Pro Survival Research Articles

PM2.5 toxicity in blood impairs cardiac redox balance and promotes mitochondrial dysfunction in rat heart that further aggravates ischemia reperfusion injury by modulating PI3K/AKT/mTOR/NF-kB signaling axis.

According to the pathophysiological mechanisms linking particulate matter (PM2.5) exposure and cardiovascular diseases, PM2.5 may directly translocate into the blood stream and remote target organs and thereby induce cardiovascular effects. The toxicity of PM2.5 is known to induce oxidative stress in pulmonary tissue, but its impact on the redox state in heart (distant organ) is unknown and how it modulates the cardiac response to ischemia reperfusion (IR) remains unclear. In the present study, we evaluated the toxic effect of PM2.5 on cardiac physiology in the presence and absence of IR after introducing PM2.5 into the blood. Female Wistar rats were injected with diesel particulate matter (DPM) via i.p & i.v routes at a concentration of 10 µg/ml. The toxic impact of PM2.5 not only adversely affects the cardiac ultra-structure (leading to nuclear infiltration, edema, irregularities in heart muscle and nuclear infiltration), but also altered the cellular redox balance, elevated inflammation and promoted the upregulation of proapoptotic mediator genes at the basal level of myocardium. The results showed alterations in cardiac ultrastructure, elevated oxidative stress and significant redox imbalance, increased inflammation and proapoptotic mediators at the basal level of myocardium. Moreover, the cardioprotective pro survival signaling axis was declined along with an increased NF-kB activation at the basal level. IR inflicted further injury with deterioration of cardiac hemodynamic indices (Heart rate [HR], Left ventricular developed pressure [LVDP], Left ventricular end-diastolic pressure [LVEDP] and rate pressure product [RPP]) along with prominent inactivation of signaling pathways. Furthermore, the levels of GSH/GSSG, NADH/NAD, NADPH/NADP were significantly low along with increased lipid peroxidation in mitochondria of PM2.5 treated IR rat hearts. This observation was supported by downregulation of glutaredoxin and peroxiredoxin genes in the myocardium. Similarly the presence of oxidative stress inducing metals was found at a higher concentration in cardiac mitochondria. Thus, the toxic impact of PM2.5 in heart augment the IR associated pathological changes by altering the physiological response, initiating cellular metabolic alterations in mitochondria and modifying the signaling molecules.

Abstract 5986: The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study

Abstract INTRODUCTION: CDKN2A, coding for p16INK4a, a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in oral cavity squamous cell carcinoma (OCSCC). Drugs like palbociclib are CDK4/6 inhibitors that phenomenally also induce senescence. Senescence can be pro-tumorigenic. Studies suggests prosurvival BCL-2 family support tumor cell survival in senescent state. Agents targeting BCL-2 family e.g. navitoclax, are “senolytics” The current study examines combining palbociclib (P) with the senolytic agent navitoclax (N) as a treatment strategy. We demonstrate efficacy of combining P+N in OCSCC, and examine senescence and apoptosis processes as related mechanistic response of combination. EXPERIMENTAL PROCEDURE: We examined cell viability after sequential treatment: 72 h P followed by 72 h N (P+N) in 2-dimensional (2D) model of immortalized cell line HN5 and 3D model of a human OCSCC patient-derived conditional reprogrammed cell line (CR18). Flow cytometry measured apoptosis with Annexin V assay in 2D. Confocal imaging in 3D spheroids corroborated pro-survival protein levels. An in vivo xenograft study was performed with HN5 in 8 weeks old male nude mice to examine tumor responses to sequential P+N treatment. 3 million cells were injected in right flank. Tumors were treated at ~300 mm3 (P-100 mg/kg o.p; N -17 mg/kg i.p). After treatment termination, Bcl-2 family proteins (BcLxl); apoptosis markers (caspase 3, Cleaved PARP) and β-galactosidase (β -gal) for senescence were evaluated with western blot. QPCR measured expression of CDK4/6 target (FOXM1) to examine senescence and pro survival genes. RESULTS: Sequential treatment of P+N (1µM each) demonstrated consistent synergy in both 2D and 3D viability assays. Bright field images of P+N revealed senescence with P followed with apoptosis on N application. β -gal levels increased to 1.5 fold with P and Caspase 3 showed 2-fold increase with P+N. Flow cytometry data revealed 2 folds increase in the apoptotic population with P+N. Confocal imaging in organoids demonstrated increased levels of BcLxL with P, and repression of BCL-xL following N administration. Sequential P+N significantly reduced tumor growth in vivo. P+N demonstrated a robust 5-fold decrease in tumor volume (TV) with a complete cure (TV<20 mm3) in 60% population in comparison to either of the cohorts post 30 days of treatment (control: 2000 mm3, P- 1200 mm3; N- 1500 mm3, p<0.05). Compared to control sequential P+N treatment in vivo i) reduced anti-apoptotic BcLxL mRNA expression to 2.5 fold ii) Cleaved PARP increased 10 folds and iii) BcLxL levels decreased 2 fold. CONCLUSION: Our results suggest that CDK4/6 inhibition makes OCSCC undergo senescence and make it more susceptible to apoptosis when combined with the BCL2-family inhibitor, navitoclax. We propose a sequential P+N as a rational and potentially effective treatment strategy in OCSCC. Citation Format: Nitisha Shrivastava, Nicole Kawachi, Michael B. Prystowsky, Evripidis Gavathiotis, Chandan Guha, Thomas J. Ow. The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5986.

