Effect of angiotensin receptor blockade on prevention and reversion of tamoxifen-resistant phenotype in MCF-7 cells.

Tamoxifen (TAM) is a standard adjuvant endocrine therapy in postmenopausal breast cancer patients, but innate or acquired TAM resistance has remained to be a therapeutic challenge for clinicians. The aim of this study was to explore the possible participation of renin-angiotensin system (RAS) in the acquisition of TAM resistance and try to prevent and regress the resistance using an angiotensin II receptor type-1 (AGTR1) blocker, losartan. Establishment of TAM-resistant (TAM-R) cells was accomplished by continuous exposure of MCF-7 cells to 1 µmol/L TAM. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to determine cell growth. Moreover, messenger RNA (mRNA) expression levels of AGTR1 and angiotensin II receptor type-2 (AGTR2) were measured by quantitative real-time polymerase chain reaction. A significant increase of AGTR1 and AGTR2 transcripts was observed in TAM-R cells compared to MCF-7 cells. Interestingly, losartan-TAM combination effectively resensitized TAM-R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest an important role of RAS in acquired TAM resistance and targeting of RAS by losartan may overcome TAM resistance phenomenon and provide a novel avenue for treatment of resistant breast cancers.

Triple blockade of HER2 by a cocktail of anti-HER2 scFv antibodies induces high antiproliferative effects in breast cancer cells.

Dual therapy targeting human epidermal growth factor receptor 2 (HER2) by pertuzumab and trastuzumab (Herceptin) resulted in the significant survival of patients with HER2-positive breast cancers. However, a number of HER2-overexpressing breast cancers escape from this combination therapy. Due to several advantages of human single-chain antibodies (single chain variable fragments (scFvs)), these molecules were approved as valuable alternatives to entire IgGs for molecular targeting. In this study, our aim was to evaluate the growth inhibitory effects of three novel human anti-HER2 scFvs on breast cancer cells either alone or in combination and to assess their influence on HER2 expression in these cells. Flow cytometry was performed to show the cell binding ability of the scFvs to HER2-overexpressing cell lines, BT-474 and SKBR-3 cells, and HER2 low-expressing cell line, HeLa cells. The antiproliferative effects of the antibodies on the cancer cells were assessed by MTT assay. The amounts of HER2 gene and protein expression after antibody treatments were determined by quantitative real-time PCR and western blotting, respectively. FACS analysis showed that the anti-HER2 scFvs bound to BT-474 and SKBR-3 cells significantly higher than HeLa cells. Growth inhibitory assessment demonstrated that the triple blockade of HER2 by a cocktail of the three anti-HER2 scFvs significantly inhibited the proliferation of the both cancer cells to a greater extent than scFvs individually, in dual combination (scFv-I and scFv-III), and Herceptin. The percentages of growth inhibition of BT-474 and SKBR-3 cells after treatment with the cocktail were up to 77.4 and 76.5 %, respectively. The three scFv antibodies also reduced HER2 expression at both the gene and protein levels individually and in combination. Our results suggest that the cocktail of the three anti-HER2 scFv-I, scFv-II, and scFv-III, which induces high growth inhibition in breast cancer cells and downregulates HER2 gene and protein expression, can be considered as a new alternative for targeting of HER2-positive breast cancers.

IL-23/IL-27 Ratio in Peripheral Blood of Patients with Breast Cancer.

BACKGROUND: Interleukin (IL)-23 and IL-27 are two IL-12-related cytokines which their function may dramatically influence the inflammatory response to tumor development. IL-12 and IL-27 seem to have antagonistic roles with IL-23 in tumor site. In this study, IL-23 and IL-27 mRNA expressions were analyzed in peripheral blood of patients with breast cancer and healthy volunteers using quantitative real-time PCR. METHODS: Peripheral blood samples were collected from 50 women with breast cancer and 50 healthy ones. The total RNA was extracted from peripheral blood after lysis with ammonium chloride and TRizol reagent and the cDNA was synthesized. The expression of IL-23 and IL-27 gene transcripts was determined with real-time polymerase chain reaction (qRT-PCR) using Syber Green PCR Master Mix. RESULTS: It is found that IL-23 and IL-27 transcripts had significantly higher expression in peripheral blood of patients compared with the healthy controls. The ratio of IL-23 transcript expression to IL-27 was 3.4 fold lower in the studied patients compared with the normal individuals. CONCLUSION: It is concluded that the over expression of IL-23 and IL-27 gene transcript in peripheral blood of breast cancer patients may be an immune response against tumor development and the inflammatory response plays a critical role in tumor development via up regulating the corresponding cytokines. However, the IL-23/IL-27 ratio may play an important role in cytokine-based immunotherapy against cancer. Further research should be carried out to assess these cytokines in a larger sample size. .

