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Objective: To investigate the effect of glycolytic enzyme pyruvate kinase type 2 (PKM2) on the proliferation and apoptosis of human leukemia HL-60 cells., Methods: si-PKM2 plasmid was transfected into HL-60 cells (set as si-PKM2 group), and blank vector transfected cells were set as control group (si-Ctl group). The expression levels of PKM2 mRNA and protein in si-Ctl group and si-PKM2 group were detected by RT-qPCR and Western blot. CCK-8 cell detection kit was used to detect the proliferation ability of the cells in the two groups. Flow cytometry was used to detect the changes of cell cycle and apoptosis. Western blot and RT-qPCR were used to detect the changes of p-Akt and p-mTOR protein levels in PI3K/Akt/mTOR signaling pathway and the changes of glycolysis-related mRNA levels of the cells in the two groups. The changes in glucose consumption and lactic acid production of the cells were assayed. Over expressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002 or galactose, the changes in cell proliferation ability, cell cycle and apoptosis, as well as changes in glucose consumption and lactic acid production were detected., Results: Interfered by si-PKM2, mRNA and protein levels of PKM2 in si-PKM2 group significantly decreased, and proliferation ability of the cells was also reduced (P<0.05). After PKM2 knockdown, the cells were significantly blocked at G 1 phase, and cell apoptosis was obviously induced (P<0.05). p-Akt and p-mTOR levels were lower in si-PKM2 group than those in si-Ctl group. The glucose consumption and lactic acid production significantly decreased in si-PKM2 cells. Overexpressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002. The glucose consumption and lactate acid production induced by overexpressed PKM2 were reduced. Overexpressed PKM2, HL-60 cells showed no significant changes in cell proliferation, cycle and apoptosis when cultured with galactose., Conclusion: PKM2 knockdown can inhibit the proliferation and induce apoptosis of HL-60 cells, and its molecular mechanism may be related to the PKM2-mediated PI3K/Akt/mTOR-glycolysis, which suggesting that PKM2 may serve as a molecular target for the prevention and treatment of leukemia.

Objective: To investigate the effects of inhibition of lncRNA PVT1 on the proliferation, apoptosis and oxidative stress of vascular endothelial cells induced by hyperglycemic. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into four groups: control group (5.5 mmol/L glucose), high glucose group (30 mmol/L glucose), high glucose + siNC group (30 mmol/L glucose +siNC, negative control group), HG + siPVT1 group (30 mmol/L glucose + siPVT1, lncRNA PVT1 silencing group). The expression of PVT1 after transfection was detected by quantitative real-time PCR. MTT assay was used to detect the effect of siPVT1 (small interfering RNA PVT1) on the proliferation of HUVECs cells induced by high glucose. Flow cytometry was used to detect ROS and apoptosis of HUVECs cells induced by siPVT1. Western blot was used to detect the expression levels of apoptotic proteins such as Bax, Bcl-2, and cleaved caspase-3 in HUVECs cells. Results: Compared with the control group, after transfection with siPVT1, the expression level of PVT1 was decreased significantly ( P ＜0.05). MTT results showed that the proliferation activity of HUVECs cells in the high-glucose group was reduced significantly after 24 h and 48 h. Compared with the HG + siNC group, the proliferation activity of HUVECs cells in the HG + siPVT1 group was increased significantly ( P ＜0.05) after 24 h and 48 h. Flow cytometry results showed that ROS and apoptosis rate of HUVECs cells in the high-glucose group were increased significantly compared with the control group. Compared with the HG + siNC (negative control) group, ROS and apoptosis rates of HUVECs cells in the HG + siPVT1 group were reduced significantly. Compared with the control group, the expression levels of cleaved-caspase-3 and Bax in the high-glucose group were significantly up-regulated, while the expression level of Bcl-2 was down-regulated. Compared with the HG + siNC group, the expression levels of cleaved-caspase-3 and Bax were down-regulated, and the expression level of Bcl-2 was up-regulated. The differences were statistically significant ( P ＜0.05). Conclusion: Inhibition of lncRNA PVT1 can significantly increase the proliferation activity of HUVECs cells induced by hyperglycemia, reduce oxidative stress and inhibit cell apoptosis.

Background: Therapeutic failure and drug resistance are common and have important implications in the poor prognosis of advanced breast cancer. It is necessary to acquire a natural product to overcome the resistance of cancer and increase the sensitivity of drug-resistant cells to anticancer agents., Purpose: To demonstrate whether the compound Vielanin K (VK) has the potential to increase the sensitivity of MCF-7 and MCF-7/MDR cells to anticancer agents., Methods: Cell viability and proliferative capacity were determined by MTT, colony formation and EdU assays. Apoptosis and Ca 2+ accumulation were evaluated by flow cytometry. Then, proteins were detected by immunoblotting, and gene expression levels were explored by qRT-PCR., Results: In MCF-7 and corresponding MDR cells, VK increased the fluorescence intensity of Rho123, but not CFDA. VK treatment did not affect the protein expression of P-gp, MRP1 or BCRP. VK treatment enhanced the DOX-induced apoptotic cascade, while VK combined with DOX increased JNK phosphorylation by activating the IRE1α-TRAF2 signaling pathway. In addition, Ca 2+ was released from the endoplasmic reticulum following combination treatment, thereby giving rise to mitochondrial apoptosis. Silencing IRE1α and JNK with small interfering RNA (siRNA) efficiently attenuated combination treatment-induced apoptosis. These effects caused mitochondrial depolarization and reduced viability in MCF-7 and corresponding MCF-7/MDR cells., Conclusion: VK combined with DOX increases the apoptosis of MCF-7 and corresponding MCF-7/MDR cells by activating ER stress and mitochondrial apoptosis via IRE1α-TRAF2-JNK signaling., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing financial interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Increased activity of the PI3K/AKT/mTOR pathway has been observed in chronic myeloid leukemia (CML). Morin, a kind of flavonoid, exhibits a significant anticancer activity by suppressing the PI3K/AKT signaling pathway. However, the effect of morin on CML and its underlying mechanisms is poorly understood. Here, we found that morin dose dependently inhibited the proliferation of CML cell lines K562 and KCL22 and induced their apoptosis, with a significant increase in cell apoptosis upon exposure of cells to 50 μmol/L morin. Moreover, morin significantly reduced CML xenograft growth in nude mice. Mechanically, morin attenuated phosphorylated AKT level by upregulating PTEN expression, thus leading to the inhibition of AKT signaling. Knockdown of PTEN by its siRNA completely abrogated morin-induced cell apoptosis, indicating that PTEN mediates the inductive effect of morin on CML cell apoptosis. More importantly, we found that miR-188-5p was significantly upregulated in CML patients and CML cell lines. Treating CML cells with morin markedly downregulated the miR-188-5p expression level. Further, we demonstrated that miR-188-5p repressed PTEN expression by directly targeting its 3'-UTR. miR-188-5p downregulation induced by morin enhanced CML cell apoptosis by relieving miR-188-5p repression of PTEN expression. In summary, morin exerts significant anticancer efficacy in CML by regulating the miR-188-5p/PTEN axis and thus repressing the PI3K/AKT signaling., (©2019 American Association for Cancer Research.)

