Higher Levels Of Apoptosis Research Articles

Cistanoside A promotes osteogenesis of primary osteoblasts by alleviating apoptosis and activating autophagy through involvement of the Wnt/β-catenin signal pathway.

BackgroundAs a phenylethanoid glycoside extracted from Cistanche deserticola, cistanoside A has been shown to have antioxidative effects. In recent years, it has been found to play an important role in osteoporosis.MethodsPrimary osteoblasts were randomly divided into a cistanoside A (Cis A)-1 group (5 µM), a Cis A-2 group (10 µM), and a Cis A-3 group (20 µM) to screen the optimal dose. Then, cells were treated with Rapamycin (Rapa), 3-MA, Dickkopf-1 (DKK-1), 3MA + Cis A (10 µM), and DKK-1 + Cis A (10 µM). After a certain period of routine culture, Alkaline Phosphatase (ALP) and Alizarin Red S Staining were performed again and the cells were collected for subsequent experiments including immunofluorescence staining, western blotting, transmission electron microscopy, mitochondrial membrane measurement, and Annexin-V-Fluorescein isothiocyanate (Annexin-V-FITC).ResultsThe optimal Cis A dose that preserved osteoblast viability and activated osteogenesis was 10 µM. It appeared that Cis A (10 µM) decreased apoptosis and augmented autophagy via increasing microtubule-associated protein light chain 3 (LC3)-I/II expressions as well as raising Wnt/β-catenin signal pathway activity. The addition of 3-MA further inhibited osteogenic differentiation and suppressed Wnt/β-catenin signal pathway activity to increase apoptosis while reducing autophagy levels. A combination of Cis A and DKK-1 resulted in higher levels of apoptosis but lower levels of autophagy.ConclusionsCis A appears to be a potent inducer of autophagy and inhibitor of apoptosis in primary osteoblasts by working through the Wnt/β-catenin signal pathway, thereby resulting in enhanced osteogenic differentiation.

Cirrhosis-Based Acute-on-Chronic Liver Failure Is Marked by Inflammation and Impaired Liver Regeneration Despite Stat3 Activation.

Acute-on-chronic liver failure (ACLF) is associated with excessive systemic inflammation, cell death, and organ failures. Yet, little is known about the hepatic histopathology of ACLF. Here, we assessed the histopathology and regenerative capacity of the liver in ACLF with or without cirrhosis. Liver specimens of patients with compensated cirrhosis (N= 37), acute decompensation (N= 40), and ACLF with (N= 18) or without (N= 10) cirrhosis were assessed for morphological features and the pro-regenerative Stat3 pathway. ACLF was associated with high levels of lobular inflammation, tissue necrosis, and apoptosis. In patients with cirrhosis, the percentage of pStat3-positive hepatocytes was increasing with disease severity (3.5%/10.4%/21% for compensated cirrhosis/acute decompensation/cirrhosis-ACLF; P < .001), but lower in noncirrhotic ACLF vs cirrhosis-ACLF (21% vs 13%; P= .02). A distinct pattern of the expression of the proliferation marker Ki-67, a downstream effector marker of pStat3, was observed. Ki-67-positive hepatocytes were more frequent in patients with cirrhosis-ACLF compared to compensated cirrhosis or acute decompensation (4.9% vs 1.3% vs 1.8%; P < .05), but much lower in cirrhosis-ACLF vs noncirrhotic ACLF (4.9% vs 13.5%; P= .01). The ratio of Ki-67-positive to pStat3-positive hepatocytes was lowest in cirrhosis-ACLF and predicted 3-month transplant-free survival accurately (area under the curve= 0.95, P < .00001). Our study identifies hepatic inflammation and Stat3 activation as hallmarks of ACLF. In cirrhosis-ACLF, Stat3 activation does not appear to translate in effective liver regeneration, which is distinct from noncirrhotic ACLF.

