Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
Adenocarcinoma of Lung/pathology* ; Apoptosis/drug effects* ; Cell Cycle Checkpoints/drug effects* ; Cell Cycle Proteins/genetics* ; Cell Line, Tumor ; Forkhead Box Protein O3/physiology* ; Gene Expression Regulation, Neoplastic/drug effects* ; Humans ; Lung Neoplasms/pathology* ; Oligopeptides/pharmacology* ; Proto-Oncogene Proteins c-akt/physiology* ; Up-Regulation

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer's disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP^751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
Amyloid beta-Protein Precursor/physiology* ; Apoptosis/drug effects* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Fluorouracil/pharmacology* ; Humans ; Liver Neoplasms/drug therapy* ; Mitochondria/physiology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; bcl-X Protein/genetics*

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
Acrylamides/pharmacology* ; Animals ; Apoptosis/drug effects* ; Cryopreservation ; Disaccharides/pharmacology* ; Electrolytes/pharmacology* ; Glutamates/pharmacology* ; Glutathione/pharmacology* ; Heart/physiology* ; Heart Transplantation/methods* ; Hepatocyte Growth Factor/antagonists & inhibitors* ; Histidine/pharmacology* ; Histone Deacetylase Inhibitors/pharmacology* ; Intracellular Signaling Peptides and Proteins/antagonists & inhibitors* ; Male ; Mannitol/pharmacology* ; Oxidative Stress/drug effects* ; Phenylenediamines/pharmacology* ; Proto-Oncogene Proteins/antagonists & inhibitors* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; YAP-Signaling Proteins

OBJECTIVE: To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. METHODS: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. RESULTS: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL (P<0.05). CONCLUSION The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Hepatic Stellate Cells ; drug effects ; physiology ; Humans ; Injections ; Liver Cirrhosis ; drug therapy ; Tumor Suppressor Proteins ; genetics

Atherosclerotic cardiovascular disease (ASCVD) is the deadliest disease in the world, with endothelial injury occurring throughout the course of the disease. Therefore, improvement in endothelial function is of essential importance in the prevention of ASCVD. Red yeast rice (RYR), a healthy traditional Chinese food, has a lipid modulation function and also plays a vital role in the improvement of endothelial reactivity and cardiovascular protection; thus, it is significant in the prevention and treatment of ASCVD. This article reviews the molecular mechanisms of RYR and its related products in the improvement of endothelial function in terms of endothelial reactivity, anti-apoptosis of endothelial progenitor cells, oxidative stress alleviation and anti-inflammation.
Apoptosis ; drug effects ; Atherosclerosis ; pathology ; physiopathology ; prevention & control ; Biological Products ; chemistry ; pharmacology ; therapeutic use ; Cardiovascular Diseases ; pathology ; physiopathology ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; therapeutic use ; Endothelium, Vascular ; cytology ; drug effects ; physiology ; Humans ; Inflammation ; prevention & control ; Lipid Metabolism ; drug effects ; Oxidative Stress ; drug effects

ObjectiveTo explore the antagonistic effect of vitamin E (VE) on male reproductive toxicity induced by di-2-ethylhexyl phthalate (DEHP) in pubertal SD rats and its underlying mechanisms.

METHODSThirty 5-week-old male SD rats were randomly divided into five groups of equal number, corn oil control, low-dose (10 mg/kg/d), medium-dose (100 mg/kg/d) and high-dose DEHP exposure (500 mg/kg/d), and VE intervention (high-dose DEHP + VE ［100 mg/kg/d］), and treated respectively for 30 successive days. At 3 days after treatment, the testes of the animals were harvested for determination of the oxidative stress index, serum reproductive hormone levels, cauda epididymal sperm parameters, and expressions of cell apoptosis-related genes and proteins.

RESULTSCompared with the control group, the rats of the medium- and high-dose DEHP groups showed significant decreases in the levels of such serum reproductive hormones as follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), sperm parameters as average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), linearity (LIN) and wobble (WOB), and the activities of superoxide dismutase (SOD) and glutathione peroxide (GSH-Px), but significant increases were observed in the latter two groups in the content of malondialdehyde (MDA)(［3.32±0.87］ nmol/mg pro vs ［2.13±0.49］ nmol/ mg pro), mRNA expressions of Bad, Bax, Cytochrome C, Caspase-3 and the Bax/Bcl-2 ratio, and protein expressions of Cytochrome C and Caspase-3. In comparison with the high-dose DEHP group, the VE intervention group exhibited remarkably increased serum LH and T levels, sperm VAP, VSL, VCL, STR and WOB, and activities of SOD and GSH-Px, but markedly decreased mRNA expressions of Bad, Bax, Cytochrome C, Caspase-3 and the Bax/Bcl-2 ratio as well as the protein expressions of Cytochrome C and Caspase-3 in the testis tissue (P<0.05).

CONCLUSIONSExposure to DEHP induces androgen secretion disorders, causes oxidative damage to the testicular tissue, activates the mitochondrial apoptosis pathway in the testis, and ultimately reduces the quality of epididymal sperm, while VE can protect the rat testis from DEHP-induced reproductive toxicity.

Animals ; Antioxidants ; pharmacology ; Apoptosis ; genetics ; Autophagy-Related Protein 5 ; metabolism ; Caspase 3 ; metabolism ; Diethylhexyl Phthalate ; antagonists & inhibitors ; Epididymis ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Malondialdehyde ; metabolism ; Mitochondria ; drug effects ; Oxidative Stress ; drug effects ; Oxidoreductases ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reproduction ; Spermatozoa ; drug effects ; physiology ; Superoxide Dismutase ; metabolism ; Testis ; drug effects ; Testosterone ; blood ; Vitamin E ; pharmacology

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.
Animals ; Antifreeze Proteins/*pharmacology ; Apoptosis/drug effects ; Cryopreservation ; Cryoprotective Agents/pharmacology ; Female ; Mice ; Ovarian Follicle/cytology/drug effects ; Ovary/cytology/drug effects/*physiology ; *Vitrification/drug effects

OBJECTIVE: To investigate the anti-leukemia effect of total saponins of Rubus parvifolius L. (TSRP) on K562 cell xenografts in nude mice and the mechanisms of action. METHODS: The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group(Ara-c) and 3 TSRP groups (20, 40 and 100 mg/kg). The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin (HE) and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 (STAT3), eukaryotic initiation factor 4E (eIF4E) and B-cell lymphoma-2 (bcl-2) were determined with immunohistochemistry tests. RESULTS: Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a signifificant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a signifificant decrease in tumor growth rate and tumor weight in comparison to the control group (all P<0.05). The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of eIF4E and STAT3 were decreased obviously after the treatment of TSRP. CONCLUSION TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.
Animals ; Apoptosis ; drug effects ; Eukaryotic Initiation Factor-4E ; physiology ; Humans ; K562 Cells ; Leukemia ; drug therapy ; pathology ; Male ; Mice ; Rubus ; chemistry ; STAT3 Transcription Factor ; physiology ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Xenograft Model Antitumor Assays

OBJECTIVE: To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes. METHODS: H9c2 cardiac myocytes were treated with HO, gastrodin, gastrodin+HO, cyclosporin A (CsA), or CsA+gas+HO group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells. RESULTS: Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in HO-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in HO-exposed cells. CONCLUSIONS Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.
Adenosine Triphosphate ; analysis ; Apoptosis ; drug effects ; Benzyl Alcohols ; antagonists & inhibitors ; pharmacology ; Caspase 3 ; analysis ; Cell Line ; Cell Survival ; drug effects ; Cyclosporine ; pharmacology ; Cytochromes c ; analysis ; Glucosides ; antagonists & inhibitors ; pharmacology ; Humans ; Hydrogen Peroxide ; antagonists & inhibitors ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondrial Membrane Transport Proteins ; physiology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

Oligodendrocytes (OLs) are myelinating glial cells that form myelin sheaths around axons to ensure rapid and focal conduction of action potentials. Here, we found that an axonal outgrowth regulatory molecule, AATYK (apoptosis-associated tyrosine kinase), was up-regulated with OL differentiation and remyelination. We therefore studied its role in OL differentiation. The results showed that AATYK knockdown inhibited OL differentiation and the expression of myelin genes in vitro. Moreover, AATYK-deficiency maintained the proliferation status of OLs but did not affect their survival. Thus, AATYK is essential for the differentiation of OLs.
Animals ; Animals, Newborn ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cell Proliferation ; drug effects ; genetics ; Cells, Cultured ; Cuprizone ; toxicity ; Demyelinating Diseases ; chemically induced ; metabolism ; pathology ; Embryo, Mammalian ; Gene Expression Regulation, Developmental ; genetics ; Ki-67 Antigen ; metabolism ; Mice ; Mice, Inbred C57BL ; Myelin Basic Protein ; metabolism ; Myelin Proteolipid Protein ; metabolism ; Myelin Sheath ; drug effects ; metabolism ; Oligodendroglia ; drug effects ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley