Chronic Low-dose Treatment Research Articles

Oral low-dose rapamycin treatment prevents aortic aneurysms in marfan mice

Abstract Background/Introduction Marfan syndrome (MFS) is a hereditary connective tissue disorder caused by fibrillin-1 (FBN1) gene mutations. Life-threatening complications are cardiovascular, e.g. thoracic aortic aneurysms and dissections. The pathophysiological cause is the increased release of transforming growth factor β-1 (TGF-β1) from the FBN1-deficient extracellular matrix (ECM) and structural disintegrity. Overactivation of the mechanistic target of rapamycin (mTOR) pathway has been demonstrated in the murine mgR/mgR model of MFS. Short-term administration of rapamycin significantly reduced aortic aneurysm formation and increased the life span in these mice. Purpose To assess the effect of an early continuous oral low-dose rapamycin treatment on the aortic aneurysm formation in the FBN1 hypomorphic mgR/mgR mouse model. Methods Male and female 3-4 week-old mgR/mgR mice were orally administered vehicle or rapamycin through diet (8mg/kg/KG) and sacrificed after 11 weeks. Rapamycin concentration was measured by liquid chromatography-mass spectrometry in whole blood. Aortic diameter was studied using echocardiography. Histopathological and immunofluorescence analyses were performed using aortic tissue sections and techniques. Results Blood rapamycin concentration following oral administration remained below detection level. Rapamycin normalized the aortic wall thickness and the diameter in female mgR/mgR mice to levels of the littermate control mice but not in male mgR/mgR mice. The circumferentially distributed elastin fibres remained significantly intact in sections of the ascending aorta of female mice. Echocardiography revealed that rapamycin-treated female mice stayed below a cut-off value for aortic aneurysms of 2 mm inner aortic diameter eight weeks after starting therapy. In contrast, vehicle-treated female animals already exceeded this value after five weeks. In female but not in male mice, the abundance of the mTOR downstream target phosphorylated ribosomal protein S6 was effectively suppressed by low-dose rapamycin up to levels detected in control animals. At the same time, extracellular matrix proteins like the proteoglycan aggrecan (ACAN) and the related chondroitin sulfate were significantly decreased in female mice. The abundance of ADAMTS5 transglutaminase-2 and xylosyltransferase-1, key enzymes involved in the turnover of proteoglycans and biosynthesis of glycosaminoglycan chains, was improved by the rapamycin treatment in female mice. No decrease in mTOR activity could be detected in male mgR/mgR animals. Here, the ACAN and chondroitin sulphate levels also remained at the level of the vehicle-treated animals. Conclusions Chronic low-dose rapamycin treatment reduced aneurysm formation only in female mgR/mgR mice, since the dose was too low for male mgR/mgR mice, by affecting the composition and organization of key ECM components essential for maintaining aortic homeostasis and mechanical properties.

Effect of chronic low-dose treatment with chitooligosaccharides on microbial dysbiosis and inflammation associated chronic ulcerative colitis in Balb/c mice

The study aimed to investigate the potential of low dose chitooligosaccharide (COS) in ameliorating dextran sodium sulfate (DSS) induced chronic colitis by regulating microbial dysbiosis and pro-inflammatory responses. Chronic colitis was induced in BALB/c mice by DSS (4% w/v, 3 cycles of 5 days) administration. The mice were divided into four groups: vehicle, DSS, DSS + mesalamine and DSS+COS. COS and mesalamine were administered orally, daily once, from day 1 to day 30 at a dose of 20 mg/kg and 50 mg/kg respectively. The disease activity index (DAI), colon length, histopathological score, microbial composition, and pro-inflammatory cytokine expression were evaluated. COS (20 mg/kg, COSLow) administration reduced the disease activity index, and colon shortening, caused by DSS significantly. Furthermore, COSLow restored the altered microbiome in the gut and inhibited the elevated pro-inflammatory cytokines (IL-1 and IL-6) in the colon against DSS-induced chronic colitis in mice. Moreover, COSLow treatment improved the probiotic microflora thereby restoring the gut homeostasis. In conclusion, this is the first study where microbial dysbiosis and pro-inflammatory responses were modulated by chronic COSLow treatment against DSS-induced chronic colitis in Balb/c mice. Therefore, COS supplementation at a relatively low dose could be efficacious for chronic inflammatory bowel disease.

