CoCl₂, a Mimic of Hypoxia, Induces Formation of Polyploid Giant Cells with Stem Characteristics in Colon Cancer

<div><p>The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs) contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl₂ induced the formation of cells with larger cell and nuclear size (PGCCs), while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl₂ treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl₂ treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47) and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs) characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.</p></div

HIF-1α, p53, and cyclin D1 expression in colon cancer cells treated with CoCl₂.

<p>A) HCT-116 and Caco-2 cells were treated with the indicated doses of CoCl₂ for 6 hours and then the expression of HIF-1α, p53 and cyclin D1 proteins was evaluated using specific antibodies. B) The corresponding densitometric analyses of the protein bands detected in the immunoblots and normalized to the signal of β-actin are also shown. Data are means ± SD of three independent experiments. (*p<0.05, compared with the control).</p

Colon cancer cells enriched in PGCCs show chemoresistance.

<p>Both HCT-116 and Caco-2 cells were preincubated for 48 hours in the presence or absence of CoCl₂ for PGCC enrichment and then they were exposed for 72 h to different doses of 5-fluorouracil (A) or oxaliplatin (B). Cell proliferation is shown as percentage of control cells unexposed to chemotherapeutic drugs. The data represent the mean ± SD of three independent experiments (* p<0.05, compared to cells without CoCl₂ treatment).</p

Alteration of cell cycle in colon cancer exposed to CoCl₂.

<p>Both HCT-116 (A) and Caco-2 (B) cells were treated with the indicated doses of CoCl₂ for 6 or 48 hours. Next, a cell cycle analysis was performed by flow cytometry. The bar graphs on the right show the relative changes to control cells in the percentage of cells in different phases of the cell cycle. Data represent the mean ± SD of three independent cultures.</p

Effect of p53 inhibition on hypoxia-induced cell cycle alterations and the formation of PGCCs in colon cancer cells.

<p>HCT-116 cells were treated with 300 µM CoCl₂ in the presence or in the absence of 10 µM pifithrin-α (PFT-α), which is a specific inhibitor of the transcriptional activity of p53. Next, a cell cycle analysis was performed by flow cytometry.(A) Data represent the mean ± SD of three independent cultures. (*p<0.05, compared with cells exposed to CoCl₂ in the absence of PFT-α), and (B) changes in cell morphology were examined using visible microscopy. Scale bars correspond to 50 microns.</p

Confirmation of induction of cell polyploidy in colon cancer cells after treatment with CoCl₂.

<p>HCT-116 cells were cultured in the presence of 300 µM CoCl₂ and then a FISH analysis using probes for chromosomes X (green), 18 (blue) and 21 (red) was performed.</p

Increase in size of secondary colonospheres formed by colon cancer cells enriched in PGCCs.

<p>The secondary colonosphere formation assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099143#pone-0099143-g006" target="_blank">Figure 6</a> legend and under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099143#s2" target="_blank">Materials and Methods</a>. (Final magnification, ×200). Scale bar corresponds to 10 microns.</p

Mechanism of action of procaine.

<p>Changes in mRNA expression of GSK3β from undifferentiated mesenchymal stem cells (UC), MSC differentiated into osteo/odontoblast (OB), MSC differentiated into osteo/odontoblast plus procaine (1μM) (OB+Proc), MSC differentiated into osteo/odontoblast plus lithium chloride (10 mM) (OB+LiCl) and the combination of osteo/odontogenic stimuli, Procaine (1μM) and Lithium Chloride (10 mM) were analyzed A). Effect of increasing concentrations of Procaine (0, 0.5, 1 and 2 μM) on GSK3βexpression B) and phospho-β-catenin C) for 7 days on MSC. * p<0.05 vs UC; # p<0.05 vs OB; + p<0.01 vs OB+LiCl; ••p<0.01 vs. 0.5 μM Proc.</p

Procaine prevents osteo/odontogenic differentiation of mesenchymal stem cells (MSC).

<p>Changes in the mRNA expression of A) early markers of oste/odontogenesis and B) specific genes of mesenchymal stem cells (MSC) were determined by RT-PCR after osteo/odontogenic differentiation (OB), OB with procaine (1 μM) (OB+Proc) during 21 days. The size and intensity of amplicon was electrophoresed on agarose gel (2%). Ribosomal 18S expression was used as housekeeping. C) Runx2 transcription factor activity was determined by a commercial TransAM^™ assay. Only OB cells showed to be significantly positive for this transcription factor (*p<0.05 vs all groups). D) Western blot for cytoplasmatic protein smooth muscle-22 alpha after 21 days of osteo/odontogenic differentiation. Images are representative of three cultures.</p