-
Molecular Medicine Reports Oct 2015Previous studies have reported the antitumor activity of N‑Myc downstream‑regulated gene 2 (NDRG2), a novel p53‑inducible gene, in several types of cancer. The...
Previous studies have reported the antitumor activity of N‑Myc downstream‑regulated gene 2 (NDRG2), a novel p53‑inducible gene, in several types of cancer. The present study aimed to investigate the effects of NDRG2 expression on the proliferation of a human bladder cancer cell line. NDRG2 and control green fluorescent protein (GFP) recombinant adenovirus plasmids were constructed and transfected into a bladder cancer cell line with mutant p53 (T24 cells). NDRG2 expression was analyzed using western blot analysis and immunofluorescence assay (IFA); in addition, the subcellular localization of NDRG2 was detected using a confocal microscope. The proliferation rate of cells was measured using colony formation and MTT assays. Furthermore, the cell cycle of transfected T24 cells was detected by flow cytometry. The results indicated that T24 cells expressed low levels of NDRG2 prior to infection with GFP‑NDRG2 recombinant adenovirus; by contrast, following infection, NDRG2 was primarily overexpressed in mitochondria. The proliferation rate of T24 cells was significantly reduced by NDRG2 expression (P<0.01). In addition, 82.1% of NDRG2‑expressing cells were in S‑phase, compared to 74.4% in the control virus‑infected cells (P<0.05). Furthermore, upregulation of NDRG2 induced an increase in oncosis, rather than apoptosis, in T24 cell. In conclusion, the results of the present study indicated that NDRG2 expression in mitochondria may arrest bladder cancer cells in S‑phase as well as decrease cell proliferation through inducing oncosis. It was therefore proposed that NDRG2 was not only a biomarker, but also a tumor suppressor for bladder cancer.
Topics: Adenoviridae; Cell Death; Cell Line, Tumor; Cell Proliferation; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Humans; Mitochondria; Osmotic Pressure; S Phase Cell Cycle Checkpoints; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Urinary Bladder
PubMed: 26239274
DOI: 10.3892/mmr.2015.4169 -
Journal of Biomedical Optics Feb 2019Optical coherence tomography (OCT) is used to provide anatomical information of biological systems but can also provide functional information by characterizing the...
Optical coherence tomography (OCT) is used to provide anatomical information of biological systems but can also provide functional information by characterizing the motion of intracellular structures. Dynamic light scattering OCT was performed on intact, control MCF-7 breast cancer cells and cells either treated with paclitaxel to induce apoptosis or deprived of nutrients to induce oncosis. Autocorrelations (ACs) of the temporal fluctuations of OCT intensity signals demonstrate a significant decrease in decorrelation time after 24 h in both the paclitaxel-treated and nutrient-deprived cell groups but no significant differences between the two groups. The acquired ACs were then used as input for the CONTIN deconvolution algorithm, and all produced CONTIN outputs with three distinct peaks for all experimental conditions. After 24 h of either paclitaxel treatment or nutrient deprivation, the area-under-the-curve (AUC) of the first peak increased significantly while the AUC of the third peak decreased significantly. These results lend strong support to the hypothesis that ACs acquired from cells are composed of multiple components that correspond to light scattered by different subcellular structures and organelles.
Topics: Algorithms; Apoptosis; Area Under Curve; Dynamic Light Scattering; Female; Humans; Light; MCF-7 Cells; Motion; Paclitaxel; Scattering, Radiation; Tomography, Optical Coherence
PubMed: 30770677
DOI: 10.1117/1.JBO.24.2.025002 -
The Journal of Biological Chemistry Aug 2015Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is...
Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.
Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Carrier Proteins; Caspases; Cell Line, Tumor; Chromatin; Colchicine; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Humans; Microfilament Proteins; Necrosis; Neurons; Nocodazole; Peptidomimetics; Quinolines; Rotenone; Signal Transduction; Staurosporine; Thapsigargin
PubMed: 26124276
DOI: 10.1074/jbc.M115.644179 -
Molecular Therapy Oncolytics Mar 2017Enadenotucirev (EnAd) is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse...
Enadenotucirev (EnAd) is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse range of carcinoma cells, shows effective anticancer activity in preclinical systems, and is currently undergoing phase I/II clinical trials. EnAd kills cells more quickly than type 5 adenovirus, and speed of cytotoxicity is dose dependent. The EnAd death pathway does not involve p53, is predominantly caspase independent, and appears to involve a rapid fall in cellular ATP. Infected cells show early loss of membrane integrity; increased exposure of calreticulin; extracellular release of ATP, HSP70, and HMGB1; and influx of calcium. The virus also causes an obvious single membrane blister reminiscent of ischemic cell death by oncosis. In human tumor biopsies maintained in ex vivo culture, EnAd mediated release of pro-inflammatory mediators such as TNF-α, IL-6, and HMGB1. In accordance with this, EnAd-infected tumor cells showed potent stimulation of dendritic cells and CD4 T cells in a mixed tumor-leukocyte reaction in vitro. Whereas many viruses have evolved for efficient propagation with minimal inflammation, bioselection of EnAd for rapid killing has yielded a virus with a short life cycle that combines potent cytotoxicity with a proinflammatory mechanism of cell death.
PubMed: 28345021
DOI: 10.1016/j.omto.2016.11.003 -
Oncotarget Oct 2015Despite advances in the development of molecularly targeted therapies, metastatic renal cell carcinoma (RCC) is still incurable. Artesunate (ART), a well-known...
Repurposing the anti-malarial drug artesunate as a novel therapeutic agent for metastatic renal cell carcinoma due to its attenuation of tumor growth, metastasis, and angiogenesis.
Despite advances in the development of molecularly targeted therapies, metastatic renal cell carcinoma (RCC) is still incurable. Artesunate (ART), a well-known anti-malarial drug with low toxicity, exhibits highly selective anti-tumor actions against various tumors through generation of cytotoxic carbon-centered free radical in the presence of free iron. However, the therapeutic efficacy of ART against metastatic RCC has not yet been fully elucidated. In the analysis on a dataset from The Cancer Genome Atlas (TCGA) (n = 469) and a tissue microarray set from Samsung Medical Center (n = 119) from a cohort of patients with clear cell RCC (ccRCC), up-regulation of transferrin receptor 1 (TfR1), which is a well-known predictive marker for ART, was correlated with the presence of distant metastasis and an unfavorable prognosis. Moreover, ART exerted potent selective cytotoxicity against human RCC cell lines (Caki-1, 786-O, and SN12C-GFP-SRLu2) and sensitized these cells to sorafenib in vitro, and the extent of ART cytotoxicity correlated with TfR1 expression. ART-mediated growth inhibition of human RCC cell lines was shown to result from the induction of cell cycle arrest at the G2/M phase and oncosis-like cell death. Furthermore, ART inhibited cell clonogenicity and invasion of human RCC cells and anti-angiogenic effects in vitro in a dose-dependent manner. Consistent with these in vitro data, anti-tumor, anti-metastatic and anti-angiogenic effects of ART were also validated in human 786-O xenografts. Taken together, ART is a promising novel candidate for treating human RCC, either alone or in combination with other therapies.
Topics: Animals; Antigens, CD; Antimalarials; Antineoplastic Agents; Artemisinins; Artesunate; Carcinoma, Renal Cell; Cell Death; Cell Line, Tumor; Cell Proliferation; Female; Human Umbilical Vein Endothelial Cells; Humans; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Prognosis; Reactive Oxygen Species; Receptors, Transferrin; Xenograft Model Antitumor Assays
PubMed: 26426994
DOI: 10.18632/oncotarget.5422 -
Toxins Feb 2015Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum...
Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host's innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process.
Topics: Animals; Bacterial Toxins; Cell Survival; Chlorocebus aethiops; Clostridium septicum; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Muscle, Skeletal; Pore Forming Cytotoxic Proteins; Proto-Oncogene Proteins c-raf; Sepsis; Spleen; Vero Cells
PubMed: 25675415
DOI: 10.3390/toxins7020516 -
Biophysical Journal Jun 2017Although it has previously been shown that the spectral analysis of ultrasound backscatter data is sensitive to the cellular changes caused by apoptosis, the sensitivity...
Although it has previously been shown that the spectral analysis of ultrasound backscatter data is sensitive to the cellular changes caused by apoptosis, the sensitivity of spectral analysis to oncosis or ischemic cell death had not previously been studied. Whereas many anticancer treatments induce apoptosis, others induce cell starvation, or oncosis. HT-29 colon adenocarcinoma cells were formed into pellets and covered in phosphate-buffered saline at room temperature for 56 h. The pellets were imaged every 8 h with high-frequency (55 MHz) ultrasound and the raw radio-frequency data processed. The lack of nutrients available to the cells induced cell death due to oncosis. The attenuation slope, speed of sound, spectral slope, and midband fit were estimated at each of the eight time points to identify changes as the cells died due to starvation. The spectral slope decreased monotonically over the 56 h, whereas the attenuation slope showed an increase between 1 and 48 h, followed by a slight decrease between 48 and 56 h. The midband fit did not vary over time. The speed of sound increased from 1514 to 1532 m/s over the first 24 h, after which time it plateaued. These in vitro results indicate different trends in ultrasound parameter changes from those of in vitro apoptotic cells, suggesting that these different methods of cell death can be identified not only by morphological markers, but also by specific ultrasound signatures.
Topics: Cell Count; Cell Culture Techniques; Cell Death; Cell Line, Tumor; Cell Nucleus Size; Cell Size; Humans; In Situ Nick-End Labeling; Signal Processing, Computer-Assisted; Ultrasonography
PubMed: 28636919
DOI: 10.1016/j.bpj.2017.05.017