Redistribution of β-catenin in response to EGF and lithium signalling in human oesophageal squamous carcinoma cell lines

BACKGROUND: The β-catenin link between membrane-bound cadherins and the actin cytoskeleton regulates cell adhesion and consequently metastasis. Abnormal stabilisation of β-catenin enhances its transcriptional activities. Factors affecting β-catenin's functions are important in understanding metastatic diseases such as oesophageal squamous cell carcinoma (SCC). RESULTS: In human oesophageal SCCs β-catenin localises predominantly to the plasma membrane. The presence of free β-catenin in the cytoplasm/nucleus was low. This indicates that β-catenin's activities are skewed towards cell-cell adhesion in these oesophageal SCCs. Exposure to EGF or Li alone, produced a slight increase in membrane concentrations but only Li induced β-catenin stabilisation in the cytoplasm. In combination, EGF and Li decreased membrane-associated β-catenin, concomitantly increasing cytoplasmic concentrations. Convergence of these signalling pathways appears to induce a β-catenin shift from the membrane into the cytoplasm. CONCLUSION: Therefore, although the adhesive role of β-catenin appears to be intact, exogenous signals increase the stability of free β-catenin thereby reducing cell-cell adhesion in these tumours

Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay

Cell death is important in physiology, and can happen as a result of structural damage, or as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies, and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V/PI apoptosis assay.The National Research Foundation (NRF) Thuthuka program.http://www.plosone.orgdm2022Immunolog

The acidic tumour microenvironment : manipulating the immune response to elicit escape

The success of cancer treatment relies on the composition of the tumour microenvironment which is comprised of tumour cells, blood vessels, stromal cells, immune cells, and extracellular matrix components. Barriers to effective cancer treatment need to be overcome, and the acidic microenvironment of the tumour provides a key target for treatment. This review intends to provide an overview of the effects that low extracellular pH has on components of the tumour microenvironment and how they contribute to immune escape. Further, potential therapeutic targets will be discussed.The National Research Foundation (NRF) Thuthuka program.http://www.elsevier.com/locate/humimmhj2023Immunolog

The effect of acute acid exposure on immunomodulatory protein secretion, cell survival, and cell cycle progression in tumour cell lines

DATA AVAILABILITY : Data will be made available on request.Cancer develops when multiple systems fail to suppress uncontrolled cell proliferation. Breast cancers and oesophageal squamous cell carcinoma (OSCC) are common cancers prone to genetic instability. They typically occur in acidic microenvironments which impacts on cell proliferation, apoptosis, and their influence on surrounding cells to support tumour growth and immune evasion. This study aimed to evaluate the impact of the acidic tumour microenvironment on the production of pro-tumorigenic and immunomodulatory factors in cancer cell lines. Multiple factors that may mediate immune evasion were secreted including IL-6, IL-8, G-CSF, IP-10, GDF-15, Lipocalin-2, sICAM-1, and myoglobin. Others, such as VEGF, FGF, and EGF that are essential for tumour cell survival were also detected. Treatment with moderate acidity did not significantly affect secretion of most proteins, whereas very low pH did. Distinct differences in apoptosis were noted between the cell lines, with WHCO6 being better adapted to survive at moderate acid levels. Conditioned medium from acid-treated cells stimulated increased cell viability and proliferation in WHCO6, but increased cell death in MCF-7. This study highlights the importance of acidic tumour microenvironment in controlling apoptosis, cell proliferation, and immune evasion which may be different at different anatomical sites. Immunomodulatory molecules and growth factors provide therapeutic targets to improve the prognosis of individuals with cancer.The National Research Foundation (NRF) Thuthuka program.https://www.elsevier.com/locate/cytokine2024-12-28hj2023Immunolog

The effect of siRNA-mediated downregulation of LRP expression on the cellular viability of WHCO1, MCF-7 and MDA-MB 231 cells.

<p>The viability of WHCO1, MCF-7 and MDA-MB 231 cells was analysed 72 h post-transfection using an MTT assay. siRNA-LAMR1 treated WHCO1, MCF-7 and MDA-MB 231 as well as esiRNA-RPSA treated MDA-MB 231 cells revealed a significant reduction in cellular viability compared to untreated cells set to 100%. 8mM PCA and siRNA-scr were used as positive and negative controls, respectively. The error bars represent the standard deviation (n = 3) and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is indicated by an asterisk.</p

LRP expression in WHCO1, MDA-MB 231 and MCF-7 cells 72 h post-transfection.

<p>WHCO1 and MCF-7 cells were transfected with siRNA-LAMR1 and MDA-MB 231 cells with siRNA-LAMR1 and esiRNA RPSA. Densitometric analysis of the western blot signals revealed significant (p < 0.05, n = 3) differences in LRP expression inWHCO1, MDA-MB 231 and MCF-7 cells, respectively (compared to non-transfected cells). Error bars represent standard deviation.</p

Western blot analysis of LRP expression in MDA-MB 231 cells 24 h and 48 h post-transfection with siRNA-LAMR1.

<p>No significant decrease in expression of the 37-kDa LRP was observed 24 h and 48 h after transfection when compared to non-transfected cells. The standard deviation is represented by error bars (n = 3).</p

Sequence of control siRNA-RLUC as well as siRNA used for downregulation of LRP/LR.

<p>Sequence of control siRNA-RLUC as well as siRNA used for downregulation of LRP/LR.</p

Changes in nuclear area after siRNA-mediated downregulation of LRP.

<p>72 h post-transfection, MCF-7, MDA-MB 231 and WHCO1 cells were stained with Hoescht 33324 and viewed by immunofluorescence microscopy. The change in nuclear morphology was quantified with ImageJ software and is represented as the average nuclear area. The error bars indicate the standard deviation and a significant difference (* p < 0.05, ** p < 0.01, *** p <0.001) between the untreated control and the treated samples is shown by an asterisk.</p