A comparison of proliferation levels in normal skin, physiological scar and keloid tissue

Proliferation is an important characteristic of life, and many signaling pathways participate in this complicated process. The MAPK/Erk pathway is a classic pathway in cell proliferation. In this study, expression levels of key factors in the MAPK/Erk pathway were measured to assess the proliferation level among normal skin, physiological scar, and keloid tissue. Thirty patients were selected randomly from the Department of Plastic Surgery at Peking Union Medical College Hospital from January 2019 to December 2020. Histological appearance and fiber tissue content were observed by Hematoxylin and eosin staining and Masson staining. Expression levels of key factors in the MAPK/Erk pathway (ATF2, c-Jun, c-Myc, p38 and STAT1) and relative proteins (HIF-1α and PCNA) in tissues were detected by immunohistochemistry and analyzed as the percentage of positively stained cells in both the tissue epidermis and dermis. Western blot was used for quantitative analysis of the above factors. In results, keloid tissue showed a significantly higher fiber and less cell content. In the immunohistochemical result, higher expression of key factors was observed in the epidermis than in the dermal layer, and the expression of all factors was increased remarkably in keloid tissue. In western blot analysis, all factors (except STAT1) showed higher expression in keloid tissue. In our former research, keloid showed similar apoptosis level as physiological scar and normal skin. On combining our former conclusion and results in this study, an imbalance condition between the high proliferation level and normal apoptosis level may lead to the growth characteristics of keloid.

NDUFC1 Is Upregulated in Gastric Cancer and Regulates Cell Proliferation, Apoptosis, Cycle and Migration.

Gastric cancer is one of the most common primary tumors of the digestive system. NADH: ubiquinone oxidoreductase subunit C1 (NDUFC1), which is an accessory subunit of the NADH dehydrogenase (complex I), is responsible for the transportation of electrons from NADH to the respiratory chain essential for the oxidative phosphorylation. However, little is known about the roles of NDUFC1 in carcinogenesis. In this study, NDUFC1 protein level in NSCLC tissues was tested by immunohistochemistry (IHC) staining. NDUFC1 mRNA level in gastric cancer cell lines was determined by qRT-PCR. MGC-803 and SGC-7901 cells were transfected with shNDUFC1 lentivirus designed to silence NDUFC1. MTT assay, CCK8 assay, wound healing assay and transwell migration assay were conducted. Cell cycle and apoptosis were detected by flow cytometry. In vivo experiments were performed using nude mice. The results indicated that overexpressed NDUFC1 in gastric cancer was related to more serious tumor infiltrates, a higher risk of lymphatic metastasis, a higher proportion of positive lymph nodes, and a more advanced tumor stage. Compared with shCtrl groups, MGC-803 and SGC-7901 of shNDUFC1 groups had lower abilities of proliferation and migration, higher levels of apoptosis. NDUFC1 knockdown also inhibited SGC-7901 cell growth in vivo and suppressed Ki67 expression in xenograft tumors. More importantly, we found that NDUFC1 downregulation made the levels of P-Akt, P-mTOR, CCND1, CDK6, PIK3CA, Bcl-2, Survivin, and XIAP decreased, and that PI3K/AKT signaling pathway agonist SC79 rescued the inhibitory effects on cell proliferation and migration, reversed the promoted effects on cell apoptosis caused by NDUFC1 knockdown. More importantly, compared with NDUFC1 knockdown group, the expression of P-Akt, Bcl-2, Survivin, and XIAP was raised in shNDUFC1 + SC79 group. Thus, our suspicion was that NDUFC1 exacerbates NSCLC progression via PI3K/Akt pathway. Taken together, our study indicated that targeting NDUFC1 could open innovative perspectives for new multi-targeting approaches in the treatment of gastric cancer.

Endoderm development requires centrioles to restrain p53-mediated apoptosis in the absence of ERK activity.

Centrioles comprise the heart of centrosomes, microtubule-organizing centers. To study the function of centrioles in lung and gut development, we genetically disrupted centrioles throughout the mouse endoderm. Surprisingly, removing centrioles from the endoderm did not disrupt intestinal growth or development but blocked lung branching. In the lung, acentriolar SOX2-expressing airway epithelial cells apoptosed. Loss of centrioles activated p53, and removing p53 restored survival of SOX2-expressing cells, lung branching, and mouse viability. To investigate how endodermal p53 activation specifically killed acentriolar SOX2-expressing cells, we assessed ERK, a prosurvival cue. ERK was active throughout the intestine and in the distal lung buds, correlating with tolerance to centriole loss. Pharmacologically inhibiting ERK activated apoptosis in acentriolar cells, revealing that ERK activity protects acentriolar cells from apoptosis. Therefore, centrioles are largely dispensable for endodermal growth and the spatial distribution of ERK activity in the endoderm shapes the developmental consequences of centriolar defects and p53 activation.

