Tumour suppressive effects of WEE1 gene silencing in breast cancer cells.

Background: WEE1 is a G2/M checkpoint regulator protein. Various studies have indicated that WEE1 could be a good target for cancer therapy. The main aim of this study was to asssess the tumor suppressive potential of WEE1 silencing in two different breast cancer cell lines, MCF7 which carries the wild-type p53 and MDA-MB468 which contains a mutant type. Materials and Methods: After WEE1 knockdown with specific shRNAs downstream effects on cell viability and cell cycle progression were determined using MTT and flow cytometry analyses, respectively. Real-time PCR and Western blotting were conducted to assess the effect of WEE1 inhibition on the expression of apoptotic (p53) and anti-apoptotic (Bcl2) factors and also a growth marker (VEGF). Results: The results showed that WEE1 inhibition could cause a significant decrease in the viability of both MCF7 and MDA-MB-468 breast cancer cell lines by more than 50%. Interestingly, DNA content assays showed a significant increase in apoptotic cells following WEE1 silencing. WEE1 inhibition also induced upregulation of the apoptotic marker, p53, in breast cancer cells. A significant decrease in the expression of VEGF and Bcl-2 was observed following WEE1 inhibition in both cell lines. Conclusions: In concordance with previous studies, our data showed that WEE1 inhibition could induce G2 arrest abrogation and consequent cell death in breast cancer cells. Moreover, in this study, the observed interactions between the pro- and anti-apoptotic proteins and decrease in the angiogenesis marker expression confirm the susceptibility to apoptosis and validate the tumor suppressive effect of WEE1 inhibition in breast cancer cells. Interestingly, the levels of the sensitivity to WEE1 silencing in breast cancer cells, MCF7 and MDA-MB468, seem to be in concordance with the level of p53 expression

Changes in lymphocytes′ telomerase activity by 4-1BB costimulation

Aim: Lymphocytes are exceptional among somatic cells as these cells can induce telomerase enzyme after antigen stimulation to compensate chromosomal loss during rapid cell division. Activation of telomerase in lymphocytes needs CD28 signal, which simultaneously costimulates T cells during activation. 4-1BB of tumor necrosis factor superfamily also has been shown to costimulate T cells. Herein, we investigated changes in telomerase activity of lymphocytes during longitudinal cultures when T cells costimulated by CD80 or 4-1BB ligand or both molecules in conjunction with anti-CD3 stimulation. Materials and Methods: Artificial antigen presenting cells (aAPCs) were produced by transduction of CD80, 4-1BB ligand or green fluorescent protein genes into A549 carcinoma cells using recombinant adenoviral vectors. Peripheral blood mononuclear cells were stimulated with anti-CD3 and co-cultured with the aAPCs. Cellular growth, expression of telomerase and production of interferon gamma (IFN-γ) were assessed at different time points and followed up to day 35. Results: 4-1BBL provided effective costimulation for lymphocytes′ activation, cytokine production or long-lasting growth where its effects exceeded over CD80 after the first week. 4-1BBL also promoted the telomerase activity and more importantly was able to re-induce the enzyme in the cells that stopped to grow with CD80 costimulation. Although combination of CD80 and 4-1BBL has additional effect on cellular growth or initial telomerase activity, it could not support telomerase activity in later time points. Conclusion: Our results underscored unique features of 4-1BB over CD28 for prolonged support of lymphocytes′ costimulation, which can be recruited for in vitro or ex vivo propagation of T cells for cancer immunotherapy purposes

IL-23 and IL-27 gene expression in three breast cancer cell lines and metastatic and non-metastatic lymph nodes in breast cancer patients

Introduction: There is a great need to identify prognostic and diagnostic cancer bio-markers which can be applied for vaccine and immunotherapy development. The aim of this study was to determine gene expression of IL-23 and IL-27 in two groups: lymph nodes of breast cancer patients and three cell lines. Materials and Methods: IL-23 and IL-27 mRNA transcript were investigated in 10 lymph node samples (consisting of 5 metastasis and 5 non metastasis ones) and three cell lines (including of SKBR3, MDA-MB-468 and MCF7) by quantitative real time PCR (Q-PCR) procedure using designed primers, Master Mix reaction containing SYBER green and β actin housekeeping gene. Results: Data showed no significant differences in IL-23 and IL-27 mRNA gene expressions among metastatic lymph node and non-metastatatic ones. Also, we did not find any significant differences in IL-23 and IL-27 mRNA expressions in cancer cell lines. Conclusions: Our findings indicate that breast cancer microenvironment has no effect on inflammation via either IL-23 gene expression or Il-27

Increased IL-17 and IL-6 Transcripts in Peripheral Blood Mononuclear Cells: Implication for a Robust Proinflammatory Response in Early Stages of Breast Cancer

Background: Several recent studies demonstrated that transforming growth factor beta (TGF-β), by stimulating T regulatory cells, and interleukins 6 and 17 (IL-6, IL-17), by inducing inflammatory reactions, may be critical factors in cancer pathogenesis. Methods: We used quantitative real-time polymerase chain reaction assays to quantify the expression of IL-17, IL-6 and TGF-β mRNA in peripheral blood mononuclear cells and lymphocytes from draining lymph nodes of 60 women with breast cancer. The results were compared according to the patients’ clinical or pathological status. Results: Higher amounts of IL-17 and IL-6 mRNA, but not TGF-β transcripts, were found in patients compared to controls. There were no significant differences between patients with negative or positive nodes or with different histological grades or stages of disease. Conclusion: Most women in this analysis had stage I or II disease. We thus conclude that IL-17, a prominent proinflammatory cytokine, may play an important role in recruiting and infiltrating antitumor immune responses in early stages of breast cancer

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

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