Kinetics of Circulating Extracellular Vesicles Over the 24-Hour Dosing Interval After Low-Dose Aspirin Administration in Patients atCardiovascular Risk.

Extracellular vesicles (EVs) are small vesicles deriving from all cell types during cell activation, involved in transcellular communication, and regarded as predictors of vascular damage and of cardiovascular events. We tested the hypothesis that, in patients on chronic low-dose aspirin treatment for cardiovascular prevention, aspirin may affect the release of EVs within the 24-hour interval. We enrolled 84 patients, mostly at high or very high cardiovascular risk, on chronic low-dose aspirin treatment. The numbers of circulating EVs (cEVs) and annexinV+ cEVs (total, platelet-derived, endothelial-derived, and leucocyte-derived) were assessed immediately before, and after 10 and 24 hours of a witnessed aspirin administration. Platelet cyclooxygenase 1 (COX-1) recovery was characterized by measuring serum thromboxane B2 (sTXB2 ) at the same timepoints. Nine healthy participants were also enrolled. In patients, daily aspirin administration acutely inhibited after 10 hours following aspirin administrations the release of cEVs (total and leukocyte-derived) and annexinV+ cEVs (total, platelet-derived, endothelial-derived, and leukocyte-derived), with a rapid recovery at 24 hours. The inhibition after 10 hours suggests a COX-1-dependent mechanism. Interestingly, the slope of platelet-derived and of annexinV+ platelet-derived cEVs were both directly related to sTXB2 slope and COX-1 messenger RNA, raising the hypothesis that vice versa, cEVs may affect the rate of COX-1 recovery and the subsequent duration of aspirin effect. In healthy participants, no circadian difference was observed, except for leukocyte-derived cEVs. Our findings suggest a previously unappreciated effect of aspirin on the kinetics of a subset of cEVs possibly contributing to the cardioprotective effects of this drug.

NMDA receptor antagonists reduce amyloid-β deposition by modulating calpain-1 signaling and autophagy, rescuing cognitive impairment in 5XFAD mice

Overstimulation of N-methyl-d-aspartate receptors (NMDARs) is the leading cause of brain excitotoxicity and often contributes to neurodegenerative diseases such as Alzheimer’s Disease (AD), the most common form of dementia. This study aimed to evaluate a new NMDA receptor antagonist (UB-ALT-EV) and memantine in 6-month-old female 5XFAD mice that were exposed orally to a chronic low-dose treatment. Behavioral and cognitive tests confirmed better cognitive performance in both treated groups. Calcium-dependent protein calpain-1 reduction was found after UB-ALT-EV treatment but not after memantine. Changes in spectrin breakdown products (SBDP) and the p25/p35 ratio confirmed diminished calpain-1 activity. Amyloid β (Aβ) production and deposition was evaluated in 5XFAD mice and demonstrated a robust effect of NMDAR antagonists on reducing Aβ deposition and the number and size of Thioflavin-S positive plaques. Furthermore, glycogen synthase kinase 3β (GSK3β) active form and phosphorylated tau (AT8) levels were diminished after UB-ALT-EV treatment, revealing tau pathology improvement. Because calpain-1 is involved in autophagy activation, autophagic proteins were studied. Strikingly, results showed changes in the protein levels of unc-51-like kinase (ULK-1), beclin-1, microtubule-associated protein 1A/1B-light chain 3(LC3B-II)/LC3B-I ratio, and lysosomal-associated membrane protein 1 (LAMP-1) after NMDAR antagonist treatments, suggesting an accumulation of autophagolysosomes in 5XFAD mice, reversed by UB-ALT-EV. Likewise, treatment with UB-ALT-EV recovered a WT mice profile in apoptosis markers Bcl-2, Bax, and caspase-3. In conclusion, our results revealed the potential neuroprotective effect of UB-ALT-EV by attenuating NMDA-mediated apoptosis and reducing Aβ deposition and deposition jointly with the autophagy rescue to finally reduce cognitive alterations in a mice model of familial AD.

