scholarly journals Exosomal ERp44 derived from ER-stressed cells strengthens cisplatin resistance of nasopharyngeal carcinoma

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tian Xia ◽  
Hui Tian ◽  
Kaiwen Zhang ◽  
Siyu Zhang ◽  
Wenhui Chen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nasopharyngeal Carcinoma ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Cisplatin Resistance ◽  
Flow Cytometric Analysis ◽  
Platinum Drug ◽  
Platinum Based Chemotherapy ◽  
The Impact

Abstract Background Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in head and neck. Platinum-based chemotherapy is an important treatment for NPC. However, the molecular mechanism of resistance to platinum drug remains unknown. Endoplasmic reticulum resident protein 44(ERp44), an unfolded protein response (UPR)-induced endoplasmic reticulum(ER) protein, is induced during ER stress. This research explored the mechanism of ERp44 in strengthening cisplatin resistance in NPC. Methods Western blot and immunohistochemistry were used to investigate the expression of ERp44 and Glucose-Regulated Protein 78(GRP78) in NPC. We took CCK8 to detect the role of ERp44 on cell chemosensitivity. Flow cytometric analysis and western blot were taken to analyze cell apoptosis. We performed differential centrifugation to isolate exosomes from serum or conditioned media of cells and analyzed the impact of exosomal ERp44 on cells cisplatin sensitivity. Finally, the results were confirmed in vivo. Results We found the increased expression of ERp44 and GRP78 in NPC and ERp44 was highly expressed in ER-stressed tissues. Cell proliferation was inhibited after cisplatin treatment when ERp44 was knocked down and ERp44 strengthened cisplatin resistance by influencing cell apoptosis and pyroptosis. Then we also collected exosomes and cell viability was increased after the addition of NPC-derived-exosomes with cisplatin treatment. More importantly, our results showed under ERS, NPC cells secreted exosomes containing ERp44 and could transfer them to adjacent cells to strengthen chemoresistance. Conclusion Our data suggested that exosomal ERp44 derived from ER-stressed NPC cells took an inevitable role in NPC chemoresistance and might act as a treatment target.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

MicroRNA-126 attenuates cell apoptosis by targeting TRAF7 in acute myeloid leukemia cells

2018 ◽  
Vol 96 (6) ◽  
pp. 840-846 ◽  
Author(s):  
Qian Ding ◽  
Qing Wang ◽  
Yi Ren ◽  
Hong Qian Zhu ◽  
ZhuYun Huang
Keyword(s):  
Acute Myeloid Leukemia ◽  
Western Blot ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Myeloid Leukemia ◽  
Flow Cytometric Analysis ◽  
Tumor Necrosis Factor Receptor ◽  
Luciferase Assay ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Acute myeloid leukemia (AML) has a 5-year survival rate of only about 30%–40% due to the self-renewal and differentiation ability of leukemia stem-like cells (LSCs). To address the potential for novel therapeutic targets in LSCs, we investigated the roles of miRNA-126 and tumor necrosis factor receptor-associated factor 7 (TRAF7) in AML. We used qRT-PCR and Western blot to investigate the expression levels of miRNA-126 and TRAF7 in AML cell lines. Then, we uncovered the effect of miRNA-126 on AML cell proliferation and apoptosis by MTT assay and flow cytometric analysis, respectively. Furthermore, dual-luciferase assay and Western blot were used to determine the target of miRNA-126 in AML and the potential mechanism by which cell apoptosis is suppressed by miRNA-126. We found that miRNA-126 was highly expressed in all of the AML cell lines, and that inhibition of miRNA-126 significantly induced cell death through apoptosis. The suppression of apoptosis in AML with high expression of miRNA-126 was caused by down-regulating TRAF7, which blocked the c-FLIP pathway. The role of miRNA-126 in AML makes it a potential therapeutic target to improve clinical outcomes for patients with AML.

scholarly journals MiR-130a-3p inhibits the viability, proliferation, invasion, and cell cycle, and promotes apoptosis of nasopharyngeal carcinoma cells by suppressing BACH2 expression

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Xin Chen ◽  
Bo Yue ◽  
Changming Zhang ◽  
Meihao Qi ◽  
Jianhua Qiu ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Down Regulation ◽  
Tissue Samples ◽  
Mesenchymal Transition

The aim of the present study was to explore the mechanism through which miR-130a-3p affects the viability, proliferation, migration, and invasion of nasopharyngeal carcinoma (NPC). Tissue samples were collected from the hospital department. NPC cell lines were purchased to conduct the in vitro and in vivo assays. A series of biological assays including MTT, Transwell, and wound healing assays were conducted to investigate the effects of miR-130a-3p and BACH2 on NPC cells. MiR-130a-3p was down-regulated in both NPC tissues and cell lines, whereas BACH2 was up-regulated in both tissues and cell lines. MiR-130a-3p overexpression inhibited NPC cell viability, proliferation, migration, and invasion but promoted cell apoptosis. The converse was true of BACH2, the down-regulation of which could inhibit the corresponding cell abilities and promote apoptosis of NPC cells. The target relationship between miR-130a-3p and BACH2 was confirmed. The epithelial–mesenchymal transition (EMT) pathway was also influenced by miR-130a-3p down-regulation. In conclusion, miR-130a-3p could bind to BACH2, inhibit NPC cell abilities, and promote cell apoptosis.

Murine Platelets Expressing a Polymorphism of Integrin β3 Associated with Acute Coronary Thrombosis in Humans.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3896-3896
Author(s):  
David A. Wilcox ◽  
Juan Fang ◽  
Bryon D. Johnson
Keyword(s):  
Platelet Function ◽  
Flow Cytometric Analysis ◽  
Thrombotic Complication ◽  
Homogeneous Population ◽  
Integrin Αiibβ3 ◽  
Integrin Β3 ◽  
Flow Cytometric ◽  
The Impact ◽  
Human Integrin

Abstract Inheritance of a naturally occurring polymorphism of the human integrin β3-subunit at amino acid 33 causes a change from Leu to Pro that has been implicated as a risk factor for thrombotic complication in humans. It is unclear; however, if Pro (also known as the platelet alloantigen PlA2 form of β3) can alter integrin activation to significantly affect platelet function in vivo, since some clinical studies do not corroborate the initial findings. Since PlA2 is a frequent variant of β3 expressed on 28% of platelets from Americans the impact of this genetic variation on platelet function could have widespread clinical relevance; therefore, we developed a murine model to determine if expression of this common polymorphism of human integrin β3 can increase platelet function and lead to an increased propensity for thrombosis within a homogeneous population of mice. This model eliminates issues arising from variation in the genetic make-up, physical environment, and/or lifestyles of humans and allows for in vivo analysis not possible with human subjects. To accomplish this, cDNA encoding each form of the human β3-subunit was subcloned into an HIV type-1 lentivirus-derived vector under the transcriptional control of the human αIIb gene promoter to direct synthesis of β3 specifically to the megakaryocyte lineage. Bone marrow was isolated from β3-deficient mice and transduced with β3 virions encoding either the PlA1 or PlA2 form of human β3, and then transplanted into lethally irradiated β3-deficient littermates. Flow cytometric analysis demonstrated stable expression of the hybrid murine/human αIIbβ3 integrin complex on the surface of circulating platelets. Immunoanalysis using monoclonal antibodies and human serum that react specifically with the PlA1 or PlA2 confirmed the identity of each alloantigen of human β3. The lentivirus contained a second transgene encoding a drug-selectable marker (P140K MGMT) that was used for in vivo enrichment of transduced cells in mice treated with two regimens of cytotoxic reagents, O6-BG/BCNU. Flow cytometric analysis showed that use of drug-selection resulted in increased expression of the integrin αIIbβ3 complex to equal levels on nearly 100% of platelets using either form of human β3. Similar to platelets from normal mice, platelets expressing each PlA form of β3 could be induced to form aggregates ex vivo upon treatment with a cocktail of physiological agonists of platelet activation (adenosine diphosphate, epinephrine and the thrombin receptor activating peptide). These results demonstrate the feasibility for targeting expression of altered forms of the human integrin β3-subunit to murine platelets and pave the way for future studies to examine and compare the effect of integrin αIIbβ3 structure on platelet function and thrombosis in vivo.

scholarly journals Hoxblinc Is Aberrantly Expressed in Acute Myeloid Leukemia and Functions As a Potent Oncogenic Long Non-Coding RNA in Leukemogenesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 886-886
Author(s):  
Ganqian Zhu ◽  
Huacheng Luo ◽  
Shi Chen ◽  
Qian Lai ◽  
Ying Guo ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Three Dimensional ◽  
Donor Cell ◽  
Lower Percentage ◽  
Leukemic Transformation ◽  
Aberrant Expression ◽  
The Impact

Abstract Aberrant expression of long non-coding RNAs (lncRNAs) might contribute to the development and progression of leukemia. However, functional studies on the actual role of lncRNAs during the development of leukemia remain scarce, and very few lncRNAs have been shown to be involved in leukemogenesis. HoxBlinc is an anterior HoxB gene-associated intergenic lncRNA. It is a cis-acting lncRNA and functions as an epigenetic regulator to coordinate anterior HoxB gene expression. Giving the dysregulation of HOXA/B genes is a dominant mechanism of leukemic transformation, HoxBlinc might be an oncogenic lncRNA of leukemia. To determine whether HOXBLINC lncRNA is aberrantly expressed in human AML samples, we performed RT-qPCR on bone marrow mononuclear cells (BMMNCs) from a cohort of 73 AML patients. A dramatic up-regulation of HOXBLINC was observed in over 60% of the patients. When TCGA-AML datasets of a cohort of 179 AML patients were analyzed for their HOXBLINC expression, a significant portion of these AML patients had high levels of HOXBLINC expression. Interestingly, AML patients with high HOXBLINC expression (the top thirty percentile of patients) had a significantly shortened survival as compared to patients with low HOXBLINC expression (the bottom thirty percentile). To investigate the impact of HoxBlinc overexpression on normal hematopoiesis and the pathogenesis of hematological malignancies in vivo, we generated a HoxBlinc transgenic(Tg) mouse model. Within 1 year of age, 67% of the HoxBlincTg mice (10 of 15) died or were sacrificed because of a moribund condition due to AML. We then assessed whether overexpression of HoxBlinc affects the pools of HSC/HPCs by flow cytometric analysis on the BM cells of young WT and HoxBlincTg mice (8-10 weeks of age). HoxBlincTg BM had a dramatically greater number of LT-HSC, ST-HSC, MPP cells, and a significantly higher percentage of GMP, but a lower percentage of MEP/CMP cell populations as compared to WT group. To determine the effect of HoxBlinc overexpression on the function of HSC/HPCs, we performed paired-daughter cell assay, replating assay and liquid culture on sorted LT-HSC, LSK or LK cells from young WT and HoxBlincTg mice, the results indicate that transgenic expression of HoxBlinc enhances HSC self-renewal and impairs HSC/HPC differentiation. To assess whether HoxBlinc overexpression-mediated changes in HSC/HPC function are cell-autonomous, we performed competitive transplantation assays to examine the repopulating capacity of HoxBlincTg BM cells. When the donor cell chimerism was analyzed kinetically in the PB of recipient mice, the CD45.2 cell population remained ~50% in mice receiving WT BM cells, whereas the CD45.2 chimerism in the recipients transplanted with HoxBlincTg BM cells steadily increased. Interestingly, mice receiving HoxBlincTg BM cells developed AML at 2-6 months after transplantation. Previous data reported that HoxBlinc can recruit the Setd1a/Mll1 histone H3K4 methyltransferase complex to mediate formation of the active topologically associated domain (TAD) in the anterior HoxB locus for transcription of the anterior HoxB genes. In this study, LSK or LK cells sorted from young WT and HoxBlincTg mice were analyzed by RNA-seq, ATAC-seq, H3K4me3 CHIP-seq and 4C analysis. Mechanistically, HoxBlinc overexpression alters HoxB locus chromatin three-dimensional organization to enhance enhancer/promoter chromatin accessibility and coordinate the expression of not only HoxB1-5 but also HoxA9, Runx1, Meis1 and so on, which are critical genes for HSC regulation and/or leukemogenesis. Our study provides novel insights into the HSC regulation by lncRNAs and identifies HOXBLINC, which coordinates to maintain an oncogenic transcription program for leukemic transformation, as a potent oncogenic lncRNA in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals MEG3 promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-361-5p/FOXO1 axis

2019 ◽  
Author(s):  
Anying Wang ◽  
Naixia Hu ◽  
Yefeng Zhang ◽  
Yuanzhen Chen ◽  
Changhui Su ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Non Coding Rna ◽  
Cartilage Cells ◽  
Osteoarthritis Chondrocytes ◽  
Oa Chondrocytes

Abstract Background: This study aimed to investigate the role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) and related molecular mechanisms, in osteoarthritis (OA). Methods: Cartilage tissues of OA patients and healthy volunteers were isolated and cultured. After transfection with the appropriate construct, chondrocytes were classified into Blank, pcDNA3.1-NC, pcDNA3.1-MEG3, si-NC, si-MEG3, pcDNA3.1-NC + mimics NC, pcDNA3.1-MEG3 + mimics NC, pcDNA3.1-NC + miR-361-5p mimics and pcDNA3.1-MEG3 + miR-361-5p mimics groups. qRT-PCR was used to detect the expression of MEG3, miR-361-5p and FOXO1 . Western blot, luciferase reporter assay, RIP, CCK-8, and flow cytometry analysis were performed to reveal the morphology, proliferation, and apoptotic status of cartilage cells. Histological analysis and immunostaining were conducted in the OA rat model. Results: Expression of MEG3 and FOXO1 was significantly decreased in OA compared with the normal group, while the expression of miR-361-5p was increased. MEG3 might serve as a ceRNA of miR-361-5p in OA chondrocytes. Moreover, using western blot analyses and the CCK-8 assay, MEG3 was shown to target miR-361-5p/FOXO1, elevate cell proliferation, and impair cell apoptosis. Functional analysis in vivo showed that MEG3 suppressed degradation of the cartilage matrix. Conclusion: MEG3 can contribute to cell proliferation and inhibit cell apoptosis and degradation of extracellular matrix (ECM) via the miR-361-5p/FOXO1 axis in OA chondrocytes.

scholarly journals Imeglimin Ameliorates β-cell Apoptosis by Modulating the Endoplasmic Reticulum Homeostasis Pathway

2021 ◽  
Author(s):  
Jinghe Li ◽  
Ryota Inoue ◽  
Yu Togashi ◽  
Tomoko Okuyama ◽  
Aoi Satoh ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Survival ◽  
Cell Apoptosis ◽  
Cell Function ◽  
Cell Mass ◽  
Β Cells ◽  
Β Cell ◽  
Akita Mice ◽  
The Impact

The effects of imeglimin, a novel anti-diabetes agent, on β-cell function remain unclear. Here, we unveiled the impact of imeglimin on β-cell survival. Treatment with imeglimin augmented mitochondrial function, enhanced insulin secretion, promoted β-cell proliferation, and improved β-cell survival in mouse islets. Imeglimin upregulated the expression of endoplasmic reticulum (ER)-related molecules including <i>Chop (Ddit3),</i> <i>Gadd34</i> (<i>Ppp1r15a</i>), <i>Atf3</i>, and <i>Sdf2l1</i>, and decreased eIF2α phosphorylation, after treatment with thapsigargin, and restored global protein synthesis in β-cells under ER stress. Imeglimin failed to protect ER stress-induced β-cell apoptosis in CHOP-deficient islets or in the presence of GADD34 inhibitor. Treatment with imeglimin showed a significant decrease in the number of apoptotic β-cells and increased β-cell mass in Akita mice. Imeglimin also protected against β-cell apoptosis in both human islets and human pluripotent stem cell (<a>hPSC)-derived β-like cells</a>. <a>Taken together, imeglimin modulates ER homeostasis pathway, which results in the prevention of β-cell apoptosis both <i>in vitro</i> and <i>in vivo</i>.</a>

scholarly journals Mel Aggravates Pyroptosis Induced by Temozolomide Through Inhibiting NRF2-are Pathway in Glioblastoma

