Synergistic effect of a nonsteroidal anti-inflammatory drug in combination with topotecan on small cell lung cancer cells.

BACKGROUND: The topoisomerase I inhibitor topotecan (TPT) is used in the treatment of recurrent small cell lung cancer (SCLC). However, the drug has a limited success rate and causes distress to patients due to its side effects, such as hematologic toxicities, including anemia and thrombocytopenia. Due to these pharmacokinetic limitations and undesirable side effects of chemotherapeutic drugs, the development of combination therapies has gained popularity in SCLC. Meclofenamic acid (MA), a nonsteroidal anti-inflammatory drug, has demonstrated anticancer effects on various types of cancers through different mechanisms. This study aims to investigate the potential synergistic effects of MA and TPT on the small cell lung cancer cell line DMS114. METHODS AND RESULTS: To assess the cytotoxic and apoptotic effects of the combined treatment of MA and TPT, trypan blue exclusion assay, Annexin V, acridine orange/propidium iodide staining, western blot, and cell cycle analysis were conducted. The results demonstrated that the combination of MA and TPT elicited synergistic effects by enhancing toxicity in DMS114 cells (P < 0.01) without causing toxicity in healthy epithelial lung cells MRC5. The strongest synergistic effect was observed when the cells were treated with 60 µM MA and 10 nM TPT for 48 h (CI = 0,751; DRI = 10,871). CONCLUSION: This study, for the first time, furnishes compelling evidence that MA and TPT synergistically reduce cellular proliferation and induce apoptosis in SCLC cells. Combinations of these drugs holds promise as a potential therapeutic strategy to improve efficacy and reduce the side effects associated with TPT.

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival.

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.

Investigation of the effect of meclofenamic acid on the proteome of LNCaP cells reveals changes in alternative polyadenylation and splicing machinery.

Prostate cancer is the most common type of cancer among men, and there is still no definitively effective drug treatment. Thus, the search for novel drug agents that may be used for the effective treatment continues. Meclofenamic acid (MA), a non-steroidal anti-inflammatory drug, with anti-tumor effects in various types of cancers was used to investigate its effects on LNCaP cells, a prostate cancer cell line, at the proteome level. The cells were treated with 80 µM MA for 24 h and a comparative proteomic analysis was performed with their untreated control cells. Proteins were extracted from the cells and then were subjected to two-dimensional gel electrophoresis. Protein spots displaying changes in their regulation ratios for more than two-fold were excised from the gels and identified with MALDI-TOF/TOF mass spectrometry. Bioinformatics analysis of the differentially regulated proteins that we identified showed that they were all associated with and took part in related pathways. Glycolytic pathway, cytoskeletal formation, transport activity, protein metabolism, and most notably an mRNA processing pathway were affected by the MA treatment. In addition to presenting a detailed information for what is happening inside the cells upon MA treatment, the proteins affected by MA treatment hold the potential to be novel targets for prostate cancer treatment provided that further in vivo experiments are carried out.

Effect of aflibercept on proliferative vitreoretinopathy: Proteomic analysis in an experimental animal model.

PURPOSE: The aim of this study was to monitor inflammatory, proliferative and progressive effects of proliferative vitreoretinopathy (PVR) and aflibercept treatment in dispase induced PVR rat model by proteomic analysis. MATERIAL AND METHODS: A total of 35 male Long Evans pigmented rats were divided into three groups, namely, PVR (dispase+saline), PVR+aflibercept (dispase+aflibercept) and control. The PVR group received 2 µl of 0.03 IU/µl dispase and 2 µl saline, the PVR+aflibercept group received 2 µl of 0.03 IU/µl and 2 µl of 40 mg/ml aflibercept at the first day of the experiment. At the end of the 6th week all retina and vitreous specimens were collected by evisceration and transferred to the proteomics laboratory for analysis. Proteomic analysis by 2D gel electrophoresis coupled with MALDI-TOF/TOF was performed. RESULTS: In the PVR and PVR+aflibercept group 16 different proteins that were identified to be differentially regulated in comparison to the control group. In the PVR+aflibercept group, ENO1, ENO2, LDH-B, PEBP-1 and GS levels were higher than the PVR group. In addition, the association of proteins such as UCHL, PEBP1, PDHB and ENO1 with PVR has been demonstrated for the first time. CONCLUSION: STRING analysis elucidated the functional protein-protein interaction among the differentially regulated proteins and highlighted that those proteins mainly played roles in carbon and nucleotide metabolisms. Functional analysis of the differentially regulated proteins indicated the presence of inflammation, gliosis and retinal damage in the PVR group. Aflibercept treatment had pronounced effect on prevention of inflammation and retinal damage while causing a slight increase in gliosis. However, aflibercept treatment was not effective enough to normalize the levels of differentially regulated proteins of the PVR group. Therefore, we predict that the treatment dose of aflibercept used in this study was below of its ideal concentration and should be increased in the future studies. The differential regulation of these structural proteins in this study should shed some light to the mechanism of glial wound formation in the retina and guide future treatment modalities.

Comparative Proteome Analyses of Ureteropelvic Junction Obstruction and Surrounding Ureteral Tissue.

Ureteropelvic junction (UPJ) obstruction is a common problem in children, but its etiology remains unclear. In this study, the proteome profiles of the obstructed segment and its surrounding distal and proximal parts were comparatively evaluated. Twelve children younger than 2 years of age with unilateral intrinsic UPJ obstruction were included. The excised operational tissue was divided into three parts immediately after resection: the obstructed part (Obst), the distal normal ureteral part (Dist), and the proximal part of the obstructed segment (Prox). Proteins extracted from the tissue samples were subjected to two-dimensional gel electrophoresis analysis to identify differentially regulated proteins. Spot analysis revealed that four proteins, namely tropomyosin beta and alpha-1 chains, actin and desmin, were upregulated in Obst in comparison to Dist. A similar analysis between Obst and Prox showed that heat shock protein beta-1 and carbonic anhydrase-1 were upregulated in Obst, while tropomyosin alpha 3 chain and ATP synthase beta were upregulated in Prox. The last comparative analysis between Dist and Prox revealed upregulation of annexin-A5 and annexin-A1 in Dist and vimentin, mitochondrial ATP synthase subunit-beta, peroxiredoxin-2, and apolipoprotein-A1 in Prox. Bioinformatics analysis using the STRING server indicated that the differentially regulated proteins, altogether, point to the changes occurring in muscle filament sliding pathway. When regulations occurring in each group were mutually compared, a change in lipase inhibition activity was detected by STRING. This is the first study scrutinizing changes occurring in protein profiles in UPJ.

Comparative Proteomics Analysis of Four Commonly Used Methods for Identification of Novel Plasma Membrane Proteins.

Plasma membrane proteins perform a variety of important tasks in the cells. These tasks can be diverse as carrying nutrients across the plasma membrane, receiving chemical signals from outside the cell, translating them into intracellular action, and anchoring the cell in a particular location. When these crucial roles of plasma membrane proteins are considered, the need for their characterization becomes inevitable. Certain characteristics of plasma membrane proteins such as hydrophobicity, low solubility, and low abundance limit their detection by proteomic analyses. Here, we presented a comparative proteomics study in which the most commonly used plasma membrane protein enrichment methods were evaluated. The methods that were utilized include biotinylation, selective CyDye labeling, temperature-dependent phase partition, and density-gradient ultracentrifugation. Western blot analysis was performed to assess the level of plasma membrane protein enrichment using plasma membrane and cytoplasmic protein markers. Quantitative evaluation of the level of enrichment was performed by two-dimensional electrophoresis (2-DE) and benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE) from which the protein spots were cut and identified. Results from this study demonstrated that density-gradient ultracentrifugation method was superior when coupled with 16-BAC/SDS-PAGE. This work presents a valuable contribution and provides a future direction to the membrane sub-proteome research by evaluating commonly used methods for plasma membrane protein enrichment.

Identification of differentially regulated deceitful proteins in SH-SY5Y cells engineered with Tet-regulated protein expression system.

Tetracycline regulated protein expression in mammalian cells is a powerful tool to predict the physiological function, cellular localization, and stability of a protein. In addition, to predict metabolic networks affected by the expression of wild-type or mutant forms of proteins, researchers generally produce a single mammalian cell clone that can express the protein of interest under tetracycline control and study the changes occurring in overall proteome before and after expression of a protein of interest. One limitation of tetracycline regulated clonal cell creation, however, is that it sometimes creates clones with changed protein levels even without the expression of the protein of interest due to the nonspecific insertion of the gene encoding the protein of interest into the genome or disruption of a metabolic pathway due to insertional silencing or activation. The aim of this study was to demonstrate the limitation of tetracycline regulated gene expression by creating clonal cell lines expressing the wild-type or the mutant forms of Fat mass and obesity-associated protein. Comparative proteome analysis of the protein extracts by two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF revealed the presence of eight proteins subjected to differential regulation even in the absence of induction. The identified proteins were 14-3-3 protein Epsilon, Vimentin, Heterogeneous nuclear ribonucleoprotein K, Tubulin beta-2C chain, Heat shock protein HSP 90-alpha, Heat shock protein HSP 90-beta, Alpha-enolase, TATA-binding protein-associated factor 2N. An ultimate care should be taken to prevent reporting of deceitful proteins generated from studies utilizing tetracycline regulated gene expression systems.

Exogenous Expressions of FTO Wild-Type and R316Q Mutant Proteins Caused an Increase in HNRPK Levels in 3T3-L1 Cells as Demonstrated by DIGE Analysis.

Fat mass and obesity-associated protein is an enzyme that oxidatively demethylates DNA. Although there are numerous studies regarding the catalytic function of FTO, the overall existence or absence of FTO on cellular proteome has not been investigated. This study investigated the changes in the soluble proteome of 3T3-L1 cells upon expression of the WT and the mutant (R316Q) FTO proteins. Protein extracts prepared from 3T3-L1 cells expressing either the WT or the mutant FTO proteins were used in DIGE experiments. Analysis of the data revealed the number of spots matched to every member and there were 350 ± 20 spots with 30.5% overall mean coefficient of variation. Eleven regulated protein spots were excised from the gels and identified by MALDI-TOF/TOF. One of the identified proteins was heterogeneous nuclear ribonucleoprotein K, which displayed more than 2.6- and 3.7-fold increases in its abundance in the WT and the mutant FTO expressing cells, respectively. Western blot analysis validated these observations. This is the first study revealing the presence of a parallel increase in expressions of FTO and HNRNPK proteins. This increase may codictate the metabolic changes occurring in the cell and may attribute a significance to HNRNPK in FTO-associated transformations.

Proteomics analysis of pleomorphic adenoma of the human parotid gland.

The objective of this study is to perform proteomic analysis of pleomorphic adenoma (PA) in the human parotid gland (PG) with comparison of normal PG. This is an individual prospective randomized controlled trial. This study was performed in a tertiary referral center. Tissue samples of PG and PA were taken after surgical excision of PG from 13 patients. Protein extracts were prepared and protein pools created from the soluble extracts were subjected to 2D-DIGE analysis. Proteins displaying regulation in their abundance were determined and identified using MALDIT-OF/TOF analysis. The identified proteins were subjected to STRING analysis for classification of the proteins based on their biological roles in metabolic pathways. Fifteen proteins, carbonic anhydrase 1, carbonic anhydrase 2, fibrinogen beta chain, alpha-amylase 1, heats hock protein hsp 90-alpha, clusterin, 78 kDa glucose-regulated protein, endoplasmin, alpha-amylase 2b, ATP synthase subunit alpha (mitochondrial), elongation factor 1-gamma, malate dehydrogenase, cytoplasmic, triosephosphate isomerase, receptor of activated protein c kinase 1, and aconitate hydratase, mitochondrial were down-regulated, whereas 11 proteins including ig kappa chain c region, serotransferrin, vimentin, annexin a5, glial fibrillary acidic protein, calreticulin, cartilage oligomeric matrix protein, microfibril-associated glycoprotein 4, 14-3-3 protein epsilon, fibulin-5, and f-box only protein 2 were up-regulated in PA samples in comparison to healthy parotid tissue. This study described the differences observed in protein expression patterns of the PA and normal PG. The results may provide new insights into the pathogenesis and diagnosis of PA in human PG. LEVEL OF EVIDENCE: 1b.

Proteomic studies associated with Parkinson's disease.

INTRODUCTION: Parkinson's disease (PD) is an insidious disorder affecting more than 1-2% of the population over the age of 65. Understanding the etiology of PD may create opportunities for developing new treatments. Genomic and transcriptomic studies are useful, but do not provide evidence for the actual status of the disease. Conversely, proteomic studies deal with proteins, which are real time players, and can hence provide information on the dynamic nature of the affected cells. The number of publications relating to the proteomics of PD is vast. Therefore, there is a need to evaluate the current proteomics literature and establish the connections between the past and the present to foresee the future. Areas covered: PubMed and Web of Science were used to retrieve the literature associated with PD proteomics. Studies using human samples, model organisms and cell lines were selected and reviewed to highlight their contributions to PD. Expert commentary: The proteomic studies associated with PD achieved only limited success in facilitating disease diagnosis, monitoring and progression. A global system biology approach using new models is needed. Future research should integrate the findings of proteomics with other omics data to facilitate both early diagnosis and the treatment of PD.

Linking a compound-heterozygous Parkin mutant (Q311R and A371T) to Parkinson's disease by using proteomic and molecular approaches.

Parkin is an E3-protein ubiquitin ligase, which plays an important role as a scavenger in cell metabolism. Since the discovery of the link between Parkin and Parkinson's disease, Parkin was placed in the center of Parkinson's disease research. Previously, we isolated a mutant form of the Parkin protein (Q311R and A371T) from a Parkinson's disease patient. In this study, we aimed at characterizing this mutant Parkin protein by using biochemical and proteomic approaches. We used neuroblastoma cells (SH-SY5Y) as our model and created two inducible cell lines that expressed the wild type and the mutant Parkin proteins. We first investigated the effect of expressing both the wild type and the mutant Parkin proteins on the overall proteome by using 2D-DIGE approach. The experiments yielded the identification of 22 differentially regulated proteins, of which 13 were regulated in the mutant Parkin expressing cells. Classification of the identified proteins based on biological process and molecular function revealed that the majority of the regulated proteins belonged to protein folding and energy metabolism. Ingenuity Pathway Analysis predicted the presence of a link between the regulated proteins of the mutant Parkin expressing cells and Parkinson's disease. We also performed biochemical characterization studies on the wild type and the mutant Parkin proteins to make sense out of the differences observed at the proteome level. Both proteins displayed biological activity, had similar stabilities and localized similarly to the cytoplasm and the nucleus in SH-SY5Y cells. The mutant protein, however, was cut by a protease and subjected to a post-translational modification. The observed differences at the proteome level might be due to the differences in processing of the mutant Parkin protein. Overall, we were able to create a possible link between a pair of Parkin mutations to its pertinent disease by using 2D-DIGE in combination with biochemical and molecular approaches.

Effect of resveratrol on chromosomal aberrations induced by doxorubicin in rat bone marrow cells.

This study investigated the effects of resveratrol (RES) on doxorubicin (DXR) induced rat bone marrow cell chromosome aberrations. RES, a polyphenolic compound, has attracted considerable attention because of its antioxidant and antimutagenic effects. DXR, a chemotherapeutic agent, is known to cause chromosomal aberrations in healthy cells in cancer patients. In this study, Wistar albino male rats were divided into 6 groups with 6 animals each. The control group received distilled water i.p. and the DXR group received an i.p. injection of doxorubicin (90mg/kgbw). For the 2 RES dose groups (12.5 and 25mg/kgbw, respectively), RES was injected i.p. 5 times during the 24h study period to coincide with the schedule for the DXR+RES groups. The DXR-RES groups received DXR (90mg/kgbw) and RES at either 12.5 or 25mg/kgbw, i.p. 30min before, concurrently, and then every 6h after DXR administration. Bone marrow collection was timed to coincide with 24h after DXR administration in all groups. RES administration alone did not induce any significant increase in frequency of chromosome aberrations or abnormal metaphases compared with controls (p>0.05) while DXR alone did (p<0.05). In the DXR-RES 12.5mg/kgbw group, frequency of chromosome aberrations and abnormal metaphases were slightly reduced compared to DXR alone, but this was not statistically significant. However, in the DXR-RES 25mg/kgbw group, RES resulted in a statistically significant reduction in the frequency of chromosome aberrations and abnormal metaphases compared to those induced by DXR alone (p<0.05). These results indicate that RES (25mg/kgbw) significantly reduces frequency of DXR induced chromosome damage in bone marrow cells.

Protective effects of increasing vitamin E and a doses on cisplatin-induced oxidative damage to kidney tissue in rats.

OBJECTIVE: Cisplatin (DDP, cis-diamminedichloroplatinium II) is one of the most potent chemotherapeutic antitumor drugs, but is able to generate reactive oxygen species (ROS) and it also inhibits the activity of antioxidant enzymes in renal tissue. In the present study, we investigated the preventive effect of 100, 200 and 400 mg/kg b.w. doses of vitamin E (VE), and 25, 50, and 100 mg/kg b.w. doses of vitamin A (VA) combination on malondialdehyde (MDA), nitric oxide (NO), and glutathione (GSH) levels and superoxide dismutase (SOD) activity in cisplatin-induced toxicity in rat kidneys. Our literature survey indicated a lack of any experimental study showing the beneficial effect of VA on cisplatin-induced MDA, NO, GSH and SOD changes. For this reason, we hoped that this study would provide a unique contribution in that respect. MATERIALS AND METHODS: 59 Wistar rats (11 to replace prematurely lost animals) were used. 48 evaluable rats were divided into 8 groups (n = 6 in each group): control group, DDP alone (5 mg/kg b.w.) group, 3 VE combination treatment groups of VE100+DDP, VE200+DDP, and VE400+DDP, and 3 VA combination treatment groups of VA25+DDP, VA50+DDP, and VA100+DDP. Kidney MDA, GSH, NO levels and SOD activities were determined for the assessment of oxidant-antioxidant balance. RESULTS: While in the DDP group the tissue levels of MDA and NO were found to be significantly higher than in the control group, GSH levels and SOD activities were significantly lower. MDA and NO levels were found to be significantly lower and GSH levels and SOD activities significantly higher in the VE200+DDP and VE400+ DDP groups when compared with the DDP alone group. MDA and NO levels were found to be significantly lower in the VA50+DDP and VA100+DDP groups when compared with the DDP alone group. However, identical comparisons with the DDP alone group showed significantly higher GSH levels and SOD activities in the VA25+DDP, VA50+DDP, and VA100+DDP groups. Among the VE100+ DDP, VE200+DDP, and VE400+DDP groups, and VA25+ DDP, VA50+DDP, and VA100+DDP groups, MDA and NO levels decreased and GSH levels and SOD activities increased steadily and significantly as the doses of VE and VA increased. CONCLUSION: These vitamins would be effective in protecting against cisplatin-induced tissue damage in rat kidneys. It is possible that the toxic effect of cisplatin is somehow minimized by a compensatory mechanism involving VE and VA via induction of antioxidant enzyme activities following intraperitoneal injection of DDP.

Effects of 10alpha,25-dihydroxyvitamin D3 on doxorubicin-induced chromosomal aberrations in rat bone marrow cells.

The present study was carried out to evaluate the effects of 1alpha,25-dihydroxyvitamin D3 (VD) on chromosomal aberrations induced by doxorubicin (DXR). Wistar rats were divided into eight experimental groups of five animals each. Control group animals were treated with i.p. distilled water. The animals in three VD groups were given only VD for 4, 6 or 8 weeks. In the DXR groups the animals were given only DXR. In the combination groups VD doses were given for 4, 6 or 8 weeks for each group and DXR was injected 24 h before sacrificing the rats. DXR (50 mg/100 g b.w.) was injected intraperitoneally and VD by gavage 3 microg/kg/day twice weekly. Animals treated with both VD and DXR showed a low frequency of chromosomal aberrations and abnormal metaphases when compared with animals treated with DXR alone (p < 0.0001). The numbers of both chromosomal aberrations and abnormal metaphases were similar in weeks 6 and 8 (p > 0.05) and lower than those in week 4 for the VD groups (p < 0.0001). Under the present experimental conditions, the efficiency of VD in protecting cells against DXR-induced chromosome damage was found to be dose dependent. The protective effects of VD on chromosome aberrations induced by DXR are discussed in the light of literature data.

Effects of vitamin A on doxorubicin-induced chromosomal aberrations in bone marrow cells of rats.

The present study was carried out to evaluate the role of vitamin A (VA) on the induction of chromosomal aberrations (CA) in rat bone marrow cells and to investigate its modulating effect on chromosomal damage induced by doxorubicin (DXR). Wistar rats were treated with VA (7.5, 15 and 30 microg/kg body wt) once a day for 2 days by gavage before injecting DXR (90 mg/kg body wt). Rats in the control group were treated with corresponding doses of water and olive oil. Animals treated with the medium dose of VA (15 microg/kg body wt) plus single dose of DXR presented a statistically significant reduction in total number of CA and in number of abnormal metaphases (P < 0.05). However, when compared with control and DXR groups, the low and high VA doses (7.5 and 30 microg/kg body wt) were found to be less efficient than the medium dose VA (15 microg/kg body wt) in terms of parameters analyzed. Furthermore, the high dose of VA group (30 microg/kg body wt) was found to be clastogenic (P < 0.05). This study concludes that the protective effect of VA against chromosome damage is dose dependent.