Keratinocytes Counteract UVB-Induced Immunosuppression in Mice through HIF-1a Signaling

The transcription factor HIF-1a regulates cellular metabolism under hypoxia but also immune responses and UVB-induced skin reactions. In keratinocytes (KCs), HIF-1a is an environmental sensor orchestrating the adaptation to environmental changes. In this study, we investigated the role of HIF-1a in KCs for skin reactions to acute and chronic UVB exposure in mice. The function of HIF-1a in KCs under UVB exposure was analyzed in KC-specific HIF-1a conditional knockout (cKO) mice. cKO mice were hypersensitive to acute high-dose UVB irradiation compared with wild-type mice, displaying increased cell death and delayed barrier repair. After chronic low-dose UVB treatment, cKO mice also had stronger epidermal damage but reduced infiltration of dermal macrophages and T helper cells compared with wild-type mice. Irradiated cKO mice revealed accumulation of regulatory lymphocytes in dorsal skin-draining lymph nodes compared with wild-type and unirradiated mice. This was reflected by an augmented IL-10 release of lymph node cells and a weaker contact hypersensitivity reaction to DNFB in UVB-exposed cKO mice than in wild-type and unirradiated controls. In summary, we found that KC-specific HIF-1a expression is crucial for adaptation to UVB exposure and inhibits the development of UVB-induced immunosuppression in mice. Therefore, HIF-1a signaling in KCs could ameliorate photoaging-related skin disorders.

Chronic low-dose intravenous immunoglobulins as steroid-sparing therapy in myasthenia gravis

Intravenous immunoglobulin (IVIg) has been proven beneficial in myasthenic crisis, but their role as maintenance therapy is unclear. The aim of this study was to determine if maintenance therapy with low-dose IVIg improves clinical outcome and may be used as a steroid-sparing agent in myasthenia gravis (MG). We retrospectively reviewed charts of all MG patients treated with IVIg from January 2006 to December 2019. Long-term treatment response to IVIg was assessed by improvement in the Myasthenia Gravis Foundation of America (MGFA) clinical classification scale as primary end point, as well as the ability to reduce the time-weighted average required dose of prednisone as secondary end-point, in a follow-up period of 36months. 109 patients were treated with IVIg. The mean follow-up time was 34.03 ± 5.5months. Sixty-seven patients (61.4%) responded to therapy with at least one-point improvement of the MGFA scale. There was no statistical difference in demographic and clinical characteristics between IVIg responders and non-responders. The mean prednisone dose decreased significantly from 33.1 ± 14.5mg at baseline to 7.2 ± 7.8mg after 36months of IVIg treatment (P < 0.0001), with the greatest effect after 6months (33.1 ± 14.5mg Vs. 17.9 ± 11.7mg; P < 0.0001). In the follow-up period of 36months, most patients (92.5%) remained clinically and pharmacologically stable under chronic IVIg treatment. This retrospective study demonstrates that chronic low-dose IVIg treatment in patients with MG improves clinical outcomes and has a prolonged and significant steroid-sparing effect over a period of 3years.

Effects of chronic low-dose aspirin treatment on tumor prevention in three mouse models of intestinal tumorigenesis.

Although early detection and treatment of colorectal cancer (CRC) have improved, it remains a significant health‐care problem with high morbidity and mortality. Data indicate that long‐term intake of low‐dose aspirin reduces the risk of CRC; however, the mechanisms underlying this chemopreventive effect are still unclear. Different mouse models for inflammation‐associated, sporadic, and hereditary CRC were applied to assess the efficacy and mechanism of low‐dose aspirin on tumor prevention. An initial dosing study performed in healthy mice indicates that aspirin at a dose of 25 mg/kg/d has a similar pharmacodynamic effect as low‐dose aspirin treatment in human subjects (100 mg/d). Chronic low‐dose aspirin treatment suppresses colitis‐associated and to a lesser extent spontaneous tumorigenesis in mice. Aspirin's antitumor effect is most pronounced in a preventive approach when aspirin administration starts before the tumor‐initiating genotoxic event and continues for the duration of the experiment. These effects are not associated with alterations in cell proliferation, apoptosis, or activation of signaling pathways involved in CRC. Aspirin‐induced reduction in tumor burden is accompanied by inhibition of thromboxane B2 formation, indicating reduced platelet activation. Aspirin treatment also results in decreased colonic prostaglandin E2 formation and tumor angiogenesis. With respect to colitis‐triggered tumorigenesis, aspirin administration is associated with a reduction in inflammatory activity in the colon, as indicated by decreased levels of pro‐inflammatory mediators, and tumor‐associated iNOS‐positive macrophages. Our results suggest that low‐dose aspirin represents an effective antitumor agent in the context of colon tumorigenesis primarily due to its well‐established cyclooxygenase inhibition effects.

