Effect of Hypoxia on motility and stemness of Non-Small-Cell Lung Cancer Cells
John C. Livesey: 0000-0001-9010-5970
Conference Title/Conference Publication
University of the Pacific’s Research Day
University of the Pacific
Date of Presentation
INTRODUCTION: Cancer cells, due to rapid cell proliferation, experience hypoxia as the tumor growth exceeds blood supply. In the process of adapting to hypoxia, cancer cells acquire increased potential for migration, invasion and metastasis, which is the leading cause of cancer related death. For the cancer cells to metastasize, they undergo epithelial-to-mesenchymal transition (EMT) by losing epithelial markers such as E-cadherin and acquiring mesenchymal markers such as N cadherin and vimentin. A majority of the tumors contain a subpopulation of cells called cancer stem cells (CSCs) that possess self-renewal properties and may be resistant to chemotherapy and radiotherapy. The population of cancer stem cells is also proposed to be increased under hypoxic conditions.
PURPOSE: Our hypothesis is that hypoxia increases the motility of non-small cell lung cancer (NSCLC) cells by inducing EMT and also increases the frequency of expression of CSC markers in NSCLC cells.
METHODS: Cell viability: Cell proliferation of NCI-H1650 cells propagated under hypoxia (1% Oxygen) was compared against cells propagated under normoxia using MTS reagent. H1650 cells propagated under hypoxia showed a significant increase in cell proliferation till 48th hour of propagation compared to cells propagated under normoxia.
RESULTS: Wound healing assay: The influence of hypoxia on the motility of non-small cell lung cancer (NSCLC) cell lines was measured with a wound healing assay. In this study, the motility of cancer cells that are propagated under hypoxia was compared against the motility of cancer cells that were propagated under normoxia. No significant difference in motility was observed between H1650 cells propagated under hypoxia and normoxia. Phenotype: No substantial difference in phenotype was observed between cells propagated under hypoxia and normoxia. PCR and Western blots: The influence of hypoxia on the expression of EMT and cancer stem cell markers in NSCLC cell lines was also measured. In this study, expression of EMT markers such as E-cadherin, vimentin and N-cadherin and cancer stem cell markers, such as Sox2 were determined using western blotting and RT-PCR. In the H1650 cell line, the expression of E-cadherin and vimentin at the mRNA level was not affected by hypoxia, while N cadherin expression was significantly down regulated and Sox2 expression was significantly upregulated under hypoxia. Hypoxia inducible factor 1α (HIF1α), which is upregulated in a majority of cell lines under hypoxic conditions was not affected by hypoxia in the H1650 cell line. E-cadherin protein expression was not affected by hypoxia, whereas vimentin and Sox2 protein expression was not observed indicating possible altered translation, stability, or post translational modification of the protein.
Significance: These studies show that hypoxia increased cell proliferation but minimally affected mesenchymal transition and stemness in H1650 cell line.
Arikatla, Swetha and Livesey, John C., "Effect of Hypoxia on motility and stemness of Non-Small-Cell Lung Cancer Cells" (2015). School of Pharmacy Faculty Presentations. 372.