FYCO1 Increase and Effect of Arimoclomol-Treatment in Human VCP-Pathology.

Dominant VCP–mutations cause a variety of neurological manifestations including inclusion body myopathy with early–onset Paget disease and frontotemporal dementia 1 (IBMPFD). VCP encodes a ubiquitously expressed multifunctional protein that is a member of the AAA+ protein family, implicated in multiple cellular functions ranging from organelle biogenesis to ubiquitin–dependent protein degradation. The latter function accords with the presence of protein aggregates in muscle biopsy specimens derived from VCP–patients. Studying the proteomic signature of VCP–mutant fibroblasts, we identified a (pathophysiological) increase of FYCO1, a protein involved in autophagosome transport. We confirmed this finding applying immunostaining also in muscle biopsies derived from VCP–patients. Treatment of fibroblasts with arimoclomol, an orphan drug thought to restore physiologic cellular protein repair pathways, ameliorated cellular cytotoxicity in VCP–patient derived cells. This finding was accompanied by increased abundance of proteins involved in immune response with a direct impact on protein clearaqnce as well as by elevation of pro–survival proteins as unravelled by untargeted proteomic profiling. Hence, the combined results of our study reveal a dysregulation of FYCO1 in the context of VCP–etiopathology, highlight arimoclomol as a potential drug and introduce proteins targeted by the pre–clinical testing of this drug in fibroblasts.

P875: ACTIVATION OF LNCRNA NEAT1 LEADS TO SURVIVAL ADVANTAGE OF MULTIPLE MYELOMA CELLS BY SUPPORTING A REGULATORY LOOP WITH DNA REPAIR PROTEINS: RATIONAL BASES FOR NEAT1 THERAPEUTIC TARGETING IN THE DISEASE