An integrative approach to cisplatin chronic toxicities in mice reveals importance of organic cation-transporter-dependent protein networks for renoprotection.

Cisplatin (CDDP) is one of the most important chemotherapeutic drugs in modern oncology. However, its use is limited by severe toxicities, which impair life quality after cancer. Here, we investigated the role of organic cation transporters (OCT) in mediating toxicities associated with chronic (twice the week for 4weeks) low-dose (4mg/kg body weight) CDDP treatment (resembling therapeutic protocols in patients) of wild-type (WT) mice and mice with OCT genetic deletion (OCT1/2-/-). Functional and molecular analysis showed that OCT1/2-/- mice are partially protected from CDDP-induced nephrotoxicity and peripheral neurotoxicity, whereas ototoxicity was not detectable. Surprisingly, proteomic analysis of the kidneys demonstrated that genetic deletion of OCT1/2 itself was associated with significant changes in expression of proinflammatory and profibrotic proteins which are part of an OCT-associated protein network. This signature directly regulated by OCT consisted of three classes of proteins, viz., profibrotic proteins, proinflammatory proteins, and nutrient sensing molecules. Consistent with functional protection, CDDP-induced proteome changes were more severe in WT mice than in OCT1/2-/- mice. Laser ablation-inductively coupled plasma-mass spectrometry analysis demonstrated that the presence of OCT was not associated with higher renal platinum concentrations. Taken together, these results redefine the role of OCT from passive membrane transporters to active modulators of cell signaling in the kidney.

Low-Dose Rapamycin Facilitates Recovery from Exercise-Induced Muscle Injury

Rapamycin has been shown to have a dose-dependent effect on multiple signaling proteins in skeletal muscle cells that influence protein synthesis and calcium handling. However, it has yet to be determined if a low-dose of rapamycin impacts skeletal muscle during recovery from an exercise-induced injury. PURPOSE: To determine if low-dose rapamycin affects the rate of isometric strength recovery, muscle ubiquitination levels and markers of autophagy compared to saline control 14 days after exercise induced injury. METHODS: Mice were injected with either saline (SAL; 0.9%) or low-dose rapamycin (RAP; 10 μg/kg body weight) every other day for 2 weeks before and after a single bout of 150 eccentric contractions of the left anterior crural muscles. The recovery of strength of the anterior crural muscles was measured in vivo immediately, 7 days, and 14 days after injury induction. The magnitude of expression of beclin-1, ubiquitin, and ubiquitinated protein in injured and contralateral control TA muscles (i.e., primary anterior crural muscle) were analyzed via Western blot at 14 days after injury. RESULTS: Isometric twitch torque values did not differ between groups at any time point. No group differences in peak isometric tetanic torque were observed pre-injury, post-injury or 7 days following injury. However at 14 days, RAP mice recovered to pre-injury peak isometric torque values (Pre= 2.30 ± .07 Nmm; 14d= 2.25 ± 0.08 Nmm) while SAL group was significantly lower than pre-injury. At 14 days, RAP mice generated 15.4% higher maximal torque than SAL group (p = 0.04). Beclin-1 and free ubiquitin expression in TA muscles were significantly increased in both SAL (1.4-fold and 2.3-fold, respectively) and RAP (2.2-fold and 8.0-fold, respectively) mice at 14 days after injury compared to the uninjured muscle. The increase in the free ubiquitin in the injured muscle was 3.3-fold greater in the RAP treatment compared to SAL (p = 0.001). There were no significant changes in the ubiquitination of proteins among the groups at 14 days post-injury. CONCLUSION: Chronic low-dose rapamycin treatment in mice enhances recovery of skeletal muscle from eccentric contraction-induced injury at the 14th day and accentuates the upregulation of free ubiquitin.

Dexamethasone Conjugation to Biodegradable Avidin-Nucleic-Acid-Nano-Assemblies Promotes Selective Liver Targeting and Improves Therapeutic Efficacy in an Autoimmune Hepatitis Murine Model.

Steroids are the standard therapy for autoimmune hepatitis (AIH) but the long-lasting administration is hampered by severe side effects. Methods to improve the tropism of the drug toward the liver are therefore required. Among them, conjugation to nanoparticles represents one possible strategy. In this study, we exploited the natural liver tropism of Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) to carry dexamethasone selectively to the liver in an AIH animal model. An acid-labile biotin-hydrazone linker was developed for reversible dexamethasone loading onto ANANAS. The biodistribution, pharmacokinetics and efficacy of free and ANANAS-linked dexamethasone (ANANAS-Hz-Dex) in healthy and AIH mice were investigated upon intraperitoneal administration. In ANANAS-treated animals, the free drug was detected only in the liver. Super-resolution microscopy showed that nanoparticles segregate inside lysosomes of liver immunocompetent cells, mainly involved in AIH progression. In agreement with these observational results, chronic low-dose treatment with ANANAS-Hz-Dex reduced the expression of liver inflammation markers and, in contrast to the free drug, also the levels of circulating AIH-specific autoantibodies. These data suggest that the ANANAS carrier attenuates AIH-related liver damage without drug accumulation in off-site tissues. The safety and biodegradability of the ANANAS carrier make this formulation a promising tool for the treatment of autoimmune liver disorders.

5-Azacytidine Induces NOXA and PUMA Expression to Prime AML Cells for Venetoclax-Mediated Apoptosis

Acute myeloid leukemia (AML) is a clonal hematologic malignancy characterized by genomic heterogeneity and epigenetic changes, including aberrant DNA hypermethylation. Phase-Ib clinical data in relapsed/refractory AML patients indicate that combining venetoclax with the hypomethylating agents (HMAs) 5-azacitidine (5-Aza) or decitabine results in an overall response (OR) of 62% (DiNardo et al. 2018) compared to the historical OR of 28-29% with HMAs treatment alone (Kantarjian et al. 2013; Dombret et al. 2015). Subsequently, a randomized phase-III clinical trial was initiated to evaluate venetoclax activity in combination with 5-Aza in treatment-naïve AML patients ineligible for standard induction therapy (M15-656, NCT02993523). However, the underlying mechanism for the combinational activity observed between venetoclax and 5-Aza is unknown.In this study, we demonstrate that both chronic low-dose 5-Aza treatment, which induced global DNA demethylation, and acute treatment (24 hours, non-epigenetic effects) can drive combinational activity with venetoclax in AML through distinct mechanisms. Chronic culture with a low-dose 5-Aza for one week sensitized AML cell lines to venetoclax in vitro. In contrast, acute treatment with 5-Aza, activated the integrated stress response (ISR) pathway to induce expression of the BH3-only proteins NOXA (PMAIP1) and PUMA (BBC3) in human AML cell lines, independent of DNA methylation. This resulted in an increase in the amount of NOXA and/or PUMA in complex with anti-apoptotic proteins like BCL-2, BCL-XL and MCL-1, thereby “priming” AML cells for induction of apoptosis by venetoclax treatment. Priming for apoptosis resulted in significant synergistic cell death in a panel of AML cell lines treated with venetoclax and 5-Aza in vitro. In this panel of cell lines, the level of the PMAIP1, BBC3, and DDIT3 gene induction correlated with the synergy observed between venetoclax and 5-Aza. Importantly, subsequent PMAIP1 deletion significantly impacted the kinetics and depth of apoptosis induced by 5-Aza or venetoclax alone or in combination. In accordance with the in vitro combinational activity, the venetoclax/5-Aza combination provided added benefit over either agent alone in two xenograft models of AML. Together, these data provide a rationale for an ongoing randomized phase-III clinical trial evaluating venetoclax activity in combination with 5-Aza (M15-656, NCT02993523).Disclosures:DC, SJ, JP, RP, NT, YX, EB, JL, and DP are employees of AbbVie. LS is a former employee of AbbVie and was employed during the duration of this study. The design, study conduct, and financial support for this research were provided by AbbVie and Genentech. AbbVie participated in the interpretation of data, review, and approval of the publication. DisclosuresCojocari:AbbVie Inc: Employment. Jin:AbbVie Inc: Employment, Equity Ownership. Purkal:AbbVie Inc: Employment, Equity Ownership. Popovic:AbbVie Inc: Employment, Equity Ownership. Talaty:AbbVie Inc: Employment, Equity Ownership. Xiao:AbbVie Inc: Employment, Equity Ownership. Solomon:AbbVie Inc: Equity Ownership. Boghaert:AbbVie Inc: Employment, Equity Ownership. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties.

PO-397 Role of IFNγ in adaptation and immunotherapy resistance in melanoma

IntroductionIFNγ is a key component of our immune system, regulating a plethora of biological processes, including antitumor immunity. Its role in tumour immunology is however contradictory, exerting anti-tumour effects early in tumour development but promoting tumour immune evasion and tumour development at later stages. Its involvement in immunotherapy resistance is also extremely complex and is potentially mediated through multiple cellular processes. In this study, we aimed to generate further insights into the role of sustained IFNγ signalling in acquired resistance to immune selection and immunotherapy.Material and methodsA panel of established human and mouse melanoma cell lines were treated with increasing doses of IFNγ for 24 hour and the levels of IFNγ pathway activation were assessed by phospho-STAT1 immunoblotting. To assess the effects of chronic low-dose IFNγ treatment in gene expression of melanoma cell lines, RNA sequencing was performed in melanoma cell lines treated with INFγ for 2 weeks, followed by pathway analysis and gene set enrichment analysis of the differentially expressed genes. Finally, we assessed the DNA damage effects of chronic low-dose IFNγ treatment in melanoma cell lines by γH2AX immunofluorescent staining.Results and discussionsWe showed that established melanoma cell lines retain responsiveness to IFNγ, as reflected by the increased levels of phospho-STAT1 after treatment with increasing doses of IFNγ. Interestingly, RNAseq in melanoma cell lines chronically treated with low-dose IFNγ revealed oxidative stress response as well as DNA damage and repair pathways among the top upregulated pathways in IFNγ treated cells. In line with this, gene set enrichment analysis showed enrichment of DNA damage response and repair related signatures in genes upregulated by IFNγ treatment. Finally, we showed that chronic low-dose IFNγ treatment of melanoma cell lines induces DNA damage, as revealed by increased levels of γH2AX foci per cell and percentage of γH2AX positive cells.ConclusionIn vitro chronic treatment of melanoma cells with low-dose IFNγ has been previously shown to make cells acquire an immunotherapy resistance phenotype. In this study we showed that chronic low-dose treatment of human and mouse melanoma cell lines induces DNA damage, as revealed by γH2AX immunofluorescent staining and RNAseq followed by pathway analysis. The role of IFNγ-driven constitutive DNA damage response in immune evasion and immunotherapy resistance merits further evaluation as an actionable therapeutic target.

N-methyl-N-nitro-N-nitrosoguanidine-mediated ING4 downregulation contributed to the angiogenesis of transformed human gastric epithelial cells

AimsAngiogenesis is associated with the progression and mortality of gastric cancer. Epidemiological evidences indicate that long-term N-nitroso compounds (NOCs) exposure predominantly contributes to the mortality of gastric cancer. Therefore, further reduced mortality of gastric cancer demands to explore the exact mechanisms of NOCs induced angiogenesis. As a tumor suppressor gene, inhibitor of growth protein 4 (ING4) plays an important role in pathological angiogenesis. In this study, we will investigate ING4 expression level in human gastric epithelial cells after the long-term low dose exposure of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and the pathological impact of MNNG-reduced ING4 on angiogenesis of transformed cells. Main methodsThe soft agar colony formation assay, Western blotting, immunofluorescence and wound healing assay were used to evaluate the characteristics of transformed cells. HUVEC growth and tube formation assays were performed to test the angiogenic abilities. EMSA, luciferase reporter gene assay, real-time PCR and Western blotting were used to explore the exact mechanism. Key findingsBy establishing transformed human gastric epithelial cells via chronic low dose treatment, a gradually ING4 downregulation was observed in the later-stage of MNNG-induced cell transformation. Moreover, we demonstrated that MNNG exposure-reduced ING4 expression significantly resulted into aggravating angiogenesis through increasing the phosphorylation level of NF-κB p65 and subsequently DAN binding activity and regulating the expressions of NF-κB p65 downstream pro-angiogenic genes, MMP-2 and MMP-9. SignificanceOur findings provided a significant mechanistic insight into angiogenesis of MNNG-transformed human gastric epithelial cell and supported the concept that ING4 may be a relevant therapeutic target for gastric cancer.

