Anti-apoptotic Molecule Bcl-2 Research Articles

Chiral Amino Acids Mediate Mitochondria-Dependent Apoptosis of Human Proximal Tubular Epithelial Cells Under Oxidative Stress.

Amino acids are the basic structural units of life, and their intake levels affect disease and health. In the case of renal disease, alterations in amino acid metabolism can be used not only as a clinical indicator of renal disease but also as a therapeutic strategy. However, the biological roles and molecular mechanisms of natural chiral amino acids in human proximal tubular epithelial cells (HK-2) remain unclear. In this study, cell viability assays revealed that chiral acidic amino acids (Glu and Asp) and aromatic amino acids (Trp and Phe) inhibited cell growth. The molecular mechanisms indicated that cell growth was closely related to ROS levels. Specifically, chiral Glu, Asp, Trp, and Phe induced oxidative stress and mitochondria-dependent apoptosis in HK-2 cells. This was manifested by elevated levels of intracellular ROS, 8-OHdG, and MDA, increased activities of antioxidant enzymes CAT, SOD, and GPx, decreased mitochondrial membrane potential, increased cytoplasmic Ca2+ concentration, and cell acidification. The expression levels of apoptosis-related molecules Caspase-9, Caspase-3, Cyt-C, and Bax were increased, and the expression level of anti-apoptotic molecule Bcl-2 was decreased. Moreover, L-Glu, D-Asp, L-Trp, and D-Phe exhibited a more pronounced inhibition of cell growth and elicited more substantial alterations in gene expression compared to the other configurations.

FHOD3 Promotes the Progression of Lung Cancer by Regulating the Caspase-3-Mediated Signaling Pathway.

Lung cancer causes hundreds of thousands of deaths each year worldwide. FHOD3 was reported to accelerate the progression of brain cancer. However, its role in lung cancer is not clear. This study aimed to investigate the role of FHOD3 in lung cancer. The clinical significance of FHOD3 in lung cancer was analyzed based on the data from the TCGA database. The expression level of FHOD3 was detected by qPCR technology. Cell proliferation was detected by CCK-8 assay, and cell invasion was detected by transwell assay. The activity of caspase-3 was determined by the ELISA method, cell apoptosis was iden-tified by TUNEL assay, and protein expression was measured by western blotting technology. Based on the TCGA data, FHOD3 was overexpressed in tumor tissues compared to the normal tissues. Patients with higher FHOD3 expression exhibited a worse survival rate. The expression levels of FHOD3 in lung cancer cell lines were much higher than that in normal cells. When FHOD3 was knocked down, the ability of cell proliferation and invasion was sig-nificantly inhibited. Cell apoptosis rate was increased reversely. The activity of caspase-3 was increased significantly. In addition, the expression level of cleaved caspase-3 was increased. The expression levels of Bax, caspase-8, and ICAD were also increased significantly. However, the expression of antiapoptotic molecule Bcl-2 was decreased reversely. This suggests that the caspase-3-mediated apoptosis signaling pathway was activated by FHOD3 knockdown. FHOD3 was overexpressed and negatively associated with survival rate in lung cancer patients. FHOD3 regulates cell proliferation, invasion, and apoptosis through the caspase-3-mediated signaling pathway. This study indicates that FHOD3 is an important gene contributing to the progression of lung cancer and might be a new drug target for the therapy of lung cancer.

Protective effect of fructooligosaccharide against oxidative stress and apoptosis induced by Aeromonas hydrophila in Megalobrama amblycephala

This research aimed to investigate the effects of dietary fructooligosaccharides (FOS) on attenuating the Aeromonas hydrophila (A. hydrophila)-induced oxidative stress and apoptosis in blunt snout bream Megalobrama amblycephala. Fish were divided into three groups as follows: C1 (Control), T1 (A. hydrophila), and T2 (A. hydrophila + 4 g/kg FOS). The results showed that the activities of antioxidant enzymes increased, the liver morphology had disorderly arrangement, and extensive cell necrosis occurred because of A. hydrophila-infection. While the dietary FOS improved the above-mentioned liver damage. Additionaly, FOS elevated mRNA levels of pro-apoptotic molecules, including caspase-8 and 9, and down-regulated mRNA levels of the anti-apoptotic molecule Bcl-2, which is triggered by A. hydrophila-infection. The transcriptome analysis showed that the oxidative stress-related DEGs pathways were activated in intestine of blunt snout bream by A. hydrophila-infection. The FOS-added group led to the enrichment of more pathways to health. Further WGCNA co-expression network analysis showed that the screened single genes were clustered into 49 modules. The two modules with the highest association to the five traits (10 hub genes) were chosen to build the network by combining the physiological and biochemical characteristic. In summary, this research offers a foundation for the exploring of A. hydrophila-restoration genes in dietary FOS, and also lays a theoretical foundation for aquaculture in the future.

