Lab Anim Res.  2015 Dec;31(4):188-197. 10.5625/lar.2015.31.4.188.

Tissue transglutaminase-interleukin-6 axis facilitates peritoneal tumor spreading and metastasis of human ovarian cancer cells

• Oh K
• Moon HG
• Lee DS
• Yoo YB

• KMID: 2312145
• DOI: http://doi.org/10.5625/lar.2015.31.4.188

Abstract

Inflammation has recently been implicated in cancer formation and progression. As tissue transglutaminase (TG2) has been associated with both inflammatory signaling and tumor cell behavior, we propose that TG2 may be an important link inducing interleukin-6 (IL-6)-mediated cancer cell aggressiveness, including cancer stem cell-like characteristics and distant hematogenous metastasis. We evaluated the effect of differential TG2 and IL-6 expression on in vivo distant metastasis of human ovarian cancer cells. IL-6 production in human ovarian cancer cells was dependent on their TG2 expression levels. The size and efficiency of tumor sphere formation were correlated with TG2 expression levels and were dependent on TG2-mediated IL-6 secretion in human ovarian cancer cells. Primary tumor growth and propagation in the peritoneum and distant hematogenous metastasis into the liver and lung were also dependent on TG2 and downstream IL-6 expression levels in human ovarian cancer cells. In this report, we provide evidence that TG2 is an important link in IL-6-mediated tumor cell aggressiveness, and that TG2 and downstream IL-6 could be important mediators of distant hematogenous metastasis of human ovarian cancer cells. Intervention specific to TG2 and/or downstream IL-6 in ovarian cancer cells could provide a promising means to control tumor metastasis.

Keyword

Tissue transglutaminase; interleukin-6; human ovarian cancer; metastasis

MeSH Terms

Axis, Cervical Vertebra*
Humans*
Inflammation
Interleukin-6
Liver
Lung
Neoplasm Metastasis*
Ovarian Neoplasms*
Peritoneum
Interleukin-6

Figure

• 

  Figure 1 TG2 expression levels in cancer cells correlated with IL-6 production. (A) TG2 expression in the two human ovarian cancer cell lines was analyzed by Western blotting. (B) IL-6 levels in culture supernatants of ovarian cancer cells were determined by enzyme-linked immunosorbent assay (ELISA). Cells (1×104) were cultured in a 24-well tissue culture plate, and the supernatants were collected 24, 48, and 72 h after culture. (C) MDAH-2774 human ovarian cancer cells (cont_2774, control empty-vector transfected; shTG2_2774, TG2-knocked-down) were stably transfected with short hairpin RNAs (shRNAs) targeting TG2. The efficiency of knockdown was assessed by Western blotting. (D) IL-6 levels in culture supernatants of control and TG2-knocked-down MDAH-2774 cells were determined by ELISA. Cells (1×104) were cultured in a 24-well tissue culture plate, and the supernatants were collected 24 and 48 h after culture. (E, F) The effect of the TG2 inhibitor cyteamine (CyM) on IL-6 secretion (E) and cell viability (F) in control MDAH-2774 cells was determined by ELISA and the MTT assay, respectively. (G) MDAH-2774 cells were stably transfected with shRNAs targeting IL-6. The efficiency of knockdown was verified by measuring IL-6 levels in the culture supernatants. TG2 expression of control and IL-6-knocked-down MDAH-2774 cells was analyzed by Western blotting. Data represent the mean±standard deviation (SD), based on three independent experiments using samples from triplicate cell cultures.

• 

  Figure 2 TG2-mediated IL-6 allows anchorage-independent survival in cancer cells. (A) Control (cont_2774) and TG2-knocked-down (shTG2_2774) MDAH-2774 cells were cultured in serum-free medium consisting of a 1:1 mixture of Ham's F-12 and DMEM, supplemented with epidermal growth factor, basic fibroblast growth factor, hydrocortisone, B-27 supplement, L-glutamine, and antibiotics. Visible spheres were counted under a microscope on day 8 post-plating. (B) Phase-contrast microscopy of day 8 spheres generated from the MDAH-2774 cell line. (C) Effect of the TG2 inhibitor cyteamine (CyM) on sphere formation. (D, E) Spheres from control (D) and TG2-knocked-down (shTG2_2774) (E) MDAH-2774 cells were generated in the presence of anti-IL-6 blocking antibody (10 mg/mL) or recombinant IL-6 (10 ng/mL), respectively. Visible spheres were counted under a microscope on day 8 post-plating. (F) cIAP2 and Bcl-2 expression was analyzed by Western blotting after culture in sphere medium. Data represent the mean±SD based on three independent experiments using samples from triplicate cell cultures.

• 

  Figure 3 TG2-knocked-down and IL-6-knocked-down ovarian cancer cells revealed reduced tumor growth and distant metastasis. (A-D) NOG mice were injected intraperitoneally with control, TG2-knocked-down, or IL-6-knocked-down MDAH-2774 cells (5×105 cells/each mouse). At 6 weeks after inoculation, the mice were killed and the primary tumor masses in the abdominal cavity and distant metastases in the liver, lung, and other organs were analyzed. (A) Tumor formation in the abdominal cavities, livers, and lungs of mice xenografted with MDAH-2774 cells. (B) Lung sections from mice xenografted with MDAH-2774 cells were obtained and stained with hematoxylin and eosin. (A, B) Data shown are representative of each group. (C) Number of masses in the mesentery, liver, and lung from mice xenografted with MDAH-2774. (D) Comparison of tumor size developed in the mesentery of mice xenografted with MDAH-2774. (C, D) Data are presented as the mean±SD) of 12-14 mice for each group from two independent experiments.

• 

  Figure 4 TG2-knocked-down or IL-6-knocked-down ovarian cancer cells revealed reduced epithelial-to-mesenchymal transition and in vitro migration. (A) Intracellular activity and expression of TG2 in the lung tissues from MDAH-2774 cells and cultivated peritoneal mass. Enzyme activity was estimated by biotinylated pentylamine (BP) incorporation probed with HRP-conjugated streptavidin (SA) in Western blot analysis. (B) Morphology of MDAH-2774 cells and cultivated peritoneal mass. Peritoneal masses from MDAH-2774-bearing mice were removed and digested with collagenase D and DNase I. Cells were suspended in 30% Percoll, layered onto the top of a 70% Percoll gradient, and centrifuged. The top layer, an enriched population of epithelial and fibroblast cells, was retained. Human cells were selected using MACS separation with antihuman MHC class I antibody (anti-HLAI). (C) Expression of TG2, fibronectin, and vimentin in MDAH-2774 cells and peritoneal mass was determined by Western blot analysis. (D) Expression of matrix metalloproteinases in MDAH-2774 cells and peritoneal mass were determined by reverse transcription-polymerase chain reaction. (E) Migration of MDAH-2774 cells into the wound areas created by scratching was induced by IL-6 (10 ng/mL) for 20 h. These results represent one of three independent experiments.

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