Transferase-mediated dUTP-biotin Nick End-labeling Assay Research Articles

The Long Noncoding RNA Gall Bladder Cancer-Associated Suppressor of Pyruvate Carboxylase Inhibits the Proliferation, Migration, and Invasion of Colorectal Cancer Cells and Induces Their Apoptosis.

This study aimed to determine the role of the long noncoding RNA (lncRNA) gall bladder cancer-associated suppressor of pyruvate carboxylase (SOD2-1) in the progression of colorectal cancer (CRC). A total of 23 pairs of specimens, including CRC tissues and adjacent normal tissues, were collected, and the expression of lncRNA SOD2-1 (lnc-SOD2-1) was measured. lnc-SOD2-1 function was examined using HCT15 and HCT116 cells. A lnc-SOD2-1 overexpression vector was designed and transfected into both cell lines. MTS and colony formation assays were used to determine cell viability. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays were performed to measure apoptosis. Cell migration and invasion were evaluated using the Transwell assay. Migration and invasion markers were validated using quantitative reverse transcription-polymerase chain reaction and western blot analysis. The results indicated that the expression of lnc-SOD2-1 was downregulated in CRC tissues. lnc-SOD2-1 overexpression evidently decreased cell viability and led to the formation of fewer cell colonies. lnc-SOD2-1 overexpression induced ~ twofold higher apoptosis than the control group. lnc-SOD2-1 overexpression reduced the proportion of migratory and invasive cells to 50% and 75% of the control group, respectively. lnc-SOD2-1 overexpression significantly decreased the expression of matrix metalloproteinase-2 and -9. In conclusion, lnc-SOD2-1 may act as a tumor suppressor that inhibits the proliferation, migration, and invasion of CRC cells and induces their apoptosis.

D-galactose-induced mitochondrial oxidative damage and apoptosis in the cochlear stria vascularis of mice

BackgroundAge-related hearing loss, known as presbycusis, is the result of auditory system degeneration. Numerous studies have suggested that reactive oxygen species (ROS) and mitochondrial oxidative damage play important roles in the occurrence and progression of aging. The D-galactose (D-gal)-induced aging model is well known and widely utilized in aging research. Our previous studies demonstrate that administration of D-gal causes mitochondrial oxidative damage and causes subsequent dysfunction in the cochlear ribbon synapses, which in turn leads to hearing changes and early stage presbycusis. Stria vascularis (SV) cells are vital for hearing function. However, it is unclear to what extent D-gal induces oxidative damage and apoptosis in the cochlear SV of mice. In addition, the source of the causative ROS in the cochlear SV has not been fully investigated.MethodsIn this study, we investigated ROS generation in the cochlear SV of mice treated with D-gal. Hearing function was measured using the auditory brainstem response (ABR). Immunofluorescence was used to examine apoptosis and oxidative damage. Transmission electron microscopy was also used to investigate the mitochondrial ultrastructure. DNA fragmentation was determined using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Mitochondrial membrane potential (MMP) and ATP were also measured.ResultsWe found that D-gal-treated mice exhibited a significant shift in the mean amplitude and latency of the ABR; a remarkable increase in the levels of NADPH oxidase (NOX-2), Uncoupling protein 2 (UCP2) and cleaved caspase-3 (c-Cas3) was observed, as well as an increase in the number of TUNEL-positive cells were observed in the SV of mice. Both the expression of the DNA oxidative damage biomarker 8-hydroxy-2-deoxyguanosine (8-OHdG) and a commonly occurring mitochondrial DNA deletion were markedly elevated in the SV of mice that had been treated with D-gal to induce aging. Conversely, the ATP level and MMP were significantly reduced in D-gal-induced aging mice. We also found alterations in the mitochondrial ultrastructure in the SV of aging mice, which include swollen and distorted mitochondrial shape, shortened and thickened microvilli, and the accumulation of lysosomes in the SV.ConclusionOur findings suggest that the impairment of cochlear SV during presbycusis may be caused by mitochondrial oxidative damage and subsequent apoptosis.

APOPTOSIS OCCURS IN ANTERIOR TALOFIBULAR LIGAMENT OF CHRONIC LATERAL ANKLE INSTABILITY: “ADD EVIDENCE FROM BENCH TO BED”

Chronic lateral ankle instability (CLAI) is treated operatively, whereas acute ligament injury is usually treated nonoperatively. Such treatments have been widely validated. Apoptosis is known to cause ligament degeneration; however, few reports have focused on the possible role of apoptosis in degeneration of ruptured lateral ankle ligaments. The aim of our study is to elucidate the apoptosis that occurs within anterior talofibular ligament (ATFL) to further validate current CLAI treatments by adducing molecular and cellular evidence.Between March 2019 and February 2021, 50 patients were prospectively enrolled in this study. Ruptured ATFL tissues were collected from 21 CLAI patients (group C) and 17 acute ankle fracture patients (group A). Apoptotic cells were counted using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Western blotting for caspases 3, 7, 8, and 9 and cytochrome c, was performed to explore intrinsic and extrinsic apoptotic pathways. Immunohistochemistry was used to detect caspases 3, 7, 8, and 9 and cytochrome c, in ligament vessel endothelial cells.More apoptotic cells were observed in group C than group A in TUNEL assay. Western blotting revealed that the apoptotic activities of group C ligaments were significantly higher than those of group A (all p < 0.001). Immunohistochemistry revealed increased expression of caspases 3, 7, 8, and 9, and cytochrome c, in group C compared to group A.The ATFL apoptotic activities of CLAI patients were significantly higher than those of acute ankle fracture patients, as revealed biochemically and histologically. Our data further validate current CLAI treatments from a molecular and cellular perspective. Efforts should be made to reverse or prevent ATFL apoptosis in CLAI patients.

In vitro high glucose increases apoptosis, decreases nerve outgrowth, and promotes survival of sympathetic pelvic neurons.

Diabetes mellitus (DM) is a common cause of erectile dysfunction (ED), yet the molecular basis of DM neurogenic ED remains unknown. In this study we examined the impact of high glucose on survival and growth of primary cultured pelvic neurons in a rat model and assessed whether coculturing with healthy Schwann cells (SCs) can rescue pelvic neuron growth in patients with DM. Major pelvic ganglia (MPGs) from adult male Sprague Dawley rats (n = 8) were dissociated and plated on coverslips. Neurons were exposed to high glucose (45mM) for 24 or 48hours and compared to time-matched controls (25mM). Neurons were stained for neuron-specific beta-tubulin, neuronal nitric oxide synthase, vesicular acetylcholine transferase, tyrosine hydroxylase, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) assay. Schwann cells were dissociated from MPGs of healthy male Sprague Dawley rats (n = 4) and grown to confluence. Additional Sprague Dawley rats were made diabetic with streptozotocin (50mg/kg, n = 4), and 5weeks later MPGs were collected from these rats, dissociated, and cocultured on healthy SCs. Neurons and SCs were stained with beta-tubulin and S100. Length, branching, and survival of nitrergic, parasympathetic, and sympathetic neurons was assessed in neurons exposed to normal or high glucose concentrations, and neuron length was measured in neuron-SC coculture. The total number of neurons and the length and number of branches were significantly decreased after 24 and 48hours of high glucose (P < .05). The percentage of nitrergic neurons decreased 10% after 24 hours and 50% after 48 hours of high glucose (P < .05). After 24 hours of high glucose, cholinergic-positive neurons were unchanged; however, these neurons decreased 30% after 48 hours (P < .05). The proportion of sympathetic neurons increased 25% after 48 hours of high glucose (P < .05). At both timepoints, there was a 2-fold increase in the total apoptotic neurons with high glucose (P < .05). Neurite outgrowth recovered to control lengths after coculture of diabetic neurons with healthy SCs (P < .05). Glucose can be used as a tool to investigate the direct effects of DM on neuritogenesis. Our data suggest that an effective treatment for DM ED protects and repairs the penile neuronal supply. Exposing MPG neurons to high glucose offers a quick and, inexpensive proxy for DM-related conditions. A limitation of our study is that our model reflects type 1 DM, whereas clinically, most diabetic ED patients have type 2 DM. Culturing pelvic neurons in high glucose can be used as a tool to elucidate how to protect proerectile neurons from cell death and may lead to new therapeutic strategies for diabetic men suffering from ED.

AEBP1 Contributes to Breast Cancer Progression by Facilitating Cell Proliferation, Migration, Invasion, and Blocking Apoptosis.

The aberrant expression of adipocyte enhancer binding protein 1 (AEBP1) has been observed in many cancers and it seems to be involved in the tumorigenesis, progression, and metastasis in numerous tumor types. However, the contribution of AEBP1 in breast cancer (BCa) remains inexplicable. Information related to the diagnostic significance and expression of AEBP1 in BCa was obtained from the public dataset Kaplan-Meier Plotter (http://kmplot.com/analysis/) and the dataset UALCAN (https://ualcan.path.uab.edu/index.html). The MTT (methyl thiazolyl tetrazolium) assay, colony formation assay, Transwell® assay, and FACS (fluorescence-activated cell sorting) assay were used to detect the proliferation, invasive and apoptotic ability of cells before and after treatment. In addition, we constructed an AEBP1 overexpression vector and silenced AEBP1, combined with Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), western blot, immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) assay to investigate the prognostic significance, biological functions and potential mechanisms of AEBP1 in BCa. Higher expression of AEBP1 mRNA (message RNA) was observed in BCa patients with later-stage, who obtained poorer overall survival. Meanwhile, compared with adjacent noncancerous tissues, AEBP1 protein expression was dramatically upregulated in the BCa ones. Furthermore, overexpressed AEBP1 enhanced cell proliferation, migration, invasion, and blocked cell apoptosis in BCa cells. Moreover, the research certificated that AEBP1 upregulated the expression of MMP (matrix metalloproteinase)-2, 9, vimentin, N-cadherin (neural-cadherin), phosphorylation of ERK (extracellular signal-regulated kinase), Smad2/3 (Abbreviated from Sma for nematode and Mad for Drosophila) and AKT (V-akt murine thymoma viral oncogene homolog), while down-regulated the expression of E-cadherin (epithelial cadherin) and PTEN (phosphatase and tensin homolog deleted on chromosome 10). To inhibit cell apoptosis, enforced expression of AEBP1 effectively blocked the cleavage of caspase 9 and p53 (protein 53) and promoted the expression of anti-apoptotic protein Bcl-2 (B-cell lymphoma-2). Finally, AEBP1 accelerated subcutaneously transplanted tumor growth in nude mice by increasing the expression of the cell proliferation biomarker ki67, the phosphorylation of AKT, and blocked apoptosis in vivo. In summary, these data suggested the important role of AEBP1 in the BCa progression, which could be used as a potential biomarker for prognostic hallmark and a novel therapeutic strategy.

Exposure to benzo[α]pyrene causes oxidative stress and cell damage in bay scallop Argopecten irradians

In the present study, bay scallop antioxidant response to acute exposure to the benzo[α]pyrene (BaP; a type of polycyclic aromatic hydrocarbon) was investigated. Scallops were exposed to doses of 0.5 and 1.0 µg/L (low-dose group) and 10 and 50 µg/L (high-dose group) for 72 h. The following oxidative stress indicators were measured in the digestive diverticula tissues or hemolymph: superoxide dismutase, catalase, hydrogen peroxide, and lipid peroxidation. The apoptotic response in the high dose group was assessed using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The stress indicators in each group increased significantly with time, except in the 0.5 µg/L group. The TUNEL assay revealed that more apoptotic cells were detected after exposure to BaP over time. These results suggest that exposure to BaP induces oxidative stress and causes tissue damage in bivalves. These findings also revealed that high concentrations of BaP exposure can have potential adverse effects (apoptosis and cell damage) in bivalves. Additionally, these results might shed some new light on the ecotoxicological effects of BaP on marine bivalves.

Effect of mistletoe extract on tumor response in neoadjuvant chemoradiotherapy for rectal cancer: a cohort study

BackgroundMistletoe extract, used as a complementary chemotherapeutic agent for cancer patients, has anticancer effects against various malignancies. The aim of the present study was to evaluate the effect of mistletoe extract (Abnoba Viscum Q®) on tumor responses to neoadjuvant chemoradiotherapy (NCRT) for locally advanced rectal cancer.MethodsThis study included patients with rectal cancer who underwent NCRT between January 2018 and July 2020. In the mistletoe group (MG), the patients were administered Abnoba Viscum Q® subcutaneously during chemoradiotherapy—maintained just before surgery. Patient demographics, clinical outcomes, histopathological outcomes, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay results were compared between the MG and non-mistletoe group (NMG). Two rectal cancer cell lines (SNU-503 and SNU-503R80Gy) were treated with Abnoba Viscum Q® to assess its mechanistic effects in vivo.ResultsOverall, the study included 52 patients (MG: n = 15; NMG: n = 37). Baseline demographics between the two groups were similar, except carbohydrate antigen 19-9 levels and tumor location from the anal verge. There was no difference in the clinical stage between the two groups. A better tumor response in the MG, relative to the NMG, was observed with respect to tumor regression grade (TRG), T stage, and overall tumor–node–metastasis stage. Tumor response was significantly better in the MG than in the NMG in terms of pathologic complete response rate (53.3% vs. 21.6%, P = 0.044), good TRG response (66.7% vs. 32.4%, P = 0.024), T downstaging (86.7% vs. 43.2%, P = 0.004), and overall downstaging (86.7% vs. 56.8%, P = 0.040). The toxicities during NCRT were minimal in both groups. More apoptotic cells were noted in MG samples than in the NMG samples on TUNEL staining. Cleaved caspase-3 level following treatment with Abnoba Viscum Q® was higher in SNU-503R80Gy cells than in SNU-503 cells.ConclusionPatients treated with chemoradiation combined with mistletoe extract showed better outcomes than patients not treated with mistletoe extract in terms of tumor responses. This diversity in treatment may improve the efficacy of NCRT, leading to better oncologic outcomes. Prospective and randomized studies with long-term follow-up are warranted to confirm and extend these results.

Indole-3-carbinol inhibits the proliferation of colorectal carcinoma LoVo cells through activation of the apoptotic signaling pathway.