Hypothesis Explaining Development of Squamous Cell Carcinoma During Infection with Schistosoma Haematobium

Schistosome worms are blood-dwelling flukes that cause chronic infection in more than 200 million people and are thought to be responsible for 500,000 deaths annually. During infection with Schistosoma haematobium, eggs are deposited in the mucosa and submucosa of the bladder and lower ureters. Squamous cell carcinoma (SCC) of the bladder is a long-term sequela of chronic infection. The mechanisms underlying the association between S. haematobium and SCC of the bladder are largely unknown, with all reports to date exclusively demonstrating epidemiological evidence linking S. haematobium infection with SCC of the bladder. Scientists hypothesised that the parasite antigens might induce alterations in epithelial cells towards cancer. Scientists used Chinese Hamster Ovary (CHO) cells and treated the cells in culture with S. haematobium total antigen (Sh). Results showed increased proliferation, increased S-phase and decreased apoptosis, as well as down-regulation of tumor suppressor p27 and up-regulation of anti-apoptotic molecule Bcl-2 (Botelho et al., 2009). Angiogenesis is defined as the formation of new blood vessels from preexisting ones and is recognized as a key event in cell proliferation and carcinogenesis and spread of malignant lesions (Dematei et al., 2017). S. haematobium is classified as a Group 1 carcinogen by the World Health Organization’s International Agency for Research on Cancer (Hodderet al., 2000). Another factor that may play a major role in bladder carcinogenesis in schistosomiasis patients is the presence of continuous physical irritation and inflammation produced by Schistosoma eggs in the bladder mucosa. The adult S. haematobium worms inhabit the veins of the perivesical plexus, where the female lays eggs. Some eggs pass through the bladder mucosa and are excreted in the urine. Other eggs are trapped in the tissue. A chronic inflammatory reaction is initiated, with the invasion of histiocytes and other inflammatory cells into the bladder, the formation of granulomas and eventually fibrosis. In addition, the eruption of the eggs through the mucosa stimulates reparative urothelial hyperplasia and cell turnover (Rosin et al., 1994). In our article we try to explain possible mechanism of conversion normal cells into tumor cells from the position of karyogamic theory.

Phase 1b trial of tagraxofusp in combination with azacitidine with or without venetoclax in acute myeloid leukemia

AbstractCD123, a subunit of the interleukin-3 receptor, is expressed on ∼80% of acute myeloid leukemias (AMLs). Tagraxofusp (TAG), recombinant interleukin-3 fused to a truncated diphtheria toxin payload, is a first-in-class drug targeting CD123 approved for treatment of blastic plasmacytoid dendritic cell neoplasm. We previously found that AMLs with acquired resistance to TAG were re-sensitized by the DNA hypomethylating agent azacitidine (AZA) and that TAG-exposed cells became more dependent on the antiapoptotic molecule BCL-2. Here, we report a phase 1b study in 56 adults with CD123-positive AML or high-risk myelodysplastic syndrome (MDS), first combining TAG with AZA in AML/MDS, and subsequently TAG, AZA, and the BCL-2 inhibitor venetoclax (VEN) in AML. Adverse events with 3-day TAG dosing were as expected, without indication of increased toxicity of TAG or AZA+/−VEN in combination. The recommended phase 2 dose of TAG was 12 μg/kg/day for 3 days, with 7-day AZA +/− 21-day VEN. In an expansion cohort of 26 patients (median age 71) with previously untreated European LeukemiaNet adverse-risk AML (50% TP53 mutated), triplet TAG-AZA-VEN induced response in 69% (n=18/26; 39% complete remission [CR], 19% complete remission with incomplete count recovery [CRi], 12% morphologic leukemia-free state [MLFS]). Among 13 patients with TP53 mutations, 7/13 (54%) achieved CR/CRi/MLFS (CR = 4, CRi = 2, MLFS = 1). Twelve of 17 (71%) tested responders had no flow measurable residual disease. Median overall survival and progression-free survival were 14 months (95% CI, 9.5-NA) and 8.5 months (95% CI, 5.1-NA), respectively. In summary, TAG-AZA-VEN shows encouraging safety and activity in high-risk AML, including TP53-mutated disease, supporting further clinical development of TAG combinations. The study was registered on ClinicalTrials.gov as #NCT03113643.

Aluminium bioaccumulation in colon cancer, impinging on epithelial-mesenchymal-transition and cell death

We investigated the presence of aluminium (Al) in human colon cancer samples and its potential association with biological processes involved in cancer progression, such as epithelial to mesenchymal transition (EMT) and cell death. 25 consecutive colon samples were collected from patients undergoing colonic resection. Both neoplastic and normal mucosa were collected from each patient and subjected to histological, ultrastructural and immunohistochemical analyses. Moreover, colon samples from two Al-positive patients underwent multi-omic analyses, including whole genome sequencing and RNA sequencing (RNAseq). Morin staining, used to identify in situ aluminium bioaccumulation, showed the presence of Al in tumor areas of 24 % of patients. Transmission electron microscopy and energy-dispersive X-ray microanalysis confirmed the presence of Al specifically in intracytoplasmic electrondense nanodeposits adjacent to mitochondria of colon cancer cells. Immunohistochemical analyses for vimentin and nuclear β-catenin were performed to highlight the occurrence of the EMT phenomenon in association to Al bioaccumulation. Al-positive samples showed a significant increase in both the number of vimentin-positive and nuclear β-catenin-positive cancer cells compared to Al-negative samples. Moreover, Al-positive samples exhibited a significant decrease in the number of apoptotic cells, as well as the expression of the anti-apoptotic molecule BCL-2. Multi-omic analyses revealed a higher tumor mutational burden (TMB) in Al-positive colon cancers (n = 2) compared to a control cohort (n = 100). Additionally, somatic mutations in genes associated with EMT (GATA3) and apoptosis (TP53) were observed in Al-positive colon cancers. In conclusion, this study provides the first evidence of Al bioaccumulation in colon cancer and its potential role in modulating molecular pathways involved in cancer progression, such as EMT and apoptosis. Understanding the molecular mechanisms underlying Al toxicity might contribute to improve strategies for prevention, early detection, and targeted therapies for the management of colon cancer patients.

Adipose tissue-derived mesenchymal stromal cells attenuate acute lung injury induced by trauma and haemorrhagic shock

BackgroundMesenchymal stromal cells (MSCs) have shown promising therapeutic options for acute lung injury (ALI) caused by multiple factors. Here, we evaluated the therapeutic potential of adipose tissue-derived mesenchymal stromal cells (ADSCs) in trauma and hemorrhagic shock (THS)-induced ALI. MethodsALI model induced by THS was constructed by fractures plus abdominal trauma plus acute hemorrhage plus fluid resuscitation. The ADSCs group rats were generated by injecting 2 × 106 ADSCs at 0 and 1 h after THS. The sham, ALI, and ADSCs group rats were sacrificed at 24 h after resuscitation. The changes in lung histopathology, total protein in bronchoalveolar lavage fluid (BALF), mRNA expression of pro-inflammatory/anti-inflammatory cytokines, antioxidant, and anti-apoptotic indicator, and the activity of Toll-like receptor 4 (TLR4) signaling in lung tissues were evaluated. ResultsAdministration of the ADSCs reversed ALI induced by THS, including lung histopathological changes/scores, and BALF total protein concentration. Additionally, ADSCs therapy also significantly down-regulated mRNA expression of pro-inflammatory TNF-α, IL-1β, and IL-6, up-regulated mRNA expression of anti-inflammatory IL-10, anti-apoptotic molecule Bcl-2, and anti-oxidative molecule HO-1 in THS rats. Furthermore, ADSCs suppressed the expression of TLR4 in lung tissue. ConclusionOur data show that ADSCs administration can exert therapeutic effects on THS-induced ALI in rats and may provide beneficial in preventative strategies for ALI.

Proximal Tubular Lats2 Ablation Exacerbates Ischemia/Reperfusion Injury (IRI)-Induced Renal Maladaptive Repair through the Upregulation of P53.

The Hippo pathway mediates renal maladaptive repair after acute kidney injury (AKI), which has been considered a driving force in the progression to chronic kidney disease (CKD). LATS2, a core kinase of the Hippo pathway, exerts non-Hippo-dependent functions in the regulation of the cell cycle and cell fate, providing new insights into AKI and further repair. However, its role remains unknown. Here, we utilized a proximal tubular Lats2 conditional knockout mouse strain (Lats2-CKO) to evaluate the effect of LATS2 deficiency on ischemia/reperfusion-induced AKI-to-CKD transition. Lats2-CKO mice presented with more severe tubular maladaptive repair, inflammatory infiltration, interstitial fibrosis, and apoptosis following AKI. Importantly, we discovered that Lats2 ablation caused the activation of p53, with increased levels of cellular apoptotic molecules (p21, Bax, and cleaved caspase-3), and decreased levels of anti-apoptotic molecules (Bcl-2 and Bcl-xL). Pifithirin-α (p53 inhibitor) effectively attenuated renal fibrosis, inflammation, and apoptosis in Lats2-CKO mice after AKI. Consistently, in vitro Lats2 overexpression decreased p53, p21, Bax and cleaved caspase 3 expression after hypoxia/reoxygenation (H/R) treatment. Of note, the phosphorylation of MDM2, which promotes the ubiquitination degradation of p53, at site Ser186 was decreased in Lats2-CKO kidneys, but increased by Lats2 overexpression in vitro. Therefore, LATS2 deficiency aggravated ischemia/reperfusion injury (IRI)-induced maladaptive repair via regulating the tubular MDM2-p53 axis in AKI-to-CKD transition.

Protective Actions of Cannabidiol on Aging-Related Inflammation, Oxidative Stress and Apoptosis Alterations in Liver and Lung of Long Evans Rats.

