RuvBL2 Is Involved in Histone Deacetylase Inhibitor PCI-24781-Induced Cell Death in SK-N-DZ Neuroblastoma Cells

<div><p>Neuroblastoma is the second most common solid tumor diagnosed during infancy. The survival rate among children with high-risk neuroblastoma is less than 40%, highlighting the urgent needs for new treatment strategies. PCI-24781 is a novel hydroxamic acid-based histone deacetylase (HDAC) inhibitor that has high efficacy and safety for cancer treatment. However, the underlying mechanisms of PCI-24781 are not clearly elucidated in neuroblastoma cells. In the present study, we demonstrated that PCI-24781 treatment significantly inhibited tumor growth at very low doses in neuroblastoma cells SK-N-DZ, not in normal cell line HS-68. However, PCI-24781 caused the accumulation of acetylated histone H3 both in SK-N-DZ and HS-68 cell line. Treatment of SK-N-DZ with PCI-24781 also induced cell cycle arrest in G2/M phase and activated apoptosis signaling pathways via the up-regulation of DR4, p21, p53 and caspase 3. Further proteomic analysis revealed differential protein expression profiles between non-treated and PCI-24781 treated SK-N-DZ cells. Totally 42 differentially expressed proteins were identified by MALDI-TOF MS system. Western blotting confirmed the expression level of five candidate proteins including prohibitin, hHR23a, RuvBL2, TRAP1 and PDCD6IP. Selective knockdown of RuvBL2 rescued cells from PCI-24781-induced cell death, implying that RuvBL2 might play an important role in anti-tumor activity of PCI-24781 in SK-N-DZ cells. The present results provide a new insight into the potential mechanism of PCI-24781 in SK-N-DZ cell line.</p></div

The differentially expressed proteins regulated by PCI-24781 in SK-N-DZ cells (Average ratio ≥1.5 and ≤0.5).

<p>The differentially expressed proteins regulated by PCI-24781 in SK-N-DZ cells (Average ratio ≥1.5 and ≤0.5).</p

Schematic representation of proposed mechanism involved in PCI-24781-induced cell death.

<p>Proteins with up arrow and down arrow represent up-regulation and down-regulation, respectively in response to PCI-24781 treatment. Symbol (−) represents no change upon PCI-24781 treatment. TF: transcription factor in the chromatin complex.</p

PCI-24781 induced G2/M cell cycle arrest and apoptosis in SK-N-DZ not HS-68 cells.

<p>SK-N-DZ and HS-68 cells were treated with the increasing doses of PCI-24781 for 24 h (A and B), and different time points of 0.5 µM PCI-24781 as indicated (C and D), respectively. Cell cycle analysis was performed by propidium iodide staining method. Values are the mean of three independently experiments. Control: no treatment; PCI*: PCI-24781.</p

PCI-24781 affected both extrinsic and intrinsic apoptotic pathways.

<p>(A) SK-N-DZ cells were untreated (control) or treated with 0.5 µM of PCI-24781 for 36 h and the cell lysates were incubated with human apoptosis arrays according to the manufacturer’s instructions. Each protein was spotted in duplicate. The spots in upper left and lower right corner are positive controls. Eight apoptotic proteins were shown below. (B) Densitometric analysis was done by ImageJ software and the fold changes of apoptotic proteins were represented by histogram. (C) The expression of DR4, p21, p53, caspase 3 and cleaved caspase 3 was confirmed by Western blotting. GAPDH was used as loading control.</p

Detection of Lung Cancer: Concomitant Volatile Organic Compounds and Metabolomic Profiling of Six Cancer Cell Lines of Different Histological Origins

In recent years, there has been an extensive search for a non-invasive screening technique for early detection of lung cancer. Volatile organic compound (VOC) analysis in exhaled breath is one such promising technique. This approach is based on the fact that tumor growth is accompanied by unique oncogenesis, leading to detectable changes in VOC emitting profile. Here, we conducted a comprehensive profiling of VOCs and metabolites from six different lung cancer cell lines and one normal lung cell line using mass spectrometry. The concomitant VOCs and metabolite profiling allowed significant discrimination between lung cancer and normal cell, nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC), as well as between different subtypes of NSCLC. It was found that a combination of benzaldehyde, 2-ethylhexanol, and 2,4-decadien-1-ol could serve as potential volatile biomarkers for lung cancer. A detailed correlation between nonvolatile metabolites and VOCs can demonstrate possible biochemical pathways for VOC production by the cancer cells, thus enabling further optimization of VOCs as biomarkers. These findings could eventually lead to noninvasive early detection of lung cancer and differential diagnosis of lung cancer subtypes, thus revolutionizing lung cancer treatment

