Effects of bromodomain and extra-terminal inhibitor JQ1 and interleukin-6 on breast cancer cells.

BACKGROUND: Bromodomain and extra-terminal (BET) proteins are recognized acetylated lysine of histone 4 and act as scaffolds to recruit many other proteins to promoters and enhancers of active genes, especially at the super-enhancers of key genes, driving the transcription process and have been identified as potential therapeutic targets in breast cancer. However, the efficacy of BET inhibitors such as JQ1 in breast cancer therapy is impeded by interleukin-6 (IL-6) through an as-yet-defined mechanism. METHODS AND RESULTS: We investigated the interplay between IL-6 and JQ1 in MCF-7 and MDA-MB-231 human breast cancer cells. The results demonstrate that the efficacy of JQ1 on the inhibition of cell growth and apoptosis was stronger in MDA-MB-231 cells than in MCF-7 cells. Further, MCF-7 cells, but not MDA-MB-231 cells, exhibited increased expression of CXCR4 following IL-6 treatment. JQ1 significantly reduced CXCR4 surface expression in both cell lines and diminished the effects of IL-6 pre-treatment on MCF-7 cells. While IL-6 suppressed the extension of breast cancer stem cells in MCF-7 cells, JQ1 impeded its inhibitory effect. In MCF-7 cells JQ1 increased the number of senescent cells in a time-dependent manner. CONCLUSION: Analysis of gene expression indicated that JQ1 and IL-6 synergistically increase SNAIL expression and decrease c-MYC expression in MCF-7 cells. So, the BET proteins are promising, novel therapeutic targets in late-stage breast cancers. BET inhibitors similar to JQ1 show promise as therapeutic candidates for breast cancers, especially when triple-negative breast cancer cells are increased and/or tumor-promoting factors like IL-6 exist in the tumor microenvironment.

CRISPR/Cas9-mediated LINC00511 knockout strategies, increased apoptosis of breast cancer cells via suppressing antiapoptotic genes.

BACKGROUND: The growing detection of long noncoding RNAs (lncRNAs) required the application of functional approaches in order to provide absolutely precise, conducive, and reliable processed information along with effective consequences. We utilized genetic knockout (KO) techniques to ablate the Long Intergenic Noncoding RNA 00,511 gene in several humans who suffered from breast cancer cells and at the end we analyzed and examined the results. RESULTS: The predictive relevance of LINC00511 expression pattern was measured by using a pooled hazard ratio (HR) with a 95% confidence interval (CI). The link among LINC00511 expression profiles and cancer metastasis was measured by using a pooled odds ratio (OR) with a 95% confidence interval. This meta- analysis was composed of fifteen studies which contained a total of 1040 tumor patients. We used three distinct CRISPR/Cas9-mediated knockdown techniques to prevent the LINC00511 lncRNA from being transcribed. RT-PCR was used to measure lncRNA and RNA expression. We used CCK-8, colony formation tests, and the invasion transwell test to measure cell proliferation and invasion. The stemness was measured by using a sphere-formation test. To validate molecular attachment, luciferase reporter assays were performed. The functional impacts of LINC00511 gene deletion in knockdown breast cancer cell lines were confirmed by using RT-qPCR, MTT, and a colony formation test. This meta-analysis was composed of 15 trials which contained a total of 1040 malignant tumors. Greater LINC00511 expression was ascribed to a lower overall survival (HR = 1.93, 95% CI 1.49-2.49, < P 0.001) and to an increased proportion of lymph node metastasis (OR = 3.07, 95% CI 2.23-4.23, P < 0.001) in the meta-analysis. It was found that the role of LINC00511 was overexpressed in breast cancer samples, and this overexpression was ascribed to a poor prognosis. The gain and loss-of-function tests demonstrated findings such as LINC00511 increased breast cancer cell proliferation, sphere-forming ability, and tumor growth. Additionally, the transcription factor E2F1 binds to the Nanog gene's promoter site to induce transcription. P57, P21, Prkca, MDM4, Map2k6, and FADD gene expression in the treatment group (LINC00511 deletion) was significantly higher than in the control group (P < 0.01). In addition, knockout cells had lower expression of BCL2 and surviving genes than control cells P < 0.001). In each of the two target alleles, the du-HITI approach introduced a reporter and a transcription termination signal. This strategy's donor vector preparation was significantly easier than "CRISPR HDR," and cell selection was likewise much easier than "CRISPR excision." Furthermore, when this approach was used in the initial transfection attempt, single-cell knockouts for both alleles were generated. CONCLUSIONS: The methods employed and described in this work could be extended to the production of LINC00511 knockout cell lines and, in theory, to the deletion of other lncRNAs to study their function.

Silencing of α-N-acetylgalactosaminidase in the gastric cancer cells amplified cell death and attenuated migration, while the multidrug resistance remained unchanged.

Although the elevated level of the α-N-acetylgalactosaminidase enzyme (encoded by the NAGA gene) is a well-recognized feature of cancer cells; little research works have been undertaken on the cancer malignancy mechanisms. The effects of NAGA gene downregulation on cancer cells' features such as drug resistance, impaired programmed cell death, and migration were analyzed in this study. The cells grew exponentially with a doubling time of 30 h in an optimal condition. Toxicity of daunorubicin chemotherapy drug on NAGA-transfected EPG85.257RDB cells was evaluated in comparison to control cells and no significant change was recorded. Quantitative transcript analyses and protein levels revealed that the MDR1 pump almost remained unchanged during the study. Moreover, the NAGA gene downregulation enhanced the late apoptosis rate in EPG85.257RDB cells at 24 h posttransfection. The investigated expression level of genes and proteins involved in the TNFR2 signaling pathway, related to cancer cell apoptosis, showed considerable alterations after NAGA silencing as well. MAP3K14 and CASP3 genes were downregulated while IL6, RELA, and TRAF2 experienced an upregulation. Also, NAGA silencing generally diminished the migration ability of EPG85.257RDB cells and the MMP1 gene (as a critical gene in metastasis) expression decreased significantly. The expression of the p-FAK protein, which is located in the downstream of the α2 ß1 integrin signaling pathway, was reduced likewise. It could be concluded that despite drug resistance, NAGA silencing resulted in augmentative and regressive effects on cell death and migration.

The relation between the ghrelin receptor and FOXP3 in bladder cancer.

Immune responses play an important role in the fate of bladder cancer tumors. Treg cells are immunosuppressive and down-regulate the proliferation of effector T cells, which favor tumor survival. Ghrelin is a hormone that stimulates release of growth hormone and anti-inflammatory response to cancer cells. Ghrelin also is a gastrointestinal hormone that regulates immune responses via the growth hormone secretagogue receptor (GHS-R1a). The relation among ghrelin, its receptor, and Treg cells that surround bladder tumors is not clear. We found that Foxp3+ T and GHS-R1a cells are increased significantly in bladder tumor tissues. Therefore, we suggest that ghrelin may increase the number of Treg cells in the tumor and suppress activity of the immune system against bladder cancer.

The functions of azurin of Pseudomonas aeruginosa and human mammaglobin-A on proapoptotic and cell cycle regulatory genes expression in the MCF-7 breast cancer cell line.

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p < 0.05) difference after transfection. The expression of BAK, FAS, and BAX genes in transformed cells compare with non-transformed and transformed MCF-7 by pBudCE4.1 were increased statistically significant (p < 0.05) increases. Although, the increase of SURVIVIN and P53 expressions in transformed cells were not statistically significant (p > 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Biodecomposition of Phenanthrene and Pyrene by a Genetically Engineered Escherichia coli.

