Elsevier

Cytokine

Volume 59, Issue 2, August 2012, Pages 228-236
Cytokine

Interleukin-6 signaling regulates anchorage-independent growth, proliferation, adhesion and invasion in human ovarian cancer cells

https://doi.org/10.1016/j.cyto.2012.04.020Get rights and content

Abstract

It has been widely reported that Interleukin-6 (IL-6) is overexpressed in the serum and ascites of ovarian cancer (OVCA) patients, and elevated IL-6 level correlates with poor prognosis and survival. However, the exact role that IL-6 plays in this malignancy or whether IL-6 can regulate tumorigenic properties has not been established. Here we demonstrate that overexpression of IL-6 in non-IL-6-expressing A2780 cells (by transfecting with plasmid encoding for sense IL-6) increases anchorage-independent growth, proliferation, adhesion and invasion, while depletion of endogenous IL-6 expression in IL-6-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-6) decreases. Further investigation indicates that IL-6 promotes OVCA cell proliferation by altering cell cycle distribution rather than inhibiting apoptosis and that IL-6-enhanced OVCA cell invasive may be associated with increased matrix metalloproteinase (MMP)-9 but not MMP-2 proteolytic activity. In addition, overexpressing or deleting of IL-6 in OVCA cells enhances or reduces its receptor (IL-6Rα and gp130) expression and basal phosphorylation levels of both ERK and Akt, and additional treatment with specific inhibitor of the ERK or Akt signaling pathway significantly inhibits the proliferation of IL-6-overexpressing A2780 cells. Our data suggest that the autocrine production of IL-6 by OVCA cells regulates tumorigenic properties of these cells by inducing IL-6 signaling pathways. Thus, regulation of IL-6 expression or its related signaling pathway may be a promising strategy for controlling the progression of OVCA.

Highlights

► We find that IL-6 secreted by OVCA cells promotes malignant behavior of these cells. ► IL-6 promotes OVCA cell proliferation by altering cell cycle distribution. ► IL-6 stimulates cell proliferation via activation of Raf/MEK/ERK and PI3K/Akt. ► IL-6-enhanced cell invasive correlates with increased MMP-9 but not MMP-2 activity. ► Regulation of IL-6 may be a promising strategy for controlling OVCA progression.

Introduction

Ovarian cancer (OVCA) is the second most common and the most deadly malignancy of the female reproductive tract [1]. Etiological factors involved in ovarian carcinogenesis remain poorly defined, and effective treatment protocols are limited. The currently most frequently used therapy for the treatment of OVCA is a combination of carboplatin and paclitaxel. Although up to 80% of patients initially respond well to therapy, the majority of patients suffer recurrent disease and often die as a result of metastatic disease [2]. New therapeutic agents are needed to improve survival rates and to eventually cure patients of this deadly disease.

Interleukin-6 (IL-6), a cytokine known as mediator of immunological and inflammatory events, has been shown to have a role in the development and progression of several types of tumors [3], [4]. In particular, clinical observations have documented increased IL-6 levels in plasma from patients with therapy-resistant metastatic disease compared to patients with earlier stages of the disease and healthy individuals [5], [6], [7]. It has been shown that IL-6 was elevated in the serum and peritoneal fluid from patients with OVCA [8], [9], [10], [11], [12], [13], [14], and high levels of IL-6 in body fluids were associated with poor prognosis and survival [10], [11], [12], [13], [14] but there is no published information as to the source of this IL-6 in tumor biopsies from patients with OVCA. Recently, Coward et al. studied IL-6 and its transmembrane receptors expression in OVCA biopsies by automated immunohistochemistry on tissue microarrays from 221 OVCA patients and confirmed that intensity of IL-6 staining in malignant cells significantly associated with poor prognosis [15]. Previous studies suggested a pathogenic role of this cytokine in the malignant transformation and progression of OVCA [16], [17], [18], [19], [20]. Our recent study has demonstrated that IL-6 secreted by OVCA cells may contribute to the refractoriness of these cells to conventional chemotherapy through down-regulation of proteolytic activation of caspase-3 [21]. However, the exact role that IL-6 plays in this malignancy or whether IL-6 can regulate tumorigenic properties has not been established.

The aim of the present study was to examine whether overexpression or deletion of IL-6 in OVCA cells affects the tumorigenic properties, including in vitro anchorage-independent growth, proliferation, adhesion and invasion. Furthermore, we also explored the mechanisms by which autocrine IL-6 involved in the regulation of the tumorigenic potential of OVCA cells.

Section snippets

Cell transfection and generation of stable cell lines

IL-6 receptor-bearing OVCA cell lines, A2780 (non-IL-6-expressing) and SKOV-3 (IL-6-overexpressing) were cultured as described previously [21]. A2780 and SKOV-3 cells were transfected with pcDNA3.1(+)-ssIL-6 (i.e., sense IL-6 vector) and pcDNA3.1(+)-asIL-6 (i.e., antisense IL-6 vector) by Lipofectamine™ 2000 (Invitrogen, San Diego, CA), respectively. Three A2780/ssIL-6 stable cell lines that produced low (A2780/ssIL-6L), middle (A2780/ssIL-6M) and high (A2780/ssIL-6H) levels of IL-6 and two

IL-6 increases transformed cell behavior of human OVCA cells

To ascertain the biological effects of IL-6 expression on transformed cell behavior, we began by assessing the extent to which overexpression or deletion of IL-6 in OVCA cells modulated anchorage-independent growth in soft agar. As shown in Fig. 1A to E, a significant increase in the number of colonies formed by sense IL-6-expressing A2780 cells compared with the control vector-transfected or parental cells, which had no difference. Whereas a significant decrease in the number of colonies

Discussion

Several studies have addressed the role of IL-6 in tumor cell growth, but its exact role remains varied and unclear. It appears that the effect of IL-6 on tumor cell growth may depend on the tumor cell type. In this study, we observe that overexpression of IL-6 in non-IL-6-expressing A2780 cells by transfecting with plasmid encoding for sense IL-6 increases anchorage-independent growth, proliferation, adhesion and invasion, while depletion of endogenous IL-6 expression in IL-6-overexpressing

Acknowledgments

We thank Dr. Allen C. Gao (Roswell Park Cancer Institute, Buffalo, NY) for providing pcDNA3.1(+)-ssIL-6 and pcDNA3.1(+)-asIL-6 plasmids. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81041071, 30901985), Tianjin Municipal Science and Technology Commission (Nos. 12JCZDJC26300, 10JCZDJC21100) and Key Program for Science and Technology in Logistics College of Chinese People’s Armed Police Forces (Nos. WHZ201202, WYM201105, WYQ201105).

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