Enhanced Anti-cancer Effects of Conditioned Medium From Hypoxic Cultured Human Adult Dermal Fibroblasts

This study investigates the anti-cancer effects of cervical cancer (HeLa) cells in a conditioned medium (CM) obtained from normoxic and hypoxic cultured human adult dermal broblasts (HDFs). The HeLa cells showed decreased cell viability, arrested cell cycles, and increased apoptosis in CM from hypoxic cultured HDFs (H-CM) compared with normoxic cultured HDFs (N-CM). In up-regulated (> 2-fold) proteins of H-CM compared with N-CM, the top enriched term of biological process of gene ontology (GO) was GO:0006955~immune response. In intracellular down-regulated (> 2-fold) proteins of HeLa cells treated with H-CM compared with N-CM, the top enriched term of biological process of GO was GO:0016579~protein deubiquitination, and the terms of the KEGG pathway were determined to be hsa05166:HTLV-I infection, hsa03410:base-excision repair, and hsa05340:primary immunodeciency. Among down-regulated hub proteins with ≥ 5 edges (ESR1 MCL1, TBP, CD19, LCK, PCNA, CHEK1, and POLA1) of HeLa cells treated with H-CM, the top enriched term of biological process of GO and the KEGG pathway were GO:0006272~leading strand elongation and hsa05166:HTLV-I infection. H-CM displayed not only enhanced anti-cancer effects on HeLa cells compared with N-CM, but also induced intracellular signaling patterns with 9 hub proteins.


Introduction
Hypoxic (low oxygen) conditions reportedly induce cancer proliferation or metastasis 1,2 or cause severe injury in a variety of diseases [3][4][5][6] . Bene cial effects of hypoxia have also been reported, in the form of enhanced wound healing 7 , angiogenesis 8 , anti-aging 9 , and anti-cancers effects 10 , depending on the kind of cells.
In the case of broblasts, hypoxia plays an important role in construction and repair of organs and tissues by secretory factors and the extracellular matrix (ECM) reorganization 11 , and in cancer initiation, progression, metastasis through direct interaction signaling 12 . When broblasts were exposed to hypoxic conditions, contradictory results have been reported. Hypoxic broblasts exhibited increased cell viability and proliferation 13 and stimulated invasive activity of cancer cells 14 , but proliferation of severely hypoxic broblast was regulated 15 , and broblast-mediated cancer stiffness and metastasis were impaired 16 .
In a previous study, epithelial cells 17 , mesenchymal stem cells 18 , embryonic stem cells 19 and immune cells 20 all suppressed cancer cells. However, the anti-cancer effects of hypoxic broblasts have not yet been studied. In this study, we investigated whether a conditioned medium (CM) from hypoxic human adult dermal broblast (HDFs) (H-CM), compared with normoxic CM (N-CM) enhanced anti-cancer effects on cervical cancer (HeLa) cells. We also pro led secretory proteins in H-CM and determined the intracellular signaling pattern in HeLa cells induced by H-CM, compared with N-CM, using protein antibody array analysis.

