Changes in gene expression pattern in hypoxia-induced MCF-7 breast cancer stem cells and the impact of their secretome on angiogenesis related genes in HUVECs

Pharmacy Practice

  • Hana M Hammad1Department of Biological Sciences, School of Science, The University of Jordan, Amman, Jordan.
  • Malek A Zihlif2Department of Pharmacology, School of Medicine, The University of Jordan, Amman, Jordan.
  • Tuqa Abu Thiab1Department of Biological Sciences, School of Science, The University of Jordan, Amman, Jordan.
  • Amer Imraish1Department of Biological Sciences, School of Science, The University of Jordan, Amman, Jordan.

Volume 23 Issue 2 Pages 1-10

DOI: 10.18549/PharmPract.2025.2.3114

Abstract

Background: Hypoxia, a hallmark of solid tumors, results from inadequate oxygen supply as tumors grow, leading to a cascade of cellular adaptations that enhance malignancy and therapeutic resistance. The research primarily investigates the hypoxia-induced gene expression changes in breast CSCs and evaluates the effects of these changes on tumor angiogenesis and metastasis. Objective: To characterize the changes in gene expression of hypoxia and angiogenesis pathways in both hypoxic CSCs and HUVEC cells exposed to the hypoxic CSCs conditioned medium, and to evaluate the effect of secreted factors by CSCs in the media on the angiogenic capability of Human umbilical vein endothelial cells (HUVEC). Methods: Chemo-resistance to doxorubicin was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) cell proliferation assay. Real-time quantitative polymerase chain reaction (qRT-PCR) assay was performed to assess gene expression pattern in hypoxia pathway in CSCs. Furthermore, the study explored the interaction between hypoxic CSCs and human umbilical vein endothelial cells (HUVEC), through assessing the expression of angiogenic pathway genes and Capillary-like tube structure formation assay in HUVECs exposed to hypoxic CSCs-conditioned medium. Results: Flow cytometric identification of CD44+/CD24- cells showed that that the sorted MCF7-CSCs acquired stemness character much higher than their parental cells, and declines over time. Hypoxia significantly increased the resistance of CSCs to doxorubicin, with a maximum of 4.78 folds enhancement after 22 episodes of 8-h hypoxia compared to normal MCF7 in 2D cultures. Gene expression analysis of hypoxic CSCs revealed significant changes in 14 genes of the hypoxic pathway. The expressions of 5 of these genes were significantly up-regulated, while those of 9 genes were significantly down-regulated (p < 0.05). Gene expression analysis of HUVECs treated with conditioned media collected from hypoxic CSCs revealed significant changes in 31 genes of Angiogenic pathway genes. The expressions of 5 of these genes were significantly up-regulated, while those of 26 genes were significantly down-regulated (p < 0.05). Conclusion: These findings underscore the critical impact of hypoxia on breast cancer progression through modulation of CSC characteristics and angiogenic responses. This study paves the way for the development of novel therapeutic approaches that could inhibit tumor progression and overcome resistance to existing treatments.

Keywords

  • Hypoxia
  • Angiogenesis
  • Stem cells
  • MCF-7
  • CSCs
  • HUVEC
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