Indole-3-carbinol (I3C) is a phytochemical that exhibits growth-inhibitory activity against various cancer cells. However, there are limited studies on the effects of I3C on colon cancer cells. In this study, the growth-inhibitory activity of I3C against the human colorectal carcinoma cell line (LoVo) was examined. The results of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, colony formation, and cell counting assays revealed that I3C suppressed the proliferation of LoVo cells. Microscopy and wound-healing analyses revealed that I3C affected the morphology and inhibited the migration of LoVo cells, respectively. I3C induced apoptosis and DNA fragmentation as evidenced by the results of fluorescein isothiocyanate-conjugated annexin V staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay, respectively. Additionally, I3C arrested the cell cycle at the G0/G1 phase and enhanced the reactive oxygen species levels. Western blotting analysis revealed that treatment with I3C resulted in the activation of apoptotic proteins, such as poly(ADP-ribose) polymerase, caspase-3, caspase-7, caspase-9, Bax, Bim, and p53 in LoVo cells. These results indicate that I3C induces apoptosis in LoVo cells by upregulating p53, leading to the activation of Bax and caspases. Taken together, I3C exerts cytotoxic effects on LoVo cells by activating apoptosis.

Xinyang Tablet inhibits MLK3-mediated pyroptosis to attenuate inflammation and cardiac dysfunction in pressure overload

Ethnopharmacological relevanceXinyang tablet (XYT) has been traditionally used in the treatment of cardiovascular diseases (CVDs). Our previous study indicated that XYT exhibited protective effects in heart failure (HF). Aim of the studyThe aim of the present study was to determine the protective effects of XYT in pressure overload induced HF and to elucidate its underlying mechanisms of action. Materials and methodsWe analyzed XYT content using high-performance liquid chromatography (HPLC.). Mice were subjected to transverse aortic constriction (TAC) to generate pressure overload–induced cardiac remodeling and were then orally administered XYT or URMC-099 for 1 week after the operation. HL1 mouse cardiomyoblasts were induced by lipopolysaccharides (LPS) to trigger pyroptosis and were then treated with XYT or URMC-099. We used echocardiography (ECG), hematoxylin and eosin (H&E) staining, Masson's trichrome staining and a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay to evaluate the effects of XYT. Messenger ribonucleic acid (mRNA) levels of collagen metabolism biomarkers and inflammation-related factors were detected. We determined protein levels of inflammation- and pyroptosis-related signaling pathway members via Western blot (WB). Caspase-1 activity was measured in cell lysate using a Caspase-1 Activity Assay Kit. Subsequently, to define the candidate ingredients in XYT that regulate mixed-lineage kinase-3 (MLK3), we used molecular docking (MD) to predict and evaluate binding affinity with MLK3. Finally, we screened 24 active potential compounds that regulate MLK3 via MD. ResultsECG, H&E staining, Masson's trichrome staining and TUNEL assay results showed that XYT remarkably improved heart function, amelorated myocardial fibrosis and inhibited apoptosis in vivo. Moreover, it reduced expression of proteins or mRNAs related to collagen metabolism, including collagen type 1 (COL1), fibronectin (FN), alpha smooth-muscle actin (α-SMA), and matrix metalloproteinases-2 and -9 (MMP-2, MMP-9). XYT also inhibited inflammation and the induction of pyroptosis at an early stage, as well as attenuated inflammation and pyroptosis levels in vitro. ConclusionOur data indicated that XYT exerted protective effects against pressure overload induced myocardial fibrosis (MF), which might be associated with the induction of pyroptosis-mediated MLK3 signaling.

Prevention of Necrotizing Enterocolitis through Milk Polar Lipids Reducing Intestinal Epithelial Apoptosis.

Neonatal necrotizing enterocolitis (NEC) is a common and devastating disease. The objective of this research was to investigate the protective mechanisms of milk polar lipids (MPLs) on the attenuation of lipopolysaccharides (LPS)-induced intestinal inflammation and apoptosis. MPLs were extracted from buttermilk and analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A neonatal NEC rat model was used to investigate the effects of MPLs on NEC and its underlying mechanisms. Hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay were used to observe intestinal morphological changes and intestinal epithelial cell apoptosis, which showed that MPLs could reduce NEC symptoms and intestinal apoptosis. The expressions of IL-6, IL-8, and TNF-α in the MPL group was significantly downregulated (P < 0.05), and the expression levels of IL-10 were significantly upregulated (P < 0.05). At the same time, MPLs also significantly reduced (P < 0.05) activation of the LPS-induced TLR4/NF-κB signaling pathway. Furthermore, MPLs inhibit apoptosis by reducing the expressions of Bax, caspase-9, and caspase-3 and by increasing the expression of Bcl-2. In conclusion, MPLs could reduce NEC symptoms in mice by inhibiting cell inflammation and protecting against intestinal apoptosis.

Metformin and rapamycin protect cells from vital dye-induced damage in retinal pigment epithelial cells and in vivo.

To evaluate the effect of autophagy inducers on damage caused by vital dye in adult human RPE (ARPE) cells and in a rat model. ARPE-19 cells were exposed to ICG or BBG (0.05mg/ml) with rapamycin (200nM) or metformin (2mM) for 30min and treated with or without 20μM chloroquine (CQ) to identify the protein levels of LC3 and SQSTM1 by immunoblotting. In vivo study was performed by injecting 10μl 0.05% ICG and 0.25% BBG into the subretinal space of the rat eyes, and/or co-treated them with metformin and rapamycin. The retinas were used to determine autophagy with the LC3-II level and apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay. In this study, both ICG and BBG inhibited autophagy flux in adult human retinal pigment epithelium cells (ARPE-19), whereas only ICG consistently reduced autophagy in the retina of rats. Moreover, rapamycin and metformin induced autophagic flux in ARPE-19 cells and increased the LC3-II level in retinal tissues exposed to vital dyes. Both ICG and BBG increased apoptosis in the retina of rats. However, both rapamycin and metformin induced autophagy and reduced the apoptosis caused by vital dyes. Taken together, these results suggest that rapamycin and metformin may diminish vital dye-induced retinal damage in vivo through activation of autophagy.

Yi-qi-yang-yin-tian-sui-fang enhances cisplatin-induced tumor eradication and inhibits interleukin-7 reduction in non-small cell lung cancer.

Traditional Chinese Medicine (TCM) has been recognized to be conducive to enhancing the efficiency and reducing the side effects in the whole course of cancer treatment. The mechanisms of TCM/chemotherapy combination involved with interleukin-7 (IL-7) potentially enhance immune responses against tumor. In the present study, we emphasized on a herbal formulation Yi-qi-yang-yin-tian-sui-fang or TCM for short, and investigated its roles in chemotherapy in non-small cell lung cancer (NSCLC). The mice bared with tumor were treated with cisplatin (DDP) and simultaneously administrated with/without low, medium and high doses of TCMs (effective content: 0.5, 2.0 and 8.0 g/per mice) via oral gavage. The results indicated that combination of TCM further elevated the therapy efficiency of DDP in a dose-dependent manner. The growth of tumor cells was estimated by Ki-67 stain and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay. The addition of TCM to the DDP treatment could significantly decrease the expression of Ki-67 and promote the apoptosis of tumor cells. In addition, the serum IL-7 level was down-regulated by DDP but restored by the treatment of TCM. The expression of IL-7 and its receptor IL-7R in tumor tissues was also recovered by TCM. Furthermore, the side effect from bone marrow suppression (myelosuppression) induced by DDP were assessed. TCM could abrogate DDP-induced apoptosis of bone marrow and also remarkably induced the expressions of IL-7 and hematopoietic growth factors including G-CSF, GM-CSF, SCF, and SDF-1 in bone marrow. These data indicated that this TCM combined with DDP showed superior anti-tumor effects with reduced myelosuppression via up-regulating IL-7.

YM155 Down-Regulates Survivin and Induces P53 Up-Regulated Modulator of Apoptosis (PUMA)-Dependent in Oral Squamous Cell Carcinoma Cells.

BackgroundYM155, which inhibits the anti-apoptotic protein survivin, is known to exert anti-tumor effects in various cancers. However, there were few reports describing the inhibitory effect of YM155 on human oral squamous cell carcinoma (OSCC) cells that highly express survivin. In this study, we investigated the anti-tumor effects of YM155 on OSCC cells and then examined its molecular mechanisms.Material/MethodsSCC9 cells of OSCC were treated with series of concentrations of YM155 (0.01, 0.1, 1, and 10 ng/ml) for 6, 12, and 24 h. The effect of YM155 on survival of SCC9 cells was detected by MTT and colony formation assay. Cell apoptosis was detected by flow cytometric analysis and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays. Western blot was used to detect the protein expression of survivin, p53, and PUMA. Caspase-3 activity was measured by cleavage of the caspase-3 substrate. To test the role of PUMA and caspase-3 on YM155-induced apoptosis and growth inhibition, the SCC9 cells was transfected with PUMA siRNA or caspase-3 siRNA or control siRNA for 16 h before YM155 (1 and 10 ng/ml) treatment for 24 h. In addition, we also investigated the effect of YM155 in an in vivo xenograft model.ResultsTreatment of YM155 efficiently reduced survivin expression and increased PUMA expression and caspase-3 activation in the SCC9 cells. YM155 treatment resulted in 18–86% decrease in cell viability, 10–60% decrease in colony numbers, and 8–40% increase in cell apoptosis (p<0.05 and p<0.01). However, the induction of cell apoptosis growth inhibition was reversed by PUMA siRNA or caspase-3 transfection. In addition, animals treated with YM155 showed more than 60% tumor growth inhibition compared to the controls (p<0.05).ConclusionsYM155 is a potent inhibitor of progression of SCC9 cells, which could be due to attenuation of survivin, and activation of the PUMA/caspase-3 cellular signaling processes. This study suggests that YM155 may be a potential molecular target with therapeutic relevance for the treatment of OSCC.

