both groups, increased macrophage infiltration was observed in H157sc cell-derived xenografts compared to H157siIL-6 cell-derived xenografts, confirming the positive IL-6 role in the radiation-induced macrophage infiltration process. In mechanistic dissection studies, radiation-induced up-regulation of CCL2 and CCL5 by IL-6 was detected, and blocking the action of CCL2/CCL5 molecules significantly reduced the number of migrated macrophages to tumor cells after irradiation. These results demonstrate that targeting the IL-6 signaling or CCL2/CCL5 molecules in combination with conventional radiotherapy potentially blocks undesired radiation-induced macrophage infiltration.

A Study of Differential Expression of Testicular Genes in Various Reproductive Phases of Hemidactylus flaviviridis (Wall Lizard) to Derive Their Association with Onset of Spermatogenesis and Its Relevance to Mammals.

Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility.

IL-6 signaling promotes DNA repair and prevents apoptosis in CD133+ stem-like cells of lung cancer after radiation.

BackgroundLocal tumor control by standard fractionated radiotherapy (RT) remains poor because of tumor resistance to radiation (radioresistance). It has been suggested that cancer stem cells (CSCs) are more radioresistant than non-CSCs. In previous studies, we have shown IL-6 promotes self-renewal of CD133+ CSC-like cells. In this study, we investigated whether IL-6 plays roles not only in promoting self-renewal of CD133+ cells after radiation, but also in conferring radioresistance of CD133+ cells in NSCLC.Materials and methodsTo compare radiation sensitivity of CSCs and non-CSCs, CD133+ CSC-like and CD133- cell populations were isolated from two NSCLC cell lines, A549 and H157, by immunomagnetic separation and their sensitivities to ionizing radiation were investigated using the clonogenic survival assay. To further study the IL-6 effect on the radiosensitivity of CD133+ CSC-like cells, CD133+ cells were isolated from A549IL-6si/sc and H157IL-6si/sc cells whose intracellular IL-6 levels were manipulated via the lentiviral transduction with IL-6siRNA. Post-irradiation DNA damage was analyzed by γ-H2AX staining and Comet assay. Molecular mechanisms by which IL-6 regulates the molecules associated with DNA repair and anti-apoptosis after radiation were analyzed by Western blot and immunofluoresecence (IF) staining analyses.ResultsNSCLC CD133+ CSC-like cells were enriched upon radiation. Survival of NSCLC CD133+ cells after radiation was higher than that of CD133- cells. Survival of IL-6 expressing NSC LC CD133+ cells (sc) was higher than that of IL-6 knocked-down cells (IL-6si) after radiation. IL-6 played a role in protecting NSCLC CD133+ cells from radiation-induced DNA damage and apoptosis.ConclusionsIL-6 signaling promotes DNA repair while protecting CD133+ CSC-like cells from apoptotic death after radiation for lung cancer. A combined therapy of radiation and agents that inhibit IL-6 signaling (or its downstream signaling) is suggested to reduce CSC-mediated radioresistance in lung cancer.

The Apoptotic Effect of HIF-1α Inhibition Combined with Glucose plus Insulin Treatment on Gastric Cancer under Hypoxic Conditions.

Gastric cancer grows under a hypoxic environment. HIF-1α is known to play an important role in controlling the production of reactive oxygen species (ROS) in the mitochondria under hypoxic conditions. We previously established HIF-1α knockdown (KD) cells and control (SC) cells in the 58As9 gastric cancer cell line. In this study, we revealed that KD cells, but not SC cells, induced apoptosis under conditions of hypoxia (1% O2) due to excessive production of ROS. A quantitative RT-PCR analysis demonstrated that the expressions of ten genes, which are involved in the control mechanisms of ROS (including the Warburg effect, mitophagy, electron transport chain [ETC] modification and ROS scavenging), were regulated by HIF-1α. Moreover, the promotion of glucose uptake by glucose plus insulin (GI) treatment enhanced the apoptotic effect, which was accompanied by further ROS production in hypoxic KD cells. A Western blot analysis showed that the membranous expression of GLUT1 in KD cells was elevated by glucose and/or insulin treatments, indicating that the GI-induced glucose uptake is mediated by the increased translocation of GLUT1 on the cell membrane. Finally, the anti-tumor effect of HIF-1α knockdown (KD) plus GI was evaluated using a tumor xenograft model, where a hypoxic environment naturally exists. As a result, the GI treatment strongly inhibited the growth of the KD tumors whereby cell apoptosis was highly induced in comparison to the control treatment. In contrast, the growth of the SC tumors expressing HIF-1α was not affected by the GI treatment. Taken together, the results suggest that HIF-1α inhibition plus GI may be an ideal therapy, because the apoptosis due to the destruction of ROS homeostasis is specifically induced in gastric cancer that grows under a hypoxic environment, but not in the normal tissue under the aerobic conditions.

Abstract 740: IL-6 signaling modulates cisplatin resistance in non-small cell lung cancer via up-regulations of anti-apoptotic and DNA repair associated molecules