Background: Multiple myeloma (MM) is a fatal malignant proliferation of antibody-secreting bone marrow plasma cells characterized by marked genomic instability. Long non-coding RNA (lncRNA) represents the largest class of non-protein coding genes in the human genome. Their crucial role in solid tumor and hematological malignances, including MM, is well documented. NEAT1 is a highly expressed nuclear lncRNA, representing the core structural component of paraspeckle organelles. We previously demonstrated that NEAT1 silencing negatively regulates proliferation and viability of MM cells, both in vitro and in vivo, highlighting its pivotal role in DNA integrity maintenance, by regulating the homologous recombination. Despite the increasing information obtained by loss-of-function approaches, there is still a lack of information regarding possible oncogenic benefits acquired by MM cells upon NEAT1 overexpression. Aims: Taking advantage of the use of the CRISPR/Cas9 SAM genome editing approach, the present study aimed to shed light on the biological and molecular implication of NEAT1 overexpression in MM. Methods: We adopted the CRISPR/Cas9 Synergistic Activation Mediator editing system to transactivate NEAT1 in AMO-1 MM cell line. LNA-gapmeR antisense oligonucleotide technology was used to silence NEAT1 in MM cells by gymnotic delivery. Hypoxia was induced by culturing the cells into a modular incubator chamber flushed with a mixture of 1% O2, 5% CO2 and 94%N2 at 37 °C. NEAT1 expression was assessed by qRT-PCR and RNA-FISH. Cell cycle phases distribution and apoptotic cells percentage were assessed by flow cytometry. Clonogenic potential was evaluated by methylcellulose assay. Protein expression was investigated by WB and IF. Cell morphological analysis was performed after May-Grunwald Giemsa staining. Transcriptomic analysis was performed by RNA sequencing, following the TruSeq Stranded Total RNA protocol. Libraries with optimal quality and quantity were run on NextSeq 500. Statistical significance was determined by Student t test analysis; differences were considered significant when P-value was <0.05. Results: We performed a transcriptomic analysis of AMO-1 cells either transactivated or silenced for NEAT1. Our data revealed a NEAT1 pivotal role in the maintenance of DNA integrity, showing a significant deregulation of almost all the crucial cellular DNA repair mechanisms for both single and double strand breaks. In particular, we found that NEAT1 transactivation affects DNA repair mechanisms through the activation of a molecular axis including two fundamental kinase proteins, i.e. ATM and DNA-PKcs, and the direct target pRPA32. Furthermore, our data strongly confirmed the activation of this NEAT1-orchestrated molecular axis whether MM cells are exposed to nutrient starvation and hypoxia, suggesting its implication in conferring oncogenic and pro-survival properties to MM cells. The disruption of this oncogenic loop leads to MM cell death and the loss of pro survival advantages, thus suggesting that NEAT1 should be considered a crucial factor for MM cells survival. Summary/Conclusion: Overall, we provided novel important insights into the role of NEAT1 in MM pathobiology, demonstrating that its deregulation strongly correlates with adaptation to stress condition, often associated with late stages of the disease. Taken together, our results suggest that NEAT1 could represent Achilles’ heel for MM cells and should be considered a potential therapeutic target for MM treatment.

38P Triple-negative breast cancers with expression of glucocorticoid receptor in immune cells show better prognosis

Glucocorticoid receptor (GR) is shown to have variable frequency of expression in invasive tumors of the breast. Investigation of additional nuclear receptors like GR in receptor negative tumors like triple negative breast cancer (TNBC) may have prognostic and therapeutic significance. Expression of GR was evaluated by immunohistochemistry in 175 tumors of invasive breast cancer with long term follow up. GR Expression was separately evaluated in invasive tumor cells, stromal cells and tumor infiltrating lymphocytes (TIL's). Staining pattern was categorised as positive when more than 1% of the cells stained in each subpopulation of cells. Disease free survival was analysed between GR positive and negative status by Kaplan Meier analysis. Of the 175 tumors, 121 (70%) were ER positive, 53 (30%) were ER negative and 29% (51) were triple negative. 74% (130/175) tumors showed expression of GR in invasive tumor cells while (84%) 147/175 had expression in TIL's. No significant difference in distribution of GR was noted between ER positive and ER negative tumors (78% vs 66%, p-0.1). Of the TNBC's 54% (28/51) and 70% (36/51) showed expression of GR in invasive tumor and TIL's respectively. Overall, GR positive tumors had significant better survival than GR negative tumors (mean survival time of 85 vs 59 months respectively, p-0.04) Contrary to the reports that GR expression in TIL's are associated with immunosuppressive activity in model systems, TNBC's with increased expression of GR in immune cells were associated with better survival (Mean survival time 74 vs 41 months, log rank test- p-0.03). TNBC tumors which were GR negative had higher lymph node metastases (p-0.04) and none of the other clinical features like age, menopausal state, tumor size and grade were different between GR positive and negative tumors within TNBC. Glucocorticoids (GC) are often used to alleviate the adverse symptoms during chemotherapy. Determining the GR status is of importance due to the pro cell survival effect of the glucocorticoids mediated through GR during chemotherapy. Though GC mediated effects on chemotherapy are controversial, our results indicate favourable effects in TNBC.