Melatonin, a neurohormone secreted by the pineal gland, has a variety of biological functions, such as circadian rhythms regulation, anti-oxidative activity, immunomodulatory effects, and anittumor, etc. At present, its antitumor effect has attracted people's attention due to its extensive tissue distribution, good tissue compatibility, and low toxic and side effects. In the gastrointestinal tract, there is high level of melatonin and many studies showed melatonin has effects of anti-gastric cancer. In this experiment, human gastric cancer cell lines AGS and MGC803 were used to investigate the intracellular molecular mechanism of melatonin against gastric cancer. After AGS and MGC803 have been treated with melatonin, the changes of cell morphology and cellular structure were observed under electron microscope. Flow cytometer and apoptosis detection kits were used to analyze the effect of apoptosis on AGS and MGC803. The alterations of apoptosis-related proteins Caspase 9, Caspase 3, and upstream regulators AKT, MDM2 including expression, phosphorylation, and activation were detected to analyze the intracellular molecular mechanism of melatonin inhibiting gastric cancer. In AGS and MGC803 cells with melatonin exposure, cleaved Caspase 9 was upregulated and Caspase 3 was activated; moreover, MDM2 and AKT expression and phosphorylation were downregulated. Melatonin promoted apoptosis of AGS and MGC803 cells by the downregulation of AKT and MDM2. Anat Rec, 302:1544-1551, 2019. © 2019 American Association for Anatomy., (© 2019 American Association for Anatomy.)

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine-rich repeat-containing G protein-coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β-geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4 +/- mice at E16.5 by β-gal staining, and Lgr4-deficiency promoted BMSC proliferation (16 ± 4 in wild-type [WT] and 28 ± 2 in Lgr4 -/- ) using colony forming units-fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4 -/- ) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4-regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4-inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs-delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm-derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy., (© 2018 Wiley Periodicals, Inc.)

Artemisianins A-D (1-4), four stereoisomers of sesquiterpenoid dimers, forming via [4+2] cycloaddition from a 1, 10-seco-guaianolide dienophile and a guaianolide diene, along with two biosynthetically related precurors 5 and 6, were isolated from the famous traditional Chinese medicine Artemisia argyi. The structures of 1-4, including their absolute configurations, were elucidated by extensive spectroscopic data and ECD/TDDFT calculation analysis. Compounds 1-4 exhibited cytotoxicity with IC 50 values ranging from 7.2 to 23.3 μM. The accumulation of Ca 2+ in cytoplasm and enlarged endoplasmic reticulum (ER) indicated that 1 mediated HT-29 cancer cell apoptosis through improvement of ER-stress, which was further proved by unfolded protein response (UPR) pathway on basis of the upregulation of IRE1α, p-PERK, ATF6, and CHOP., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Giant clams are one of the most important animals in coral reef ecosystem, and its growth and reproduction are being threatened by heat stress due to global warming. In the present study, the symbiont density, the crucial enzyme activities and the transcriptome were investigated in the outer mantle of giant clam Tridacna crocea after the acute exposure of high temperature. The density of symbiotic zooxanthellae decreased significantly during 12-24 h, with the minimum level (7.75 × 10 5  cell cm -2 , p < 0.05) at 12 h after heat stress. The activities of superoxide dismutase in the heat stress group was significantly lower than that in the control group at 24 h after heat stress, while no significant change in the activities of catalase was observed during the entire stress process. The activation level of caspase3 began to increase significantly at 12 h (1.22-fold, p < 0.05), and reached the highest level at 24 h (1.38-fold, p < 0.05) after heat stress. Six paired-end libraries were sequenced in two groups, including the heat stress and control group at 12 h after heat stress. Through the assembling of 187,116,632 paired-end reads with lengths of 2 × 150 bp, a total of 26,676 genes were obtained which derived from giant clam. Bioinformatics analysis revealed 47 significantly upregulated and 88 significantly downregulated genes at 12 h after the treatment. There were 12 overrepresented GO terms for significantly upregulated genes, mostly related to unfolded protein binding and ATP binding, whereas no GO term was overrepresented for significantly downregulated genes. These results collectively suggest high temperature could induce excessive oxidative stress through the repressed antioxidant ability, the apoptosis activated by the unfolded protein response, and further the collapse of the symbiosis between host and symbiont, which has been threatening the growth and reproduction of the giant clam T. crocea., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Apoptosis plays a critical role in normal embryonic development and tissue homeostasis regulation. EndophilinA2 (EndoA2) is widely reported to regulate endocytosis. Additionally, EndoA2 has been demonstrated to be involved in tumor metastasis, neuroregulation and vascular function. In this study, we used siRNA and Ad-EndoA2 transfection strategy to investigate whether EndoA2 provides a protective effect against apoptosis induced by H 2 O 2 in H9C2 cardiomyocytes and the underlying mechanisms. We found that EndoA2 siRNA knockdown promoted H 2 O 2 -induced apoptosis in H9C2 cardiomyocytes, evidenced by decreased cell number, increased apoptotic cells, and activation of caspase-3. In contrast, EndoA2 overexpression showed the opposite effects and inhibited H 2 O 2 -induced apoptosis in H9C2 cardiomyocytes. Further studies revealed that EndoA2 overexpression strengthened autophagy, evidenced by the increased LC3 II/I ratio and P62 degradation, whereas EndoA2 siRNA knockdown produced the opposite effects. Furthermore, we revealed that there was an interaction between Bif-1 and Beclin-1. Upon H 2 O 2 treatment, the association of Bif-1 and Beclin-1 remarkably increased. EndoA2 overexpression further promoted the binding of Bif-1 with Beclin-1, whereas EndoA2 siRNA knockdown reduced this association. These data strongly suggested that EndoA2 inhibited H 2 O 2 -induced apoptosis in H9C2 cardiomyocytes, possibly by promoting Bif-1 to form a complex with Beclin-1 and strengthening autophagy. This study provides a novel target for heart diseases., (Copyright © 2018. Published by Elsevier Inc.)

Physapubenolide (PB) is a cytotoxic withanolide isolated from Physalis angulata that was used as a traditional Chinese medicine. In this study, we investigated the role of TIGAR and ROS in PB-induced apoptosis and autophagosome formation in human breast carcinoma MDA-MB-231 and MCF-7 cells. PB induced apoptosis by decreasing mitochondrial membrane potential and elevating the Bax/Bcl-2 protein expression ratio in MDA-MB-231 and MCF-7 cells. Caspase inhibitor Z-VAD-FMK treatment partly blocked PB induced cytotoxicity, suggesting that apoptosis serves as an important role in the anti-proliferative effect of PB. Meanwhile, PB induced autophagosome formation, as characterized by increased acridine orange-stained positive cells, accumulation of punctate LC3B fluorescence and a greater number of autophagic vacuoles under electron microscopy. Furthermore, PB inhibited autophagic flux as reflected by the overlapping of mCherry and GFP fluorescence when MDA-MB-231 cells were transfected with GFP-mCherry-LC3 plasmid. Depletion of LC3B, ATG5 or ATG7 reduced PB-induced cytotoxicity, indicating that autophagosome associated cell death participated in the anti-cancer effect of PB. Moreover, PB-induced apoptosis and autophagosome formation were linked to the generation of intracellular ROS, and pre-treatment with the antioxidant NAC obviously mitigated the effects. Interestingly, PB treatment slightly increased TIGAR expression at low concentrations but decreased TIGAR expression drastically at high concentrations. Downregulation of TIGAR by small interfering RNA augmented low concentrations of PB-induced apoptosis and autophagosome formation, which contributed to the observed anti-cancer effect of PB and were reversed by NAC pre-treatment. Consistently, in MDA-MB-231 or MCF-7 xenograft mouse model, PB suppressed tumor growth through ROS induced apoptosis and autophagosome associated cell death accompanied with the downregulation of TIGAR. Taken together, these results indicate that downregulation of TIGAR increased PB-induced apoptosis and autophagosomes associated cell death through promoting ROS generation in MDA-MB-231 and MCF-7 cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of this work was to investigate the core structure of a Dendrobium huoshanense polysaccharide DHPD1 required for the inhibition of lens epithelial cell apoptosis. In order to obtain the fragments containing the core domain, pectinase was employed to hydrolyze DHPD1. After 24h reaction, it is interesting that the hydrolyzation seemed to be stopped, leading to a final enzymatic fragment DHPD1-24 with molecular weight about 1552Da. Compared to DHPD1, although the bioactivity is decreased, DHPD1-24 remained the ability to inhibit the H 2 O 2 -induced apoptosis of human lens epithelial (HLE) cells via suppressing the MAPK signaling pathways. These results suggested that DHPD1-24 might be the core domain required for DHPD1 to inhibit HLE cell apoptosis. Methylation analysis showed DHPD1-24 was composed of (1→5)-linked-Araf, (1→3,6)-linked-Manp, 1-linked-Glcp, (1→4)-linked-Glcp, (1→6)-linked-Glcp, (1→4,6)-linked-Glcp, (1→6)-linked-Galp and 1-linked-Xylp in a molar ratio of 1.06:1.53:2.11:2.04:0.93:0.91:0.36:1.01. Moreover, the primary structural features of DHPD1-24 were characterized by NMR spectrum., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cancer cells of epithelial and mesenchymal phenotypes exhibit different sensitivities to apoptosis stimuli, but the mechanisms underlying this phenomenon remain partly understood. We constructed a novel recombinant adenovirus expressing Ad12 E1A (Ad-E1A12) that can strongly induce apoptosis. Ad-E1A12 infection of epithelial cancer cells displayed dramatic detachment and apoptosis, whereas cancer cells of mesenchymal phenotypes with metastatic propensity were markedly more resistant to this virus. Notably, forced detachment of epithelial cells did not further sensitize them to Ad-E1A12-induced apoptosis, suggesting that cell detachment is a consequence rather than the cause of Ad-E1A12-induced apoptosis. Ad-E1A12 increased phosphorylation of AKT1 and ribosomal protein S6 through independent mechanisms in different cell types. Ad-E1A12-induced AKT1 phosphorylation was PI3K-dependent in epithelial cancer cells, and mTOR-dependent in mesenchymal cancer cells. Epithelial cancer cells upon Ad-E1A12-induced detachment could not sustain AKT activation due to AKT1 degradation, but AKT1 activation was maintained in mesenchymal cancer cells. Expression of epithelial cell-restricted miR-200 family in mesenchymal cells limited mTOR signaling and sensitized them to Ad-E1A12-induced cell killing. Thus, epithelial cancer cells rely on the canonical PI3K-AKT signaling pathway for survival, while mesenchymal cancer cells deploy the PI3K-independent mTORC2-AKT axis in response to strong death stimuli.

