Epidermal growth factor potentiates in vitro metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel

<p>Abstract</p> <p>Background</p> <p>Although a high level of functional voltage-gated sodium channel (VGSC) expression has been found in strongly metastatic human and rat prostate cancer (PCa) cells, the mechanism(s) responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF), a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion.</p> <p>Results</p> <p>Exogenous EGF enhanced the cells' <it>in vitro </it>metastatic behaviours (migration, endocytosis and invasion). Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa) mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX) suppressed the effect of EGF on all three metastatic cell behaviours studied.</p> <p>Conclusion</p> <p>1) EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2) VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.</p

Workshops for subject-specific teachers’ training: A case study for teaching cancer biology

Teachers’ training in higher education institutions widely serves general purposes. However, recent dialogues and research highlight the importance of teachers’ deep understanding of the material being taught and the ways students think about the content as critical components of great teaching. We explored the novelty of providing a one-day workshop entitled, ‘Effective strategies for teaching cancer biology’. The Biochemical Society supported the event and marketed it throughout the UK – not with any targeted level of university teaching experience and attendees therefore ranged from those who had never taught to those at the level of Senior Fellow of the Higher Education Academy. The day included various short talks, the sharing of good practice and the opportunity to experience a demonstration lesson as a student. Twelve out of thirteen who provided feedback had not received previous subject-specific teacher-training. Half of the attendees gave feedback with the highest score out of five, having found the event ‘very valuable’. This experience suggests that subject-specific training may be beneficial and applicable to other subject areas

In Vitro Investigations of miR-33a Expression in Estrogen Receptor-Targeting Therapies in Breast Cancer Cells

Background: Increased fatty acid synthesis leads to the aggressive phenotype of breast cancer and renders efficiency of therapeutics. Regulatory microRNAs (miRNAs) on lipid biosynthesis pathways as miR-33a have potential to clarify the exact mechanism. (2) Methods: We determined miR-33a expression levels following exposure of MCF-7 and MDA-MB-231 breast cancer cells to estrogen receptor (ER) activator (estradiol-17β, E2) or anti-estrogens (ICI 182,780, Fulvestrant, FUL) at non-cytotoxic concentrations. We related miR-33a expression levels in the cells to cellular lipid biosynthesis-related pathways through immunoblotting. (3) Results: miR-33a mimic treatment led to significantly downregulation of fatty acid synthase (FASN) in MCF-7 cells but not in MDA-MB-231 cells in the presence of estradiol-17β (E2) or Fulvestrant (FUL). In contrast to the miR-33a inhibitor effect, miR-33a mimic co-transfection with E2 or FUL led to diminished AMP-activated protein kinase α (AMPKα) activity in MCF-7 cells. E2 increases FASN levels in MDA-MB-231 cells regardless of miR-33a cellular levels. miR-33a inhibitor co-treatment suppressed E2-mediated AMPKα activity in MDA-MB-231 cells. (4) Conclusions: The cellular expression levels of miR-33a are critical to understanding differential responses which include cellular energy sensors such as AMPKα activation status in breast cancer cells

Wnt-11 promotes neuroendocrine-like differentiation, survival and migration of prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Wnt-11 is a secreted protein that modulates cell growth, differentiation and morphogenesis during development. We previously reported that Wnt-11 expression is elevated in hormone-independent prostate cancer and that the progression of prostate cancer from androgen-dependent to androgen-independent proliferation correlates with a loss of mutual inhibition between Wnt-11- and androgen receptor-dependent signals. However, the prevalence of increased expression of Wnt-11 in patient tumours and the functions of Wnt-11 in prostate cancer cells were not known.</p> <p>Results</p> <p>Wnt-11 protein levels in prostate tumours were determined by immunohistochemical analysis of prostate tumour tissue arrays. Wnt-11 protein was elevated in 77/117 of tumours when compared with 27 benign prostatic hypertrophy specimens and was present in 4/4 bone metastases. In addition, there was a positive correlation between Wnt-11 expression and PSA levels above 10 ng/ml. Androgen-depleted LNCaP prostate cancer cells form neurites and express genes associated with neuroendocrine-like differentiation (NED), a feature of prostate tumours that have a poor prognosis. Since androgen-depletion increases expression of Wnt-11, we examined the role of Wnt-11 in NED. Ectopic expression of Wnt-11 induced expression of NSE and ASCL1, which are markers of NED, and this was prevented by inhibitors of cyclic AMP-dependent protein kinase, consistent with the known role of this kinase in NED. In contrast, Wnt-11 did not induce NSE expression in RWPE-1 cells, which are derived from benign prostate, suggesting that the role of Wnt-11 in NED is specific to prostate cancer. In addition, silencing of Wnt-11 expression in androgen-depleted LNCaP cells prevented NED and resulted in apoptosis. Silencing of Wnt-11 gene expression in androgen-independent PC3 cells also reduced expression of NSE and increased apoptosis. Finally, silencing of Wnt-11 reduced PC3 cell migration and ectopic expression of Wnt-11 promoted LNCaP cell invasion.</p> <p>Conclusions</p> <p>These observations suggest that the increased level of Wnt-11 found in prostate cancer contributes to tumour progression by promoting NED, tumour cell survival and cell migration/invasion, and may provide an opportunity for novel therapy in prostate cancer.</p

Nickel’s Role in Pancreatic Ductal Adenocarcinoma: Potential Involvement of microRNAs

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types with a limited overall survival rate due to the asymptomatic progression of symptoms in metastatic stages of the malignancy and the lack of an early reliable diagnostic biomarker. MicroRNAs (miRs/miRNAs) are small (~18–24 nucleotides), endogenous, non-coding RNAs, which are closely linked to the development of numerous malignancies comprising PDAC. Recent studies have described the role of environmental pollutants such as nickel (Ni) in PDAC, but the mechanisms of Ni-mediated toxicity in cancer are still not completely understood. Specifically, Ni has been found to alter the expression and function of miRs in several malignancies, leading to changes in target gene expression. In this study, we found that levels of Ni were significantly higher in cancerous tissue, thus implicating Ni in pancreatic carcinogenesis. Hence, in vitro studies followed by using both normal and pancreatic tumor cell lines and increasing Ni concentration increased lethality. Comparing LC50 values, Ni-acetate groups demonstrated lower values needed than in NiCl2 groups, suggesting greater Ni-acetate. Panc-10.05 cell line appeared the most sensitive to Ni compounds. Exposure to Ni-acetate resulted in an increased phospho-AKT, and decreased FOXO1 expression in Panc-10.05 cells, while NiCl2 also increased PTEN expression in Panc-10.05 cells. Specifically, following NiCl2 exposure to PDAC cells, the expression levels of miR-221 and miR-155 were significantly upregulated, while the expression levels of miR-126 were significantly decreased. Hence, our study has suggested pilot insights to indicate that the environmental pollutant Ni plays an important role in the progression of PDAC by promoting an association between miRs and Ni exposure during PDAC pathogenesis

The interaction of Wnt-11 and signalling cascades in prostate cancer

Prostate cancer (PCa) is the second most common cancer among the male population. Conventional therapies target androgen signalling, which drives tumour growth; however, they provide limited survival benefits for patients. It is essential, therefore, to develop a more specific biomarker than the current gold standard, PSA testing. The Wnt signalling pathway induces expression of target genes through cell surface receptors. A non-canonical member of this family, Wnt-11, is evolutionarily highly conserved and is normally expressed by various cells in the developing embryo, as well as in the heart, liver and skeletal muscle of adult humans. We comprehensively review several cell signalling pathways to explain how they interact with Wnt-11, demonstrating its use as a potential biomarker for PCa. Several studies have shown that the expression of Wnt-11 is associated with gastric, renal and colorectal adenocarcinomas and PCa. Moreover, Wnt-11 affects extracellular matrix composition and cytoskeletal rearrangement, and it is required for proliferation and/or survival during cell differentiation. It was found that PCa cell lines express high levels of Wnt-11, which allows differentiation of the epithelial prostate tumour cells to neuron-like (NE) cells. The NE cells produce additional factors that can cause regression after treatment. Accumulating evidence shows that Wnt-11 could be a potential biomarker in diagnosing PCa. Many studies have shown both non-canonical and canonical Wnts interact with several signalling cascades such as PKC, JNK, NF-κB, Rho, PKA and PI3K. In particular, evidence demonstrates Wnt-11 is involved in the progression of PCa, thus it could have the potential to become both a specific disease marker and an important therapeutic target

Functional evidence for EGF-induced enhancement of metastatic cell behaviours via VGSC expression/activity in PC-3M cells

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> (A) Migration index (MiI), expressed relative to the control level (Cont), fixed as 100 %. Effects of EGF (50 ng/ml), TTX (500 nM) and EGF+TTX are shown. (B) Dose dependence of the effect of EGF on MiI. ΔMiI denotes the percentage change (increase) in MiI induced by increasing concentrations of EGF, expressed relative to the maximum (fixed as 100 %) seen for 50 ng/ml. (C) Endocytosis index (EI), expressed as percentage of the control level (Cont). Effects of EGF (20 ng/ml), TTX (500 nM) and EGF+TTX are shown. (D) Dose dependence of the effect of EGF on EI. ΔEI denotes the change (increase) in EI induced by given concentrations of EGF. (E) Boyden chamber invasion assay data. Effects of EGF (100 ng/ml), TTX (500 nM), EGF+TTX and AG1478 (100 nM) are shown. Invasion index (InvI) denotes the percentage of cells crossing the membrane in Transwell assays. Each data point or histobar denotes mean ± standard error (n = 4)

Effects of TTX (500 nM) and TTX+EGF on respective relative levels of total and plasma membrane (PM) VGSC protein expression, presented as a percentages of respective controls (Cont)

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> Two concentrations of EGF were used: 50 ng/ml (EGF1) and 100 ng/ml (EGF2). Relative levels of total VGSC were deduced from Western blots (as in Fig. 2). Relative levels of PM expression were obtained from immunocytochemistry/digital analysis(as in Fig. 4). Each histobar denotes mean ± standard error (n = 3–6). Light bars, total VGSC protein. Shaded bars, VGSC protein expressed in plasma membrane (PM)

Confocal microscopy and densitometric analysis of VGSC protein expression in PC-3M cells

<p><b>Copyright information:</b></p><p>Taken from "Epidermal growth factor potentiates metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel"</p><p>http://www.molecular-cancer.com/content/6/1/76</p><p>Molecular Cancer 2007;6():76-76.</p><p>Published online 24 Nov 2007</p><p>PMCID:PMC2211503.</p><p></p> (A). Typical confocal images. (i) Control. (ii) EGF (100 ng/ml). (iii) AG1478 (100 nM). Each treatment was for 24 h. Scale bar, 20 μm (applicable to all panels). (B) Signal density, ie optical density of plasma membrane (PM) VGSC immunocytochemistry, corresponding to images such as (A). Each histobar denotes mean ± standard error (n = 50 cells/3 separate experiments). (C) Effects of EGF (similar treatment as in A) on total and PM VGSC expression. The PM fraction was immunoprecipitated by biotin labelling. Key: 1) Control total VGSC protein. (2) EGF-treated total VGSC protein. (3) Control PM VGSC protein. (4) EGF-treated PM VGSC protein. Note the change in the molecular size of the biotinylated fractions (lanes 3 & 4)