Computational modeling and experimental analysis for the diagnosis of cell survival/death for Akt protein

BackgroundSignalling systems that control cell decisions allow cells to process input signals by apprehending the information of the cell to give one of these two feasible outputs: cell death or cell survival. In this paper, a well-structured control design methodology supported by a hierarchical design system was developed to examine signalling networks that control cell decisions by considering a combinations of three primary signals (input proteins): the pro survival growth factors, epidermal growth factor (EGF), insulin, and the pro death cytokine, tumour necrosis factor-α (TNF), for AKT/protein kinase B. The AKT actions were examined by using the three input proteins for cell survival/apoptosis for a period of 0–24 h in 13 different slices for ten different combinations. ResultsExperimental analysis was performed to consider the reactions that were essential to explain the action of AKT. Furthermore, pre-processing and data normalization were performed by using standard deviation, plotting histograms, and scatter plots. Feature extraction and selection were performed using correlation matrix. Radial basis function (RBF) and multiple-layer perceptron (MLP) were used for cell survival/death classification. For all the ten combinations of the three input proteins, 42.85, 347.22, 153.13 were obtained as the minimum value, maximum value, and mean value, respectively, and 126.11 was obtained as the standard deviation for 5-0-5 ng/ml combinations of TNF-EGF-Insulin. The results obtained with MLP 10-8-1 were found to outperform other techniques. ConclusionThe results from the experimental analysis indicate that it is possible to build self-consistent compendia cell-signalling data based on AKT protein which were simulated computationally to yield important insights for the control of cell survival/death.

Mechanism of Action of the Tumor Vessel Targeting Agent NGR-hTNF: Role of Both NGR Peptide and hTNF in Cell Binding and Signaling.

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug’s activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.

Understanding the role of calcium-mediated cell death in high-frequency irreversible electroporation

High-frequency irreversible electroporation (H-FIRE) is an emerging electroporation-based therapy used to ablate cancerous tissue. Treatment consists of delivering short, bipolar pulses (1–10μs) in a series of 80–100 bursts (1 burst/s, 100μs on-time). Reducing pulse duration leads to reduced treatment volumes compared to traditional IRE, therefore larger voltages must be applied to generate ablations comparable in size. We show that adjuvant calcium enhances ablation area in vitro for H-FIRE treatments of several pulse durations (1, 2, 5, 10μs). Furthermore, H-FIRE treatment using 10μs pulses delivered with 1mM CaCl2 results in cell death thresholds (771±129V/cm) comparable to IRE thresholds without calcium (698±103V/cm). Quantifying the reversible electroporation threshold revealed that CaCl2 enhances the permeabilization of cells compared to a NaCl control. Gene expression analysis determined that CaCl2 upregulates expression of eIFB5 and 60S ribosomal subunit genes while downregulating NOX1/4, leading to increased signaling in pathways that may cause necroptosis. The opposite was found for control treatment without CaCl2 suggesting cells experience an increase in pro survival signaling. Our study is the first to identify key genes and signaling pathways responsible for differences in cell response to H-FIRE treatment with and without calcium.

Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3.

BackgroundThe population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature.ObjectivesThus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation.Materials and methodsThawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 μM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor).ResultsRosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (p<0.01), both of which were prevented by incubation in the presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (P<0.01).ConclusionWe provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology.

Abstract 4397: Tak-1 inhibition re-sensitizes cancer cells to TNFα

Abstract Identifying new pharmaceutical targets in cancer has been a long lasting goal of academia. Complex genomic changes coupled with a cancer cell's ability to adapt make tumors particularly difficult to target. To this end, we have identified the TAK1 kinase in the TNFα pathway as a potential mediator of cell survival or apoptosis. Here we evaluate the apoptotic effects of a novel TAK1 inhibitor, Takinib. The selectivity of Takinib was established with an IC50 of 9nm for TAK1, followed by an IC50 for IRAK4 and IRAK1 at 120nm and 390nm respectively. Treatment of MDA-MB-231 cells with Takinib on its own is not sufficient to induce consequential cell death. However, combination therapy with TNFα induces caspase 3/7 activity and subsequent cell death in a dose-dependent manner. Genetic knock out of TAK1 expression in MDA-MB-231 cells with the CRISPR/Cas9 system significantly sensitized previous resistant cells to TNFα. Furthermore, we have shown that activation of human THP-1 cells with LPS is sufficient to produce TNFα levels necessary to facilitate Takinib mediated cell death, indicating that potential immunotherapy applications may be possible. Together these findings support the idea that TAK1 may be an effective target in the TNFα pathway, shifting the cellular mechanisms from pro survival to pro apoptosis. Citation Format: Scott Scarneo, Madeline Sell, Juliane Totzke, David Carlson, Phillip Hughes, Tim Haystead. Tak-1 inhibition re-sensitizes cancer cells to TNFα [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4397.

Thyroid hormones act as mitogenic and pro survival factors in rat ovarian follicles.