RLj-RGD3, a Novel Recombinant Toxin Protein from Lampetra japonica, Protects against Cerebral Reperfusion Injury Following Middle Cerebral Artery Occlusion Involving the Integrin-PI3K/Akt Pathway in Rats.

BackgroundThe RGD-toxin protein Lj-RGD3 is a naturally occurring 118 amino acid peptide that can be obtained from the salivary gland of the Lampetra japonica fish. This unique peptide contains 3 RGD (Arg-Gly-Asp) motifs in its primary structure. Lj-RGD3 is available in recombinant form (rLj-RGD3) and can be produced in large quantities using DNA recombination techniques. The pharmacology of the three RGD motif-containing peptides has not been studied. This study investigated the protective effects of rLj-RGD3, a novel polypeptide, against ischemia/reperfusion-induced damage to the brain caused by middle cerebral artery occlusion (MCAO) in a rat stroke model. We also explored the mechanism by which rLj-RGD3 acts by measuring protein and mRNA expression levels, with an emphasis on the FAK and integrin-PI3K/Akt anti-apoptosis pathways.MethodsrLj-RGD3 was obtained from the buccal secretions of Lampetra japonica using gene recombination technology. Sprague Dawley (SD) rats were randomly divided into the following seven groups: a sham group; a vehicle-treated (VT) group; 100.0 μg·kg-1, 50.0 μg·kg-1 and 25.0 μg·kg-1 dose rLj-RGD3 groups; and two positive controls, including 1.5 mg·kg-1 Edaravone (ED) and 100.0 μg·kg-1 Eptifibatide (EP). MCAO was induced using a model consisting of 2 h of ischemia and 24 h of reperfusion. Behavioral changes were observed in the normal and operation groups after focal cerebral ischemia/reperfusion was applied. In addition, behavioral scores were evaluated at 4 and 24 h after reperfusion. Brain infarct volumes were determined based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining. Moreover, neuronal apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays. We determined the expression levels of focal adhesion kinase (FAK), phosphatidyl inositol 3-kinase (PI3K), protein kinase B (Akt, PKB), caspase-3 and Bcl-2 in the brain using western blot analysis and RT-PCR assays. The research protocol was approved by the Institutional Ethics Committee of Dalian Medical University.ResultsThe behavioral scores and cerebral infarct volumes of the rLj-RGD3 groups were markedly lower at 4 and 24 h/RF. The rLj-RGD3 protein significantly ameliorated pathological changes in the brain and reduced the number of apoptotic neurons. Furthermore, the FAK and PI3K/Akt pathways were activated. rLj-RGD3 significantly increased the expression of FAK, p-FAK and Bcl-2 proteins. In contrast, caspase-3 expression was inhibited.Conclusion/SignificanceWe conclude that recombinant Lampetra japonica RGD-peptide (rLj-RGD3) exerts a protective effect against cerebral ischemia/reperfusion injury in the brain. In addition, the mechanism of this protection is associated with the activation of the integrin-PI3K/Akt pathway. These results provide a theoretical foundation and an experimental basis for using RGD peptides as novel drugs for treating ischemic cerebral vascular diseases in addition to promoting the research and development of marine biotechnology drugs.

Reduced apurinic/apyrimidinic endonuclease activity enhances the antitumor activity of oxymatrine in lung cancer cells.

Lung cancer is the leading cause of cancer-related deaths worldwide and is associated with a very poor outcome. Oxymatrine exerts antitumor effects by inducing apoptosis and inhibiting the proliferation of different cancer cells; however, the anticancer effects and mechanism of action of oxymatrine have not been evaluated sufficiently in human lung cancer cells. Thus, the present study aimed to investigate the anticancer effects of oxymatrine in human lung cancer cells and identify the molecular mechanisms underlying these effects. MTT assays demonstrated that oxymatrine significantly inhibited the proliferation of A549 and H1299 cells in a time- and dose-dependent manner. In addition, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays suggested that oxymatrine treatment may induce lung cancer cell apoptosis in a dose-dependent manner. Furthermore, we detected that oxymatrine induced a significant increase in DNA damage and the expression of PARP and phosphorylated H2AX, and a significant decrease in that of nuclear APE1 and AP endonuclease activity in A549 cells. APE1 knockdown cells (APE1shRNA) plus oxymatrine treatment reduced cells proliferation and induced apoptosis more seriously than control shRNA cells. This appeared to be a consequence of an increase in the number of apurinic/apyrimidinic (AP) sites, DNA damage, PARP and H2AX phosphorylation, which together resulted in the induction of apoptosis. In contrast, the sensitizing effects of APE1 overexpression plus oxymatrine treatment did not occur in APEOE cells. These findings reveal a potential mechanism of action for oxymatrine-induced apoptosis and suggest that oxymatrine is a promising potential therapeutic agent for the treatment of lung cancer.

Mitochondrial Fission Increases Apoptosis and Decreases Autophagy in Renal Proximal Tubular Epithelial Cells Treated with High Glucose

The aim of this study was to examine the effect of mitochondrial morphogenesis changes on apoptosis and autophagy of high-glucose-treated proximal tubular epithelial cells (HK2). Cell viability, apoptosis, and mitochondrial morphogenesis were examined using crystal violet, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and mitotracker staining, respectively. High glucose inhibited cell viability and induced mitochondrial fission in HK2 cells. After depleting mitofusin 1 (MFN1), the MFN1(-) HK2 cells (fission type) became more susceptible to high-glucose-induced apoptosis and mitochondrial fragmentation observed by TUNEL and mitotracker assays. In siMFN2 HK2 cells (fission type), mitochondria were highly fragmented (>80% fission rate) with or without high-glucose treatment; however, siFIS1 (mitochondrial fission protein 1) HK2 cells (fusion type) exhibited little fragmentation (<13%). High-glucose treatment induced autophagy, characterized by the formation of autophagosome and microtubule-associated protein light chain 3 (LC3) B-II, as observed by transmission electron microscopy and western blotting, respectively. LC3B-II levels decreased in both MFN1(-) and siMFN2 HK2 cells, but increased in siFIS1 HK2 cells. Moreover, autophagy displays a protective role against high-glucose-induced cell death based on cotreatment with autophagy inhibitors (3-methyladenine and chloroquine). Mitochondrial fission may increase apoptosis and decrease autophagy of high-glucose-treated HK2 cells.

Ursodeoxycholic acid induces apoptosis in hepatocellular carcinoma xenografts in mice

To evaluate the efficacy of ursodeoxycholic acid (UDCA) as a chemotherapeutic agent for the treatment of hepatocellular carcinoma (HCC). BALB/c nude mice were randomized into four groups 24 h before subcutaneous injection of hepatocarcinoma BEL7402 cells suspended in phosphate buffered saline (PBS) into the right flank. The control group (n = 10) was fed a standard diet while treatment groups (n = 10 each) were fed a standard daily diet supplemented with different concentrations of UDCA (30, 50 and 70 mg/kg per day) for 21 d. Tumor growth was measured once each week, and tumor volume (V) was calculated with the following equation: V = (L × W(2)) × 0.52, where L is the length and W is the width of the xenograft. After 21 d, mice were killed under ether anesthesia, and tumors were excised and weighed. Apoptosis was evaluated through detection of DNA fragmentation with gel electrophoresis and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. Western blot analysis was performed to determine the expression of apoptosis-related proteins BAX, BCL2, APAF1, cleaved caspase-9, and cleaved caspase-3. UDCA suppressed tumor growth relative to controls. The mean tumor volumes were the following: control, 1090 ± 89 mm(3); 30 mg/kg per day, 612 ± 46 mm(3); 50 mg/kg per day, 563 ± 38 mm(3); and 70 mg/kg per day, 221 ± 26 mm(3). Decreased tumor volumes reached statistical significance relative to control xenografts (30 mg/kg per day, P < 0.05; 50 mg/kg per day, P < 0.05; 70 mg/kg per day, P < 0.01). Increasing concentrations of UDCA led to increased DNA fragmentation observed on gel electrophoresis and in the TUNEL assay (control, 1.6% ± 0.3%; 30 mg/kg per day, 2.9% ± 0.5%; 50 mg/kg per day, 3.15% ± 0.7%, and 70 mg/kg per day, 4.86% ± 0.9%). Western blot analysis revealed increased expression of BAX, APAF1, cleaved-caspase-9 and cleaved-caspase-3 proteins, which induce apoptosis, but decreased expression of BCL2 protein, which is an inhibitor of apoptosis, following administration of UDCA. UDCA suppresses growth of BEL7402 hepatocellular carcinoma cells in vivo, in part through apoptosis induction, and is thus a candidate for therapeutic treatment of HCC.

Phagocytosis (cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas.

To identify those with a micropapillary pattern, ascertain relative frequency and document clinicopathological characteristics by reviewing gastric carcinomas. One hundred and fifty-one patients diagnosed with gastric cancer who underwent gastrectomy were retrospectively studied and the presence of a regional invasive micropapillary component was evaluated by light microscopy. All available hematoxylin-eosin (HE)-stained slides were histologically reviewed and 5 tumors were selected as putative micropapillary carcinoma when cancer cell clusters without a vascular core within empty lymphatic-like space comprised at least 5% of the tumor. Tumor tissues from these 5 invasive gastric carcinomas were immunostained using an anti-mucin 1 (MUC1) antibody (clone MA695) to detect the characteristic inside-out pattern and with D2-40 antibody to determine the presence of intratumoral lymph vessels. Detection of intraepithelial neutrophil apoptosis was evaluated in consecutive histological tissue sections by three independent methods, namely light microscopy with HE staining, the conventional terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemistry for activated caspase-3 (clone C92-605). Among 151 gastric cancers resected for cure, 5 (3.3%) were adenocarcinomas with a micropapillary component. Four of the patients died of disease from 6 to 23 mo and one patient was alive with metastases at 9 mo. All patients had advanced-stage cancer (≥ pT2) and lymph node metastasis. Positive MUC1 immunostaining on the stroma-facing surface (inside-out pattern) of the carcinomatous cluster cells, together with negative immunostaining for D2-40 in the cells limiting lymphatic-like spaces, confirmed the true micropapillary pattern in these gastric neoplasms. In all five cases, several micropapillae were infiltrated by neutrophils. HE staining, TUNEL assay and immunostaining for caspase-3 demonstrated apoptotic neutrophils within cytoplasmic vacuoles of tumor cells. These data suggest phagocytosis (cannibalism) of apoptotic neutrophils by micropapillary tumor cells. Tumor cell cannibalism is usually found in aggressive tumors with anaplastic morphology. Our data extend these observations to gastric micropapillary carcinoma: a tumor histotype analogously characterized by aggressive behavior and poor prognosis. The results are of interest because they raise the intriguing possibility that neutrophil cannibalism by tumor cells may be one of the mechanisms favoring tumor growth in gastric micropapillary carcinomas. This is the first study showing phagocytosis (cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas.

Evaluation of apoptosis using TUNEL assay and caspase-3 immunostaining in NMU induced rat mammary tumours

Avoidance of apoptosis is an important event of tumourigenesis. Tumour progression is dependent on balance between cell proliferation and apoptosis. The present study was carried out to evaluate apoptosis in N-methyl nitrosourea (NMU) induced rat mammary tumours by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL) assay in correlation with caspase-3 and PCNA immunostaining. A total of 22 female virgin Wistar rats of 28–30 days of age were selected for the experiment. NMU was injected at 50th, 80th and 110th days of age in 16 rats and 6 control group rats received the vehicle only. Six numbers of mammary tumours were successfully induced in 4 (25%) rats with average latency period of 87±2.63 days. The mean counts of PCNA and caspase-3 immunostaining were 57.67±9.45 and 1.82±0.21, respectively. The mean count of the TUNEL positive cells were 2.44±0.69. It was observed that apoptosis was evident by TUNEL assay and caspase-3 immunostaining in highly proliferative and more aggressive NMU induced rat mammary tumours, similar to that observed in human breast tumours.

Comparison of anti‐inflammatory potential of four different dibenzocyclooctadiene lignans in microglia; action via activation of PKA and Nrf‐2 signaling and inhibition of MAPK/STAT/NF‐κB pathways

The aim of our study was to determine the signaling pathways associated with the antineuroinflammatory and neuroprotective responses induced by dibenzocyclooctadiene lignans in microglia. We employed ELISA, gelatin zymography, transient transfection, Western blot, chromatin immunoprecipitation, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assays to characterize the effects of dibenzocyclooctadiene lignans on microglia. We found that dibenzocyclooctadiene lignans suppress TLR 2/4 agonist-induced pro-inflammatory cytokines and chemokines, PGE2 , nitric oxide, reactive oxygen species (ROS), and MMP-9 enzymatic activity through the suppression of MAPK, NF-κB, and JAK-STAT activation. We next demonstrated that dibenzocyclooctadiene lignans induced the expression of phase II detoxifying/antioxidant enzymes and suppressed the iNOS and ROS activation induced by TLR 2/4 agonists. Interestingly, we also found that dibenzocyclooctadiene lignans induced PKA/CREB/Nrf-2 activation in microglia and that activation of phase II detoxifying/antioxidant enzymes via stimulation of the PKA/CREB/Nrf-2 pathway attenuated TLR 2/4 agonist-induced iNOS and ROS activation. Furthermore, dibenzocyclooctadiene lignans protected primary cortical neurons against microglia-mediated neurotoxicity. Our findings indicate that phase II detoxifying/antioxidant enzymes and their upstream effectors, PKA/CREB/Nrf-2, play a pivotal role in the antineuroinflammatory and neuroprotective effects of dibenzocyclooctadiene lignans in TLR 2/4 agonist-stimulated microglia.