Aging is characterised by the progressive accumulation of oxidative damage which leads to inflammation and apoptosis in cells. This affects all tissues in the body causing the deterioration of several organs. Previous studies observed that cannabidiol (CBD) could extend lifespan and health span by its antioxidant, anti-inflammatory and autophagy properties. However, research on the anti-aging effect of CBD is still in the beginning stages. This study aimed to investigate the role of cannabidiol (CBD) in the prevention of age-related alterations in liver and lung using a murine model. 15-month-old Long Evans rats were treated with 10 mg/kg b.w./day of CBD for 10 weeks and compared to animals of the same age as old control and 2-month-old animals as young control. Gene and/or protein expressions, by RT-qPCR and Western blotting, respectively, were assessed in terms of molecules related to oxidative stress (GST, GPx, GR and HO-1d), inflammation (NFκB, IL-1β and TNF-α) and apoptosis (BAX, Bcl-2, AIF, and CASP-1). In addition, MDA and MPO levels were measured by colorimetric assay. Results were analysed by ANOVA followed by Tukey-Kramer test, considering statistically significant a p < 0.05. GST, GPx and GR expressions were significantly reduced (p < 0.01) in liver samples from old animals compared to young ones and CBD treatment was able to revert it. A significant increase was observed in old animals compared to young ones in relation to oxidative stress markers (MDA and HO-1d), proinflammatory molecules (NFκB, IL-1β and TNF-α), MPO levels and proapoptotic molecules (BAX, AIF and CASP-1), while no significant alterations were observed in the antiapoptotic molecules (Bcl-2). All these changes were more noticeable in the liver, while the lung seemed to be less affected. In almost all the measured parameters, CBD treatment was able to revert the alterations caused by age restoring the levels to those observed in the group of young animals. Chronic treatment with CBD in 15-month-old rats showed beneficial effects in lung and more significantly in liver by reducing the levels of inflammatory, oxidative and apoptotic mediators, and hence the cell damage associated with these three processes inherent to aging.

Peripheral blood lymphocytes apoptosis role in rheumatoid arthritis progressing

Rheumatoid arthritis (RA) is an autoimmune, chronic, and genetically linked inflammatory lesion of joint tissues that is accompanied by extra-articular systemic pathologies. The disease progression leads to joints immobilization, and eventually, the patient's disability occurs approximately ten years from the first clinical manifestation. RA pathogenesis involves various mechanisms: specific joint-related damage, nonspecific adaptive, and vessel-related pathological changes. Our research aimed to study the role of peripheral blood lymphocyte apoptosis in RA pathogenesis. We have analyzed research data from Google Scholar, PubMed, Web of Science, and Scopus databases to investigate the role of lymphocyte apoptosis in RA progression. Clinical manifestations in RA are caused by autoreactive T- and B-lymphocyte activity supported by humoral and cellular immune factors activity. Disease pathogenesis is caused by an imbalance in the process of programmed cell death (apoptosis): a proportion of immune cells are rapidly destroyed. In contrast, apoptosis is inhibited in the other classes of immune cells. High infiltration of the joint by autoreactive sensitized lymphocytes worsens the patient's condition. Apoptosis inhibition is especially noticeable in the early stages of RA and correlates with the concentration of the anti-apoptotic molecule Bcl-2 in the synovia. Activating the apoptotic destruction of lymphocytes (by drug action) allows a positive therapeutic effect and sustained remission. However, it should be noted that genetic factors play a significant role in the onset, progression and drug response of RA. In addition, environmental and behavioral factors can activate RA progression and influence treatment efficacy.

Pyrroloquinoline Quinone (PQQ) Improves Long-term Survival of Fat Grafts by Alleviating Oxidative Stress and Promoting Angiogenesis During the Early Phase After Transplantation.

Reducing absorption after autologous fat grafting is a current challenge. Pyrroloquinoline quinone (PQQ) is the strongest known catalyst of redox reactions, which can scavenge reactive oxygen species (ROS) and alleviate oxidative stress. The aim of this study was to establish an in vivo model of PQQ-assisted lipotransfer and clarify the role of PQQ in reducing oxidative stress, alleviating apoptosis, and promoting angiogenesis during the acute hypoxic phase after grafting. In addition the study was performed to assess whether this intervention would have a positive effect on the improvement of long-term volume retention. Different concentrations of PQQ (low: 10 μM, medium: 100 μM, and high: 1000 μM) were mixed with human adipose tissue and transplanted subcutaneously into nude mice. Meanwhile, a control group of phosphate-buffered saline in an equal volume to PQQ was set up. On the third day after grafting, whole mount fluorescence staining was applied to detect ROS, mitochondrial membrane potential (MMP), apoptosis, adipocyte activity, and angiogenesis. Graft volume retention rate and electron microscopic morphology were evaluated at the third month. Immunohistochemistry and polymerase chain reaction (PCR) were further employed to elucidate the mechanism of action of PQQ. PQQ-assisted fat grafting improved the long-term volume retention, promoted the quality and viability of the adipose tissue, and reduced the level of fibrosis. The underlying mechanism of PQQ assisted in scavenging the accumulated ROS, restoring MMP, enhancing adipocyte viability, alleviating tissue apoptosis, and promoting timely angiogenesis during the hypoxia stress phase. The most effective concentration of PQQ was 100 μM. Immunohistochemistry and PCR experiments confirmed that PQQ reduced the expression of Bax and cytochrome c in the mitochondrial apoptotic pathway and increased the level of the antiapoptotic molecule Bcl-2. PQQ could improve the long-term survival of adipocytes by alleviating hypoxic stress and promoting timely angiogenesis in the early phase following lipotransfer.

PCTAIRE Protein Kinase 1 (PCTK1) Suppresses Proliferation, Stemness, and Chemoresistance in Colorectal Cancer through the BMPR1B-Smad1/5/8 Signaling Pathway.

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality worldwide. Even with advances in therapy, CRC mortality remains high. Therefore, there is an urgent need to develop effective therapeutics for CRC. PCTAIRE protein kinase 1 (PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family, and the function of PCTK1 in CRC is poorly understood. In this study, we found that patients with elevated PCTK1 levels had a better overall survival rate in CRC based on the TCGA dataset. Functional analysis also showed that PCTK1 suppressed cancer stemness and cell proliferation by using PCTK1 knockdown (PCTK1-KD) or knockout (PCTK1-KO) and PCTK1 overexpression (PCTK1-over) CRC cell lines. Furthermore, overexpression of PCTK1 decreased xenograft tumor growth and knockout of PCTK1 significantly increased in vivo tumor growth. Moreover, knockout of PCTK1 was observed to increase the resistance of CRC cells to both irinotecan (CPT-11) alone and in combination with 5-fluorouracil (5-FU). Additionally, the fold change of the anti-apoptotic molecules (Bcl-2 and Bcl-xL) and the proapoptotic molecules (Bax, c-PARP, p53, and c-caspase3) was reflected in the chemoresistance of PCTK1-KO CRC cells. PCTK1 signaling in the regulation of cancer progression and chemoresponse was analyzed using RNA sequencing and gene set enrichment analysis (GSEA). Furthermore, PCTK1 and Bone Morphogenetic Protein Receptor Type 1B (BMPR1B) in CRC tumors were negatively correlated in CRC patients from the Timer2.0 and cBioPortal database. We also found that BMPR1B was negatively correlated with PCTK1 in CRC cells, and BMPR1B expression was upregulated in PCTK1-KO cells and xenograft tumor tissues. Finally, BMPR1B-KD partially reversed cell proliferation, cancer stemness, and chemoresistance in PCTK1-KO cells. Moreover, the nuclear translocation of Smad1/5/8, a downstream molecule of BMPR1B, was increased in PCTK1-KO cells. Pharmacological inhibition of Smad1/5/8 also suppressed the malignant progression of CRC. Taken together, our results indicated that PCTK1 suppresses proliferation and cancer stemness and increases the chemoresponse of CRC through the BMPR1B-Smad1/5/8 signaling pathway.

Antrodin C Isolated from Antrodia Cinnamomea Induced Apoptosis through ROS/AKT/ERK/P38 Signaling Pathway and Epigenetic Histone Acetylation of TNFα in Colorectal Cancer Cells.

Antrodin C, a maleimide derivative compound isolated from the ethanol extract of the mycelium of Antrodia cinnamomea, is an endemic fungus of Taiwan and a potential chemoprotective agent. However, the molecular mechanisms underlying the mode of action of antrodin C on cancer cells, especially in human colorectal cancer (CRC), remain unclear. The cell death and ROS of the antrodin-C-treated HCT-116 cells were measured by annexin V-FITC/propidium iodide staining, DCFDA, and Fluo-3 fluorescence staining assays. Moreover, signaling molecules regulating TNFα cell death pathways and ROS/AKT/ERK/P38 pathways were also detected in cells treated with antrodin C by Western blotting and chromatin immunoprecipitation. The effects of antrodin C were determined in HCT-116 cell xenograft animal models in terms of tumor volumes and histopathological evaluation. Treatment with antrodin C triggered the activation of extrinsic apoptosis pathways (TNFα, Bax, caspase-3, and -9), and also suppressed the expression of anti-apoptotic molecules Bcl-2 in HCT-116 cells in a time-dependent manner. Antrodin C also decreased cell proliferation and growth through the inactivation of cyclin D1/cyclin for the arrest of the cell cycle at the G1 phase. The activation of the ROS/AKT/ERK/P38 pathways was involved in antrodin-C-induced transcriptional activation, which implicates the role of the histone H3K9K14ac (Acetyl Lys9/Lys14) of the TNFα promoters. Immunohistochemical analyses revealed that antrodin C treatment significantly induced TNFα levels, whereas it decreased the levels of PCNA, cyclin D1, cyclin E, and MMP-9 in an in vivo xenograft mouse model. Thus, antrodin C induces cell apoptosis via the activation of the ROS/AKT/ERK/P38 signaling modules, indicating a new mechanism for antrodin C to treat CRC in vitro and in vivo.