Giant cell tumor of bone (GCTB) is a rare and highly osteolytic bone tumor that usually leads to an extensive bone lesion. The purpose of this study was to discover novel therapeutic targets and identify potential agents for treating GCTB. After screening the serum cytokine profiles in 52 GCTB patients and 10 normal individuals using the ELISA assay, we found that NF-κB signaling-related cytokines, including TNFα, MCP-1, IL1α, and IL17A, were significantly increased in GCTB patients. The results were confirmed by IHC that the expression and activity of p65 were significantly increased in GCTB patients. Moreover, all of the NF-κB inhibitors tested suppressed GCTB cell growth, and bortezomib (Velcade), a well-known proteasome inhibitor, was the most potent inhibitor in blocking GCTB cells growth. Our results showed that bortezomib not only induced GCTB neoplastic stromal cell (NSC) apoptosis, but also suppressed GCTB NSC-induced giant cell differentiation, formation, and resorption. Moreover, bortezomib specifically suppressed GCTB NSC-induced preosteoclast recruitment. Furthermore, bortezomib ameliorated GCTB cell-induced bone destruction in vivo As a result, bortezomib suppressed NF-κB-regulated gene expression in GCTB NSC apoptosis, monocyte migration, angiogenesis, and osteoclastogenesis. Particularly, the inhibitory effects of bortezomib were much better than zoledronic acid, a drug currently used in treating GCTB, in our in vitro experimental paradigms. Together, our results demonstrated that NF-κB signaling pathway is highly activated in GCTB, and bortezomib could suppress GCTB and osteolysis in vivo and in vitro, indicating that bortezomib is a potential agent in the treatment of GCTB. Mol Cancer Ther; 15(5); 854-65. ©2016 AACR., (©2016 American Association for Cancer Research.)

Objective: To investigate the effect of rapamycin on the expression of survivin and caspase-3 at mRNA level in K562 cells and the influence of rapamycin on K562 cell ultrastructure., Methods: The effects of rapamycin at various concentration on K562 cell proliferation were analyzed by CCK8; the morphological characteristics of K562 cells was observed by transmission electron microscopy; the expression of survivin and caspase-3 at mRNA level in K562 cells treated with rapamycin was detected by RT-PCR., Results: The proliferation of K562 cells was significantly inhibited by rapamycin. The apoptosis level of K562 cells increased with increase of rapamycin concentration, the expression of survivin at mRNA level decreased with increase of rapamycin concentration (P < 0.05). The expression of caspase-3 at mRNA level increased with increase of rapamycin concentration., Conclusion: Rapamycin can prornote K562 cell apoptosis through up-regulating caspase-3 level and reduceing survivin level.

Objective: To investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of multiple myeloma H929 cell line and its mechanisms., Methods: H929 cells were treated with different concentrations of GI254023X, the proliferation-inhibitive curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V/7-AAD double staining. The cleavage of Notch1 protein (cleaved notch1) was determined by Western blot. The transcripts of Notch1 target gene Hes-1 were detected by real-time PCR., Results: The GI254023X inhibited the proliferation of H929 cells in the time- and dose- dependent manners. As compared with the control group, the apoptosis of cells increased along with enhancement of GI254023X concentration; The expression of cleaved Notch1 was down-regulated after the treatment with GI254023X. The levels of Hes-1 mRNA transcripts in H929 cells was reduced in GI254023X treated group., Conclusion: GI254023X can remarkably inhibit the proliferation and induce the apoptosis of H929 cells. Its mechanism may be associated with inbihition of Notch1 activation.

Objective: To investigate the effects of Bortezomib on proliferation, apoptosis and SHP-2 gene expression of lymphoma Jurkat cells and Raji cells., Methods: Methylthiazoly tetrazolium assay (MTT) was used to observe the proliferation of Jurkat cells and Raji cells treated with bortezomib in different doses. Cell apoptosis was detected by morphological examination and flow cytometry. The level of SHP-2 mRNA expression before and after the treatment with bortezomib was measured by RT-PCR., Results: Bortezomib could inhibit the proliferation of Jurkat and Raji cells and induce their apoptosis with time-and dose-dependent manner. After treatment with 5-100 nmol/L bortezomib, the expression of SHP-2 in Jurkat cells and Raji cells was upregulated., Conclusion: Bortezomib can inhibit the proliferation and induc the apoptosis of Jurkat and Raji cells obviously, upregulate the expression of SHP-2 mRNA, suggesting that the SHP-2 may participate in regulation of bortezomib induced apoptosis of Jurkat cells and Raji cells.

The apoptotic effects of maslinic acid (MA) at 4, 8, 16, 32 and 64 μmol/L on human lung cancer A549 cells under normoxic and hypoxic conditions were examined. MA at 4-64 and 16-64 μmol/L lowered Bcl-2 expression under normoxic and hypoxic conditions, respectively (p < 0.05). This agent at 4-64 μmol/L decreased Na+-K+-ATPase activity and increased caspase-3 expression under normoxic conditions, but at 8-64 μmol/L it caused these changes under hypoxic conditions (p < 0.05). MA up-regulated caspase-8, cytochrome c and apoptosis-inducing factor expression under normoxic and hypoxic conditions at 8-64 μmol/L and 32-64 μmol/L, respectively (p < 0.05). MA down-regulated hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), survivin and inducible nitric oxide synthase (iNOS) expression under normoxic and hypoxic conditions at 8-64 and 16-64 μmol/L, respectively (p < 0.05). After cells were pre-treated with YC-1, an inhibitor of HIF-1α, MA failed to affect the protein expression of HIF-1α, VEGF, survivin and iNOS (p > 0.05). MA at 8-64 and 32-64 μmol/L reduced reactive oxygen species and nitric oxide levels under both conditions (p < 0.05). These findings suggest that maslinic acid, a pentacyclic triterpenic acid, exerted its cytotoxic activities toward A549 cells by mediating mitochondrial apoptosis and the HIF-1α pathway.