2020 ◽  
Author(s):  
Hao Pan ◽  
Handong wang ◽  
Qiang Wang ◽  
Wenhao Niu ◽  
Qi Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Combined Treatment ◽  
Flow Cytometric Analysis ◽  
Glioma Cells ◽  
Mtor Pathway ◽  
Tumor Growth Inhibition ◽  
Dependent Manner ◽  
Related Protein

Abstract Background:Glioblastoma(GBM) is a common malignant tumor of the brain. It has been verified that melatonin(MEL) can inhibit glioma proliferation. But its mechanism has not been fully elucidated. We intend to examine the mechanism of MEL on glioma cells from the perspective of pyroptosis and Nrf2. Methods:Expression of MEL receptor in glioma was detected by western blot. GBM cell viability treated with temozolomide(TMZ) plus MEL was detected by CCK-8. Pyroptosis rate was determinate by flow cytometric analysis. Western blot was used to detect the Nrf2 and pyroptosis related protein level after MEL treatment. Orthotopic tumor growth inhibition study was performed to further investigate the tumor inhibition effect of TMZ plus MEL.Results:We first confirmed MEL receptor was abundant in glioma tissue and cell lines. After combined treatment of TMZ and MEL, cell viability decreased significantly as compared to those of TMZ treatment alone. Also, the ratio of pyroptosis and ROS level increased, followed by elevated expression of pyroptosis related protein. Furthermore, MEL can induce a diminution of Nrf2 expression in glioma in dose- and time-dependent manner. TMZ can increase Nrf2-ARE pathway expression, which also can be deprived by MEL. Its inhibition of Nrf2 depends on dephosphorylation of IGF-1/AKT/mTOR pathway. More importantly, after overexpression of Nrf2 in glioma cells, the level of pyroptosis-related protein elevated by MEL decreased, suggesting that the effect of MEL on promoting pyroptosis is dependent on its inhibition of Nrf2. In vivo results further confirmed that MEL plus TMZ induced significantly decreased tumor size and increased pyroptosis rate, but had no significant effect on mouse body weight, ALT, AST.Conclusion:MEL can inhibit the phosphorylation of IGF-1/AKT/mTOR pathway at millimol level, which further reduces the expression of Nrf2 and promotes pyroptosis of glioma cells. Considering the modest efficacy of TMZ chemotherapy, MEL can be considered as a potential chemotherapy sensitizer to improve the chemotherapy effect of glioma.

scholarly journals PTEN mutant NSCLC require ATM to suppress pro-apoptotic signalling and evade radiotherapy

2021 ◽  
Author(s):  
Thomas Fischer ◽  
Oliver Hartmann ◽  
Michaela Reissland ◽  
Cristian Prieto-Garcia ◽  
Kevin Klann ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dna Damage ◽  
Western Blot ◽  
Colony Formation ◽  
Ex Vivo ◽  
Radiation Sensitivity ◽  
Genetic Alterations ◽  
Altered Expression ◽  
The Impact

Background: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy. Methods: Using CRISPR genome editing, we deleted PTEN in a human tracheal stem cell-like cell line as well generated primary murine NSCLC, proficient or deficient for Pten, in vivo. These models were used to verify the impact of PTEN loss in vitro and in vivo by immunohistochemical staining, western blot and RNA-Sequencing. Radiation sensitivity was assessed by colony formation and growth assays. To elucidate putative treatment options, identified via the molecular characterisation, PTEN pro- and deficient cells were treated with PI3K/mTOR/DNA-PK-inhibitor PI-103 or the ATM-inhibitors KU-60019 und AZD 1390. Changes in radiation sensitivity were assessed by colony-formation assay, FACS, western-blot, phospho-proteomic mass spectrometry and ex vivo lung slice cultures. Results: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model. Conclusion: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.

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