Phosphoinositide 3-Kinase Gamma Inhibition Protects From Anthracycline Cardiotoxicity and Reduces Tumor Growth.

Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition. Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models. PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Chronic low-dose pro-oxidant treatment stimulates transcriptional activity of telomeric retroelements and increases telomere length in Drosophila

It has been proposed that oxidative stress, elicited by high levels of reactive oxygen species, accelerates telomere shortening by erosion of telomeric DNA repeats. While most eukaryotes counteract telomere shortening by telomerase-driven addition of these repeats, telomeric loss in Drosophila is compensated by retrotransposition of the telomeric retroelements HeT-A, TART and TAHRE to chromosome ends. In this study we tested the effect of chronic exposure of flies to non-/sub-lethal doses of paraquat, which is a redox cycling compound widely used to induce oxidative stress in various experimental paradigms including telomere length analyses. Indeed, chronic paraquat exposure for five generations resulted in elevated transcriptional activity of both telomeric and non-telomeric transposable elements, and extended telomeric length in the tested fly lines. We propose that low oxidative stress leads to increased telomere length within Drosophila populations. For a mechanistic understanding of the observed phenomenon we discuss two scenarios: adaption, acting through a direct stimulation of telomere extension, or positive selection favoring individuals with longer telomeres within the population.

Chronic low-dose glucocorticoid treatment increases subcutaneous abdominal fat, but not visceral fat, of male Wistar rats

AimMost studies developed to investigate the effects of glucocorticoids chronic treatment on white adipose tissue uses high doses of these hormones. This study analyzes some effects of a chronic, continuous and steady infusion of low-dose hydrocortisone and the relationship with lipid accumulation in white adipose depots in rats. Main methodsNineteen male Wistar rats were divided into control (CON) and cortisol (CORT) groups. Along six weeks CORT group received continuous infusion of 0.6mg/kg/day of hydrocortisone, while CON group received saline. After euthanasia, subcutaneous and visceral (retroperitoneal and mesenteric) fat pads were excised, weighted and analyzed for: lipogenic enzymes activity; molecular changes of 11-hydroxysteroid dehydrogenase type 1 (11βHSD1) enzyme; enzymes involved in lipid uptake, incorporation, and metabolism and in fatty acids esterification. Besides, morphometric cell analysis was performed. Key findingsCORT group showed increased triglycerides, changes in lipoprotein profile and 26,8% increment in central subcutaneous (SC) mass, while visceral fat pads masses remained unchanged. Adipocytes from SC, only, presented increased fatty acid synthase, ATP-citrate lyase and glucose-6-phosphate dehydrogenase activity, in addition to reduced AMP-activated protein kinase and 11βHSD1 enzymes content. SignificanceChronic low-dose hydrocortisone treatment consequences seem to be different from those commonly seen in long term hypercortisolism. While high doses promote lipid accumulation in visceral depots, a low dose showed an increase in central SC depot only. This appears to involve an increment in lipid storage and in de novo lipogenesis enzymes activity.

Effect of MPTP on mRNA expression of PGC-1α in mouse brain

The peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) is a key regulator of mitochondrial biogenesis, respiration and adaptive thermogenesis. Besides the full-length protein (FL-PGC-1α), several other functionally active PGC-1α isoforms were identified as a result of alternative splicing (e.g., N-truncated PGC-1α; NT-PGC-1α) or alternative promoter usage (e.g., central nervous system-specific PGC-1α isoforms; CNS-PGC-1α). Achieving neuroprotection via CNS-targeted pharmacological stimulation is limited due to poor penetration of the blood brain barrier (BBB) by the proposed pharmaceutical agents, so preconditioning emerged as another option. The current study aimed to examine of how the expression levels of FL-, NT-, CNS- and reference PGC-1α isoforms change in different brain regions following various 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment regimens, including chronic low-dose treatment for preconditioning. Ninety minutes following the acute treatment regimen, the expression levels of FL-, NT- and CNS-PGC-1α isoforms increased significantly in the striatum, cortex and cerebellum. However, this elevation diminished 7days following the last MPTP injection in the acute treatment regimen. The chronic low-dose administration of MPTP, which did not cause significant toxic effects in light of the relatively unaltered dopamine levels, did not result in any significant change of PGC-1α expression. The elevation of PGC-1α levels following acute treatment may demonstrate a short-term compensatory mechanism against mitochondrial damage induced by the complex I inhibitor MPTP. However, drug-induced preconditioning by chronic low-dose MPTP seems not to induce protective responses via the PGC-1α system.