A novel electrochemiluminescence (ECL) cytosensors was developed for the detection of early apoptotic cells by the specific interaction between Annexin V and phosphatidylserine(PS) based on ECL signal of CdS-QDs. Immobilization of Annexin V on a L-cysteine-capped CdS-QDs/Polyaniline nanofibers (PANI-NF) resulted in the stable and high loading of Annexin V on the sensor surface and the possibility of sensitivity enhancement. Early apoptotic cells showed an increased exposure of PS on the cell membrane caused by physiological and pathological response reactions, leading to a strong interaction between the apoptotic cells and the sensor surface, which could be probed by the ECL. Using a real of early apoptotic HepG2 cell induced by resveratrol (RVL), the proposed novel strategy has demonstrated its simplicity, high sensitivity, good selectivity and high reproducibility and label-free capability which might hold a great potential for rapid detection of cell apoptosis and drug screening. The results from this approach have showed good agreement with those obtained using inverted microscope, flow cytometry(FCM) and Atomic force microscopy(AFM). The linear range for early apoptotic cells detection ranged from 500 to 1.0 × 10(6) cells mL(-1) with a detection limit of 500 cells mL(-1). The reported strategy has provided a promising platform for highly sensitive cytosensing and convenient screening of some clinically anticancer drugs., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

The roots of Gypsophila oldhamiana are rich in triterpenoid saponins with antitumor properties. Although previous reports have revealed the anticancer potency of some Gypsophila species, the underlying molecular mechanisms of this activity have not been studied in detail. The purpose of the present study was to prepare a triterpenoid saponin-rich G. oldhamiana root extract (TGOE) determined by LC-electrospray ionization (ESI)-MS(n) for biological studies and to evaluate the different anti-proliferative activities and apoptotic effects of TGOE on human hepatoma SMMC-7721 and normal human hepatic L02 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that TGOE selectively inhibited the proliferation of SMMC-7721 cells in a dose-dependent manner with IC50 value of 19.50±3.63 µg/mL, while the cytotoxic effects of TGOE on L02 cells were much lower with IC50 value of 40.48±3.74 µg/mL. Analysis of apoptotic morphological changes and flow cytometry indicated that TGOE might preferentially induce apoptosis in SMMC-7721 cells, while exhibited much lower effects on L02 cells. Western blot analysis showed that the different apoptotic effects of TGOE on SMMC-7721 and L02 cells were due to different protein regulation of caspase-3 and mitogen activated protein kinases (MAPKs). TGOE significantly activated caspase-3 and increased the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while decreased the phosphorylation of p38 in SMMC-7721 cells. However, the expression of these proteins was not statistically changed in L02 cells, except for the up-regulation of p38 phosphorylation. These results suggest that TGOE may have potential beneficial effects against hepatocellular carcinoma.

Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3(x73c), a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer.

Several studies have reported the change of EphB2 in a variety of carcinomas and suggested a functional relation between EphB2 and tumor progression. However, its role in human pancreatic carcinoma has not been described. The aim of this study was to evaluate the significance of EphB2 in human pancreatic carcinoma CFPAC-1 cells. A lentivirus-based RNA interference (RNAi) vector was designed, synthesized and transfected into CFPAC-1 cells to inhibit EphB2 expression. WST-8 based Colorimetric Assay Cell Counting kit 8 (CCK-8) in vitro and xenograft transplantation model in nude mice was used to evaluate cell proliferation and growth respectively. Cell-cycle and apoptosis were analyzed by flow cytometry (FCM). RT-PCR and Western blot were used to assess mRNA expression and protein levels. EphB2 expression was significantly suppressed both in mRNA and protein levels using the lentivirus-based EphB2 RNAi in CFPAC-1 cells (P<0.01, P<0.01). Silencing EphB2 stimulated cell growth in vitro (P<0.05) and proliferation in vivo (P<0.01) versus Control RNAi. EphB2 RNAi significantly increased S phase cells from 18.15 to 27.18% (P<0.05), and significantly decreased G1 phase cells from 72.93 to 57.61% compared with Control RNAi (P<0.05). In addition, decreased apoptosis was observed in CFPAC-1 EphB2 RNAi cells compared with Control RNAi cells (P<0.01). The apoptosis rate was 1.63% and 7.44%, respectively. Silencing EphB2 increased CyclinD1, cyclindependent kinase 6 (CDK6) and Bcl-2 expression in both mRNA and protein levels compared with Control RNAi. A lentivirus-based EphB2 RNAi efficiently inhibited EphB2 gene and its protein expression. Silencing EphB2 stimulated pancreatic carcinoma growth by increasing cell proliferation through G1/S phase breakthrough, which relied on a CyclinD1/CDK6 cell-cycle regulated signal. Similarly, EphB2 inhibition also reduced CFPAC-1 cells apoptosis by up-regulating Bcl-2 expression. Thus, at least in the context of pancreatic carcinoma CFPAC-1 cells, EphB2 plays a tumor suppressor role in cell proliferation and apoptosis., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Dauricine, a bioactive component of Asiatic Moonseed Rhizome, has been widely used to treat a large number of inflammatory diseases in traditional Chinese medicine. In our study, we demonstrated that dauricine inhibited colon cancer cell proliferation and invasion, and induced apoptosis by suppressing nuclear factor-kappaB (NF-kappaB) activation in a dose- and time-dependent manner. Addition of dauricine inhibited the phosphorylation and degradation of IkappaBalpha, and the phosphorylation and translocation of p65. Moreover, dauricine down-regulated the expression of various NF-kappaB-regulated genes, including genes involved cell proliferation (cyclinD1, COX2, and c-Myc), anti-apoptosis (survivin, Bcl-2, XIAP, and IAP1), invasion (MMP-9 and ICAM-1), and angiogenesis (VEGF). In athymic nu/nu mouse model, we further demonstrated that dauricine significantly suppressed colonic tumor growth. Taken together, our results demonstrated that dauricine inhibited colon cancer cell proliferation, invasion, and induced cell apoptosis by suppressing NF-kappaB activity and the expression profile of its downstream genes. These findings provide evidence for a novel role of dauricine in preventing or treating colon cancer through modulation of NF-kappaB singling pathway., ((c) 2010 Wiley-Liss, Inc.)

Integrins are a family of receptors for extracellular matrix proteins that have critical roles in human tissue development. Previous studies identified down-regulation and/or mutations of integrin alpha7 (ITGA7) in prostate cancer, liver cancer, soft tissue leiomyosarcoma, and glioblastoma multiforme. Here we report that expression of ITGA7 induced apoptosis in the human prostate cancer cell lines PC3 and DU145. Yeast two-hybrid analysis revealed that the C-terminus of ITGA7 interacts with high temperature requirement A2 (HtrA2), a serine protease with a critical role in apoptosis. Expression of ITGA7 increases the protease activity of HtrA2 both in vitro and in vivo. Deletion of the HtrA2 interaction domain abrogates the cell death activity of ITGA7, whereas down-regulation of HtrA2 dramatically reduced cell death mediated by ITGA7. In addition, site-directed protease-null mutant HtrA2S306A expression blocked apoptosis induced by ITGA7. Interestingly, interaction between ITGA7 and its ligand laminin 2 appears to protect against cell death, since depleting laminin beta2 with a small-interfering RNA significantly exacerbated apoptosis induced by ITGA7 expression. This report provides a novel insight into the mechanism by which ITGA7 acts as a tumor suppressor.

(-)-Epigallocatechin-3-gallate (EGCG) is one of the major polyphenol components in green tea. It effectively induces apoptosis in prostate cancer cells. The anticancer effect of this reagent is appealing because it is a natural component of a popular daily beverage that has proven harmless for thousands of years, making it a good candidate chemopreventive agent. EGCG suppresses cell growth and causes cell death, but the mechanisms are not well characterized, especially in androgen-independent prostate cancer cells. In the present study, using Affymetrix genechip Hu133 2.0, we analyzed the gene expression patterns of the androgen-independent prostate cancer cell line Du145, treated with or without EGCG, and found 40 genes whose expression levels were altered (>twofold, either upregulated or downregulated, P < 0.01) upon treatment with EGCG. These gene products are involved in the functions of transcription, RNA processing, protein folding, phosphorylation, protein degradation, cell motility, and ion transport. Among them, inhibitor of DNA binding 2 (ID2), known as a dominant anti-retinoblastoma (Rb) helix-loop-helix protein, was found to be downregulated fourfold by EGCG treatment. Forced expression of ID2 in Du145 cells reduced apoptosis and increased cell survival in the presence of EGCG, and knockdown ID2 expression in Du145 cells using a morpholino oligonucleotide specific for ID2 mimicked the apoptosis effect generated by EGCG treatment, although it was milder. To our knowledge, this is the first report indicating that ID2 is one of the critical factors in the signaling pathway of Du145 cell death induced by EGCG.

The present study was purposed to investigate the inhibition effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on growth of RPMI8226 cells and adhesion between RPMI8226 cells and bone marrow stroma cells (BMSC), and to explore its mechanism as well. The inhibition effects of TRAIL on cells growth and adhesion were assayed by MTT; cell apoptosis was detected by Annexin V and PI; expression of genes bax, bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP were determined by semi-quantitative RT-PCR; apoptosis-related protein expression was detected by Western blot. The results showed that TRAIL inhibited the proliferation of RPMI8226 cells in dose- and time-dependent manners. TRAIL induced apoptosis in RPMI8226 cells, the expression level of genes bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP decreased, while the expression level of Bax increased, but the expression level of caspase-3 and NF-kappaB P65(RelA) proteins decreased. Moreover, TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly. It is concluded that TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly, and induced the apoptosis of RPMI8226 cells. Growth inhibition effect of TRAIL on RPMI8226 cells is in dose- and time-dependent manners.

The src homology 2 (SH2)-domain containing inositol-5-phosphatase (SHIP) is another recently identified lipid phosphatase after phosphatase and tensin homology deleted on chromosome ten gene (PTEN). It plays an important role in negatively regulating the proliferation of hematopoietic cells. The relationship between SHIP and the inhibition of tumor proliferation is rarely reported. The purpose of this study is to evaluate the apoptosis induced by SHIP gene in K562 cell line and to explore the involved signaling pathway. The K562 cells were transfected with human SHIP gene by using the lentiviral vector containing SHIP, and the transfection was verified by fluorescent quantitative PCR (FQ-PCR) and Western blot. Then the effects of SHIP protein expression on cell growth and apoptosis were measured. The levels of p-Akt, bcl-2 family, caspase and the activity of NFkappaB were assayed by Western blot and ELISA, respectively. The results are as follows: (1) Human leukemia cell line K562 was SHIP-negative; (2) Transfection with SHIP gene led to the re-expression of SHIP mRNA and protein in K562, as shown by FQ-PCR and Western blot; (3) The expression of SHIP protein inhibited cell growth and significantly increased apoptosis in K562 cells; (4) Compared to that in control group, the expression level of p-Akt-308 and p-Akt-473 in SHIP-expressing cell group decreased significantly (P<0.01); SHIP activated caspase-9, caspase-3, up-regulated protein levels of bad, p27, down-regulated expression of bcl-xL, while it had no effect on the expression of bcl-2 and bax. Furthermore, the inhibition of NF-kappaB was achieved along with the inactivation of Akt. These data suggest that SHIP gene has potential abilities to inhibit K562 leukemic cell proliferation and induce its apoptosis via inactivating PI3K/Akt pathway. The loss of SHIP might be the explanation of aberrant high-level p-Akt in human leukemia. It may be at least one of the mechanisms by which the loss of SHIP expression contributes to leukemia progression.

The aim of this study was to investigate the regulation of 5-aza-CdR on transcription of SHP-1 gene and effects on the proliferation and apoptosis of K562 cells. Methylation-specific PCR (MSP) was used to detect CpG island methylation in SHP-1 promoter. MTT and flow cytometry were used to detect the growth and apoptosis of K562 cells after treatment with different concentration of 5-aza-CdR. The expressions of SHP-1 mRNA and protein were determined by FQ-PCR and Western blot. The expression of p-JAK2 was assayed by Western blot. The result showed that methylation of SHP-1 gene promoter was detected in K562 cells, and the SHP-1 mRNA and protein were expressed again in K562 cells after treatment with 5-aza-CdR, meanwhile the expression of phosphorylated P-JAK2 was down-regulated; 5-aza-CdR significantly inhibited the cell growth in dose and time dependent manners. AG490 inhibited the cell proliferation. 5-aza-CdR increased the apoptosis rate of K562 cells also in dose- and time-dependent manners. The apoptosis rates of K562 cells treated with 5-aza-CdR for 1, 3 and 5 days were 9.3%, 24.2% and 37.7% respectively. After treatment with 2 micromol/L 5-aza-CdR for 24 hours, cells in G(0)/G(1) phase increased gradually, cells in G(2)/M phase decreased gradually, cells were arrested in G(0)/G(1) phase. The cell ratios in G(2)/M phase at 1, 3 and 5 days after treatment with 5-aza-CdR were 30.7%, 23.45 and 19.3% respectively. It is concluded that the 5-aza-CdR, inhibitor of specific methylation transferase, can re-express the silent SHP-1 gene in K562 cells, inhibits the proliferation of leukemia cells and induces cell apoptosis by activating JAK/STAT pathway.

We have previously shown that local administration of polyethylene glycol (PEG, MW: 2000 Da, 50% by weight), a known membrane repair agent, immediately after trauma in guinea pig spinal cord repairs neuronal membrane disruptions and reduces oxidative injury. Here we report that a similar application of PEG resulted in marked decreases in apoptotic cell death and caspase-3 activity. We suggest that PEG may suppress apoptosis through interactions with mitochondria. This is based on our current findings that in isolated mitochondria, PEG improves mitochondrial function and reduces the release of cytochrome c, a pro-apoptotic cell death factor. This hypothesis is further supported by our previous observation that PEG enters injured cells after spinal cord injury, placing PEG in a position to directly interact with mitochondria. In summary, we conclude that PEG reduces both necrosis and apoptosis through two distinct yet synergistic pathways: repair of disrupted plasma membranes and protection of mitochondria through direct interaction.

The aim of study was to investigate the combined effect of recombinant mutant human TRAIL (rmhTRAIL) with daunorubicin (DNR) or alone on K562 and U937 leukemia cell lines and its mechanism. The fibroblasts (MRC-5) of normal-human embryonic lung were used as control cells. After being treated with rmhTRAIL and DNR or only with rmTRAIL, the cytotoxic effect and the apoptosis rate in K562, U937 cells were measured by MTT assay. The expression levels of TRAIL death receptor and TRAIL decoy receptor mRNA in these three cell lines were assayed by semiquantitive RT-PCR before and after treatment with DNR. The results indicated that K562 and U937 were sensitive to rmhTRIAL. DNR had synergistic inhibitory effect with rmhTRAIL on the growth of K562 and U937 cell lines (P < 0.05). The expression level of DR4 and DR5 mRNA was significantly higher in K562 and U937 with combined treatment of rmhTRAIL and DNR than that in those alone, while the expressions of DcR1 and DcR2 mRNA were not influenced. It is concluded that in vitro, rmhTRAIL alone or in combination with DNR can obviously inhibit the growth of leukemia cell lines and induce cell apoptosis, DNR and rmhTRAIL have a synergistic inhibitory effect on growth of K562 and U937. The mechanism may correlate with the up-regulation of DR4 and DR5 of K562 and U937.

The study was aimed to investigate the expression of midkine (MK) in bone marrow mononuclear cells (BM MNC) from 65 acute myeloid leukemia patients and 15 normal controls. The method of RT-PCR was used to examine the expression of MK mRNA in BM MNC. Parts of samples were incubated for 24 hours and the gene expression of MK in the BM MNC was detected by means of Western blot. The results showed that the expression of MK of BM MNCs in 50 newly diagnosed AML patients (0.331 +/- 0.436) and 15 AML patients in relapse (0.374 +/- 0.463) were markedly higher than that in 15 CR cases (0.067 +/- 0.190), and 15 normal controls (0), respectively. The complete remission in MK positive patients (63.16%) was significantly lower than that in MK negative group (93.55%). The patients with positive MK expression had a higher relapse rate than those with negative MK expression. The positive rate of MK gene expression in drug-resistant patients and drug-sensitive patients were 57.69% and 25.64% respectively and there was positive correlation between the gene expressions of MK and bcl-2 (P < 0.01) (r = 0.0556, P < 0.001). It is concluded that MK can be secreted by AML cells and involved in drug-resistant, its positive expression may be associated with the poor prognosis in newly diagnosed AML patients. The inhibitory effect of MK on apoptosis of leukemic cells is induced by upregulating bcl-2 expression.

In order to investigate the anti-tumor activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and the mechanism underlying the cell proliferation and apoptosis modulated in myeloma cells, the effects of mevastatin, an HMG-CoA reductase inhibitor, on cell growth, cell cycle progression and apoptosis in U266 human multiple myeloma (MM) cell line in vitro were explored by MTT colorimetric assay, morphologic observation, flow cytometry, DNA gel electrophoresis, and RT-PCR. The results demonstrated that mevastatin inhibited the growth of U266 cells in time- and dose-dependent manners. Cell cycle analysis showed that U266 cells underwent G(0)/G(1) arrest under exposure to mevastatin, but it did not affect p27 expression at both mRNA and protein level. Morphologic observations revealed cytoplasm shrinkage, nuclear condensation and fragmentation in mevastatin-treated cells, and fraction of annexin V(+)PI(-) cells increased significantly in the presence of the agent as determined by flow cytometric assay. In addition, mevastatin caused the collapse of mitochondrial transmembrane potential (Deltapsim), induced DNA fragmentation, and down-regulated the mRNA expression of bcl-2. The growth-inhibitory, cell cycle arresting, and proapoptotic effects of mevastatin in U266 cells could be effectively reversed by the addition of mevalonate (MVA), the immediate endproduct of the reaction catalyzed by HMG-CoA reductase. It is concluded that mevastatin suppresses proliferation by inducing G(0)/G(1) phase arrest and triggering apoptosis via down-regulation of bcl-2 and reduction of Deltapsim, which may be attributed to the inhibition of MVA pathway by mevastatin. Statins including mevastatin may find their future application in the treatment of MM.

Objective: To study the mechanism of kaempferol in the intervention of knee osteoarthritis (KOA) in mice by inhibiting cartilage apoptosis. Methods: Firstly, the target genes of kaempferol were retrieved via TCMSP, and the genes related to KOA were obtained by GeneCards, OMIM, PharmGKB, TTD, and Drugbank databases. Then GO enrichment analysis and KEGG signaling pathway analysis were also performed. Subsequently, 18 male C57 mice were randomly divided into the sham operation group, the model group, and the kaempferol group (50 mg/kg). Except for the sham operation group, the KOA mouse model was induced by destabilization of medial meniscus surgery. The sham operation group and model group were given the same amount of normal saline daily for 8 weeks while the kaempferol group was given 50 mg/kg kaempferol intragastrically. Results: A total of 63 targets of kaempferol were found that included 35 common target genes with KOA. GO and KEGG analyses showed that biological processes such as extrinsic apoptotic signaling pathway and response to oxidative stress, as well as signaling pathways such as cell apoptosis and regulation of TNF were closely related to common target genes. Molecular docking results also showed kaempferol has good binding properties with predicted targets Bcl-2, BAX, and CASP3. Compared with the model group, the pathological changes of cartilage in the kaempferol group were significantly reduced, OARSI scores were significantly decreased (P<0.001), and cartilage area was increased (P<0.01). In addition, Western blot analysis showed that kaempferol significantly decreased the protein expression of BAX and CASP3 (P<0.01, P<0.05), and increased the protein expression of BCL-2 (P<0.05). Conclusion: The treatment of KOA with kaempferol has the characteristics of multi-target and multi-pathway, and the mechanism may be related to the regulation of key genes such as Bcl-2, BAX, and CASP3 to inhibit cartilage apoptosis. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are crucial for the pathogenesis of human diseases, including osteoarthritis (OA). Here, we set to elucidate the biological action of circ-LRP1B in OA pathogenesis. Human C28/I2 chondrocytes were stimulated by lipopolysaccharide (LPS). Circ-LRP1B, nuclear factor, erythroid 2 like 1 (NRF1) and microRNA (miR)-34a-5p were quantified by quantitative real-time PCR (qRT-PCR) or immunoblotting. Cell viability, proliferation, and apoptosis abilities were gauged by MTT, 5-Ethynyl-2'-Deoxyuridine (EdU) staining, and flow cytometry assays, respectively. Direct relationship between miR-34a-5p and circ-LRP1B or NRF1 was validated by RNA pull-down and dual-luciferase reporter assays. Circ-LRP1B was found to be underexpressed in OA cartilage and LPS-stimulated C28/I2 chondrocytes. Enforced expression of circ-LRP1B promoted cell proliferation, and repressed apoptosis and oxidative stress, as well as impacted OA-specific hallmarks expression of LPS-stimulated C28/I2 cells. NRF1 was identified as a downstream effector of circ-LRP1B function. Moreover, NRF1 was identified as a miR-34a-5p target in LPS-stimulated C28/I2 cells. Circ-LRP1B acted as a competing endogenous RNA (ceRNA) for miR-34a-5p to involve the post-transcriptional regulation of NRF1 expression. Furthermore, the effects of circ-LRP1B overexpression partly depended on the reduction of available miR-34a-5p. These findings demonstrate that circ-LRP1B functions as a ceRNA to regulate the proliferation, apoptosis and oxidative stress of LPS-stimulated human C28/I2 chondrocytes by miR-34a-5p/NRF1 network. [ABSTRACT FROM AUTHOR]

Gastric cancer is a common lethal malignancy and causes great cancer-related mortality worldwide. MicroRNA (miR)-328-3p is implicated in the progression of various human cancers; however, its role and mechanism in the progression of gastric cancer remain unclear.Human gastric cancer cells were incubated with miR-328-3p mimic, inhibitor or the matched negative control. Cell viability, colony formation, migrative and invasive capacity, cell apoptosis and oxidative stress were measured. To clarify the involvement of nuclear factor-E2-related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1), small interfering RNA was used. miR-328-3p was upregulated in human gastric cancer cells and tissues, and its level positively correlated with the progression of gastric cancer. miR-328-3p promoted cell viability, colony formation, migration and invasion, thereby facilitating the progression of gastric cancer. miR-328-3p mimic reduced, while miR-328-3p inhibitor increased apoptosis and oxidative stress of human gastric cancer cells. Mechanistically, miR-328-3p upregulated NRF2 via targeting KEAP1to attenuate excessive free radical production and cell apoptosis. miR-328-3p functions as an oncogenic gene and inhibiting miR-328-3p may help to develop novel therapeutic strategies of human gastric cancer. [ABSTRACT FROM AUTHOR]

