Int J Stem Cells.  2019 Jul;12(2):291-303. 10.15283/ijsc19002.

Enhanced Anti-Cancer Effects of Conditioned Medium from Hypoxic Human Umbilical Cord–Derived Mesenchymal Stem Cells

• Han KH
• Kim AK
• Jeong GJ
• Jeon HR
• Bhang SH
• Kim DI

• KMID: 2465899
• DOI: http://doi.org/10.15283/ijsc19002

Abstract

BACKGROUND AND OBJECTIVES
There have been contradictory reports on the pro-cancer or anti-cancer effects of mesenchymal stem cells. In this study, we investigated whether conditioned medium (CM) from hypoxic human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) (H-CM) showed enhanced anti-cancer effects compared with CM from normoxic hUC-MSCs (N-CM).
METHODS AND RESULTS
Compared with N-CM, H-CM not only strongly reduced cell viability and increased apoptosis of human cervical cancer cells (HeLa cells), but also increased caspase-3/7 activity, decreased mitochondrial membrane potential (MMP), and induced cell cycle arrest. In contrast, cell viability, apoptosis, MMP, and cell cycle of human dermal fibroblast (hDFs) were not significantly changed by either CM whereas caspase-3/7 activity was decreased by H-CM. Protein antibody array showed that activin A, Beta IG-H3, TIMP-2, RET, and IGFBP-3 were upregulated in H-CM compared with N-CM. Intracellular proteins that were upregulated by H-CM in HeLa cells were represented by apoptosis and cell cycle arrest terms of biological processes of Gene Ontology (GO), and by cell cycle of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In hDFs, negative regulation of apoptosis in biological process of GO and PI3K-Akt signaling pathway of KEGG pathways were represented.
CONCLUSIONS
H-CM showed enhanced anti-cancer effects on HeLa cells but did not influence cell viability or apoptosis of hDFs and these different effects were supported by profiling of secretory proteins in both kinds of CM and intracellular signaling of HeLa cells and hDFs.

Keyword

Anti-cancer; Conditioned medium; Fibroblasts; Hypoxia; Mesenchymal stem cells

MeSH Terms

Activins
Anoxia
Apoptosis
Biological Processes
Cell Cycle
Cell Cycle Checkpoints
Cell Survival
Culture Media, Conditioned*
Fibroblasts
Gene Ontology
Genome
HeLa Cells
Humans*
Insulin-Like Growth Factor Binding Protein 3
Membrane Potential, Mitochondrial
Mesenchymal Stromal Cells*
Tissue Inhibitor of Metalloproteinase-2
Uterine Cervical Neoplasms
Activins
Culture Media, Conditioned
Insulin-Like Growth Factor Binding Protein 3
Tissue Inhibitor of Metalloproteinase-2

Figure

• 

  Fig. 1 H-CM induced enhanced anti-cancer effects in HeLa cells. Cell viability of HeLa cells was strongly decreased at 48 hours in the presence of H-CM compared with N-CM or control medium (A). The proportion of live cells [Annexin(−)/PI(−)] was decreased, and that of late apoptotic cells [Annexin(+)/PI(+)] at 48 hours was strongly increased, in the presence of H-CM compared with N-CM or control medium (B, C). Caspase-3/7 activity was strongly increased at 12 and 24 hours in the presence of H-CM compared with N-CM or control medium (D). Mitochondrial membrane potential was decreased at 12 and 24 hours in the presence of H-CM compared with N-CM or control medium (E). Cell cycle was arrested with an increase in G0/G1 and decrease in S and G2/M phases at 24 hours in the presence of H-CM compared with N-CM or control medium (F, G). Results are expressed as mean±S.E.M. from three independent experiments (n=3, *p<0.05, **p<0.01, t-test). C-CM: serum-free medium as control, N-CM: conditioned medium from normoxic hUC-MSCs, H-CM: conditioned medium from hypoxic hUC-MSCs.

• 

  Fig. 2 H-CM did not affect cell viability of hDFs. Cell viability of hDFs at 48 hours was not significantly different in the presence of H-CM, N-CM, or C-CM (A). Apoptosis at 48 hours was not significantly different in the presence of H-CM, N-CM, or C-CM (B, C). Caspase-3/7 activity at 12 and 24 hours was decreased in the presence of H-CM compared with N-CM or C-CM (D). Mitochondrial membrane potential was not changed at 12 and 24 hours in the presence of H-CM compared with N-CM or control medium (E). The proportion of cells in G0/G1 phase of the cell cycle was decreased, and S and G2/M phases were increased, after 24 hours in the presence of H-CM or N-CM compared with control medium (F, G). There was no significant difference in cell cycle distribution only between cells treated with N-CM and H-CM, (F, G). Results are expressed as mean±S.E.M. from three independent experiments (n=3, *p<0.05, **p<0.01, t-test). C-CM, serum-free medium as control; N-CM, conditioned medium from normoxic hUC-MSCs; H-CM, conditioned medium from hypoxic hUC-MSCs.

• 

  Fig. 3 Protein profiling of H-CM compared with N-CM. Proteins that were upregulated (red) and downregulated (green) in H-CM compared with N-CM were analyzed using protein antibody array (>1.5 fold) (A). Biological process of GO analysis for upregulated proteins in H-CM compared with N-CM (B). Biological process of GO analysis for downregulated proteins in H-CM compared with N-CM (C). Molecular function of GO analysis for downregulated proteins in H-CM compared with N-CM (D). GO data were represented by the –log10 p value (p<0.01) and top 10 enriched terms, and single sample analysis was performed.

• 

  Fig. 4 Intracellular signaling induced by H-CM in HeLa cells. Upregulated proteins (red) involved in intracellular signaling of HeLa cells treated with H-CM compared with N-CM according to protein antibody array (>1.5 fold) (A). Biological process of GO analysis for upregulated intracellular proteins in HeLa cells treated with H-CM compared with N-CM (B). KEGG pathway analysis of upregulated intracellular proteins in HeLa cells treated with H-CM compared with N-CM (C). Downregulated proteins (green) involved in intracellular signaling of HeLa cells treated with H-CM compared with N-CM according to protein antibody array (>1.5 fold) (D). Biological process of GO analysis for downregulated intracellular proteins in HeLa cells treated with H-CM compared with N-CM (E). KEGG pathway analysis of downregulated intracellular proteins in HeLa cells treated with H-CM compared with N-CM (F). GO and KEGG pathway results were represented as the –log10 p value (p<0.01) and top 10 enriched terms, and single sample analysis was performed.

• 

  Fig. 5 Intracellular signaling induced by H-CM in hDFs. Upregulated proteins (red) involved in intracellular signaling of hDF cells treated with H-CM compared with N-CM according to protein antibody array (>1.5 fold) (A). Biological process of GO analysis for upregulated intracellular proteins in hDFs treated with H-CM compared with N-CM (B). KEGG pathway analysis of upregulated intracellular proteins in hDFs treated with H-CM compared with N-CM (C). Downregulated proteins (green) involved in intracellular signaling of hDFs treated with H-CM compared with N-CM according to protein antibody array (>1.5 fold) (D). Biological process of GO analysis for downregulated intracellular proteins in hDFs treated with H-CM compared with N-CM (E). KEGG pathway analysis of downregulated intracellular proteins in hDFs treated with H-CM compared with N-CM (F). GO and KEGG pathway results were represented by the –log10 p value (p<0.01) and top 10 enriched terms, and single sample analysis was performed.

Reference

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