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Enhanced reduction in cell viability of HeLa cells with H-CM. The ratio of proliferation cell viability between normoxic and hypoxic HDFs at passage 6 did not change (Fig. 1A, B). Viability of HeLa cells was signi cantly reduced by H-CM treatment compared with C-CM or N-CM at 48 to 72 h (Fig. 1C). In contrast, viability of HUVECs was signi cantly increased by H-CM compared with C-CM or N-CM at 72 h (Fig. 1D).
In the case of HUC-MSCs, viability was increased by both N-CM and H-CM compared with C-CM at 48 h,  but increased only by N-CM compared with C-CM at 72 h, with no statistically signi cant different evident between N-CM and H-CM (Fig. 1E).  Table 1). To categorize up-and down-regulated proteins in H-CM, a GO analysis using DAVID was performed (p < 0.01), and the data were described as the -log 10 p value.
In up-regulated proteins, the highest enriched term of biological process of GO was GO:0006955 ~ immune response (8.062). In the next, GO:0006954 ~ in ammatory response (6. Table 4). These proteins were categorized by GO analysis and the KEGG pathway using DAVID, and the data were described as the -log 10 p value. In up-regulated proteins, the biological process of GO and the KEGG pathway were not determined. In the GO analysis (p < 0.01) of down-regulated proteins, the top enriched term of biological process was GO:0016579 ~ protein deubiquitination (2.448), and the next enriched terms were GO:0042981 ~ regulation of apoptotic process  Table 5). In the case of the KEGG pathway (p < 0.01) of down-regulated proteins, hsa05166:HTLV-I infection (2.422), hsa03410:base-excision repair (2.078), and hsa05340:primary immunode ciency were determined (2.053). (Fig. 5C, Supplemental Table 5).
PPI network and hub protein selection. To identify PPI and select hub proteins in HeLa cells treated with H-CM compared with N-CM, the STRING database and Cytoscape software were used. A total of 47 nodes (proteins) and 74 edges (protein interaction lines) were determined in PPI of up-and down-regulated intracellular proteins in HeLa cells treated with H-CM compared with cells treated with N-CM (Fig. 5D, Table 1). When the interacting proteins had more than 5 edges in PPI, they were de ned as hub proteins. Based on this de nition, TNF (3.002 + fold), ESR1 (2.142-fold), MCL1 (2.035-fold), TBP (2.355-fold), CD19 (2.257-fold), LCK (2.030-fold), PCNA (2.172-fold), CHEK1 (2.205-fold), and POLA1 (2.022-fold) were determined to be hub proteins (Fig. 5E, Table 1). GO and the KEGG pathway were applied to these hub proteins to determine the signal pathway patterns, and the data are described as the -log 10 p value.

Discussion
We demonstrated that H-CM treatment resulted in enhanced anti-cancer effects in HeLa cells, pro led secretory proteins in H-CM compared with N-CM, and determined intracellular signaling patterns and hub proteins related to the enhanced anti-cancer effects.
In a previous study, hypoxic culture conditions (1% or 5% O 2 ) increased cell viability and proliferation of human pulmonary broblasts 13  Our study makes it clear that CM from hypoxic HDFs not only enhances anti-cancer effects but also induces anti-cancer-related intracellular signaling patterns and hub proteins related with these effects in HeLa cells. It also suggests that hypoxic culture conditions for HDFs offer a useful alternative approach to developing effective anti-cancer therapies.
Human umbilical vein endothelial cells (HUVECs) (Lonza, Warkerville, MD, USA) were cultured in endothelial cell growth medium-2 (PromoCell GmbH, Heidelberg, Germany). An oxygen level of 21% as a normoxic condition and 1% O 2 as a hypoxic condition were applied to the culture condition of HDFs from passage 4 to passage 6 10 . When cell con uency of all cells reached 90%, the cells were passaged using 0. Analysis of intracellular signaling pathways by protein antibody array. HeLa cells (2 × 10 5 ) cells were cultured in 100-mm culture plates with complete medium. When cell con uency reached 90%, the culture medium was removed and 1× PBS was added to wash the cells. After removing the PBS, 10 mL of N-CM and H-CM was applied to the cells for 24 h. The cells were then harvested and intracellular proteins analyzed with a Signaling Explorer antibody array (Full Moon BioSystems, Sunnyvale, CA, USA) by Ebiogen (Kyung Hee Business Center, Kyung Hee University, Seoul, Korea) 10 . The data were analyzed using Genowiz 4.0), and up-and down-regulated proteins were described using UniProt DB. GO and the KEGG pathways of up-and down-regulated proteins were analyzed using DAVID (p < 0.01) 10 . Protein-protein interaction (PPI) was analyzed using the STRING database (string-db.org) and Cytoscape software (www.cytoscape.org). The number of nodes (protein) and edge (protein interaction line) were analyzed, and nodes with more than 5 edges in PPI were de ned as hub proteins.
Statistical analysis. All experimental data were analyzed by the t test. A p value < 0.05 was considered statistically signi cant. All analyses were carried out using GraphPad Prism version 6.01 (San Diego, CA, USA).