Apoptosis in lupus nephritis patients: a study of Bcl-2 to assess glomerular and tubular damage

BackgroundSystemic lupus erythematosus (SLE) is an immune-mediated disease, due to exposure of self-antigens, through impairment of apoptosis and failure of lymphocytic tolerance. Impaired regulation of the pro- and anti-apoptotic gene products which coordinate programmed cell death may result in autoreactive B and T cells and autoimmunity. Genetically engineered mice that over-express the anti-apoptotic molecule Bcl-2, B cell lymphoma 2 (Bcl2) in B-lymphocytes advance a lupus-like illness. Lupus nephritis (LN) is one of the most serious manifestations of this autoimmune disorder. Glomerulonephritis (GN) is caused by either impaired regulation of apoptosis and/or clearance of apoptotic cells leading to a T cell-mediated autoimmune reaction with initiation of pathological immune complex deposits.ObjectiveTo evaluate the correlation between Bcl2 glomerular and tubular expression and pathological findings and laboratory data in different types of SLE GN.ResultsCompared to the control group, patients with lupus nephritis have significantly higher glomerular, interstitial and tubular expression level (P value < 0.001). BCL2 expression was positively correlated with serum anti-ds-DNA, urine 24-h protein and with the chronicity index. All LN patients had significant glomerular, interstitial and tubular deposits of BCL2, P value < 0.001, P value 0.004, and P value 0.03, respectively.ConclusionThe intrinsic pathway of apoptosis interferes not only with the pathogenesis of lupus glomerulonephritis but also interferes with the pathogenesis of tubulointerstitial lupus nephritis. tubulointerstitial lesions may not only be a result of glomerular injury but also a significant factor in lupus nephritis.

Multiple low-dose radiation-induced neuronal cysteine transporter expression and oxidative stress are rescued by N-acetylcysteine in neuronal SH-SY5Y cells

Recently, several studies have demonstrated that low-dose radiation (LDR) therapy has positively impacts on the treatment of Alzheimer’s disease (AD). LDR suppresses the production of pro-neuroinflammation molecules and improves cognitive function in AD. However, it is unclear whether direct exposure to LDR causes beneficial effects and what mechanism is involved in neuronal cells. In this study, we first determined the effect of high-dose radiation (HDR) alone on C6 cells and SH-SY5Y cells. We found that SH-SY5Y cells were more vulnerable than C6 cells to HDR. Moreover, in neuronal SH-SY5Y cells exposed to single or multiple LDR, N-type cells showed decreased cell viability with increasing radiation exposure time and frequency, but S-type cells were unaffected. Multiple LDR increased proapoptotic molecules such as p53, Bax and cleaved caspase-3, and decreased anti-apoptotic molecule (Bcl2). Multiple LDR also generated free radicals in neuronal SH-SY5Y cells. We detected a change in the expression of the neuronal cysteine transporter EAAC1. Pretreatment with N-acetylcysteine (NAC) rescued the increased in EAAC1 expression and the generation of ROS in neuronal SH-SY5Y cells after multiple LDR. Furthermore, we verified whether the increased in EAAC1 expression induces cell defense or cell death promotion signaling. We showed that transient overexpression of EAAC1 reduced the multiple LDR-induced p53 overexpression in neuronal SH-SY5Y cells. Our results indicate that neuronal cells can be injured by increased production of ROS not only by HDR but also by multiple LDR, which suggests that combination treatment with anti-free radical agents such as NAC may be useful in multiple LDR therapy.

P012 The administration of mm-miR-378a-3p exacerbates chronic inflammation and fibrosis in a murine intestinal model

Abstract Background Fibrosis constitute an important complication of CD. MicroRNAs (miRNAs), which are small RNA molecules that regulate gene expression, have been shown to participate in the molecular interactions of both inflammation and fibrosis. Lower levels of miR-378a-3p have been reported associated to murine liver fibrosis (Hyun et al. DOI: 10.1038/ncomms10993) and we analyze here the relevance of miR-378a-3p on intestinal fibrosis. Methods B57BL/6 mice were intravenously injected with 2,5mg/kg of negative control (NC) or mm-miR-378a-3p mimic, twice a week and received vehicle or Dextran Sulfate Sodium (DSS) for 2 cycles (7 days drinking DSS 2% in water solution followed by 10 days drinking water). Body weight and DAI score was obtained every day and the colon was collected after sacrifice. Sirius and hematoxylin-eosin dyes were employed to determine the fibrosis and structural state in 5µm slides of intestinal tissue. Gene expression and miRNA profiles were analyzed by RT-qPCR. Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were transfected with 20nM of NC or hsa-miR-378a-3p mimic during 24h. Results No significant changes in body weight, DAI score, and colon length were detected between mice receiving NC and those receiving mimic, all along the two DSS cycles. Colon of mice treated with DSS, exhibited a significant diminution in the mRNA expression of mm-miR-378a-3p compared with naïve samples. In DSS-treated mice, the iv administration of the mimic compared with the NC: a) significantly increased levels of miR-378a-3p in the colon; b) increased the number of neutrophils, and heightened changes in the glandular epithelia and architectural distortion (figure 1a) C) decreased the number of lymphoid follicles (figure 1a); d) slightly increased collagen deposition, as analyzed by sirius red (figure 1b) and e) significantly increased the mRNA expression of Tgfb1, Il1b, and Mmp2. Treatment of human intestinal fibroblasts with hsa-miR-378a-3p mimic did not significant modify the mRNA expression of markers of fibrosis but it significantly increased the mRNA expression of two antiapoptotic molecules BCL2 and MCL1. Conclusion Levels of mm-miR-378a-3p are diminished in a murine model of intestinal fibrosis; The exogenous administration of miR-378a-3p, increased the acute inflammatory response and the expression of fibrosis markers in murine fibrotic colon and the expression of antiapoptotic molecules in human intestinal fibroblasts.

Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

Background: B-cell acute lymphoblastic leukemia (ALL) harboring KMT2A-rearrangements (KMT2A-R) is a high-risk ALL subtype with significant relapse risk and poor overall survival across the age spectrum. While new antibody-based and cellular immunotherapies have revolutionized the therapeutic paradigm for many patients with relapsed ALL, application of these therapies has proven particularly challenging to date in infants given their immune system immaturity and in older adults prone to high-grade immunotherapy toxicities. Unlike other ALL subtypes, KMT2A-R ALL has an extremely low frequency of co-mutations, which makes it particularly difficult to find new treatment options. To identify targetable kinases in KMT2A-R ALL, we performed a domain-specific kinome-wide CRISPR screen and identified the serine/threonine kinase DYRK1A as required for the survival of KMT2A-R ALL. Importantly, DYRK1A appears to be non-essential for normal tissues per the Cancer Dependency MAP, suggesting potential for a therapeutic window via DYRK1A targeting. Results:In vitro treatment studies using three different menin inhibitors (MI-2-2 / MI-503 / VTP50469) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A at the RNA and the protein level. We further analyzed ChIP-Seq data and identified that oncogenic KMT2A-R directly binds to the DYRK1A promoter, indicating that DYRK1A is directly regulated by the oncogenic KMT2A fusion. Interestingly, the DYRK1A RNA and protein levels were elevated in KMT2A-R ALL cell lines and patient-derived xenograft (PDX) samples compared to non-KMT2A-R ALL cases. To test the consequence of direct pharmacologic DYRK1A inhibition on cell proliferation and apoptosis, we treated KMT2A-R ALL cells with the DYRK1A inhibitors EHT1610 and harmine. Interestingly, we observed that pharmacologic inhibition of DYRK1A with both drugs did not induce apoptosis but induced potent leukemic cell growth inhibition in vitro and in vivo. In preliminary studies, we further observed that pharmacologic inhibition of DYRK1A or genetic deletion resulted in MYC upregulation and hyperphosphorylation of ERK. It has been demonstrated that premature signaling activation in developing B cells induces cell cycle arrest as a protective mechanism to prevent the development of autoimmune diseases. We hypothesized that DYRK1A inhibitor-induced ERK hyperactivation and consequent cell cycle arrest remains a conserved mechanism in KMT2A-R ALL. Supporting our hypothesis, combining DYRK1A inhibitors with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibition does not induce apoptosis and cells restart to proliferate after DYRK1A inhibitor withdrawal, we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. Interestingly, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. BIM is a pro-apoptotic molecule that is inactive when bound by the anti-apoptotic molecule BCL2. Given that DYRK1A inhibition results in MYC upregulation, we performed a new Western blot analysis and validated that EHT1610 and harmine both induce BIM overexpression in KMT2A-R ALL cell lines. To test if combined DYRK1A and BCL2 inhibition will lead to enhanced KMT2A-R ALL apoptosis, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, combining both drugs synergistically killed KMT2A-R ALL cells in vitro. More importantly, we validated the anti-leukemic effect of harmine in combination with venetoclax in mice injected with a KMT2A-R PDX case. Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ammonia-induced excess ROS causes impairment and apoptosis in porcine IPEC-J2 intestinal epithelial cells

Ammonia is one of the most important toxic metabolites in the intestine of animals. It can cause intestinal damage and associated intestinal diseases through different endogenous or exogenous stimuli. However, the definition of harmful ammonia concentration and the molecular mechanism of ammonia - induced intestinal epithelial injury remain unclear. In this study, we found that the viability of porcine IPEC-J2 intestinal epithelial cells significantly decreased with the increase of NH4Cl dose (20–80 mM). Ammonia (40 mM NH4Cl) increased the expression level of ammonia transporter RHCG and disrupted the intestinal barrier function of IPEC-J2 cells by reducing the expression levels of the tight junction molecules ZO-1 and Claudin-1. Ammonia caused elevated levels of ROS and apoptosis in IPEC-J2 cells. This was manifested by decreased activity of antioxidant enzymes SOD and GPx, decreased mitochondrial membrane potential, and increased cytoplasmic Ca2+ concentration. In addition, the expression levels of apoptosis-related molecules Caspase-9, Caspase-3, Fas, Caspase-8, p53 and Bax were increased, the expression level of anti-apoptotic molecule Bcl-2 was decreased. Moreover, the antioxidant NAC (N-acetyl-L-cysteamine) effectively alleviated ammonia-induced cytotoxicity, reduced ROS level, Ca2+ concentration, and the apoptosis of IPEC-J2 cells. The results suggest that ammonia-induced excess ROS triggered apoptosis through mitochondrial pathway, death receptor pathway and DNA damage. This study can provide reference and theoretical basis for the definition of harmful ammonia concentration in pig intestine and the effect and mechanism of ammonia on pig intestinal health.

Effect of Extracellular Signal-Regulated Protein Kinase 5 Inhibition in Clear Cell Renal Cell Carcinoma

(1) Background: Extracellular signal-regulating kinase 5 (ERK5) has been implicated in many cellular functions, including survival, proliferation, and vascularization. Our objectives were to examine the expression and effect of ERK5 in clear cell renal cell carcinoma (ccRCC). (2) Methods: The expressions of ERK5 and its regulating micro-RNA miR-143 were investigated using immunohistochemistry and quantitative reverse transcriptase PCR in surgical specimens of ccRCC patients. With invitro and in vivo studies, we used pharmacologic ERK5 inhibitor XMD8-92, RNA interference, pre-miR-143 transduction, Western blotting, MTS assay, apoptosis assay, and subcutaneous xenograft model. (3) Results: A strong ERK5 expression in surgical specimen was associated with high-grade (p = 0.01), high-recurrence free rate (p = 0.02), and high cancer-specific survival (p = 0.03). Expression levels of ERK5 and miR-143 expression level were correlated (p = 0.049). Pre-miR-143 transduction into ccRCC cell A498 suppressed ERK5 expression. ERK5 inhibition enhanced cyclin-dependent kinase inhibitor p21 expression and decreased anti-apoptotic molecules BCL2, resulting in decreased cell proliferation and survival both in ccRCC and endothelial cells. In the xenograft model, ERK5 inhibitor XMD8-92 suppressed tumor growth. (4) Conclusions: ERK5 is regulated by miR-143, and ERK5 inhibition is a promising target for ccRCC treatment.

P53-Related Anticancer Activities of Drimia calcarata Bulb Extracts Against Lung Cancer.

Current lung cancer treatment strategies are ineffective, and lung cancer cases continue to soar; thus, novel anticancer drugs and targets are needed, and medicinal plants are promising to offer better alternatives. This study was aimed at analysing two p53 splice variants during the potential anticancer activities of Drimia calcarata (Dc) methanol and water extracts against different human lung cancer cell lines of varying p53 mutation status, and these included mutant H1573 and mutant H1437 and p53-wild type (A549) cells. The anticancer activities of the Dc extracts were assessed by establishing the cytotoxic effect and the apoptosis-inducing capacity of these extracts, using the MTT assay and Annexin V analysis, respectively, with the latter confirmed using fluorescence microscopy. The molecular mechanisms induced by these extracts were further evaluated using cell cycle analysis and RT-PCR. Both extracts demonstrated safety against noncancerous lung MRC-5 fibroblasts and exhibited significant anticancer potency (p < 0.001) against the H1437 (IC50 values: 62.50μg/ml methanol extract and 125μg/ml WE), H1573 (IC50 value: 125μg/ml for both extracts) and A549 (IC50 value: 500μg/ml ME). The water extract had no effect on the viability of A549 cells. Treated H1437 cells underwent p53-dependent apoptosis and S-phase cell cycle arrest while H1573 treated cells underwent p53-independed apoptosis and G0/G1 cell cycle arrest through upregulation of p21 mRNA expression levels. The expression levels of STAT1, STAT3, STAT5A and STAT5B genes increased significantly (p < 0.001) following the treatment of H1573 cells with ME and WE. Treatment of H1437 cells with ME upregulated the STAT1, STAT3, STAT5A and STAT5B mRNAs. Our results indicate that the proliferative inhibitory effect of D. calcarata extracts on A549 and H1573 cells is correlated with the suppression of Bcl-2, STAT3 and STAT5B while that is not the case in H1437 cells. Thus, our results suggest that the dysregulation of anti-apoptotic molecules Bcl-2, STAT3, STAT5A and STAT5B in H1437 may play a role in cancer cell survival, which may consequently contribute to the development of p53-mutated non-small human lung cancer. Our results indicate that D. calcarata is a promising source of anticancer agents for the treatment of p53-mutant human non-small lung cancer cells than the p53-wild type human non-small lung cancer cells.