Thyroid disorders are clinically associated with impaired fertility in women, and these abnormalities can be improved by restoring the euthyroid state. The exact mechanisms of thyroid effect on female fertility are not well known; however, it is conceivable that thyroid hormones (THs) might act on ovarian physiology via receptors in granulosa cells. This work is aimed at evaluating the effects of THs on non-tumoral granulosa cells and follicles. Freshly isolated rat ovarian follicles and granulosa cells were exposed to T3 or T4 (THs). Cell growth and viability were evaluated by cell counting and the MTT assay, respectively, follicle growth was evaluated by volume measurements. Apoptosis was evaluated by the TUNEL assay and active Caspase 3 staining. rGROV cells were exposed to T3, and apoptosis was induced by serum deprivation. Bcl2, Bcl-2-associated X protein (BAX), Akt and pAkt expression were evaluated by western blot. T3 induced a 40% increase in follicle volume (after 7days). This increase was presumably due to the observed decrease (33%) in the apoptotic rate of the granulosa cell population. Both T3 and T4 caused a dose-dependent increase in rat granulosa cell number and viability. In addition, THs decreased the cell apoptotic rate in a dose-dependent manner. In both conditions, T3 appeared to be more efficient. In rGROV cells, 100nM T3 induced cell growth and, in the absence of growth factors, reduced cell apoptosis by 40%, downregulating Caspase 3 and BAX. This effect was associated with an increase in pAkt levels. The involvement of the PI3K pathway was confirmed by the ability of the PI3K specific inhibitor (LY-294,002) to abolish T3 pro-survival action. THs influence cell survival of ovarian granulosa cells. This effect likely contributes to the TH-induced follicle volume increase.

The role of Indoleamine 2,3-dioxygenase (IDO) in the survival of bone marrow resident long lived Plasma cells

Abstract Long lived plasma cells (LLPCs) are essential for sustained antibody responses and protective humoral immunity. How these cells maintain longevity and a durable antibody response is largely dependent on the complex nature of the bone marrow microenvironment in which these cells reside, and the pro survival factors produced in this niche. Previous work in our lab has shown that CD28 is required for LLPC survival, and we know that CD28 back signaling to CD80/86 induces IDO production from dendritic cells (DCs) within the bone marrow microenvironment. IDO is classically known to contribute to an immunosuppressive environment (specifically with respect to T cell activity). However, upon investigation of IDO in the normal setting, we observed fewer plasma cells present in the bone marrow of IDO knockout mice in comparison to wild type mice, as well as fewer antigen-specific plasma cells in the bone marrow following vaccination. Additionally, knowing the role of DCs in IDO production in the bone marrow, we employed the use of CD11c-DTR mice to observe the effects of PC survival upon DC elimination. We found that depletion of DCs significantly decreases the amount of antigen-specific plasma cells in the bone marrow. This leads us to propose a model where CD28, through back signaling to CD80/86 induces IDO production in DCs, a mechanism to maintain the bone marrow niche for long lived plasma cell survival and sustained antibody production.

Reactive nitrogen species control apoptosis and autophagy in K562 cells: implication of TAp73α induction in controlling autophagy

The biological outcome of nitric oxide (NO) and reactive nitrogen species (RNS) in regulating pro survival and pro death autophagic pathways still demand further investigation. In the present study, we investigated the effect of nitrosative stress in K562 cells using NO donor compound DETA-NONOate, peroxynitrite, and SIN-1. Exposure to NO, peroxynitrite, and SIN-1 caused decrease in K562 cell survival. NO induced autophagy but not apoptosis or necrosis in K562 cells. In contrast, peroxynitrite and SIN-1 treatment induced apoptosis in K562 cells. Surprisingly, inhibition of autophagic response using 3-methyladenine led to the induction of apoptosis in K562 cells. Increase in 5’adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was only observed in the presence of NO donor indicated that AMPK was crucial to induce autophagy in K562 cells. We for the first time discovered a novel role of p73 in autophagy induction under nitrosative stress in K562 cells. TAp73α was only induced upon exposure to NO but not in the presence of peroxynitrite. Reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio remained unaltered upon NO exposure. Our data suggest a complex network of interaction and cross regulations between NO and p73. These data open a new path for therapies based on the abilities of RNS to induce autophagy-mediated cell death.

Interplay between P-Glycoprotein Expression and Resistance to Endoplasmic Reticulum Stressors.

Multidrug resistance (MDR) is a phenotype of cancer cells with reduced sensitivity to a wide range of unrelated drugs. P-glycoprotein (P-gp)—a drug efflux pump (ABCB1 member of the ABC transporter gene family)—is frequently observed to be a molecular cause of MDR. The drug-efflux activity of P-gp is considered as the underlying mechanism of drug resistance against P-gp substrates and results in failure of cancer chemotherapy. Several pathological impulses such as shortages of oxygen and glucose supply, alterations of calcium storage mechanisms and/or processes of protein N-glycosylation in the endoplasmic reticulum (ER) leads to ER stress (ERS), characterized by elevation of unfolded protein cell content and activation of the unfolded protein response (UPR). UPR is responsible for modification of protein folding pathways, removal of misfolded proteins by ER associated protein degradation (ERAD) and inhibition of proteosynthesis. However, sustained ERS may result in UPR-mediated cell death. Neoplastic cells could escape from the death pathway induced by ERS by switching UPR into pro survival mechanisms instead of apoptosis. Here, we aimed to present state of the art information about consequences of P-gp expression on mechanisms associated with ERS development and regulation of the ERAD system, particularly focused on advances in ERS-associated therapy of drug resistant malignancies.

Increased mitochondrial turnover in the skeletal muscle of fasted, castrated mice is related to the magnitude of autophagy activation and muscle atrophy.

Androgen-deficiency promotes muscle atrophy in part by increasing autophagy-mediated muscle protein breakdown during the fasted state, but factors contributing to this remain undefined. To identify novel factors, mice were subjected to sham or castration surgery. Seven-weeks post-surgery, mice were fasted overnight, refed for 30 min, and fasted another 4.5 h before sacrifice. BNIP3-mediated turnover of mitochondria was increased within the atrophied tibialis anterior (TA) of castrated mice and related to the magnitude of muscle atrophy and autophagy activation (i.e. decreased p62 protein content), thus linking turnover of potentially dysfunctional mitochondria with autophagy-mediated atrophy. Autophagy induction was likely facilitated by AMPK activation as a stress survival mechanism since phosphorylation of AMPK (Thr172), as well as the pro survival kinases Akt (Thr308) and (ERK1/2 Thr202/Tyr204), were increased by castration. Together, these data identify a novel relationship between mitochondrial turnover in the fasted state with autophagy activation and muscle atrophy following androgen depletion.

Anticarcinogenic activities of sulforaphane are influenced by Nerve Growth Factor in human melanoma A375 cells

Melanoma is a severe form of cancer, resistant to conventional therapies. According to in vitro studies, sulforaphane, a dietary component, has been considered a promising antineoplastic candidate.The present study analyzes the in vitro biological effects of sulforaphane in A375 melanoma cell line with or without the addition of Nerve Growth Factor.For the first time, our results show that a supplementation of Nerve Growth Factor partially reverses the sulforaphane-induced: i) inhibition of cell migration, ii) pro apoptotic changes in cell cycle and iii) modulation of active caspase-3. Furthermore, we report the sulforaphane-induced modulation in the expression of Nerve Growth Factor receptors TrKA and p75NTR, shifting their ratio from pro survival to pro apoptotic.In conclusion, the present study evidences that in vivo the antineoplastic effects of sulforaphane may be reduced by the contemporaneous presence of other biological elements such as Nerve Growth Factor and it contributes to a better definition of the real in vivo potentiality of sulforaphane as antineoplastic candidate.

EDHSS206, an Exquisitely Selective Inhibitor of TAK1, Targets Pro Survival TNFFFDependent Signaling, Inducing Apoptosis in Rheumatoid Arthritis and Breast Cancer Models

Tumor necrosis factor alpha (TNFα) administration has shown limited therapeutic utility in cancer and inflammatory disorders due to dose‐limiting side effects. We propose an alternative approach to targeting TNFα‐mediated diseases by selectively inducing the TNFα apoptotic response in TNFα‐rich microenvironments. TAK1 acts as a key mediator between survival and cell death in TNFα signaling. We report a novel potent and exquisitely selective TAK1 inhibitor (EDHS‐206, IC50 9.5nM) that induces apoptosis in a TNFα‐dependent manner in models of metastatic breast cancer and rheumatoid arthritis. Co‐crystallization studies demonstrated that EDHS‐206 inhibits the kinase in a DFG‐in conformation. Kinetic analysis of EDHS‐206‐TAK1 interactions revealed a substrate‐like intermolecular autophosphorylation mechanism for TAK1 activation, during which EDHS‐206 delays the proceedings of the rate‐limiting step. EDHS‐206 represents an attractive starting point for the development of a novel generation of inhibitors that greatly sensitize cells to TNFα‐induced cell death, potentially broadening the therapeutic efficacy of TNFα.

Serum from CCl4-induced acute rat injury model induces differentiation of ADSCs towards hepatic cells and reduces liver fibrosis

Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl4 injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl4 which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl4-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson’s trichome staining was observed.

LKB1 and its importance for survival in EpCAM negative circulating tumor cells (CTC) in metastatic breast cancer.

e23061 Background: To reach distant sites, CTC overcome metabolic stress and acquire a mesenchymal phenotype. So we assessed the pro survival function of liver kinase B1 (LKB1) a cellular metabolic stress controller, in combination with the EMT transcription factor Zinc finger E-box binding homeobox factor 1 (ZEB1) and stem cell markers e.g. CD44 in EpCAM negative CTC. Tumor spheroids and targeted si-RNA knockdown of LKB1 were analyzed in two breast cancer cell lines to support transcriptomic data. Methods: CTC were isolated from whole blood of 27 metastatic breast cancer patients. 17 age matched healthy donors were negative control. All patients were analyzed with CellSearch system to detect EpCAM positive CTC, which were eliminated by immunodepletion (AdnaTest BreastCancerSelect Kit, TATAA Biocenter) together with CD45 positive white blood cells (Miltenyi Biotec). RNA transcripts were quantified by qPCR (TaqMan Gene Expression Assay, ThermoFischer Scientific) and the relative expression was calculated by the 2-DCtmethod. EpCAM negative CTC and spheroids were analyzed by immunofluorescence. Data were analyzed by non-parametric statistical tests. Results: According to the CellSearch system, most patients (74%, n = 20) were CTC negative. 20% of this sub-group showed enhanced levels of ZEB1, 15% of LKB1, and 40% of stem cell markers. ZEB1 and LKB1 were never co-expressed, while there was an almost significant association between enhanced ZEB1 and stem cell marker expression (p = 0.053). Immunofluorescence analysis on single CTC confirmed the transcriptomic result. MDA-MB 231 cell lines, normally negative for LKB1 and unable to grow in suspension, showed the ability to generate spheroids only if LKB1 expression was activated. Targeted knockdown of LKB1 confirmed these results. Conclusions: CellSearch system negative patients present a heterogeneous CTC population with enhanced levels of ZEB1, CD44, or LKB1. While ZEB1 and CD44 might be highly expressed in cells that already have entered EMT, LKB1 overexpression could represent the response to metabolic stress induced in the early phase of intravasation Hence, LKB1 might represent a novel therapeutic target to chase CTC and prevent metastasis.

The role of Indoleamine 2,3-dioxygenase (IDO) in the survival of bone marrow resident long lived Plasma cells

Abstract Long lived plasma cells (LLPCs) are essential for sustained antibody responses and protective humoral immunity. How these cells maintain longevity and a durable antibody response is largely dependent on the complex nature of the bone marrow microenvironment in which these cells reside, and the pro survival factors produced in this niche. Work in our lab has shown that CD28 is required for LLPC survival, and that Indoleamine 2,3-dioxygenase (IDO), an enzyme that catabolizes tryptophan, is produced as a result of back signaling from CD28:CD80/86 interactions between plasma cells and bone marrow myeloid derived dendritic cells (DC). IDO is classically known to contribute to an immunosuppressive environment through its depletion of tryptophan (specifically with respect to T cells). However, to our great surprise we found that there are fewer LLPCs present in the bone marrow of IDO knockout mice in comparison to wild type mice. Additionally, live cell numbers of purified plasma cells in vitro in tryptophan free media and IDO conditioned media are greater than in media alone. These conditions also caused autophagy to occur in these plasma cells, a process shown to be required for LLPC survival. This leads us to propose a model where CD28, through back signaling to CD80/86, induces IDO production which leads to depletion of tryptophan and causes induction of autophagy, a mechanism LLPCs use to compete and survive within the bone marrow microenvironment.