Effect of follicular fluid and platelet-activating factor on lactate dehydrogenase C expression in human asthenozoospermic samples.

BACKGROUND: Application of follicular fluid (FF) and platelet-activating factor (PAF) in artificial insemination improves sperm motility. Lactate dehydrogenase C (LDH-C) is a key enzyme for sperm motility. In this study, the effects of FF and PAF on the sperm motility index and LDH-C expression were investigated. Moreover, LDH-C expression was compared between asthenozoospermic and normozoospermic samples. METHODS: The expression of LDH-C was examined by quantitative real-time polymerase chain reaction (q-RT PCR) and western blotting after it was treated with optimized concentrations of FF and PAF in twenty asthenozoospermic samples. Also, LDH-C expression was evaluated in five normozoospermic samples. RESULTS: Samples with 75% FF and 100 nM of PAF had an increase in their percentages of progressive and slowly motile sperms and a decrease in their percentages of non-progressive and non-motile sperms. Moreover, LDH-C mRNA transcripts were not changed following PAF and FF treatment, and LDH-C protein was detected in highly progressive motile specimens treated with FF in the asthenozoospermic samples. Furthermore, LDH-C expression was more detectable in the normal sperms. CONCLUSION: Our results indicated that PAF had more beneficial effects than FF on sperm motility in the asthenozoospermic samples (P=0.0001), although the LDH-C expressions of the sperms were not changed significantly in both groups. We found no association between LDH-C expression and sperm motility after FF and PAF actions. This finding, however, requires further investigation. The fact that LDH-C protein was detected in the normozoospermic, but not asthenozoospermic, samples could be cited as a reason for the infertility in these patients.

Combined 4-1BB and CD28 costimulation could unleash lymphocytes from immunosuppression induced by adipose derived stem cell soluble products.

Adipose derived stem cells (ASCs) have the potential to differentiate into multiple cell lineages with the capacity to suppress immune cells. However, the exact mechanism of this suppression is not fully understood. We hypothesized that supplying additional lymphocyte costimulation through CD28 and 4-1BB could overturn the inhibitory effect of ASCs. To that end, PHA-activated human PBMCs were cocultured with ASCs or with conditioned media (CM) prepared from cultured ASCs. Growth was analyzed in the presence or absence of anti-CD28 and anti-4-1BB antibodies. Results from CFSE dilution analysis with flow cytometry showed that significant and dose-dependent suppression of PHA-activated lymphocytes occurred in the presence of ASC-like cells or ASC's CM. However, additional costimulation of T cells through CD28 and 4-1BB was able to fully recover lymphocyte proliferative capacity in the presence of ASC's CM. Neither of the costimulatory antibodies could fully recover lymphocyte proliferation following coculture with ASCs. Reversal of ASC's immunosuppression through costimulation suggests that further investigation of ASC suppression mechanisms is warranted, since many clinical applications of ASCs are based on this feature. Moreover, such findings have the potential to boost the usefulness of ASCs in the treatment of autoimmune disease.

Regulation of carcinoembryonic antigen release from colorectal cancer cells.

Clinical and experimental evidence suggest that circulating carcinoembryonic antigen (CEA) released from tumor cells has an instrumental role in colorectal cancer-liver metastasis. However, the precise mechanism of the regulation of the CEA release from cancer cells is not known. We investigated if the rate of CEA and another GPI-anchored protein, alkaline phosphatase (AP) release is correlated with cellular glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) expression. We also evaluated the effects of phosphatidic acid (PA), a compound known to inhibit GPI-PLD activity, on the CEA and AP release from colon cancer cells. The expression of CEA, GPI-PLD, and AP in five colon carcinoma cells (LS180, Caco2, SW742, SW1116, and HT29/219) was verified by immunoblot and real-time RT-PCR analysis. The amounts of CEA and AP released into cell culture media were determined using ELISA and a colorimetric assay, respectively. We examined the effects of PA (20-100 µM) on CEA and AP release from LS180 cells. All five cancer cell lines analyzed expressed GPI-PLD protein. While there was a positive relationship between AP release and the levels of GPI-PLD transcript expression, we found no direct correlation between CEA released from cancer cells and the GPI-PLD mRNA expression level. However, the rate of CEA release was positively associated with the level of CEA transcript expression. In comparison to controls, the release of GPI-anchored CEA and AP, but not CA19-9 was inhibited significantly by both crude and pure phosphatidic acid (by 56 and 54.5%, respectively). Using PA for inhibiting CEA release from cancer cells may have therapeutic application in preventing CRC-liver metastasis.

Adipose derived stem cells (ASCs) isolated from breast cancer tissue express IL-4, IL-10 and TGF-ß1 and upregulate expression of regulatory molecules on T cells: do they protect breast cancer cells from the immune response?

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-ß1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-ß1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-ß1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25(high)Foxp3(+) T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells.

Bcl-2 and Fas expressions correlate with proliferative specificity of adipose-derived stem cells (ASCs) in breast cancer.

The tumor microenvironment consists of a dynamic interaction with several cell types including infiltrating cells from the immune system, fibroblasts, adipose derived stem cells (ASCs), and an appropriate milieu for tumor cell growth, expansion and spreading. The aim of this study was to focus on ASCs function by isolating and characterizing them from both breast cancer and normal breast adipose tissues. Therefore, the expressions of Bcl-2 and Fas mRNAs in these cells were determined using real-time quantitative PCR (Q-PCR). Also the proliferative properties of ASCs were compared among different pathological states and normal ASCs utilizing the MTT assay. It was observed that, Bcl-2 mRNA had 5-fold more expression in ASCs of patients than in controls. In contrast, Fas had a lower expression of mRNA in ASCs of patients compared to the controls. ASCs isolated from patients with stage 3 breast cancer had a statistically significant higher rate of proliferation compared to stage 2 and normal ASCs (P-value < 0.001). Based on these results, ASCs of the tumor microenvironment may contribute to tumor growth and progression and consequently protect tumor cells from the host immune response. Therefore these cells may be considered as therapeutic targets of cancer immunotherapy.

Quantification of regulatory T cells in peripheral blood of patients with systemic lupus erythematosus.

Regulatory T cells (Tregs) are supposed to stop immune responses in the course of immune activation. However, chronic activation of immune system in systemic lupus erythematosus (SLE) and many other autoreactive disorders are evidence of malfunction of this system. Therefore, it is plausible to quantify presence of these cells in different diseases. Forty-one patients with diagnosis of SLE were enrolled in this study. Patients were divided into two groups of patients with active and inactive disease based on the disease activity score. Flow cytometry analysis was used to determine the frequency of regulatory T cells in peripheral blood according to high expression of CD25 and intracellular Forkhead/winged-helix (Foxp3). Further 30 healthy individuals considered as control group. Significantly less CD4+CD25hi regulatory T cells were detected in active patients (P < 0.001) compared to healthy individuals. The percentage of CD4+CD25hi cells were inversely correlated with the SLEDAI disease score in patients with active disease (r = -0.837, P < 0.0001). Patients with active disease had lower frequencies of CD4+Foxp3+ cells. However, increased frequencies of CD4+Foxp3+ T cells were observed in peripheral blood of patients with inactive disease compared with active patients or healthy individuals (P < 0.010). Moreover, a significant difference between the proportion of CD4+CD25-Foxp3+ population in healthy controls and patients with active disease was shown (P < 0.0005). Presence of lower frequencies of Tregs in patients with SLE could be evaluated as an immune turbulence and could be employed as a target for immunotherapeutic manipulation. However, controversies need to be resolved.

Upregulation of Cytotoxic T-Lymphocyte-Associated Protein 4 and Forkhead Box P3 Transcripts in Peripheral Blood of Patients with Bladder Cancer.

Background: Regulatory T cells (Tregs) play a key role in the progression of tumors. These cells express forkhead box P3 (FOXP3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), which are the potential targets for cancer immunotherapy. The present study aimed to evaluate FOXP3 and CTLA4 transcripts in patients with bladder cancer (BC) compared with healthy individuals. Methods: Transcripts of CTLA4 and FOXP3 genes in the peripheral blood mononuclear cells (PBMCs) of 50 patients with histologically confirmed BC and 50 healthy individuals were assessed at the Institute for Cancer Research, Shiraz University of Medical Sciences (Shiraz, Iran) during 2014-2016. RNA was extracted from PBMCs, then cDNA was synthesized and subjected to quantitative real-time PCR (qRT-PCR) using appropriate primers. Statistical analysis was performed using SPSS software (version 21.0). Results: Significantly higher amounts of CTLA4 and FOXP3 gene transcripts were found in the peripheral blood of BC patients compared with healthy individuals. The expression of both genes was significantly higher in patients with non-invasive and grade I/II BC. The median of CTLA4 and FOXP3 transcript expressions was 3.74 and 5.39, respectively, in non-invasive BC patients, which was significant compared with the control group (P=0.0016 and P=0.009, respectively). The median of target gene mRNA expression in grade I/II BC patients was 2.9 for CTLA4 and 6.61 for FOXP3, which was significant compared with the controls (P=0.013 and P=0.0037, respectively). Conclusion: This study highlights the functional activity of Tregs in early stages of bladder cancer and showed the importance of CTLA4 and FOXP3, when it comes to screening BC.

Expression profile of IL-8 and growth factors in breast cancer cells and adipose-derived stem cells (ASCs) isolated from breast carcinoma.

Adipose-derived stem cells (ASCs) are regarded as a major player of breast cancer microenvironment. By production of various growth factors and expression of regulatory molecules, it is postulated that ASCs protect breast cancer cells from the host immune responses. In this study, the expressions of insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), CXCL8 (IL-8) in breast cancer cells and adipose-derived stem cells isolated from breast tissue of women with breast cancer were investigated. The results were analyzed comparatively in normal ASCs isolated from healthy normal women. In case of breast cancer tissues, results were analyzed between high stage and low stage patients. The expressions of extracted mRNAs were determined using real-time quantitative RT-PCR. As a result, in breast cancer tissues, IGF-1 and IL-8 mRNAs had 28.6 and 56-fold more expressions in high stage compared to low stage patients. In ASCs, relative quantifications (RQ) of VEGF, IL-8, HGF and IGF-1 was about 2-fold higher in patients than controls. Data of this study conclude that presence of resident ASCs within the scaffold of breast tissue may support breast tumor growth and progression through the expressions of tumor promoting factors.

Expression Pattern of Telomerase Reverse Transcriptase (hTERT) Variants and Bcl-2 in Peripheral Lymphocytes of Systemic Lupus Erythematosus Patients.

BACKGROUND & OBJECTIVE: It is not clear whether activated lymphocytes of patients with systemic lupus erythematosus (SLE) are more proliferative or less apoptotic. We aimed to delineate potential differences between B and T cells of SLE patients compared to healthy controls regarding the telomerase activity and apoptosis status. METHODS: In this cross-sectional case control study, Blood samples were taken from 10 SLE patients and 10 healthy controls. B and T cells were separated using magnetic cell sorting system. Telomeric repeat amplification protocol (TRAP) assay and real-time PCR were used to determine the telomerase activity and the expression of alternatively spliced variants. RESULTS: Four patients under treatment showed significant telomerase activity in their T cells. Four of the newly diagnosed patients showed telomerase activity in their B cells (20% of all patients and 40% of new onset patients). There was no specific pattern of human telomerase reverse transcriptase variant expression within the patients' lymphocytes. A significantly reduced expression of Bcl-2 was detected in B cells (P=0.018) and a trend toward lower Bcl-2 expression in T cells was seen in SLE patients compared to healthy controls. CONCLUSION: Although not definitive, our results may suggest that B cells may have more active roles during the earlier phases of the disease attack, while T cells take over when the disease reaches its chronic stages.