Aims: SUMOylation is a post-translational modification that plays a crucial role in the cellular stress response. We aimed to demonstrate whether and how the SUMO E2 conjugation enzyme Ubc9 affects acute myocardial ischemic (MI) injury. Methods and Results: Adenovirus expressing Ubc9 was administrated by multipoint injection in the border zone of heart immediately after MI in C57BL/6 mice. Neonatal rat cardiomyocytes (NRCMs) were also infected, followed by oxygen and glucose deprivation (OGD). In vivo , Ubc9 adenovirus-injected mice showed decreased cardiomyocyte apoptosis, reduced myocardial fibrosis, and improved cardiac function post-MI. In vitro , overexpression of Ubc9 decreased cardiomyocyte apoptosis, whereas silence of Ubc9 showed the opposite results during OGD. We next found that Ubc9 significantly decreased the accumulation of autophagy marker p62/SQSTM, while the LC3 II level hardly changed. When in the presence of bafilomycin A1 (BAF), the Ubc9 adenovirus plus OGD group presented a higher level of LC3 II and GFP-LC3 puncta than the OGD group. Moreover, the Ubc9 adenovirus group displayed increased numbers of yellow plus red puncta and a rising ratio of red to yellow puncta on the mRFP-GFP-LC3 fluorescence assay, indicating that Ubc9 induces an acceleration of autophagic flux from activation to degradation. Mechanistically, Ubc9 upregulated SUMOylation of the core proteins Vps34 and Beclin1 in the class III phosphatidylinositol 3-kinase (PI3K-III) complexes and boosted the protein assembly of PI3K-III complex I and II under OGD. Moreover, the colocalization of Vps34 with autophagosome marker LC3 or lysosome marker Lamp1 was augmented after Ubc9 overexpression, indicating a positive effect of Ubc9-boosted protein assembly of the PI3K-III complexes on autophagic flux enhancement. Conclusions: We uncovered a novel role of Ubc9 in protecting cardiomyocytes from ischemic stress via Ubc9-induced SUMOylation, leading to increased PI3K-III complex assembly and autophagy-positioning. These findings may indicate a potential therapeutic target, Ubc9, for treatment of myocardial ischemia. [ABSTRACT FROM AUTHOR]

Background/Aims: Giant cell tumor of bone (GCTB), one of the most common primary bone tumors, leads to extensive bone destruction. However, the mechanisms underlying GCTB progression remain elusive and prognostic factors and treatment targets are required. In the current study, we explored the function of the chemokine family member CCL20 in GCTB progression. Methods: We explored the expression of CCL20 in stromal cells (GCTSCs) using microarray. Clinical analyses of the role of CCL20 in tumor progression were performed based on the patient cohort of our institution. The role of CCL20 in tumor proliferation was evaluated by MTS assay, migration ability was measured by a Transwell assay, and osteoclastogenesis was induced by CCL20 or GCTSC-conditioned medium. Quantitative PCR and western blot were used to measure the expression levels of mRNAs and proteins related to tumor progression. Results: CCL20 was upregulated in GCTSCs and correlated with tumor progression and prognosis. CCL20 induced GCTSC proliferation and migration in an autocrine manner. In addition, CCL20 recruited mononuclear cells and induced osteoclastogenesis by overactivating the AKT and NF-κB signaling pathways. Antibody blockade of CCL20 abolished the exacerbated osteoclastogenesis. Conclusion: Taken together, our data indicate that GCTSC secretion of CCL20 acts as a key modulator in the pathological progression of GCTB. It can promote GCTSC proliferation and migration in an autocrine manner and can recruit bone marrow monocytes to the tumor microenvironment and enhance osteoclastogenesis in a paracrine manner. These findings strongly indicate the potential prognostic and therapeutic value of CCL20 in GCTB. [ABSTRACT FROM AUTHOR]

Globally, microplastics (MPs) are highly prevalent, especially in coastal areas. Unfortunately, golden pompano as a major marine fish in China is typically raised in floating marine cages near coasts, facing these MPs sources. However, toxicological studies on Golden Pompano which farm in coastal areas and face actual microplastic exposure are rare. Therefore, golden pompano were exposed to 10.0 μg/L, 100.0 μg/L, and 1000.0 μg/L polystyrene MPs (PS-MPs) for 14 days to study the potential impact of the microplastics on the Golden Pompano. Fish show slowed growth after 14 days of exposure. Histopathology shows irregular shaped nuclei and nuclear and cytoplasmic vacuolation traits in liver. Oxidative stress-related enzyme activity and gene expression data show that oxidative damage occurs in the high-concentrations (100.0 μg/L and 1000.0 μg/L) of PS-MPs exposures. Up-regulation of Grp78 , Xbp-1 , Eif-2α and chop gene expression indicates the occurrence of endoplasmic reticulum stress, and the western blot results also confirmed this. Severe oxidative stress also caused ERS, which ultimately increased BAX/Bcl-2 ratios and induces apoptosis. Furthermore, up-regulated anaerobic respiration, altered lipid metabolism, and immune disturbance were exhibited during PS-MPs stress. Therefore, oxidative stress appeared to be the main toxicity issue caused by MPs, while ERS-mediated apoptosis, metabolic alterations, and immune responses were induced by this stress. Notably, endoplasmic reticulum stress and apoptosis are a self-protective mechanism, which may be an intermediate link in the toxicity of microplastics. This study highlights the role of endoplasmic reticulum stress in MPs toxicology and assesses the adverse effects of microplastics on Golden Pompano. • Microplastics will affect the physiological status of large cultured fishes. • Microplastics alter liver structure and function in golden pompano. • Oxidative stress induced by microplastics mediates endoplasmic reticulum stress leading to apoptosis of liver. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Aconitum carmichaelii Debeaux is a well-known Chinese herb that has been used to treat liver diseases for many years in China. We investigated the effects of aqueous extract from Aconitum carmichaelii Debeaux (AEACD) on acute liver failure and identified the possible mechanisms of these effects. Material and methods Specific pathogen-free (SPF) male Wistar rats were used to establish acute liver failure model by intraperitoneal injection of D-galactosamine (D-GalN) and treated with Stronger Neo-Minophagen C (SNMC) and AEACD by gavage. Then, the serum biochemical parameters, the pathological scores in the liver tissue, the mRNA expressions of toll- like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), high mobility group box 1 (HMGB1) and caspase-3, the proliferating cell nuclear antigen (PCNA) positive rates were analyzed. Results The liver function was improved, the pathological scores were decreased, the expressions the TLR4, NF-κB, HMGB1, and caspase-3 were inhibited, and the PCNA positive rates were increased by both SNMC and AEACD, but AEACD induced greater effects. Conclusions AEACD protected liver function by inhibiting inflammatory reaction, apoptosis and promoting liver tissue regeneration in the acute liver failure rats induced by D-galactosamine. [ABSTRACT FROM AUTHOR]

This study investigated the effects of dietary valine on tight junction protein transcription, antioxidant status and apoptosis on grass carp gills ( Ctenopharyngodon idella ). Fish were fed six different experimental diets containing graded levels of valine (4.3, 8.0, 10.6, 13.1, 16.7, 19.1 g/kg). The results indicated that valine deficiency decreased Claudin b, Claudin 3, Occludin and ZO-1 transcription and increased Claudin 15 expression in the fish gill ( P < 0.05). These effects were partly due to the down-regulation of interleukin 10 (IL-10), transforming growth factor β1 (TGF-β1) and IκB α and the up-regulation of relative mRNA expression of interleukin 1β (IL-1β), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and nuclear factor κB P65 (NF-κB P65) (P < 0.05). However, valine deficiency and valine supplementation did not have a significant effect on Claudin c and Claudin 12 expression in grass carp gills ( P > 0.05). Valine deficiency also disrupted antioxidant status in the gill by decreasing anti-superoxide radicals and hydroxyl radical capacity, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) ( P < 0.05). These results may be ascribed to the down-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the up-regulation of Kelch-like-ECH-associated protein 1 (Keap1) ( P < 0.05). Additionally, valine deficiency induced DNA fragmentation via the up-regulation of Caspase 3, Caspase 8 and Caspase 9 expressions ( P < 0.05). These results may be ascribed to the improvement in ROS levels in the fish gill ( P < 0.05). Taken together, the results showed that valine deficiency impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gill. [ABSTRACT FROM AUTHOR]