BACKGROUND: Genetically engineered microorganisms (GEMs) can be used for bioremediation of the biological pollutants into nonhazardous or less-hazardous substances, at lower cost. Polycyclic aromatic hydrocarbons (PAHs) are one of these contaminants that associated with a risk of human cancer development. Genetically engineered E. coli that encoded catechol 2,3- dioxygenase (C230) was created and investigated its ability to biodecomposition of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC) measurement. We revised patents documents relating to the use of GEMs for bioremediation. This approach have already been done in others studies although using other genes codifying for same catechol degradation approach. OBJECTIVE: In this study, we investigated biodecomposition of phenanthrene and pyrene by a genetically engineered Escherichia coli. METHODS: Briefly, following the cloning of C230 gene (nahH) into pUC18 vector and transformation into E. coli Top10F, the complementary tests, including catalase, oxidase and PCR were used as on isolated bacteria from spiked soil. RESULTS: The results of HPLC measurement showed that in spiked soil containing engineered E. coli, biodegradation of phenanthrene and pyrene comparing to autoclaved soil that inoculated by wild type of E. coli and normal soil group with natural microbial flora, were statistically significant (p<0.05). Moreover, catalase test was positive while the oxidase tests were negative. CONCLUSION: These findings indicated that genetically manipulated E. coli can provide an effective clean-up process on PAH compounds and it is useful for bioremediation of environmental pollution with petrochemical products.

Ghrelin induces autophagy and CXCR4 expression via the SIRT1/AMPK axis in lymphoblastic leukemia cell lines.

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent malignancies in children, and the CXCR4 receptor plays an important role in the metastasis of this malignancy. Ghrelin is a hormone with various functions including stimulation of the release of growth hormone and autophagy in cancer cells. Moreover, SIRT1 and AMPK (AMP-activated protein kinase) stimulate expression of proteins involved in autophagy. On the other hand, autophagic cell death can be an alternative target for cancer therapy, in the absence of apoptosis. The relationship between ghrelin and the SIRT1/AMPK axis and the resulting effects on autophagy, apoptosis, proliferation, and expression of CXCR4 and the ghrelin receptor (GHS-R1a), in Jurkat and Molt-4 human lymphoblastic cell lines was not previously clear. Here we demonstrate that SIRT1 expression is upregulated during the induction of autophagy by ghrelin, an effect that is inhibited by inactivation of SIRT1/AMPK axis. In addition, ghrelin can affect CXCR4 and GHS-R1a expression. In conclusion, this work reveals that ghrelin induces autophagy, invasion, and downregulation of ghrelin receptor expression via the SIRT1/AMPK axis in lymphoblastic cell lines. However, in these cell lines ghrelin-induced autophagy does not lead to cell death due to weak induction of apoptosis.

The expression of Cysteine-Rich Secretory Protein 2 (CRISP2) and miR-582-5p in seminal plasma fluid and spermatozoa of infertile men.

Cysteine-Rich Secretory Protein 2 (CRISP2) plays an important role in the morphology and motion of male ejaculated spermatozoa. The association of its expression with some miRNAs is also well known. The aim of this study was to determine the expression of CRISP2 and mir-582 in the seminal plasma fluid and spermatozoa of three groups of infertile men and the possible association of their expressions. In this experimental study, the expression of CRISP2 in seminal plasma fluid and spermatozoa of 17 men with asthenozoospermia, 15 men with teratozoospermia, 17 men with teratoasthenozoospermia, and 18 infertile individuals with normozoospermia were measured using western blotting. Then by using bioinformatics studies, miR-582-5p was nominated as a CRISP2-associated miRNA, and its expression was evaluated by means of Real-Time PCR. Comparison of expression of CRISP2 and miRNA-582 in the studied groups was analyzed by t-test and Mann-Whitney U test. The expression of CRISP2 showed a significant reduction in the spermatozoa and seminal plasma fluid of all three groups, (p < 0.05). MiR-582-5p expression significantly increased in teratozoospermia patients (<0.05), and significantly decreased in teratoasthenozoospermia patients (p < 0.05). Meanwhile, changes in the expression of miR-582-5p in teratoasthenozoospermia individuals was associated with a decrease in the expression of CRISP2, which could represent the potential role of miR-582-5p in regulation of CRISP2 expression in teratoasthenozoospermia individuals.

The concurrent effects of azurin and Mammaglobin-A genes in inhibition of breast cancer progression and immune system stimulation in cancerous BALB/c mice.

In the present study, the simultaneous application of azurin gene of P. aeruginosa and MAM-A antigen on the induction of immune responses against breast cancer tumors was investigated in BALB/c mice. The pBudCE4.1-azurin-MAM-A recombinant vector was generated and prepared at a large scale. This recombinant vector alone or combined with chitosan nanoparticles was infused into the hip muscle of animals. Animals were divided into the "prevention" and "therapy" categories. The animals of prevention category were first, immunized by a recombinant vector and then exposed to chemical cancer inducers; while the animals in the therapy category were first treated with chemical compounds and then infused by a recombinant plasmid. The tumor tissues, infusion sites, and blood specimens were collected and examined by serological, molecular, and histological tests. The breast tumor incidence in the infused animals by recombinant plasmid alone or combined with nanoparticles (in both prevention and therapy categories) compared with infused mice by empty pBudCE4.1 vector was significantly decreased (p < 0.05). These results were supported by histological studies using H&E staining. The ELISA and q-real-time PCR techniques showed the range of IFN-γ, IL-12, IL-4, and IL-17A cytokines in the infused mice by recombinant vector alone or combined with nanoparticles compared to the healthy mice and infused animals by intact pBudCE4.1 were significantly increased (p < 0.05). Accordingly, the expression of the tumor markers CEA, Krt20, and Muc1 were significantly decreased in treated mice either by the sole recombinant vector or combined with nanoparticles (p < 0.05). These findings indicated that pBudCE4.1-azurin-MAM-A recombinant vector plays an essential role against the formation and expansion of breast tumors in the animal model. In addition, this recombinant vector is safe and has the proper ability to stimulate the immune system. In addition, the chitosan nanoparticle represents a promising adjuvant for DNA vaccine delivery, which improves the immune system stimulation and boosts the vaccine performance.

The Effect of the MicroRNA-183 Family on Hair Cell-Specific Markers of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Hearing loss is considered the most common sensory disorder across the world. Nowadays, a cochlear implant can be an effective treatment for patients. Moreover, it is often believed that sensorineural hearing loss in humans is caused by loss or disruption of the function of hair cells in the cochlea. In this respect, mesenchymal cells can be a good candidate for cell-based therapeutic approaches. To this end, the potential of human bone marrow-derived mesenchymal stem cells to differentiate into hair cells with the help of transfection of microRNA in vitro was investigated. MicroRNA mimics (miRNA-96, 182, and 183) were transfected to human bone marrow-derived mesenchymal stem cells using Lipofec-tamine as a common transfection reagent following the manufacturer's instructions at 50 nM for microRNA mimics and 50 nM for the scramble. The changes in cell morphology were also observed under an inverted microscope. Then, the relative expression levels of SOX2, POU4F3, MYO7A, and calretinin were assayed using real-time polymerase chain reaction according to the ΔΔCt method. The ATOH1 level was similarly measured via real-time polymerase chain reaction and Western blotting. The results showed that increased expression of miRNA-182, but neither miRNA-96 nor miRNA-183, could lead to higher expression levels in some hair cell markers. The morphology of the cells also did not change in this respect, but the evaluation of gene expression at the levels of mRNA could promote the expression of the ATOH1, SOX2, and POU4F3 markers. Furthermore, miRNA-182 could enhance the expression of ATOH1 at the protein level. According to the results of this study, it was concluded that miRNA-182 could serve as a crucial function in hair cell differentiation by the upregulation of SOX2, POU4F3, and ATOH1 to promote a hair cell's fate.

Molecular Mechanisms behind Free Radical Scavengers Function against Oxidative Stress.

Accumulating evidence shows that oxidative stress is involved in a wide variety of human diseases: rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, cancers, etc. Here, we discuss the significance of oxidative conditions in different disease, with the focus on neurodegenerative disease including Parkinson's disease, which is mainly caused by oxidative stress. Reactive oxygen and nitrogen species (ROS and RNS, respectively), collectively known as RONS, are produced by cellular enzymes such as myeloperoxidase, NADPH-oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and nitric oxide synthase (NOS). Natural antioxidant systems are categorized into enzymatic and non-enzymatic antioxidant groups. The former includes a number of enzymes such as catalase and glutathione peroxidase, while the latter contains a number of antioxidants acquired from dietary sources including vitamin C, carotenoids, flavonoids and polyphenols. There are also scavengers used for therapeutic purposes, such as 3,4-dihydroxyphenylalanine (L-DOPA) used routinely in the treatment of Parkinson's disease (not as a free radical scavenger), and 3-methyl-1-phenyl-2-pyrazolin-5-one (Edaravone) that acts as a free radical detoxifier frequently used in acute ischemic stroke. The cell surviving properties of L-DOPA and Edaravone against oxidative stress conditions rely on the alteration of a number of stress proteins such as Annexin A1, Peroxiredoxin-6 and PARK7/DJ-1 (Parkinson disease protein 7, also known as Protein deglycase DJ-1). Although they share the targets in reversing the cytotoxic effects of H2O2, they seem to have distinct mechanism of function. Exposure to L-DOPA may result in hypoxia condition and further induction of ORP150 (150-kDa oxygen-regulated protein) with its concomitant cytoprotective effects but Edaravone seems to protect cells via direct induction of Peroxiredoxin-2 and inhibition of apoptosis.

MicroRNAs: effective elements in ear-related diseases and hearing loss.

miRNAs are important factors for post-transcriptional process that controls gene expression at mRNA level. Various biological processes, including growth and differentiation, are regulated by miRNAs. miRNAs have been demonstrated to play an essential role in development and progression of hearing loss. Nowadays, miRNAs are known as critical factors involved in different physiological, biological, and pathological processes, such as gene expression, progressive sensorineural hearing loss, age-related hearing loss, noise-induced hearing loss, cholesteatoma, schwannomas, and inner ear inflammation. The miR-183 family (miR-183, miR-96 and miR-182) is expressed abundantly in some types of sensory cells in inner ear specially mechanosensory hair cells that exhibit a great expression level of this family. The plasma levels of miR-24-3p, miR-16-5p, miR-185-5p, and miR-451a were upregulated during noise exposures, and increased levels of miR-21 have been found in vestibular schwannomas and human cholesteatoma. In addition, upregulation of pro-apoptotic miRNAs and downregulation of miRNAs which promote differentiation and proliferation in age-related degeneration of the organ of Corti may potentially serve as a helpful biomarker for the early detection of age-related hearing loss. This knowledge represents miRNAs as promising diagnostic and therapeutic tools in the near future.

Comparison of Three Types of Mesenchymal Stem Cells (Bone Marrow, Adipose Tissue, and Umbilical Cord-Derived) as Potential Sources for Inner Ear Regeneration.

In this review, we compared the potential of mesenchymal stem cells derived from bone marrow, adipose tissue and umbilical cord as suitable sources for regeneration of inner ear hair cells and auditory neurons. Our intensive literature search indicates that stem cells in some of adult mammalian tissues, such as bone marrow, can generate new cells under physiological and pathological conditions. Among various types of stem cells, bone marrow-derived mesenchymal stem cells are one of the most promising candidates for cell replacement therapy. Mesenchymal stem cells have been reported to invade the damaged area, contribute to the structural reorganization of the damaged cochlea and improve incomplete hearing recovery. We suggest that bone marrow-derived mesenchymal stem cells would be more beneficial than other mesenchymal stem cells.

In Vitro Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Hair Cells Using Growth Factors.

OBJECTIVE: In this study, we attempted to differentiated human bone marrow-derived mesenchymal stem cells (hBMSCs) to auditory hair cells using growth factors. METHODS: Retinoic acid (RA), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) were added to hBMSCs cell culture medium. The cells were evaluated morphologically and the expression of SOX2, POU4F3, MYO7A, and Calretinin at mRNA level and ATOH1 mRNA and protein expression. RESULTS: After treatment with the growth factors, the morphology of the cells did not change, but evaluation of gene expression at the mRNA level increased the expression of the ATOH1, SOX2, and POU4F3 markers. Growth factors increased the expression of ATOH1 at the protein level. The expression of calretinin showed decreased and MYO7A no significant change in expression. CONCLUSION: hBMSCs have the potential to differentiate to hair cell-like using the RA, bFGF, and EGF.

Edaravone leads to proteome changes indicative of neuronal cell protection in response to oxidative stress.

Neuronal cell death, in neurodegenerative disorders, is mediated through a spectrum of biological processes. Excessive amounts of free radicals, such as reactive oxygen species (ROS), has detrimental effects on neurons leading to cell damage via peroxidation of unsaturated fatty acids in the cell membrane. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been used for neurological recovery in several countries, including Japan and China, and it has been suggested that Edaravone may have cytoprotective effects in neurodegeneration. Edaravone protects nerve cells in the brain by reducing ROS and inhibiting apoptosis. To gain further insight into the cytoprotective effects of Edaravone against oxidative stress condition we have performed comparative two-dimensional gel electrophoresis (2DE)-based proteomic analyses on SH-SY5Y neuroblastoma cells exposed to oxidative stress and in combination with Edaravone. We showed that Edaravone can reverse the cytotoxic effects of H2O2 through its specific mechanism. We observed that oxidative stress changes metabolic pathways and cytoskeletal integrity. Edaravone seems to reverse the H2O2-mediated effects at both the cellular and protein level via induction of Peroxiredoxin-2.

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches.

Proteome analysis reveals roles of L-DOPA in response to oxidative stress in neurons.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative movement disorder, caused by preferential dopaminergic neuronal cell death in the substantia nigra, a process also influenced by oxidative stress. L-3,4-dihydroxyphenylalanine (L-DOPA) represents the main treatment route for motor symptoms associated with PD however, its exact mode of action remains unclear. A spectrum of conflicting data suggests that L-DOPA may damage dopaminergic neurons due to oxidative stress whilst other data suggest that L-DOPA itself may induce low levels of oxidative stress, which in turn stimulates endogenous antioxidant mechanisms and neuroprotection. RESULTS: In this study we performed a two-dimensional gel electrophoresis (2DE)-based proteomic study to gain further insight into the mechanism by which L-DOPA can influence the toxic effects of H2O2 in neuronal cells. We observed that oxidative stress affects metabolic pathways as well as cytoskeletal integrity and that neuronal cells respond to oxidative conditions by enhancing numerous survival pathways. Our study underlines the complex nature of L-DOPA in PD and sheds light on the interplay between oxidative stress and L-DOPA. CONCLUSIONS: Oxidative stress changes neuronal metabolic routes and affects cytoskeletal integrity. Further, L-DOPA appears to reverse some H2O2-mediated effects evident at both the proteome and cellular level.

Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness.

BACKGROUND: KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. METHODS: We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. RESULTS: KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. CONCLUSIONS: Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it may represent a novel target for biomarker development and a novel therapeutic target for breast cancer.

Proteome analysis of the penicillin producer Penicillium chrysogenum: characterization of protein changes during the industrial strain improvement.

Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by "blue silver" colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin biosynthesis in the high producer strains. These results provide useful information to improve the production of many other bioactive secondary metabolites.

Association between the length of a CA dinucleotide repeat in the EGFR and risk of breast cancer.

We studied the association of breast cancer with the polymorphic CA repeat in 108 cases of breast cancer and 108 matched controls from Isfahan city of Iran. The most common genotype in controls and patients was homozygous with allele length of 16. Our findings demonstrate that Women with two short CA repeat (< 19) are at a significantly higher risk of breast cancer, at an estimated odds ratio of 1.86. We have also found that women with short alleles (< 19) had much greater risk of developing cancer before the age of 55 (OR, 3.36).