Abstract Background: Tumors often demonstrate initial responses to cisplatin-based chemotherapy in treating non-small cell lung cancer (NSCLC), but eventually develop resistance and limit the success of therapy. We investigated whether IL-6 signaling is important in developing cisplatin-resistance of NSCLC and explore if targeting IL-6 signaling may increase tumor sensitivity to cisplatin. Methods: We performed in vitro cytotoxicity test using the IL-6 knocked down and scramble (sc) control A549 and H157 NSCLC cell lines (A549IL-6si/sc and H157IL-6si/sc). We also performed in vivo mice studies by establishing xenografts using the A549IL-6si/sc cell set. We next examined the mechanism by which IL-6 mediates cisplatin resistance by performing studies of qPCR, Western analyses, and applying inhibitors of intracellular signaling molecules. Finally, we independently developed the cisplatin resistant A549 (A549CisR) and H157(H157CisR) cell lines and investigated whether targeting IL-6 signaling using a neutralizing antibody of IL-6 could prime these cicplatin-resistant cells to increase the sensitivity to cisplatin. Results: The in vitro survival tests revealed that cisplatin was more cytotoxic to the IL-6 knocked down A549IL-6si and H157IL-6si cells than sc control cells. In in vivo mice studies, we consistently observed more effective tumor regression in A549IL-6si cells-derived xenografts than sc cells-derived tumors (control) after cisplatin treatments. In mechanistic studies, we found that expressions of the anti-apoptotic proteins (Bcl-2 and Mcl-1) and the molecules associated with DNA repair (ATM, CHK1, TP73, p53, and ERCC1) were significantly increased upon cisplatin treatment in A549sc and H157sc cells, but not in IL-6si cells of A549 and H157. The molecular inhibitor studies further revealed that the activation of signaling pathways, such as Stat3, Akt, MAPK, and Erk, was responsible for the up-regulation of these molecules. Further, when we treated cisplatin resistant-A549 and H157 cell lines (A549CisR and H157CisR) with a neutralizing antibody of IL-6, we observed increased cell cytotoxicity. Conclusions and Impact: Our data support that the IL-6 signaling may be critical in conferring NSCLC cell resistance to cisplatin. In addition, we demonstrated that targeting IL-6 signaling could overcome the established acquired cisplatin resistance. Our observation supports a possible strategy to overcome cisplatin resistance through targeting IL-6 signaling or its downstream molecule(s) in treating NSCLC. Citation Format: Shan-zhou Duan, Ying Tsai, Peter Keng, Yongbing Chen, Soo Ok Lee, Yuhchyau Chen. IL-6 signaling modulates cisplatin resistance in non-small cell lung cancer via up-regulations of anti-apoptotic and DNA repair associated molecules. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2015-740

IL-6 signaling contributes to cisplatin resistance in non-small cell lung cancer via the up-regulation of anti-apoptotic and DNA repair associated molecules.

Cisplatin-based chemotherapy is currently the most effective treatment regimen for non-small cell lung cancer (NSCLC), but eventually tumor resistance develops which limits its success. The potential implication of IL-6 signaling in the cisplatin resistance of NSCLC was explored by testing whether NSCLC cells with different levels of intracellular IL-6 show different responses to the cytotoxic treatment of cisplatin. When the cisplatin cytotoxicity of the IL-6 knocked down human NSCLC cells (A549IL-6si and H157IL-6si) were compared with their corresponding scramble control cells (A549sc and H157sc), higher cisplatin cytotoxicity was found in IL-6 si cells than sc cells. Subcutaneous xenograft mouse models were developed using a pair of A549sc and A549IL-6si cells. When the tumor grew to about 400 mm2, mice were treated with cisplatin and tumor regression was monitored. Higher tumor regression was detected in the A549IL-6si xenografts compared to A549sc xenografts following cisplatin treatment. Immunostaining study results from tumor tissues also supported this finding. Expression of anti-apoptotic proteins Bcl-2 and Mcl-1 and DNA repair associated molecules ATM, CHK1, TP73, p53, and ERCC1 were significantly up regulated in cisplatin-treated A549sc and H157sc cells, but no increase was detected in A549IL-6si and H157IL-6si cells. Further inhibitor studies revealed that up regulation of these molecules by IL-6 may be through activation of IL-6 downstream signaling pathways like Akt, MAPK, Stat3, and Erk. These results provide potential for combining cisplatin and inhibitors of IL-6 signaling or its downstream signaling pathway as a future therapeutic approach in preventing development of cisplatin resistant NSCLC tumors.

Glucose 6-phosphate dehydrogenase knockdown enhances IL-8 expression in HepG2 cells via oxidative stress and NF-κB signaling pathway.

BackgroundThis study was designed to investigate the effect of glucose 6-phosphate dehydrogenase (G6PD) deficiency on pro-inflammatory cytokine secretion using a palmitate-induced inflammation HepG2 in vitro model. The modulation of cellular pro-inflammatory cytokine expression under G6PD deficiency during chronic hepatic inflammation has never been investigated before.MethodsThe culture medium of untreated and palmitate-treated G6PD-scramble (Sc) and G6PD-knockdown (Gi) HepG2 cells were subjected to cytokine array analysis, followed by validation with ELISA and qRT-PCR of the target cytokine. The mechanism of altered cytokine secretion in palmitate-treated Sc and Gi HepG2 cells was examined in the presence of anti-oxidative enzyme (glutathione peroxidase, GPX), anti-inflammatory agent (curcumin), NF-κB inhibitor (BAY11-7085) and specific SiRNA against NF-κB subunit p65.ResultsCytokine array analysis indicated that IL-8 is most significantly increased in G6PD-knockdown HepG2 cells. The up-regulation of IL-8 caused by G6PD deficiency in HepG2 cells was confirmed in other G6PD-deficient cells by qRT-PCR. The partial reduction of G6PD deficiency-derived IL-8 due to GPX and NF-κB blockers indicated that G6PD deficiency up-regulates pro-inflammatory cytokine IL-8 through oxidative stress and NF-κB pathway.ConclusionsG6PD deficiency predisposes cells to enhanced production of pro-inflammatory cytokine IL-8. Mechanistically, G6PD deficiency up-regulates IL-8 through oxidative stress and NF-κB pathway. The palmitate-induced inflammation in G6PD-deficient HepG2 cells could serve as an in vitro model to study the role of altered redox homeostasis in chronic hepatic inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-015-0078-z) contains supplementary material, which is available to authorized users.