Comparative Metabolomics in <i>Glycine max</i> and <i>Glycine soja</i> under Salt Stress To Reveal the Phenotypes of Their Offspring

Metabolomics is developing as an important functional genomics tool for understanding plant systems’ response to genetic and environmental changes. Here, we characterized the metabolic changes of cultivated soybean C08 (<i>Glycine max</i> L. Merr) and wild soybean W05 (<i>Glycine soja</i> Sieb.et Zucc.) under salt stress using MS-based metabolomics, in order to reveal the phenotypes of their eight hybrid offspring (9H0086, 9H0124, 9H0391, 9H0736, 9H0380, 9H0400, 9H0434, and 9H0590). Total small molecule extracts of soybean seedling leaves were profiled by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–Fourier transform mass spectrometry (LC–FT/MS). We found that wild soybean contained higher amounts of disaccharides, sugar alcohols, and acetylated amino acids than cultivated soybean, but with lower amounts of monosaccharides, carboxylic acids, and unsaturated fatty acids. Further investigations demonstrated that the ability of soybean to tolerate salt was mainly based on synthesis of compatible solutes, induction of reactive oxygen species (ROS) scavengers, cell membrane modifications, and induction of plant hormones. On the basis of metabolic phenotype, the salt-tolerance abilities of 9H0086, 9H0124, 9H0391, 9H0736, 9H0380, 9H0400, 9H0434, and 9H0590 were discriminated. Our results demonstrated that MS-based metabolomics provides a fast and powerful approach to discriminate the salt-tolerance characteristics of soybeans

Comparative Metabolomics in <i>Glycine max</i> and <i>Glycine soja</i> under Salt Stress To Reveal the Phenotypes of Their Offspring

Metabolomics is developing as an important functional genomics tool for understanding plant systems’ response to genetic and environmental changes. Here, we characterized the metabolic changes of cultivated soybean C08 (<i>Glycine max</i> L. Merr) and wild soybean W05 (<i>Glycine soja</i> Sieb.et Zucc.) under salt stress using MS-based metabolomics, in order to reveal the phenotypes of their eight hybrid offspring (9H0086, 9H0124, 9H0391, 9H0736, 9H0380, 9H0400, 9H0434, and 9H0590). Total small molecule extracts of soybean seedling leaves were profiled by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–Fourier transform mass spectrometry (LC–FT/MS). We found that wild soybean contained higher amounts of disaccharides, sugar alcohols, and acetylated amino acids than cultivated soybean, but with lower amounts of monosaccharides, carboxylic acids, and unsaturated fatty acids. Further investigations demonstrated that the ability of soybean to tolerate salt was mainly based on synthesis of compatible solutes, induction of reactive oxygen species (ROS) scavengers, cell membrane modifications, and induction of plant hormones. On the basis of metabolic phenotype, the salt-tolerance abilities of 9H0086, 9H0124, 9H0391, 9H0736, 9H0380, 9H0400, 9H0434, and 9H0590 were discriminated. Our results demonstrated that MS-based metabolomics provides a fast and powerful approach to discriminate the salt-tolerance characteristics of soybeans

Identifying Early Urinary Metabolic Changes with Long-Term Environmental Exposure to Cadmium by Mass-Spectrometry-Based Metabolomics

Cadmium (Cd) is a common environmental pollutant, and urinary Cd (UCd) is generally used as a marker of exposure; however, our understanding on the related urinary metabolic changes caused by Cd exposure is still not clear. In this study, we applied a mass-spectrometry-based metabolomic approach to assess the urinary metabolic changes in human with long-term environmental Cd exposure, aimed to identify early biomarkers to assess Cd nephrotoxicity. Urine samples from 94 female never smokers aged 44–70 with UCd in the range of 0.20–68.67 μg/L were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-ToF-MS) and gas chromatography–mass spectrometry (GC–MS). It was found that metabolites related to amino acid metabolism (l-glutamine, l-cystine, l-tyrosine, <i>N</i>-methyl-l-histidine, l-histidinol, taurine, phenylacetylglutamine, hippurate, and pyroglutamic acid), galactose metabolism (d-galactose and <i>myo</i>-inositol), purine metabolism (xanthine, urea, and deoxyadenosine monophosphate), creatine pathway (creatine and creatinine), and steroid hormone biosynthesis (17-α-hydroxyprogesterone, tetrahydrocortisone, estrone, and corticosterone) were significantly higher among those with a UCd level higher than 5 μg/L. Moreover, we noticed that the level of <i>N</i>-methyl-l-histidine had already started to elevate among individuals with a UCd concentration of ≥2 μg/L. The overall findings illustrate that metabolomics offer a useful approach for revealing metabolic changes as a result of Cd exposure