An Autosomal Dominant Form of Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2) Is Associated with Haematopoiesis Failure

Severe combined immunodeficiencies (SCIDs) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. Reticular dysgenesis (RD) is an autosomal recessive form of human Severe Combined Immunodeficiency (SCIDs) characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, encoding for a mitochondrial protein which regulates the homeostasis of adenine nucleotides.In three newborns presenting a RD-like clinical phenotype as frequent infections and a profound leukopenia, we identified an heterozygous dominant missense mutation in the gene coding for Rac Family Small GTPase 2 (RAC2) protein (p.G12R). Hematopoietic stem cell transplantation (HSCT) performed soon after birth was successful in two out of the three patients attesting that the inherited defect was intrinsic and not micro-environmental. RAC2 protein belongs to the Rac subfamily of RHO small GTPases. In the inactive GDP-bound state, RAC2 is located in the cytosol and upon stimulation, the active RAC2-GTP-bound form translocates to the plasma membrane. Unlike the other members of the Rac subfamily (RAC1 and RAC3), RAC2 is mostly expressed on hematopoietic cells and during T cell differentiation.To gain insight into the disease, we transduced human hematopoietic stem and progenitor cells (HSPCs) with a lentiviral construct containing the RAC2 mutated form. The mutation inhibits HSPCs proliferation and differentiation toward the myeloid and lymphoid lineages reproducing the patients' clinical phenotype. In this condition, we also observed high apoptosis level and an alteration of mitochondrial activity and Ros production. In a biochemical model, we demonstrated that the substitution of the glycine (G) amino-acid by a bulky flexible arginine (R) may prevent GTP hydrolysis. Lastly, our findings suggest that RAC2 gene sequencing must be considered in newborn screening programs for SCID detection.To decipher the mechanisms regulating RAC2 functions, we studied the impact of the p.G12R mutation on a human AML cell line expressing RAC2 protein. Our preliminary data highlight that the cell cycle and mitochondrial activity are disrupted by G12R mutation. By holotomography, we also observed morphological changes and accumulation of lipid droplets into the cells. All these data suggest that RAC2 defective signalling pathway is linked to cell metabolism imbalance and further investigations are ongoing to better understand how RAC2 controls cell energy demand, especially during differentiation. DisclosuresCavazzana: Smart Immune: Other: co-founder.

Impacts of CAD/CAM Metallic Materials on Trace Metals and Biocompatibilities: An in vivo Study in Beagle Dogs

CAD/CAM (computer-aided design and computer-aided manufacturing) technology has been widely applied in clinical dentistry, but the material safety remains a concern. To investigate the impacts of CAD/CAM metallic materials on trace metals and biocompatibilities, selective laser melted (SLM) cobalt-chromium (Co-Cr) alloys and computer numeric controlled milled (CNC milled) commercially pure titanium (CP-Ti) were placed on the maxilla of beagle dogs for 6 months. The trace metals in the oral mucosa, blood, liver, kidney, and hair were then determined by inductively coupled plasma mass spectrometry (ICP-MS). The histopathologic changes and biocompatibilities of tissues were examined by hematoxylin and eosin (H&amp;amp;E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) method, immunohistochemistry (IHC) assay, Western blot analysis, and liver and kidney function tests. Our results showed that trace metals released from these two CAD/CAM metallic materials accumulated transiently in the oral mucosa and whole blood. The trace metals released from SLM Co-Cr alloys could also transiently accumulate in the plasm and hair. In addition, these two CAD/CAM metallic materials both induced apoptosis and histopathologic changes in the oral mucosa, with SLM Co-Cr alloys inducing a higher level of apoptosis. In contrast, both materials did not exert autophagic effects on the oral mucosa or affect the trace metals, functions, or biocompatibilities of the liver and kidney. Therefore, this study suggests that CAD/CAM metallic materials should be selected carefully, especially in patients with metal- and apoptosis-related diseases, and CNC-milled CP-Ti can be recommended to patients on account of its better biocompatibility and safety.

Vitamin D3/VDR inhibits inflammation through NF-κB pathway accompanied by resisting apoptosis and inducing autophagy in abalone Haliotis discus hannai.

Vitamin D3 is believed to be a contributing factor to innate immunity. Vitamin D receptor (VDR) has a positive effect on inhibiting nuclear factor κB (NF-κB)-mediated inflammation. The underlying molecular mechanisms remain unclear, particularly in mollusks. Consequently, this study will investigate the process of vitamin D3/VDR regulating NF-κB pathway and further explore their functions on inflammation, autophagy, and apoptosis in abalone Haliotis discus hannai. Results showed that knockdown of VDR by using siRNA and dsRNA of VDR in vitro and in vivo led to more intense response of NF-κB signaling to lipopolysaccharide and higher level of apoptosis and autophagy. In addition, 1,25(OH)2D3 stimulation after VDR silencing could partially alleviate apoptosis and induce autophagy. Overexpression of VDR restricted the K48-polyubiquitin chain-dependent inhibitor of κB (IκB) ubiquitination and apoptosis-associated speck-like protein containing CARD (ASC) oligomerization. Besides, VDR silencing resulted in increase of ASC speck formation. In further mechanistic studies, we showed that VDR can directly bind to IκB and IKK1 in vitro and in vivo. In the feeding trial, H&E staining, TUNEL, and electron microscope results showed that vitamin D3 deficiency (0IU/kg) could recruit more basophilic cells and increase more TUNEL-positive apoptotic cells and lipid droplets (LDs) than vitamin D3 supplement (1000IU/kg and 5000IU/kg). In summary, abalone VDR plays a negative regulator role in NF-κB-mediated inflammation via interacting with IκB and inhibiting ubiquitin-dependent degradation of IκB. Vitamin D3 in combination with VDR is essential to establish a delicate balance between autophagy and apoptosis in response to inflammation.

Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy—RC) and nine cervical cancer patients (treatment with radiotherapy alone—R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

Transcription factor EB mediates oxidative stress-induced intervertebral disc degeneration via the NF-κB signaling pathway.

BackgroundIt is well known that the intervertebral disc is aggravated by a significant increase in the number of senescent cells, and oxidative stress (OS) is related to the deterioration of this tissue. Transcription factor EB (TFEB) can protect cells from OS. Accordingly, we investigated whether TFEB can prevent OS in human nucleus pulposus (NP) cells.MethodsFirst, TFEB expression was investigated in human NP tissue samples with different degrees of degeneration. NP cells were treated with different concentrations of hydrogen peroxide (H2O2). The expression of collagen 2, aggrecan, and P65 was detected by quantitative real-time polymerase chain reaction (PCR) and Western blotting. We overexpressed and knocked out the TFEB gene to detect the expression of collagen 2, aggrecan, and P65.ResultsWe found that the expression of TFEB decreased stepwise as the degree of intervertebral disc degeneration (IDD) increased. When the NP cells were treated with H2O2, the expression of TFEB, collagen 2, and aggrecan decreased gradually as H2O2 concentration increased. In addition, the expression of collagen2 and aggrecan increased following TFEB overexpression. However, nuclear factor-kappa B (NF-κB) decreased in NP cells after TFEB overexpression. We also found that the previously low cell viability increased and the high level of apoptosis decreased.ConclusionsThis study suggests that OS is associated with the development of IDD. TFEB mediates OS-induced IDD via the NF-κB signaling pathway. The TFEB gene can potentially be used as a diagnostic biomarker and therapeutic target.

Enzymatic Spermine Metabolites Induce Apoptosis Associated with Increase of p53, caspase-3 and miR-34a in Both Neuroblastoma Cells, SJNKP and the N-Myc-Amplified Form IMR5.

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down- or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis.