Background: MiRNA-21 was previously reported to be up-regulated in many kinds of cancer. In the present study, we want to investigate the potential role of miRNA-21 in non-small cell lung cancer. Materials and methods: Expression of miRNA-21 was detected in 60 non-small cell lung cancer (NSCLC) samples and adjacent histologically normal tissue using RT-qPCR, Correlation between miRNA-21 expression and clinicopathological features of NSCLC was analyzed using statistical software. The effect of miRNA-21 expression on the growth and apoptosis of A549 cells induced by irradiation was examined. Results: miRNA-21 expression increased in non-small cell lung cancer. Expression of miRNA-21 was positively associated with lymph node metastasis, clinical stage and poor prognosis. Multivariate Cox regression analysis showed that miRNA-21 was an independent prognostic factor for patients. Down-regulation of miRNA-21 inhibited proliferation and cell cycle progress of A549 cells and sensitized cells to radiation. Decreased miRNA-21 expression promoted the apoptosis of A549 cells induced by irradiation. Conclusions: miRNA-21 may be considered as a potential novel target for future development of specific therapeutic interventions in NSCLC. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is the most frequently diagnosed cancer in men, causing considerable morbidity and mortality. The P2X4 receptor (P2X4R) is the most ubiquitously expressed P2X receptor in mammals and is positively associated with tumorigenesis in many cancer types. However, its involvement in PCa progression is less understood. We hypothesized that P2X4R activity enhanced tumour formation by PCa cells. We showed that P2X4R was the most highly expressed, functional P2 receptor in these cells using quantitative reverse transcription PCR (RT-PCR) and a calcium influx assay. The effect of inhibiting P2X4R on PCa (PC3 and C4-2B4 cells) viability, proliferation, migration, invasion, and apoptosis were examined using the selective P2XR4 antagonists 5-BDBD and PSB-12062. The results demonstrated that inhibiting P2X4R impaired the growth and mobility of PCa cells but not apoptosis. In BALB/c immunocompromised nude mice inoculated with human PC3 cells subcutaneously, 5-BDBD showed anti-tumourigenic effects. Finally, a retrospective analysis of P2RX4 expression in clinical datasets (GDS1439, GDS1746, and GDS3289) suggested that P2X4R was positively associated with PCa malignancy. These studies suggest that P2X4R has a role in enhancing PCa tumour formation and is a clinically targetable candidate for which inhibitors are already available and have the potential to suppress disease progression. [ABSTRACT FROM AUTHOR]

The article discusses a study on the effects of high dietary calcium on renal tubular cells in growers with gout.

The transient receptor potential canonical (TRPC) channels have been implicated in various types of malignancies including gastric cancer (GC). However, the detailed mechanisms of TRPC channels underlying cell proliferation and apoptosis of GC cells remain largely unknown. Here, we report that TRPC3 was highly expressed in clinical GC specimens and correlated with GC malignant progression and poor prognosis. Forced expression of TRPC3 in GC cells enhanced both receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) and promoted the nuclear factor of activated T cell 2 (NFATc2) nuclear translocation by AKT/GSK-3β and CNB2 signaling. Pharmacological inhibition of TRPC3 or CRISPR/Cas9-mediated TRPC3 knockout effectively inhibited the growth of GC cells both in vitro and in vivo. These effects were reversible by the rescue of TRPC3 expression. Furthermore, we confirmed the role of TRPC3 and the ROCE-AKT/GSK3β-CNB2/NFATc2 signaling cascade in regulating cell cycle checkpoint, apoptosis cascade, and intracellular ROS production in GC. Overall, our findings suggest an oncogenic role of TRPC3 in GC and may highlight a potential target of TRPC3 for therapeutic intervention of GC and its malignant progression. [ABSTRACT FROM AUTHOR]

Brahma-related gene 1 (BRG1) regulates the chromatin structure and expression of cardiac genes. Although BRG1 is downregulated in adult cardiomyocytes, it is reactivated during cardiac stress. The role of BRG1 in acute myocardial infarction (AMI) has not been clearly defined. This study assessed the protective role of BRG1 in AMI using cell cultures and an animal model and explored the underlying molecular events. The results showed that in the peri-infarct zone, expression of BRG1 protein was significantly increased relative to the sham group, which was accompanied by NRF2 and HO1 upregulation and KEAP1 downregulation. BRG1 overexpression through adenoviral intramyocardial injection into AMI mice reduced the infarct size and improved cardiac functions with upregulation of NRF2 and its target HO1 and attenuated oxidative damage and cell apoptosis. However, shRNA-mediated Brg1 knockdown had the opposite effects. These results were further confirmed in cultured primary neonatal rat cardiomyocytes (NRCMs) with oxygen-glucose deprivation (OGD). Moreover, the selective NRF2 inhibitor brusatol could partially reverse cardiomyocyte antioxidant ability and BRG1 overexpression-induced cardiac protection in vitro. In addition, co-immunoprecipitation and immunofluorescence data showed that BRG1 overexpression significantly promoted the BRG1/NRF2 co-localization in cardiomyocytes. The chromatin immunoprecipitation-qPCR revealed BRG1 interaction with the Ho1 promoter and BRG1 overexpression could induce BRG1 binding to the Ho1 promoter during the OGD. In conclusion, this study demonstrated that BRG1 upregulation during AMI in vitro and in vivo increased the NRF2 level and NRF2 nuclear accumulation for HO1 expression to alleviate cardiac myocyte oxidative stress and upregulate cardiomyocyte viability. The BRG1-NRF2-HO1 pathway may represent a novel therapeutic target in the prevention of cardiac dysfunction in AMI patients. Image 1 • Brahma-related gene 1 (BRG1), regulates chromatin structure and plays critical roles in cardiac gene regulation. • However, to date, BRG1 in acute myocardial infarction (AMI) is poorly understood. • This study revealed that AMI enhanced BRG1 expression in ischemic and hypoxic cardiomyocytes in vitro and in vivo. • NRF2/HO1 signaling could mediate BRG1 protective effects on cardiomyocytes from ischemia and hypoxia-induced damage and apoptosis. • These findings may provide novel insights into future therapeutic strategies for the AMI-induced cardiomyocytes injury. [ABSTRACT FROM AUTHOR]

In the present study, a novel nanocarrier was developed for the delivery of anticancer drug to the cancer tissues. For this purpose, 5-FU loaded methoxy poly(ethylene glycol)–poly(lactide) (mPEG–PLA) (5-FU NP) based polymeric nanoparticles was designed to increase the chemotherapeutic efficacy against breast cancers. Nanoprecipitation method was used to prepare the drug-loaded nanoparticles. The nanoparticle was evaluated in terms of DLS, TEM, in vitro release kinetics, and in vivo parameters. The average particle diameter of drug loaded NP was ∼110 nm and exhibited a sustained drug release pattern for up to 120 h. The NP exhibited a pH-response drug release pattern with accelerated release at acidic media. The in vitro cytotoxicity assay showed the enhanced cytotoxicity effect of NP formulations in comparison to free drug. The NP system showed remarkable G2/M phase cell cycle arrest with significant amount of apoptosis cells in late and early phase of flow cytometer analysis. Consistently, NP formulation greatly decreased the tumor burden of mice with no sign of adverse effect. TUNEL assay further confirmed the superior anticancer effect of NP formulations which showed a high number of apoptotic cells. The favorable results obtained from this study highlights the potential application of encapsulated 5-FU NP in the treatment of breast cancers. The remarkable anticancer therapeutic efficacy with negligible toxicity profile of 5-FU NP makes it one of the possible alternative for the successful breast cancer therapy. [ABSTRACT FROM AUTHOR]