Novel Pyrimidine-5-Carbonitriles as potential apoptotic and antiproliferative agents by dual inhibition of EGFRWT/T790M and PI3k enzymes; Design, Synthesis, biological Evaluation, and docking studies.

A new series of 6-(4-fluorophenyl)-2-(methylthio) pyrimidine-5-carbonitrile derivatives were designed and synthesized as EGFR/PI3K dual inhibitors, and potential antiproliferative agents. The new 22 compounds were screened by DTP-NCI against all NCI60 cell lines. Almost all compounds showed cytotoxic activity. Compound 7c showed a promising antitumour activity on CNS cancer (SNB-75), and ovarian cancer (OVAR-4) with IC50 < 0.01, and 0.64 µM, respectively. Fortunately, 7c exhibited a better safety profile on normal cells (WI-38) than doxorubicin by 2.2-fold. Compound 7c displayed selective inhibitory activity on EGFRt790m over EGFRWT with IC50 = 0.08, and 0.13 µM, respectively, wherefore it might overcome EGFR-TKIs resistance. In addition to its remarkable inhibitory activity on all PI3K isoforms, specifically PI3K-δ with IC50 = 0.64 µM Compared with LY294002 IC50 = 7.6 µM. Compound 7c arrested the cell cycle of SNB-75 & OVAR-4 at the G0-G1 phase coupled with apoptosis induction. The western blotting analysis approved decreasing the expression level of p-AKT coupled with an increase in Casp3, Casp9, and BAX proteins in the SNB-75 & OVAR-4 after being treated with 7c which may support the suggested mechanism of action of 7c as EGFR/PI3K dual inhibitor. Physicochemical parameters were forecasted using SwissADME online tool. MD showed the interaction of 7c with the crucial amino acids of the active domain of both EGFR/PI3K which may explain its potent inhibitory activities. In vivo study disclosed a significant decrease in tumor weight and the number of nodules in the group of mice treated with 7c compared with the control group.

Histological evaluation of monopolar and bipolar radiofrequency microneedling treatment in a porcine model.

BACKGROUND AND OBJECTIVE: Fractional radiofrequency microneedling (FRM) is widely used as an option for skin rejuvenation, however there is a lack of histological evidence for the various energy delivery systems available. The objective was to assess thermal denaturation of tissue and the wound healing response in monopolar mode versus bipolar mode. Histological analysis was performed to demonstrate the efficacy of automatic impedance feedback system in monopolar mode. STUDY DESIGN AND METHODS: In this study, the acute thermal effects caused by monopolar FRM treatment to the dorsal skin of pigs were assessed histologically by hematoxylin & eosin (H&E) staining. Then, one session of either monopolar or bipolar FRM was used to treat one or the other side of the pig using varying power levels and pulse widths. The acute and chronic tissue reactions were assessed using H&E, immunofluorescence, and western blot analysis at 0, 14, 30, and 90 days after treatment. The efficacy of the impedance feedback system was also monitored histologically. RESULTS: High-energy FRM treatment produced tissue loss and necrosis. The power level and pulse duration significantly affected the coagulation amount. Histopathology at 0, 14, 30, and 90 days showed that the skin tissue reaction was more pronounced for bipolar compared to monopolar FRM. Immunofluorescence showed the expression of TGF-ß, Ki67, MMP3, and elastin increased dramatically with both modes, but were higher in the bipolar FRM treated side. The automatic impedance feedback system could effectively adjust the output energy. CONCLUSIONS: We found that bipolar FRM produced greater thermal effects, more collagen coagulation, and more pronounced molecular changes compared with monopolar mode in a porcine animal model.

Anti-Tumor Activity and Mechanism of Silibinin Based on Network Pharmacology and Experimental Verification.

Silibinin is a flavonoid compound extracted from the seeds of Silybum marianum (L.) Gaertn. It has the functions of liver protection, blood-lipid reduction and anti-tumor effects. However, the potential molecular mechanism of silibinin against tumors is still unknown. This study aimed to assess the anti-tumor effects of silibinin in adenoid cystic carcinoma (ACC2) cells and Balb/c nude mice, and explore its potential mechanism based on network pharmacology prediction and experimental verification. A total of 347 targets interacting with silibinin were collected, and 75 targets related to the tumor growth process for silibinin were filtrated. Based on the PPI analysis, CASP3, SRC, ESR1, JAK2, PRKACA, HSPA8 and CAT showed stronger interactions with other factors and may be the key targets of silibinin for treating tumors. The predicted target proteins according to network pharmacology were verified using Western blot analysis in ACC2 cells and Balb/c nude mice. In the pharmacological experiment, silibinin was revealed to significantly inhibit viability, proliferation, migration and induce the apoptosis of ACC2 cells in vitro, as well as inhibit the growth and development of tumor tissue in vivo. Western blot analysis showed that silibinin affected the expression of proteins associated with cell proliferation, migration and apoptosis, such as MMP3, JNK, PPARα and JAK. The possible molecular mechanism involved in cancer pathways, PI3K-Akt signaling pathway and viral carcinogenesis pathway via the inhibition of CASP3, MMP3, SRC, MAPK10 and CDK6 and the activation of PPARα and JAK. Overall, our results provided insight into the pharmacological mechanisms of silibinin in the treatment of tumors. These results offer a support for the anti-tumor uses of silibinin.

Synthesis, characterization, anticancer efficacy evaluation of ruthenium(II) and iridium(III) polypyridyl complexes toward A549 cells.

A new ligand DFIP (2-(dibenzo[b,d]furan-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its two complexes iridium(III) [Ir(ppy)2(DFIP)](PF6) (ppy = 2-phenylpyridine, Ir1) and ruthenium(II) [Ru(bpy)2(DFIP)](PF6)2 (bpy = 2,2'-bipyridine, Ru1) were synthesized and characterized. The anticancer effects of the two complexes on A549, BEL-7402, HepG2, SGC-7901, HCT116 and normal LO2 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Complex Ir1 shows high cytotoxic activity on A549, BEL-7402, SGC-7901 and HepG2, Ru1 exhibits moderate anticancer activity toward A549, BEL-7402 and SGC-7901 cells. The IC50 values of Ir1 and Ru1 toward A549 are 7.2 ± 0.1 and 22.6 ± 1.4 µM, respectively. The localization of complexes Ir1 and Ru1 in the mitochondrial, intracellular accumulation of reactive oxygen species (ROS) levels, and the changes of mitochondrial membrane potential (MMP) and cytochrome c (cyto-c) were investigated. Apoptosis and cell cycle were detected by flow cytometry. Immunogenic cell death (ICD) was used to detect the effects of Ir1 and Ru1 on the A549 using a confocal laser scanning microscope. The expression of apoptosis-related proteins was detected by western blotting. Ir1 and Ru1 can increase the intracellular ROS levels and release cyto-c, reduce the MMP, leading to the apoptosis of A549 cells and blocking the A549 cells at the G0/G1 phase. Additionally, the complexes caused a decrease of the expression of polyADP-ribose polymerase (PARP), caspase 3, Bcl-2 (B-cell lymphoma-2), PI3K (phosphoinositide-3 kinase) and upregulated the expression of Bax. All these findings indicated that the complexes exert anticancer efficacy to induce cell death through immunogenic cell death, apoptosis, and autophagy.

Porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV): A threat for xenotransplantation?

The potential for a donor-derived transmission of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) to the recipient has been recognized since pigs were considered candidate donors for xenotransplantation. This review gives a short description of the viral properties and summarizes the current evidence of the effects of PCMV/PRV transmission in preclinical xenotransplantation. Despite evidence that PCMV/PRV does not infect human and non-human primate cells, activation in the transplanted organ and detrimental systemic complications have been described. As PCMV/PRV is a herpesvirus able to establish latency, the importance of adequate screening of donor pigs is emphasized, as no efficient treatment is available. Furthermore, easy and successful ways of elimination of PCMV/PRV from pig herds are indicated.

Exploring Bacillus thuringiensis as a model for endospore adhesion and its potential to investigate adhesins in Pasteuria penetrans.

AIMS: Phytonematodes are a constraint on crop production and have been controlled using nematicides; these are highly toxic and legislation in Europe and elsewhere is prohibiting their use and alternatives are being sought. Pasteuria penetrans is a hyperparasitic bacterium that form endospores and have potential to control root-knot nematodes (Meloidogyne spp.), but their attachment to the nematode cuticle is host-specific. Understanding host specificity has relied upon endospore inhibition bioassays using immunological and biochemical approaches. Phylogenetic analysis of survey sequences has shown P. penetrans to be closely related to Bacillus and to have a diverse range of collagen-like fibres which we hypothesise to be involved in the endospore adhesion. However, due to the obligately hyperparasitic nature of Pasteuria species, identifying and characterizing these collagenous-like proteins through gain of function has proved difficult and new approaches are required. METHODS AND RESULTS: Using antibodies raised to synthetic peptides based on Pasteuria collagen-like genes we show similarities between P. penetrans and the more easily cultured bacterium Bacillus thuringiensis and suggest it be used as a gain of function platform/model. Using immunological approaches similar proteins between P. penetrans and B. thuringiensis are identified and characterized, one >250 kDa and another ~72 kDa are glycosylated with N-acetylglucosamine and both of which are digested if treated with collagenase. These treatments also affected endospore attachment and suggest these proteins are involved in adhesion of endospores to nematode cuticle. CONCLUSION: There are conserved similarities in the collagen-like proteins present on the surface of endospores of both P. penetrans and B. thuringiensis. SIGNIFICANCE AND IMPACT OF STUDY: As B. thuringiensis is relatively easy to culture and can be transformed, it could be developed as a platform for studying the role of the collagen-like adhesins from Pasteuria in endospore adhesion.

Species Difference in the Metabolism of Mulberrin in Vitro and Its Inhibitory Effect on Cytochrome P450 and UDP-Glucuronosyltransferase Enzymes.

This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.

Synthesis and evaluation of iridium(III) complexes on antineoplastic activity against human gastric carcinoma SGC-7901 cells.

The study was intended to determine the antineoplastic effects of two new iridium(III) complexes [Ir(ppy)2(PTTP)](PF6) (1) (ppy = 2-phenylpyridine) and [Ir(piq)2(PTTP)](PF6) (2) (piq = 1-phenylisoquinoline, PTTP = 2-phenoxy-1,4,8,9-tetraazatriphenylene). In MTT assay, the ligand PTTP displayed ineffective inhibition on cell growth in SGC-7901, BEL-7402, HepG2 as well as NIH3T3 cell lines, while complexes 1 and 2 showed high cytotoxic activity on SGC-7901 cells with an IC50 value of 0.5 ± 0.1 µM and 4.4 ± 0.6 µM, respectively. Cellular uptake, cell cloning experiments, wound healing assay and cell cycle arrest indicated that the two complexes can inhibit the cell proliferation in SGC-7901 and induce cell cycle arrest at G0/G1 phase. Additionally, reactive oxygen species (ROS) and mitochondrial membrane potential suggested that the two complexes induced cell apoptosis through disrupting mitochondrial functions. Further, western blot analysis illustrated that the two complexes caused apoptosis via regulating expression levels of Bcl-2 family proteins. Moreover, complex 1 could suppress tumor growth in vivo with an inhibitory rate of 49.41%. Altogether, these results demonstrated that complexes 1 and 2 exert a potent anticancer effect against SGC-7901 cells via mitochondrial apoptotic pathway and have a potential to be developed as antineoplastic drug candidates for human gastric cancer.

MTSS1 hypermethylation is associated with prostate cancer progression.

This study was conducted to evaluate the influence of DNA methylation of metastasis suppressor 1 (MTSS1) on prostate cancer (PCa) progression. Forty-nine paired PCa tissue samples and normal tissue samples from The Cancer Genome Atlas were analyzed. Methylome analysis, CpG island arrays and Hierarchical clustering were used to analyze methylation profiles of PCa tissues. MTSS1 methylation level was detected by methylation-specific PCR. Relative messenger RNA and the expression level of MTSS1 protein were identified by quantitative real-time PCR (qRT-PCR) and western blot analysis. The migration, invasion, proliferation, and cell cycle were detected separately by wound-healing assay, transwell chamber assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry. The roles of MTSS1 in PCa progression were demonstrated in vivo by tumor formation assays in nude mice. MTSS1 expression was decreased in PCa tissues in comparison with paired adjacent normal prostate tissues. Compared to the methylation of MTSS1 in normal prostate tissues based on the MethHC website, the MTSS1 in PCa tissues was hypermethylated. The expression of MTSS1 detected by qRT-PCR and western blot analysis was found to be downregulated in PCa cells and tissues. The reduced expression of MTSS1 by small interfering RNA-MTSS1 was recovered by 5-aza-2'-deoxycytidine treatment. Besides, MTSS1 demethylation inhibited migration, invasion, and proliferation of PCa cells, and induced cell cycle to be arrested at G0/G1 phase. Furthermore, it was shown by tumor xenograft assay that MTSS1 inhibited the growth of tumor in vivo. Hypermethylated MTSS1 promoted PCa cells migration, invasion, and proliferation, and suppressed cell cycle arrest at the G0/G1 phase.

Chaperones in sporadic inclusion body myositis-Validation of proteomic data.

INTRODUCTION: Sporadic inclusion body myositis (sIBM) is characterized by myopathological features including rimmed vacuoles (RVs) and proteins associated with protein aggregation, autophagy, and inflammation. Previous proteomic studies of RV areas revealed an overrepresentation of several chaperones and subunits of the T-complex protein 1 (TCP-1), which is involved in prevention of protein aggregation. METHODS: To validate our proteomic findings, immunofluorescence analyses of selected chaperones and quantitative Western blot analysis of TCP-1 proteins were performed in five sIBM patients and five healthy controls. RESULTS: Immunofluorescence studies confirmed increased immunoreactivity for VCP, UNC45B, GRP-75, αB-crystallin, LAMP-2, Rab-7a, and TCP-1α and TCP-θ in RVs. Quantitative Western blot analysis revealed a significantly higher level of TCP-1 in sIBM muscle tissue when compared with healthy controls. DISCUSSION: Our study findings validate new insights in protein quality control and degradation processes that seem to be relevant in sIBM. These data provide an important basis for future functional and therapeutic studies.

Sensitive detection systems for infectious agents in xenotransplantation.

Xenotransplantation of pig cells, tissues, or organs may be associated with transmission of porcine microorganisms, first of all of viruses, to the transplant recipient, potentially inducing a disease (zoonosis). I would like to define detection systems as the complex of sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls along with the specific detection methods, either PCR-based, cell-based, or immunological methods. Some xenotransplantation-relevant viruses have already been defined; others are still unknown. The PCR-based methods include PCR and real-time PCR for DNA viruses, and RT-PCR and real-time RT-PCR for RNA viruses as well as for virus expression studies at the RNA level. Furthermore, droplet digital PCR (ddPCR) can be used for the determination of virus and provirus copies. To detect expression at the protein level, immunofluorescence, immunohistochemistry, and Western blot analyses can be used. To detect virus production and to detect infectious viruses, electron microscopy and infection assays can be used. Furthermore, immunological methods such as Western blot analysis or ELISA can be used to detect virus-specific antibodies. Detection of antiviral antibodies is a reliable and sensitive indirect detection method. For these immunological methods, purified viruses, recombinant viral proteins, or synthetic peptides are used as antigens and control sera and control antigens are needed. All these methods have been used in the past for the characterization of different pig breeds including genetically modified pigs generated for xenotransplantation and for the screening of recipients in preclinical and clinical xenotransplantations. Whereas in preclinical trials a few porcine viruses have been transmitted to the non-human primate recipients, in first clinical trials no such transmissions to humans were observed. Further improvement of the detection systems and their application in virus elimination programs will lead to clean donor animals and a safe xenotransplantation.

MMP-2 and MMP-9 as prognostic markers for the early detection of urinary bladder cancer.

The present study assessed protein and gene expression levels of tissue inhibitor of metalloproteinase-2 (TIMP-2), matrix metalloproteinase-2 (MMP-2), and MMP-9 in urine and blood samples of 50 patients with bladder carcinoma. The expression of TIMP-2, MMP-2, and MMP-9 levels with tumor stage and grade was also assessed. Results showed that the expression levels of MMP-2 and MMP-9 in both blood and urine were significantly elevated in group 1 when compared with groups 2 and 3 healthy subjects. The discriminatory ability in the diagnosis of bladder carcinoma of MMP-2 and MMP-9 expression was confirmed by receiver operating characteristic curve analysis that revealed a sensitivity and specificity of 100%. MMP-2 and MMP-9 levels were not correlated with grade or stage of the tumor. With respect to TIMP-2 blood and urine levels, results showed a significant decrease in gene expression levels in bladder carcinoma group, whereas, TIMP-2 protein showed a significant increase in bladder carcinoma.

Design, synthesis and in vitro apoptotic mechanism of novel pyrrolopyrimidine derivatives.

In this work we described the synthesis and evaluation of cytotoxic and apoptotic activity of novel pyrrolopyrimidine derivatives against A549, PC3 and MCF-7 cells. Among the synthesized compounds, 6b, 8a, 9a and 7a, 8b displayed the significant cytotoxic activities against A549 and PC3 cells with IC50 value of 0.35, 1.48, 1.56 and 1.04, 1.89 µM, respectively. It was found that A549 cells were more sensitive to synthesized compounds than PC3 and MCF-7 cells. In order to evaluate the mechanism of cytotoxic activity in A549, compounds 6b, 8a and 9a were selected for further studies. Annexin V binding assay and western blot analysis results revealed that 6b, 8a and 9a induced apoptosis in A549 cells by intrinsic apoptotic pathway through the activation pro-apoptotic proteins such as Bim, Bax, Bak, Puma and deactivation of anti-apoptotic proteins including Bcl-2, Mcl-1 and Bcl-XL accompanied by the activation of caspase-3, caspase-9 and cleavage of PARP. Also, compounds 6b, 8a and 9a triggered apoptosis in HCT116 wt cells via activation of caspase-3 and caspase-9, but not in HCT116 Bax/Bak KO cells, indicating resistance to 6b, 8a and 9a treatment.

The use of serum free light chain dimerization patterns assist in the diagnosis of AL amyloidosis.

The discrimination between benign and malignant forms of plasma cell dyscrasia (PCD) is often difficult. Free light chain monomer-dimer pattern analysis (FLC-MDPA) may assist in solving this dilemma and distinguish between AL amyloidosis and benign PCD. Serum samples of patients with AL amyloidosis and benign PCD were analysed in a blinded manner. Quantitative Western blotting was performed to estimate dimerization and clonality indices, and thereby determine the source of the tested samples, as derived either from benign or malignant PCD. The findings obtained by the FLC-MDPA were compared with the actual diagnosis. Of 37 samples from patients with active AL amyloidosis, 34 (91·9%) fulfilled dimerization criteria for diagnosis of AL amyloidosis. Of the 45 samples from patients with benign PCD, 10 (21·2%) tested falsely positive or gave an inconclusive result. Thus, the sensitivity of the analysis was 92·5% with a remarkable negative predictive value of 91·9%. In addition, of 20 patients who were in complete or very good partial remission, only one tested positive. By multivariate analysis, FLC-MDPA was the best independent marker predicting AL amyloidosis (odds ratio of 84). The FLC-MDPA offers a highly effective tool in the diagnostic assessment of patients with PCD.

Effect of Paclitaxel+Hirudin on the TLR4-MyD88 Signaling Pathway During Inflammatory Activation of Human Coronary Artery Smooth Muscle Cells and Mechanistic Analysis.

BACKGROUND/AIMS: Approximately 10%-20% of patients with acute cardiovascular disease who have received coronary intervention suffer restenosis and high inflammation. The stent compound paclitaxel+hirudin was prepared for the treatment of post-intervention restenosis. This study aimed to explore the anti-inflammatory and anti-restenosis mechanisms of paclitaxel+hirudin with regard to the TLR4/MyD88/NF-κB pathway. METHODS: Human coronary artery smooth muscle cells (HCASMCs) at 4-6 generations after in vitro culture were used as a model. Lipopolysaccharide (LPS) was used as an inducer to maximally activate the TLR4/MyD88/NF-κB inflammation pathway. After MyD88 knockdown and selective blocking of MyD88 degradation with epoxomicin, the effects of paclitaxel+hirudin stenting on key sites of the TLR4/MyD88/NF-κB pathway were detected using ELISA, Q-PCR, and western blot analysis. RESULTS: LPS at 1 µg/mL for 48 h was the optimal modeling condition for inflammatory activation of HCASMCs. Paclitaxel+hirudin inhibited the levels of key proteins and the gene expression, except for that of the MyD88 gene, of the TLR4-MyD88 pathway. The trend of the effect of paclitaxel+hirudin on the pathway proteins was similar to that of MyD88 knockdown. After epoxomicin intervention, the inhibitory effects of paclitaxel+hirudin on the key genes and proteins of the TLR4-MyD88 pathway were significantly weakened, which even reached pre-intervention levels. Paclitaxel+hirudin affected the MyD88 protein in a dosage-dependent manner. CONCLUSION: The paclitaxel+hirudin compound promotes MyD88 degradation in the TLR4/MyD88/NF-κB pathway to reduce the activity of TLR4 and NF-κB p65 and to weaken the LPS-initiated inflammatory reactions of IL-1ß, IL-6, and TNF-α.

Syntheses and Biological Evaluation of Novel Hydroxamic Acid Derivatives Containing Purine Moiety as Histone Deacetylase Inhibitors.

The novel hydroxamates containing purine scaffold were designed, synthesized and screened for their biological activities as histone deacetylase (HDAC) inhibitors. Some of them exhibited excellent acti-HDACs activities and antiproliferative activities, the most promising compound was 7m'. Western blot analysis indicated the compounds 7f', 7l', 7m', 7o' could increase histone H3 acetylation levels in HCT116 and K562 cell lines, and 7m' increased the level of acetyl histone H3 in a dose-dependent manner, which is similar to the behavior of suberoylanilide hydroxamic acid (SAHA). Molecular docking study revealed that the conformation of 7m' in the active site of HDAC2 was similar to positive drug SAHA, which were oriented with the hydroxamic acid towards the catalytic center and formed metal binding with zinc ion.

2-aryl benzimidazole conjugate induced apoptosis in human breast cancer MCF-7 cells through caspase independent pathway.

Apoptosis is a representative form of programmed cell death, which has been assumed to be critical for cancer prevention. Thus, any agent that can induce apoptosis may be useful for cancer treatment and apoptosis induction is arguably the most potent defense against cancer promotion. In our previous studies, 2-aryl benzimidazole conjugates were synthesized and evaluated for their antiproliferative activity and one of the new molecule (2f) was considered as a potential lead. This lead molecule showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The results of the present study revealed that this compound arrested the cell cycle at G2/M phase. Topoisomerase II inhibition assay and Western blot analysis suggested that this compound effectively inhibits topoisomerase II activity which leads to apoptotic cell death. Apoptosis induction in MCF-7 cells was further confirmed by loss of mitochondrial membrane potential (∆Ψm), release of cytochrome c from mitochondria, an increase in the level of apoptosis inducing factor (AIF), generation of reactive oxygen species (ROS), up regulation of proapoptotic protein Bax and down regulation of anti apoptotic protein Bcl-2. Apoptosis assay using Annexin V-FITC assay also suggested that this compound induced cell death by apoptosis. However, compound 2f induced apoptosis could not be reversed by Z-VAD-FMK (a pan-caspase inhibitor) demonstrated that the 2f induced apoptosis was caspase independent. Further, 2f treatment did not activate caspase-7 and caspase-9 activity, suggesting that this compound induced apoptosis in breast cancer cells via a caspase independent pathway. Most importantly, this compound was less toxic towards non-tumorigenic breast epithelial cells, MCF-10A. Furthermore, docking studies also support the potentiality of this molecule to bind to the DNA topoisomerase II.

Generation and Applications of a DNA Aptamer against Gremlin-1.

Gremlin-1, a highly conserved glycosylated and phosphorylated secretory protein, plays important roles in diverse biological processes including early embryonic development, fibrosis, tumorigenesis, and renal pathophysiology. Aptamers, which are RNA or DNA single-stranded oligonucleotides capable of binding specifically to different targets ranging from small organics to whole cells, have potential applications in targeted imaging, diagnosis and therapy. In this study, we obtained a DNA aptamer against Gremlin-1 (G-ap49) using in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Binding assay and dot-blot showed that G-ap49 had high affinity for Gremlin-1. Further experiments indicated that G-ap49 was quite stable in a cell culture system and could be used in South-Western blot analysis, enzyme-linked aptamer sorbent assay (ELASA), and aptamer-based cytochemistry and histochemistry staining to detect Gremlin-1. Moreover, our study demonstrated that G-ap49 is capable of revealing the subcellular localization of Gremlin-1. These data indicate that G-ap49 can be used as an alternative to antibodies in detecting Gremlin-1.

State-of-the-art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome.

Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, ß-tubulin and ß-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.

Investigation of the apoptotic pathway induced by benzimidazole-oxindole conjugates against human breast cancer cells MCF-7.

In our previous studies, benzimidazole-oxindole conjugates were synthesized and evaluated by National Cancer Institute (NCI) for their cytotoxic activity and the new molecules like 5c and 5p were considered as potential leads. These conjugates arrested the cell cycle at G2/M phase and inhibited tubulin polymerization. These observations prompted us to investigate the apoptotic mechanism induced by these lead molecules against human breast cancer cells (MCF-7). Studies like measurement of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species (ROS) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis in human breast cancer cells. It was further confirmed by western blot analysis of pro apoptotic protein Bax, anti apoptotic protein Bcl-2, cytochrome c release, caspase-9 activity and cleavage of PARP.