Nucleophilic and redox properties of polybrominated diphenyl ether derived-quinone/hydroquinone metabolites are responsible for their neurotoxicity

Polybrominated diphenyl ethers (PBDEs) are a category of brominated flame retardants, which were widely used in industrial products since the 1970 s. Our previous studies indicated quinone-type metabolites of PBDEs (PBDE-Qs) cause neurotoxicity, however, their inherent toxicological mechanism remains unclear. Here, we first synthesized PBDE-Qs and corresponding reduced hydroquinone homologous (PBDE-HQs) with different pattern of bromine substitution. Their nucleophilic and redox properties were investigated. PBDE-Qs react with reduced glutathione (GSH) via Michael addition and bromine displacement reaction, whilst PBDE-HQs lack the ability of reacting with GSH. Of note, the displacement reaction only occurs with bromine on the quinone ring of PBDE-Qs but not phenyl ring. Next, electron paramagnetic resonance (EPR) analysis revealed the generation of SQ•−, along with their downstream hydroxyl radical (HO•) and methyl radical (•CH3) through a PBDE quinone/semiquinone/hydroquinone (Q/SQ•−/HQ) futile cycle. In addition, a structure-dependent cytotoxicity pattern was found, the exposure of PBDE-Q/HQ with bromine substitution on the quinone ring resulted in higher level of apoptosis and autophagy in BV2 cells. In conclusion, this work clearly demonstrated that the nucleophilic and redox properties of PBDE-Qs/HQs are responsible for their neurotoxicity, and this finding provide better understanding of neurotoxicity of PBDEs.

Synergistic effects of the combined treatment of U251 and T98G glioma cells with an anti‑tubulin tetrahydrothieno[2,3‑c]pyridine derivative and a peptide nucleic acid targeting miR‑221‑3p.

In the development of novel and more effective anti-cancer approaches, combined treatments appear to be of great interest, based on the possibility of obtaining relevant biological or therapeutic effects using lower concentrations of single drugs. Combination therapy may prove to be of utmost significance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer cases of the central nervous system, with a median survival rate of 15 months. As regards novel therapeutic approaches, the authors have recently demonstrated that peptide nucleic acids (PNAs) that target microRNA (miRNA/miR)-221 are very active in inducing the apoptosis of glioma cells. Furthermore, in a recent study, the authors described two novel series of tubulin polymerization inhibitors based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene scaffold, which exerted a potent anti-proliferative effect on a variety of tumor cell lines. The present study aimed to verify the activity on glioblastoma cancer cell lines of one of the most active compounds tested, corresponding to 2-(3′, 4′, 5′-trimethoxyanilino)-3-cyano/alkoxycarbonyl-6-substituted-4 5,6,7-tetrahydrothiene[2,3-c] pyridine (compound 3b), used in combination with an anti-miR-221-3p PNA, already demonstrated to be able to induce high levels of apoptosis. To the best of our knowledge, the results obtained herein demonstrate for the first time a 'combination therapy' performed by the combined use of a PNA targeting miR-221 and the tetrahydrothiene[2,3-c]pyridine derivative 3b, supporting the concept that the combined treatment of GBM cells with a PNA against a specific upregulated oncomiRNA (in the present study a PNA targeting miR-221-3p was used) and anti-tubulin agents (in the present study derivative 3b was used) is an encouraging strategy which may be used to enhance the efficacy of anticancer therapies and at the same time, to reduce side-effects.