Chronic low-dose melatonin treatment maintains nigrostriatal integrity in an intrastriatal rotenone model of Parkinson’s disease

Parkinson׳s disease is a major neurodegenerative disorder which primarily involves the loss of dopaminergic neurons in the substantia nigra and related projections in the striatum. The pesticide/neurotoxin, rotenone, has been shown to cause systemic inhibition of mitochondrial complex I activity in nigral dopaminergic neurons, with consequent degeneration of the nigrostriatal pathway, as observed in Parkinson׳s disease. A novel intrastriatal rotenone model of Parkinson׳s disease was used to examine the neuroprotective effects of chronic low-dose treatment with the antioxidant indoleamine, melatonin, which can upregulate neurotrophic factors and other protective proteins in the brain. Sham or lesioned rats were treated with either vehicle (0.04% ethanol in drinking water) or melatonin at a dose of 4µg/mL in drinking water. The right striatum was lesioned by stereotactic injection of rotenone at three sites (4μg/site) along its rostrocaudal axis. Apomorphine administration to lesioned animals resulted in a significant (p<0.001) increase in ipsilateral rotations, which was suppressed by melatonin. Nine weeks post-surgery, animals were sacrificed by transcardial perfusion. Subsequent immunohistochemical examination revealed a decrease in tyrosine hydroxylase immunoreactivity within the striatum and substantia nigra of rotenone-lesioned animals. Melatonin treatment attenuated the decrease in tyrosine hydroxylase in the striatum and abolished it in the substantia nigra. Stereological cell counts indicated a significant (p<0.05) decrease in dopamine neurons in the substantia nigra of rotenone-lesioned animals, which was confirmed by Nissl staining. Importantly, chronic melatonin treatment blocked the loss of dopamine neurons in rotenone-lesioned animals. These findings strongly support the therapeutic potential of long-term and low-dose melatonin treatment in Parkinson׳s disease.

Effects of chronic cerebral hypoperfusion and low-dose progesterone treatment on apoptotic processes, expression and subcellular localization of key elements within Akt and Erk signaling pathways in rat hippocampus

The present study attempted to investigate how chronic cerebral hypoperfusion (CCH) and repeated low-dose progesterone (P) treatment affect gene and protein expression, subcellular distribution of key apoptotic elements within protein kinase B (Akt) and extracellular signal-regulated kinases (Erk) signal transduction pathways, as well as neurodegenerative processes and behavior. The results revealed the absence of Erk activation in CCH in cytosolic and synaptosomal fractions, indicating a lower threshold of Akt activation in brain ischemia, while P increased their levels above control values. CCH induced an increase in caspase 3 (Casp 3) and poly (ADP-ribose) polymerase (PARP) gene and protein expression. However, P restored expression of examined molecules in all observed fractions, except for the levels of Casp 3 in synapses which highlighted its possible non-apoptotic or even protective function. Our study showed the absence of nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) response to this type of ischemic condition and its strong activation under the influence of P. Further, the initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by CCH was significantly reduced by P. Finally, P reversed the CCH-induced reduction in locomotor activity, while promoting a substantial decrease in anxiety-related behavior. Our findings support the concept that repeated low-dose post-ischemic P treatment reduces CCH-induced neurodegeneration in the hippocampus. Neuroprotection is initiated through the activation of investigated kinases and regulation of their downstream molecules in subcellular specific manner, indicating that this treatment may be a promising therapy for alleviation of CCH-induced pathologies.

Continuous activation of Nrf2 and its target antioxidant enzymes leads to arsenite-induced malignant transformation of human bronchial epithelial cells

Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuous low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation.