Prevalence and prognostic role of PIK3CA E545K mutation in Iranian colorectal cancer patients.

AIM: This study aimed to evaluate the distribution of PIK3CA E545K mutation in Iranian CRC patients and explored its roles in disease prognosis. BACKGROUND: Deregulation of the phosphoinositide 3-kinase (PI3K) pathway contributes to the progression of tumors. The p110a (PIK3CA), a catalytic subunit of PIK3, is mutated in many types of cancers. Exon 9 (E545K) is the most frequently mutated hotspot in PIK3CA in colorectal cancer (CRC). However, the prognostic role of PIK3CA E545K mutation needs to be elucidated. METHODS: Tumors from 187 CRC patients were retrospectively collected from the Taleghani and Shohada Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran, between 2010 and 2017. PIK3CA E545K status was detected in Formalin-fixed paraffin-embedded (FFPE) tissues using PCR-RFLP methods, and validated by pyrosequencing. Correlations between PIK3CA E545K mutation clinicopathological features were analyzed. RESULTS: The frequency of PIK3CA E545K gene mutations in CRC patients was 10.7%. Significant correlations were observed in PIK3CA E545K mutation with tumor differentiation and TNM stage (p < 0.042 and p = 0.033, respectively). Kaplan-Meier analysis showed a worse prognosis in overall survival (OS) in patients with PIK3CA E545K mutation (p < 0.001). Multivariate analysis indicated that PIK3CA E545K mutation was a detrimental factor for OS (HR = 6.497, 95% CI: 2.859-14.768, p < 0.021). CONCLUSION: A high frequency of PIK3CA E545K mutation was detected in Iranian CRC patients. The results of the present study suggested that PIK3CA E545K mutation may be associated with poor prognosis. These findings require further confirmation via prospective studies with larger samples.

High expression of Snail1 is associated with EMAST and poor prognosis in CRC patients.

AIM: This study aimed to determine the link between Snail1 expression and CRC patients' survival as well as its significant association with EMAST status. BACKGROUND: Snail1 is an evolutionary preserved zinc-finger transcription protein which contributes to Epithelial-to-mesenchymal transition (EMT). EMT initiates invasion and proliferation in many tumors. Elevated microsatellite alteration at selected tetranucleotide repeats (EMAST) is a marker of poor prognosis in patients with colorectal cancer (CRC). We hypothesized that Snail1 overexpression is an important mediator of metastasis and decreased survival in CRCs that characteristically have EMAST phenotype. METHODS: Quantitative real-time polymerase chain reactions were carried out to analyze the expression levels of Snail1 in both normal and tumor specimens from a total of 122 paraffin-embedded tissues (FFPE) of CRC sample with known EMAST status. The correlation between Snail1 expression and clinicopathological characteristics, survival, and EMAST status were examined. RESULTS: Snail1 overexpression was detected in tumor tissues in 32% of all examined patients and its positive expression was related to metastasis (p=0.001) and EMAST+ phenotype (P=0.017). Further, positive Snail1 expression correlates with poor overall survival in CRC patients (P=0.01). CONCLUSION: Our findings suggest that Snail1 overexpression is not only associated with EMAST but also with clinicopathological variables of poor prognosis. These results indicate that Snail1 expression levels may be useful for establishing novel therapeutic strategies and could help survival improvement in CRC patients.

Detection of gene expression in sentinel lymph node of primary breast cancer patients.

Sentinel lymph node (SLN) micrometstasis detection improves outcome for breast cancer follow up procedure. The aim of the present study was to identify gene profiles that accurately predicted the outcome of breast cancer patients. Fifty tumor sample from breast cancer patients were analyzed for the expression of 3 genes using quantitative-PCR. Also clinical verification for recurrence to distant organs was performed. Three gene signature were confirmed based on tumor's stage, grade, ER status, using conditional logistic regression. Based on this findings, the negative reported lymph nodes for metastasis, had micro metastasis in significant values. There was a significant difference between normal and cancer samples in 3 gene expression marker and also there was meaningful relationship between three gene expression with tumor's grade, stage according to progression of tumor. A novel gene expression signature predictive of micro metastatic patients was evaluated. In this assessment, relationship between this gene with tumor's features   that finding clear role for these genes with tumor's outcome, needs to be established.

Natural Killer Cells from the Subcutaneous Adipose Tissue Underexpress the NKp30 and NKp44 in Obese Persons and Are Less Active against Major Histocompatibility Complex Class I Non-Expressing Neoplastic Cells.

There are many types of leukocytes reside in subcutaneous adipose tissue (SAT), and among them, natural killer cells (NKs) comprise a major part. We show that the NKs that reside in the SAT (adipose tissue-derived NK cells; ADNKs) of the abdominal region found with phenotypic differences from the NKs circulating in the peripheral blood derived NK cells (PBNKs). In this survey, flow cytometry phenotyping was used to study the differences between the natural cytotoxicity receptor expression on ADNKs and PBNKs of both obese and lean persons. Also, their cytotoxicity and cytokine production patterns were evaluated. The activation experiments on isolated and expanded NKs with IL-2, IL-15, and IL-21 cytokines revealed the main population of the CD56dim within the total ADNKs of obese persons has an under-expression of NKp30 and NKp44 despite the unchanged levels of NKG2D. The data suggest the suppressive condition of the adipose tissue niche on the NKs response against sensitive major histocompatibility complex class I non-expressing neoplastic cells. As the NKs are the first line of the body's defense vs tumor formation, this change may lead to the development of transformed cells into the tumors.

Differentiation of human endometrial stem cells into urothelial cells on a three-dimensional nanofibrous silk-collagen scaffold: an autologous cell resource for reconstruction of the urinary bladder wall.

Reconstruction of the bladder wall via in vitro differentiated stem cells on an appropriate scaffold could be used in such conditions as cancer and neurogenic urinary bladder. This study aimed to examine the potential of human endometrial stem cells (EnSCs) to form urinary bladder epithelial cells (urothelium) on nanofibrous silk-collagen scaffolds, for construction of the urinary bladder wall. After passage 4, EnSCs were induced by keratinocyte growth factor (KGF) and epidermal growth factor (EGF) and seeded on electrospun collagen-V, silk and silk-collagen nanofibres. Later we tested urothelium-specific genes and proteins (uroplakin-Ia, uroplakin-Ib, uroplakin-II, uroplakin-III and cytokeratin 20) by immunocytochemistry, RT-PCR and western blot analyses. Scanning electron microscopy (SEM) and histology were used to detect cell-matrix interactions. DMEM/F12 supplemented by KGF and EGF induced EnSCs to express urothelial cell-specific genes and proteins. Either collagen, silk or silk-collagen scaffolds promoted cell proliferation. The nanofibrous silk-collagen scaffolds provided a three-dimensional (3D) structure to maximize cell-matrix penetration and increase differentiation of the EnSCs. Human EnSCs seeded on 3D nanofibrous silk-collagen scaffolds and differentiated to urothelial cells provide a suitable source for potential use in bladder wall reconstruction in women.

Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells.

Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was significantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These findings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

Downregulation of E-cadherin expression in breast cancer by promoter hypermethylation and its relation with progression and prognosis of tumor.

Breast cancer is the most common cancer in women around the world, and novel prognosis strategies is needed to control more accurate and effective of this malignant disease. Among the latest prognostic markers is E-cadherin, which mediates cell-cell adhesion by associating with catenins. Loss of E-cadherin gene (CDH1) function by genetic or epigenetic alteration leads to tumorigenesis. The aim of our study was to investigate E-cadherin gene promoter methylation in breast cancer, and its correlation with E-cadherin protein expression. Fifty primary breast cancers tissue with ductal type and 50 normal breast sample from the same patients that was located adjacent to tumor region as controls were provided by Imam Reza-based referral and teaching hospital affiliated to Tabriz University of Medical Sciences, Tabriz, Iran. CDH1 promoter region CpG sites methylation and E-cadherin protein expression were determined by bisulfite-specific polymerase chain reaction and Western blot analysis, and the resulting products were sequenced on an ABI automated sequencer for firm conclusion. CDH1 hypermethylation in breast tumor specimen (ductal type) was observed in 94 % (47 of 50) comparing with normal samples methylation, and the significant difference was (p = 0.000). Protein expression in tumor samples tends to diminish with the CDH1 promoter region methylation. In the group of 50 ductal carcinomas cases, most of the cases showing CDH1 hypermethylation correlated inversely with the reduced levels of expression of E-cadherin proteins (95 % of full-methylated tumor samples had no protein expression, and 4.5 % of them had weak expression levels). Possible association was observed between CDH1 methylation and its protein expression (p = 0.000). The results of methylation analysis in promoter region in ten CpG sites (863, 865, 873, 879, 887, 892, 901, 918, 920, and 940) suggested that abnormal CDH1 methylation occurs in high frequencies in ductal breast tumors probably sounds the process of carcinogenesis progression.

λ Phage nanobioparticle expressing apoptin efficiently suppress human breast carcinoma tumor growth in vivo.

Using phages is a novel field of cancer therapy and phage nanobioparticles (NBPs) such as λ phage could be modified to deliver and express genetic cassettes into eukaryotic cells safely in contrast with animal viruses. Apoptin, a protein from chicken anemia virus (CAV) has the ability to specifically induce apoptosis only in carcinoma cells. We presented a safe method of breast tumor therapy via the apoptin expressing λ NBPs. Here, we constructed a λ ZAP-CMV-apoptin recombinant NBP and investigated the effectiveness of its apoptotic activity on BT-474, MDA-MB-361, SKBR-3, UACC-812 and ZR-75 cell lines that over-expressing her-2 marker. Apoptosis was evaluated via annexin-V fluorescent iso-thiocyanate/propidium iodide staining, flow-cytometric method and TUNEL assay. Transfection with NBPs carrying λ ZAP-CMV-apoptin significantly inhibited growth of all the breast carcinoma cell lines in vitro. Also nude mice model implanted BT-474 human breast tumor was successfully responded to the systemic and local injection of untargeted recombinant λ NBPs. The results presented here reveal important features of recombinant λ nanobioparticles to serve as safe delivery and expression platform for human cancer therapy.

Endometrial stem cell differentiation into smooth muscle cell: a novel approach for bladder tissue engineering in women.

OBJECTIVE: To investigate manufacturing smooth muscle cells (SMCs) for regenerative bladder reconstruction from differentiation of endometrial stem cells (EnSCs), as the recent discovery of EnSCs from the lining of women's uteri, opens up the possibility of using these cells for tissue engineering applications, such as building up natural tissue to repair prolapsed pelvic floors as well as building urinary bladder wall. MATERIALS AND METHODS: Human EnSCs that were positive for cluster of differentiation 146 (CD146), CD105 and CD90 were isolated and cultured in Dulbecco's modified Eagle/F12 medium supplemented with myogenic growth factors. The myogenic factors included: transforming growth factor ß, platelet-derived growth factor, hepatocyte growth factor and vascular endothelial growth factor. Differentiated SMCs on bioabsorbable polyethylene-glycol and collagen hydrogels were checked for SMC markers by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), western blot (WB) and immunocytochemistry (ICC) analyses. RESULTS: Histology confirmed the growth of SMCs in the hydrogel matrices. The myogenic growth factors decreased the proliferation rate of EnSCs, but they differentiated the human EnSCs into SMCs more efficiently on hydrogel matrices and expressed specific SMC markers including α-smooth muscle actin, desmin, vinculin and calponin in RT-PCR, WB and ICC experiments. The survival rate of cultures on the hydrogel-coated matrices was significantly higher than uncoated cultures. CONCLUSIONS: Human EnSCs were successfully differentiated into SMCs, using hydrogels as scaffold. EnSCs may be used for autologous bladder wall regeneration without any immunological complications in women. Currently work is in progress using bioabsorbable nanocomposite materials as EnSC scaffolds for developing urinary bladder wall tissue.

Recombinant λ bacteriophage displaying nanobody towards third domain of HER-2 epitope inhibits proliferation of breast carcinoma SKBR-3 cell line.

Phage display of many nanobodies via filamentous phage in combination with helper phage has been reported by many scientists. The aim of this study was to produce lambda (λ) bacteriophage displaying high-affinity nanobody against HER-2 expressing breast carcinoma cells. Bacteriophage λ is a temperate phage with inherent biological safety in mammalian cells. Here we report the construction of a recombinant λ phage that efficiently expresses specific nanobody towards third domain of HER-2 target on SKBR-3 and MCF-7 cell lines in vitro. We constructed recombinant λ phage particles containing a mammalian expression cassette, C-Myc tagged, encoding VHH gene of camelid anti HER-2 third domain epitope using λ ZAP-cytomegalic virus (CMV) vector. The SKBR-3, MCF-7 and human endometrial stem cells were treated by the nanobody displayed recombinant λ phage. The cell growth inhibition assay was performed by MTT Cell Viability Assay Kit. After the fourth round of biopanning there was a significant enrichment in the phage specifically binding to the antigen. The ratio of targeted phage increased approximately 1,000-fold in the fifth round. The nanobody expressed by λ ZAP-CMV-VHH phagemid cloned in λ bioparticles significantly inhibited the proliferation of HER-2 positive SKBR-3 and MCF-7 cells. Recombinant bacteriophage λ ZAP-CMV-VHH-cDNA could be used efficiently for construction of nanobodies to mortify HER-2 positive breast carcinoma cells as a nanomedical therapeutic.

Human endometrial stem cell neurogenesis in response to NGF and bFGF.

The potential of cell therapy is promising in nerve regeneration, but is limited by ethical considerations about the proper and technically safe source of stem cells. We report the successful differentiation of human EnSCs (endometrial stem cells) as a rich source of renewable and safe progenitors into high-efficiency cholinergic neurons. The extracellular signals of NGF (nerve growth factor) and bFGF (basic fibroblast growth factor) could induce cholinergic neuron differentiation. ChAT (choline acetyltransferase), MAP2 (microtubule associated protein 2) and NF-l (neurofilament L) increased after administration of bFGF and NGF to the EnSC cultures. trkC and FGFR2 (fibroblast growth factor receptor 2), which belong to the NGF and bFGF receptors respectively, were determined in populations of EnSCs. NGF, bFGF and their combination differentially influenced human EnSCs high efficiency differentiation. By inducing cholinergic neurons from EnSCs in a chemically defined medium, we could produce human neural cells without resorting to primary culture of neurons. This in vitro method provides an unlimited source of human neural cells and facilitates clinical applications of EnSCs for neurological diseases.

DHEA provides a microenvironment for endometrial stem cells neurogenesis.

Recently, transplantation of adult stem cells over embryonic stem cells increased in regenerative medicine. Among the adult stem cells, human endometrium stromal (hEnS) cells are under the strict control of the steroid hormones and have the potential to differentiate into other cell lineages including neural cells. Unfortunately these cells may lose their neurogenic differentiation ability upon extended expansion in cultures. To avoid the back-differentiation, it is important to establish growth conditions that support the rapid proliferation and stable differentiation of hEnS cells over extended periods of time without compromising their neuronal phenotype. Differentiation of transplanted cells is strongly influenced by environmental signals. The steroidal microenvironment of the stem cells plays a major role in controlling neurogenesis in the cultures. Dehydroepiandrosterone (DHEA) administration to the cultures could support this propose. DHEA enhance survival rates of dissociated neurons in cultures. It can activate AKT protein kinase pathway as well as nerve growth factor (NGF) that enhances neurogenesis efficiently. On the other hand it seems that DHEA increase survival rate of neural cells via production of brain derived neurotrophic factor (BDNF), indirectly. BDNF is a mediator product of the DHEA that promotes the differentiation and survival of neurons. Here, we offer that DHEA is a suitable candidate that could provide a microenvironment to stimulate neurogenesis and enhanced survival of newly formed neurons derived from hEnS cells. From the point that DHEA is the most abundant steroid in the body, marketed as a supplement and is increasingly self-prescription we hypothesized that it could be the safe and high available choice. This provides a better insight into the maintenance of neural cells for treatment of a wide variety of neurological diseases such as Alzheimer's and Parkinson's by non-invasively autologous cell therapy by hEnS cells especially in women.