An Unbalanced Monocyte Macrophage Polarization in the Bone Marrow Microenvironment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundPoor graft function (PGF) remains a life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the underlying mechanisms have not yet been elucidated. Considerable evidence from murine studies has demonstrated that effective hematopoiesis depends on the specific bone marrow (BM) microenvironment, where hematopoietic stem cells (HSCs) reside. In this regard, we previously reported that PGF patients had impaired BM endosteal cells and endothelial progenitor cells, as well as dysregulated T cell responses in the BM microenvironment, which may be involved in the occurrence of PGF (BBMT 2013; BMT2016; Oncotarget 2016; BBMT 2016; Blood 2016; J Transl Med2017). Macrophage is one of the important components of BM immune microenvironment. Meanwhile, Murine studies suggest that macrophages regulate the retention of HSCs by regulating osteoblastic cell activity and nestin-expressing cells. Increasing evidence shows that BM resident macrophages are indispensable for HSCs function and BM erythroid output. However, little is known about the quantity and function of BM macrophages and whether the unbalanced BM macrophages directly interact with HSCs in PGF patients post allo-HSCT.AimsTo compare the number and function of BM macrophages between patients with PGF and GGF after allo-HSCT. Moreover, to investigate whether the unbalanced BM macrophages directly interact with HSCs in PGF patients.MethodsThis prospective nested case-control study enrolled 30 patients with PGF, 60 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 30 healthy donors (HD). Standard monocyte subsets, defined by cluster of differentiation CD14 and CD16, were quantified by flow cytometry. In addition, based on the phenotype of polarized macrophages in vitro, classically activated inflammatory macrophage (M1:CD68+CCR2+) and alternatively activated anti-inflammatory macrophage (M2:CX3CR1+CD163+/CD206+) from BM samples were analyzed by flow cytometry. BM CD14+ monocytes were isolated from BM mononuclear cell (BMMNCs) and were cultured with 100 ng/ml recombinant human M-CSF for 10 days to obtain macrophages. The functions of BM derived macrophages were evaluated by migration assay and phagocytosis assay. CD34+ cells from healthy donors were co-cultured with BM derived macrophages from PGF patients, and the levels of reactive oxygen species (ROS) and apoptosis in CD34+ cells were analyzed by flow cytometry after 5 days co-culture.ResultsAlterations in standard monocyte subsets (classical, intermediate and non-classical) were found when comparing subjects among PGF, GGF and normal controls. In particular, elevated intermediate and non-classical monocyte subsets were observed in PGF patients when compared with those in GGF patients. Moreover, PGF patients displayed an imbalanced M1/M2 ratio compared with the GGF and normal controls, attributable to a reduction in M2 and an increase in M1. Dysfunctional BM derived macrophages, which were characterized by impaired proliferation, migration, phagocytosis and higher level of apoptosis, were revealed in patients with PGF. BM CD34+ cells from healthy donors were co-cultured respectively with BM derived macrophages from patients with PGF and normal controls. Apoptosis and intracellular levels of ROS were markedly increased in CD34+ bone marrow cells which were co-cultured with PGF patients' BM derived macrophages compared with normal controls.Summary/ConclusionIn summary, the current study demonstrated the abnormal number and impaired function of BM macrophages and unbalanced BM macrophages directly interact with HSCs in PGF patients. We speculate that reduction of anti-inflammatory M2 monocytes while increase of pro-inflammatory M1 monocytes in PGF patient BM have a negative impact on HSCs in BM microenvironment. Thus, our results indicate that unbalanced BM M1/M2 and dysfunctional BM macrophages may play an important role in the pathogenesis of PGF following allo-HSCT. Therefore, it would be of value to investigate the appropriate approach to balance the M1/M2 and enhance function of BM macrophages, which may be a promising therapeutic approach for PGF patients. DisclosuresNo relevant conflicts of interest to declare.

Novel CD44-targeting and pH/redox-dual-stimuli-responsive core\u2013shell nanoparticles loading triptolide combats breast cancer growth and lung metastasis

BackgroundThe toxicity and inefficient delivery of triptolide (TPL) in tumor therapy have greatly limited the clinical application. Thus, we fabricated a CD44-targeting and tumor microenvironment pH/redox-sensitive nanosystem composed of hyaluronic acid-vitamin E succinate and poly (β-amino esters) (PBAEss) polymers to enhance the TPL-mediated suppression of breast cancer proliferation and lung metastasis.ResultsThe generated TPL nanoparticles (NPs) had high drug loading efficiency (94.93% ± 2.1%) and a desirable average size (191 nm). Mediated by the PBAEss core, TPL/NPs displayed a pH/redox-dual-stimuli-responsive drug release profile in vitro. Based on the hyaluronic acid coating, TPL/NPs exhibited selective tumor cellular uptake and high tumor tissue accumulation capacity by targeting CD44. Consequently, TPL/NPs induced higher suppression of cell proliferation, blockage of proapoptotic and cell cycle activities, and strong inhibition of cell migration and invasion than that induced by free TPL in MCF-7 and MDA-MB-231 cells. Importantly, TPL/NPs also showed higher efficacy in shrinking tumor size and blocking lung metastasis with decreased systemic toxicity in a 4T1 breast cancer mouse model at an equivalent or lower TPL dosage compared with that of free TPL. Histological immunofluorescence and immunohistochemical analyses in tumor and lung tissue revealed that TPL/NPs induced a high level of apoptosis and suppressed expression of matrix metalloproteinases, which contributed to inhibiting tumor growth and pulmonary metastasis.ConclusionCollectively, our results demonstrate that TPL/NPs, which combine tumor active targeting and pH/redox-responsive drug release with proapoptotic and antimobility effects, represent a promising candidate in halting breast cancer progression and metastasis while minimizing systemic toxicity.Graphic

Clinical Complications of Iron Overload in Patients with Myelodysplastic Syndromes: a literature review

Myelodysplastic Syndromes (MDS) are a group of heterogeneous hematologic disorders characterized by bone marrow involvement. Iron overload is the result of multiple transfusions and high levels of apoptosis. Based on a literature review, ferritin was identified as an important marker of iron accumulation in organs such as liver, heart and pancreas in patients with MDS. Some studies also demonstrated that iron overload caused cell cycle arrest causing apoptosis and progression to leukemia, high levels of reactive oxygen species and low levels of HIF-1a, causing apoptosis, being related to the presence of cytopenia in MDS.

Long Circulation of PEG-TRAIL Improves Anti-Hepatic Fibrosis Effect of TRAIL Via Targeting Activated Hepatic Stellate Cells

Background: The short half-life of TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) greatly limits its clinical application. This study was aimed to improve its potency on liver fibrosis through PEG (polyethylene glycol) modification prolonging the half-life of TRAIL.Methods: PEG, TRAIL, and the chemically synthesized complex PEG-TRAIL were used to treat 3T3 and LX-2 cells and liver fibrotic mice. In vitro, cell viability, apoptosis, and fibrosis were investigated using CCK-8 (cell counting kit-8) assay, flow cytometry, and Western blotting, respectively. In vivo, Sirius red staining, immunohistochemistry, and α-SMA (α-smooth muscle actin)/TUNEL (terminal deoxynucleotidyl transferase dUTP [2'-deoxyuridine 5'-triphosphate] nick end labeling) double-labeling immunofluorescence (IF) were performed after various treatments for liver fibrotic mice. The fibrotic liver was subjected to DR4 (death receptor 4)/TRAIL double-labeling IF to assess the retention of TRAIL enhanced by PEGylation.Results: The cells treated with PEG-TRAIL showed lower cell viability, higher apoptosis level, and stronger anti-fibrotic effect compared with PEG or TRAIL treatment. In vivo, PEGylated TRAIL exhibited a longer circulation than TRAIL did. Compared with TRAIL treatment, PEG-TRAIL caused a significant reduction of α-SMA and a markedly increase of apoptotic aHSCs. PEGylation is more likely to prolong the retention of TRAIL in circulation and enhance the possibility to target aHSCs and DR4-positive (DR4+) cells in the liver.Conclusion: PEG-TRAIL presents better anti-fibrotic and proapoptotic effects, for which, the prolonged circulation half-life in vivo may account. The PEG-TRAIL may serve as a new clinical therapeutic for liver fibrosis in the future.

Biparatopic Protein Nanoparticles for the Precision Therapy of CXCR4+ Cancers.

Simple SummaryAimed at minimizing side toxicities cancer therapies require appropriate functional vehicles at the nanoscale, for receptor-mediated tumor-targeted drug delivery. The aim of the present study was to explore the human peptide EPI-X4 as a CXCR4-targeting agent in self-assembled, protein-only nanoparticles. While the systemic tumor biodistribution of EPI-X4-based materials is modest, this peptide shows potent proapoptotic effects on CXCR4+ cancer cells. Interestingly, the in vivo selectivity of EPI-X4 was dramatically improved, once combined into biparatopic nanoparticles, with a second CXCR4 ligand, the peptide T22. Biparatopic nanoparticles promote a highly selective tumor destruction in a mouse model of human colorectal cancer, probably associated to the CXCR4 antagonist role of EPI-X4. This study not only validates a new human ligand of the tumoral marker CXCR4, but it also offers a novel strategy for the combination, in protein nanoparticles, of dual acting ligands of tumoral markers for highly selective drug delivery. The accumulated molecular knowledge about human cancer enables the identification of multiple cell surface markers as highly specific therapeutic targets. A proper tumor targeting could significantly avoid drug exposure of healthy cells, minimizing side effects, but it is also expected to increase the therapeutic index. Specifically, colorectal cancer has a particularly poor prognosis in late stages, being drug targeting an appropriate strategy to substantially improve the therapeutic efficacy. In this study, we have explored the potential of the human albumin-derived peptide, EPI-X4, as a suitable ligand to target colorectal cancer via the cell surface protein CXCR4, a chemokine receptor overexpressed in cancer stem cells. To explore the potential use of this ligand, self-assembling protein nanoparticles have been generated displaying an engineered EPI-X4 version, which conferred a modest CXCR4 targeting and fast and high level of cell apoptosis in tumor CXCR4+ cells, in vitro and in vivo. In addition, when EPI-X4-based building blocks are combined with biologically inert polypeptides containing the CXCR4 ligand T22, the resulting biparatopic nanoparticles show a dramatically improved biodistribution in mouse models of CXCR4+ human cancer, faster cell internalization and enhanced target cell death when compared to the version based on a single ligand. The generation of biparatopic materials opens exciting possibilities in oncotherapies based on high precision drug delivery based on the receptor CXCR4.

Exposure to Copper Compromises the Maturational Competency of Porcine Oocytes by Impairing Mitochondrial Function.

Copper (Cu) is an essential trace element for animals, and also an important nutritional component for the normal physiology and metabolism of animal reproductive systems. An excess or lack of Cu will directly or indirectly affect animal reproductive activities. However, the effect of Cu, in particular excessive Cu, on the reproductive performance of sows has not been studied. Here, we report that excessive Cu had negative effects on oocyte maturation and organelle functions. We showed that Cu exposure perturbed porcine oocyte meiotic maturation and impaired spindle/chromosome structure, resulting in a defective spindle assembly, as well as the abnormal distribution of actin dynamics and cortical granules. In addition, single-cell transcriptome analysis identified the target effectors of Cu actions in porcine oocytes, further demonstrating that Cu exposure affects the mitochondrial distribution and function, leading to the high levels of reactive oxygen species, DNA damage, and early apoptosis of porcine oocytes. These findings demonstrate that Cu exposure causes abnormalities in the mitochondrial distribution and function, resulting in the increased oxidative stress and levels of reactive oxygen species, DNA damage, and apoptosis, ultimately leading to a decreased porcine oocyte quality.

Direct surface modification of mesoporous silica nanoparticles by DBD plasma as a green approach to prepare dual-responsive drug delivery system

This paper deals with uniformly surface modification of mesoporous silica nanoparticles by applying dielectric barrier discharge (DBD) plasma as an energy-efficient and green approach. First, the mesoporous silica nanoparticles were synthesized from rice husk (RMSN-D) as an economic and biocompatible natural source. The surface modification stage by DBD plasma was performed in two methods: i) Direct mode and ii) Direct hybrid mode. The structure and physicochemical properties of the nanoparticles were characterized by XRD, FT-IR, Fe-SEM/EDS, TEM, EDS, BET, and HNMR techniques. Doxorubicin (Dox) was exploited as a model drug, and in-vitro Dox release was investigated. The biocompatibility of nanocarriers was accepted by MTT test on HFF-2 cell line as a normal modal cell. For the advanced cellular study, apoptosis of the MCF-7 cell line treated with Dox-loaded nanocarriers was investigated by flow cytometric analysis and morphological study by fluorescent microscopy. The results showed that Dox-loaded RMSN-D modified by direct hybrid mode induced a high level of apoptosis in the MCF-7 cells.