Cytotoxicity of 11-epi-Sinulariolide Acetate Isolated from Cultured Soft Corals on HA22T Cells through the Endoplasmic Reticulum Stress Pathway and Mitochondrial Dysfunction.

Natural compounds from soft corals have been increasingly used for their antitumor therapeutic properties. This study examined 11-epi-sinulariolide acetate (11-epi-SA), an active compound isolated from the cultured soft coral Sinularia flexibilis, to determine its potential antitumor effect on four hepatocellular carcinoma cell lines. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the results demonstrated that 11-epi-SA treatment showed more cytotoxic effect toward HA22T cells. Protein profiling of the 11-epi-SA-treated HA22T cells revealed substantial protein alterations associated with stress response and protein synthesis and folding, suggesting that the mitochondria and endoplasmic reticulum (ER) play roles in 11-epi-SA-initiated apoptosis. Moreover, 11-epi-SA activated caspase-dependent apoptotic cell death, suggesting that mitochondria-related apoptosis genes were involved in programmed cell death. The unfolded protein response signaling pathway-related proteins were also activated on 11-epi-SA treatment, and these changes were accompanied by the upregulated expression of growth arrest and DNA damage-inducible protein (GADD153) and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), the genes encoding transcription factors associated with growth arrest and apoptosis under prolonged ER stress. Two inhibitors, namely salubrinal (Sal) and SP600125, partially abrogated 11-epi-SA-related cell death, implying that the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-activating transcription factor (ATF) 6-CHOP or the inositol-requiring enzyme 1 alpha (IRE1α)-c-Jun N-terminal kinase (JNK)-cJun signal pathway was activated after 11-epi-SA treatment. In general, these results suggest that 11-epi-SA exerts cytotoxic effects on HA22T cells through mitochondrial dysfunction and ER stress cell death pathways.

A panel of tumor markers, calreticulin, annexin A2, and annexin A3 in upper tract urothelial carcinoma identified by proteomic and immunological analysis.

BACKGROUND: Upper tract urothelial carcinoma (UTUC) is a tumor with sizable metastases and local recurrence. It has a worse prognosis than bladder cancer. This study was designed to investigate the urinary potential tumor markers of UTUC. METHODS: Between January 2008 and January 2009, urine was sampled from 13 patients with UTUC and 20 healthy adults. The current study identified biomarkers for UTUC using non-fixed volume stepwise weak anion exchange chromatography for fractionation of urine protein prior to two-dimensional gel electrophoresis. RESULTS: Fifty five differential proteins have been determined by comparing with the 2-DE maps of the urine of UTUC patients and those of healthy people. Western blotting analysis and immunohistochemistry of tumor tissues and normal tissues from patients with UTUC were carried out to further verify five possible UTUC biomarkers, including zinc-alpha-2-glycoprotein, calreticulin, annexin A2, annexin A3 and haptoglobin. The data of western blot and immunohistochemical analysis are consistent with the 2-DE data. Combined the experimental data in the urine and in tumor tissues collected from patients with UTUC, the crucial over-expressed proteins are calreticulin, annexin A2, and annexin A3. CONCLUSIONS: Calreticulin, annexin A2, and annexin A3 are very likely a panel of biomarkers with potential value for UTUC diagnosis.

Identification of low-abundance proteins via fractionation of the urine proteome with weak anion exchange chromatography.

BACKGROUND: Low-abundance proteins are difficultly observed on the two-dimensional gel electrophoresis (2-DE) maps of urine proteome, because they are usually obscured by high-abundance proteins such as albumin and immunoglobulin. In this study, a novel fractionation method was developed for enriching low-abundance proteins by removing high-abundance proteins and progressive elution with salts of various concentrations. RESULTS: Stepwise weak anion exchange (WAX) chromatography, which applied DEAE-Sephacel resin with non-fixed volume elution, was used to fractionate urine proteome prior to performing 2-DE. Urine proteome was separated into four fractions by progressively eluting the column with 0 M, 50 mM, 100 mM, and 1 M NaCl solutions. Most of the heavy and light immunoglobulin chains appeared in the eluent. After the high-abundance proteins were removed, various low-abundance proteins were enriched and could be easily identified. The potential of this method for obtaining diversified fractionations was demonstrated by eluting the column separately with Na2SO4 and MgCl2 solutions. The 2-DE maps of the fractions eluted with these different salt solutions of identical ionic strength revealed markedly different stain patterns. CONCLUSION: The present study demonstrated that this fractionation method could be applied for purposes of enriching low-abundance proteins and obtaining diversified fractionations of urine, and potentially other proteomes.

Proteomic analysis of anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells.

The anti-tumor effects of 11-dehydrosinulariolide, an active ingredient isolated from soft coral Sinularia leptoclados, on CAL-27 cells were investigated in this study. In the MTT assay for cell proliferation, increasing concentrations of 11-dehydrosinulariolide decreased CAL-27 cell viability. When a concentration of 1.5 µg/mL of 11-dehydrosinulariolide was applied, the CAL-27 cells viability was reduced to a level of 70% of the control sample. The wound healing function decreased as the concentration of 11-dehydrosinulariolide increased. The results in this study indicated that treatment with 11-dehydrosinulariolide for 6 h significantly induced both early and late apoptosis of CAL-27 cells, observed by flow cytometric measurement and microscopic fluorescent observation. A comparative proteomic analysis was conducted to investigate the effects of 11-dehydrosinulariolide on CAL-27 cells at the molecular level by comparison between the protein profiling (revealed on a 2-DE map) of CAL-27 cells treated with 11-dehydrosinulariolide and that of CAL-27 cells without the treatment. A total of 28 differential proteins (12 up-regulated and 16 down-regulated) in CAL-27 cells treated with 11-dehydrosinulariolide have been identified by LC-MS/MS analysis. Some of the differential proteins are associated with cell proliferation, apoptosis, protein synthesis, protein folding, and energy metabolism. The results of this study provided clues for the investigation of biochemical mechanisms of the anti-tumor effects of 11-dehydrosinulariolide on CAL-27 cells and could be valuable information for drug development and progression monitoring of oral squamous cell carcinoma (OSCC).

Proteomic profiling of the 11-dehydrosinulariolide-treated oral carcinoma cells Ca9-22: effects on the cell apoptosis through mitochondrial-related and ER stress pathway.

An oral squamous cell carcinoma Ca9-22 cell line was treated with 11-dehydrosinulariolide, an active compound isolated from the soft coral Sinularia leptoclados, in order to evaluate the effect of this compound on cell growth and protein expression. Cell proliferation was strongly inhibited by 11-dehydrosinulariolide treatment. The 2-DE master maps of control and treated Ca9-22 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 23 proteins, of which 14 were upregulated and 9 were downregulated. The proteomic studies described here have identified some proteins, which are involved in the mitochondrial dysfunction and ER-stress pathway and imply that 11-dehydrosinulariolide induces cell apoptosis through either mitochondrial dysfunction-related or ER stress pathway. Based on this observation, several proteins related to apoptosis pathway were explored for the potential roles involved in this drug-induced cytotoxicity. Furthermore, Salubrinal, an ER stress inhibitor, is able to protect the cell from 11-dehydrosinulariolide-induced apoptosis in a physiological dosage. The significance of these studies illustrates the potential development of anticancer drugs from the natural derivatives of soft coral.

Cholesteatoma growth and proliferation: relevance with serpin B3.

OBJECTIVES/HYPOTHESIS: The mechanisms of cholesteatoma proliferation and growth remain unclear. The objective of this study is to discover the potential mechanisms of the proliferation and growth of cholesteatoma by direct experimental assessments on cholesteatoma tissues from patients. STUDY DESIGN: Retrospective study by the comparisons between cholesteatoma tissues and retroauricular skin tissues from the patients. METHODS: Two-dimensional gel electrophoresis, LC-MS/MS analysis and immunohistochemistry were performed to investigate specific protein expression in cholesteatoma tissues compared with retroauricular skin tissues collected from the patients. Western blotting analysis was conducted to verify the regulation of specific proteins found by 2-DE, and to determine the changes of associated potential modulators in cholesteatoma proliferation and growth. RESULTS: Twelve serpin B3 isoforms were found by 2-DE and identified by LC-MS/MS analysis, which is coherent with the results exhibited by immunohistochemistry and western blot. Up-regulation of STAT3 and down-regulations of cathepsin K and cathepsin L were represented using western blot. CONCLUSIONS: The data in this study suggested serpin B3, STAT3, cathepsin K, and cathepsin L are associated with the proliferation and growth of cholesteatoma, and these proteins may be influential factors in cholesteatoma growth.