Steady-state and stopped-flow kinetic studies of three Escherichia coli NfsB mutants with enhanced activity for the prodrug CB1954.

The enzyme nitroreductase, NfsB, from Escherichia coli has entered clinical trials for cancer gene therapy with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, CB1954 is a poor substrate for the enzyme. Previously we made several NfsB mutants that show better activity with CB1954 in a cell-killing assay in E. coli. Here we compare the kinetic parameters of wild-type NfsB with CB1954 to those of the most active single, double, and triple mutants isolated to date. For wild-type NfsB the global kinetic parameters for both k(cat) and K(m) for CB1954 are about 20-fold higher than previously estimated; however, the measured specificity constant, k(cat)/K(m) is the same. All of the mutants are more active with CB1954 than the wild-type enzyme, the most active mutant showing about 100-fold improved specificity constant with CB1954 over the wild-type protein with little effect on k(cat). This enhancement in specificity constants for the mutants is not seen with the antibiotic nitrofurazone as substrate, leading to reversed nitroaromatic substrate selectivity for the double and triple mutants. However, similar enhancements in specificity constants are found with the quinone menadione. Stopped-flow kinetic studies suggest that the rate-determining step of the reaction is likely to be the release of products. The most active mutant is also selective for the 4-nitro group of CB1954, rather than the 2-nitro group, giving the more cytotoxic reduction product. The double and triple mutants should be much more effective enzymes for use with CB1954 in prodrug-activation gene therapy.

4-1BBL costimulation retrieves CD28 expression in activated T cells.

Binding of CD80/86 to CD28 is regarded as the main T cell costimulatory interaction. However, CD28 downregulates soon after T cell activation. To investigate potential cross-interaction between CD137 (4-1BB) and CD28, we stimulated T cells with anti-CD3 in the presence of A549 lung carcinoma cells expressing CD80/CD86 and 4-1BBL molecules, transduced into the cells using recombinant non-replicating adenoviruses. Following initial T cell proliferation, the proportion of CD28(+) cells in both CD4(+) and CD8(+) populations was rapidly reduced by CD80/86 costimulation, whereas cultures costimulated with just 4-1BBL continued to express CD28. CD28 was also downregulated in cultures costimulated with both CD80/86 and 4-1BBL. Interestingly, in cells costimulated with CD80/86 that had downregulated CD28 expression and ceased to proliferate, reactivation of proliferation by 4-1BBL costimulation also restored their CD28 expression. These findings show a positive effect of CD137 signalling on CD28 expression, similar to the effect of CD28 engagement on 4-1BB expression during the initial phases of T cell activation. Moreover, they point to the importance of signals through 4-1BB for the purposes of ex-vivo T cell activation and expansion.

Heparin/Collagen 3D Scaffold Accelerates Hepatocyte Differentiation of Wharton's Jelly-Derived Mesenchymal Stem Cells.

Both mature and stem cell-derived hepatocytes lost their phenotype and functionality under conventional culture conditions. However, the 3D scaffolds containing the main extracellular matrix constitutions, such as heparin, may provide appropriate microenvironment for hepatocytes to be functional. The current study aimed to investigate the efficacy of the differentiation capability of hepatocytes derived from human Wharton's jelly mesenchymal stem cells (WJ-MSCs) in 3D heparinized scaffold. In this case, the human WJ-MSCs were cultured on the heparinized and non-heparinized 2D collagen gels or within 3D scaffolds in the presence of hepatogenic medium. Immunostaining was performed for anti-alpha fetoprotein, cytokeratin-18 and -19 antibodies. RT-PCR was performed for detection of hepatic nuclear factor-4 (HNF-4), albumin, cytokeratin-18 and -19, glucose-6-phosphatase (G6P), c-met and Cyp2B. The results indicated that hepatogenic media induced the cells to express early liver-specific markers including HNF4, albumin, cytokeratin-18 and 19 in all conditions. The cells cultured on both heparinized culture conditions expressed late liver-specific markers such as G6P and Cyp2B as well. Besides, the hepatocytes differentiated in 3D heparinized scaffolds stored more glycogen that indicated they were more functional. Non-heparinized 2D gel was the superior condition for cholangiocyte differentiation as indicated by higher levels of cytokeratin 19 expression. In conclusion, the heparinized 3D scaffolds provided a microenvironment to mimic Disse space. Therefore, 3D heparinized collagen scaffold can be suggested as a good vehicle for hepatocyte differentiation.

Tumour suppressive effects of WEE1 gene silencing in neuroblastomas.

AIM OF STUDY: WEE1, a member of serine/threonine protein kinase family is the master inhibitor of cyclin-dependent kinase 1 in cell cycle. Over-expression of WEE1 in glioblastomas (GBMs) and some other cancers has been shown. Here, we investigated the expression of WEE1 in 13 brain samples from GBM patients and two GBM cell lines. Further to that, we asked whether if knocking down WEE1 expression in the cell lines change tumor cells' reaction. MATERIALS AND METHODS: All brain tumor samples were collected after confirmed pathological diagnosis. Western blotting was used to screen the expression of WEE1 and a panel of tumor markers. As a model of WEE1 gene silencing with small hairpin RNA (shRNA) technology in GBMs, A172, and U373GM cell lines were transfected with four WEE1 specific shRNAs. The growth characteristics of the cells and the expression of a panel of downstream genes were investigated after gene suppression. RESULTS: All GBMs and both cell lines over-expressed WEE1. Transduction of the cell lines with different shRNAs suppressed WEE1 expression with different extent and pooling of four shRNAs together resulted in additive effect. Suppression of WEE1 not only repressed cellular growth but also changed the profile of gene expression of the cells. Quantitative real-time polymerase chain reaction showed also reduced expression of genes such as hypoxia-inducible factor-1, B-cell lymphoma-2, vascular endothelial growth factor, and p53 with crucial roles in tumor survival and invasiveness. CONCLUSION: These results highlight the key role of WEE1 suppression to combat GBMs. Moreover, it showed beneficial possibilities of WEE1 suppression with different anticancer approaches for neurological malignancies.

The influence of fibroblast growth factor 4 on hepatogenic capacity of Wharton's jelly mesenchymal stromal cells.

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) derived from human umbilical cords could be an appropriate candidate for hepatocyte replacement therapy. Improvement of the efficiency of the cell expression of liver specific genes can be considered in finding new transplantation resources. The present study aimed to differentiate WJ-MSCs toward hepatocyte-like cells on collagen film in the presence of hepatogenic factors, including fibroblast growth factor 4 (FGF4), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). MSCs derived from Wharton's jelly explants were characterized by flow cytometry. Then, the cells were cultured in the presence of hepatogenic media with or without FGF4 on 2D collagen films for 21 days. The expression of liver-specific genes was evaluated by real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. The functional assays were performed by Periodic Acid-Schiff (PAS) staining and Indocyanin Green (ICG) uptake. The cultures pre-exposed to FGF4 expressed higher levels of endodermal markers, such as albumin, compared to the control cultures. Also, cytokeratin 18 expression was significantly increased in FGF4-treated cells. However, the expression level of other liver-specific markers was not influenced by exposure to hepatogenic media with or without FGF4. In conclusion, it was demonstrated that FGF4 could induce the differentiation of WJ-MSCs toward endoderm. Despite the morphological changes and increase in PAS reaction, WJ-MSCs could not differentiate into hepatocytes by hepatogenic media consisting of IGF-1.

Expression Pattern of Alternative Splicing Variants of Human Telomerase Reverse Transcriptase (hTERT) in Cancer Cell Lines Was not Associated with the Origin of the Cells.

Telomerase and systems controlling their activity have been of great attention. There are controversies regarding the role of the alternative splicing forms of the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Therefore, the correlation between telomerase enzyme activity, the abundance of alternatively spliced variants of hTERT and doubling time of a series of cancer cell lines originated from hematopoietic, breast, colorectal, neural, ovarian, lung, kidney, bladder, prostate and head and neck cancers were investigated. Expression levels of four different variants of hTERT (the full length, α-deletion, ß-deletion and α/ß-deletion) were quantitatively measured by real time PCR. Telomerase activity was determined by the telomerase repeat amplification protocol (TRAP) while doubling time of the cells measured by plotting growth curves. Results showed high diversity in the relative proportions of hTERT transcripts while the majority of the cells expressed the full length variant as the main transcript. Telomerase activity could not be detected in all cells. Relative assessment of hTERT expression showed greater expression of the α-deleted variant in the telomerase negative cells (P= 0.04). Those cells possessed the α/ß-deleted variant to a smaller extent when compared to the cells with telomerase activity. Greater association between full length spliced variant and ß-variant expression was observed in cells presenting telomerase activity (P= 0.0007, r= 0.74). High degrees of variation among the studied cells regarding the pattern of hTERT expression were present. In spite that, the regulatory roles of hTERT on telomerase activity is still a potential to be utilized as targets for cancer therapies.

Tumor suppressive effects of WEE1 gene silencing could not enhance immunopotentiation effects of CD80 and 4-1BBL co-stimulation in human T cells.

BACKGROUND: Activation of T cells against tumors by recruiting co-stimulatory molecules has been an attractive approach for cancer immunotherapy. Reports suggested that targeting different genes in tumors might also boost T cell-mediated tumor destruction. AIMS: We investigated whether in vitro WEE1 gene silencing in MDA-MB-468 and MCF7 breast cancer cell lines could enhance immunopotentiating effects of CD80 and 4-1BBL co-stimulation in human T cells. MATERIALS AND METHODS: WEE1 gene was specifically silenced in the cancer cells using shRNA technology. The co-stimulatory molecules were over-expressed on the surface of the cancer cells by recombinant non-replicative adenoviruses. The immune reaction of T cells in the co-culture with tumor cells was studied. IFN-g production was assessed by intracellular staining of T cells. To assess cytotoxic activity of CD8+ T cells, the CD107a mobilization-degranulation assay was performed. Expression of granzyme B, perforin and fasl were examined by real time PCR. RESULTS: T cell dual co-stimulation led to a significant increase in the frequency of IFN-g producing cells and higher percentages of degranulation in CD8+ T cells. It also resulted in higher expression levels of the cytotoxicity-related genes. WEE1 gene silencing in the target cells alone however, could not produce significant immune reactivation in the cultured T cells. Likewise, the immune responses of T cells neither improved nor suppressed when dually co-stimulated PBMCs were exposed to the cancer cells with silenced WEE1. CONCLUSIONS: In spite of antitumor effects of WEE1 silencing, combination of this approach with immune co-stimulation could not boost the reactivity of cultured T cells against the tested breast cancer cells.

Comparison of hepatic nuclear factor-4 expression in two- and three-dimensional culture of Wharton's jelly-derived cells exposed to hepatogenic medium.

The research on Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from the umbilical cord suggests promising therapeutic use for hepatocyte replacement therapy. One of the highly conserved members of the nuclear receptor superfamily in the liver is hepatocyte nuclear factor-4α (HNF4α), involved in hepatocyte differentiation. The objectives of this study were to determine the effects of two- and three-dimensional (2D and 3D) cultures of WJ-MSCs on hepatocyte differentiation. MSCs were isolated from human Wharton's jelly, characterized by flow cytometry, and differentiated toward osteogenic and adipogenic lineage. WJ-MSCs were cultured in 2D collagen films and 3D collagen scaffolds in the presence of hepatogenic media with or without pre-treatment with fibroblast growth factor-4 (FGF4) for 21 days. The expression of HNF4α was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). According to flow cytometry data, the cells isolated from Wharton's jelly were shown to express MSC markers. HNF4α expression analysis revealed that pre-exposing the cells with FGF4 was more effective in hepatocyte differentiation. 3D cultures also improve the expression of HNF4α compared with 2D culture system. In conclusion, the combination of FGF4 and 3D culture improved hepatocyte differentiation. It seems 3D interaction of the cells improved the hepatogenesis.