An optical labeling-based proliferation assay system reveals the paracrine effect of interleukin-6 in breast cancer

Abstract

Proliferation analysis is one of the basic approaches to characterize various cell types. In conventional cell proliferation assays, the same sample cannot be observed over time, nor can a specific group within a heterogeneous population of cells, for example, cancerous cells, be analyzed separately. To overcome these limitations, we established an optical labeling-based proliferation assay system with the Kaede protein, whose fluorescence can be irreversibly photoconverted from green to red by irradiation. After a single non-toxic photoconversion event, the intensity of red fluorescence in each cell is reduced by cell division. From this, we developed a simple method to quantify cell proliferation by monitoring reduction of red fluorescence over time. This study shows that the optical labeling-based proliferation assay is a viable novel method to analyze cell proliferation, and could enhance our understanding of mechanisms regulating cell proliferation machinery. We used this newly established system to analyze the functions of secreted interleukin-6 (IL-6) in cancer cell proliferation, which had not been fully characterized. Reduction in proliferation was observed following IL-6 knockdown. However, after co-culturing with IL-6-expressing cells, the proliferation of Kaede-labeled IL-6-knockdown cells was restored. These data indicate that in basal-like breast cancer cells, IL-6 exhibits a paracrine effect to positively regulate cell proliferation. Our results thus demonstrate that cancer cells can secrete signaling molecules, such as IL-6, to support the proliferation of other cancer cells.

Paraneoplastic neuromyelitis optica spectrum disorders: three new cases and a review of the literature

“…Once in the CNS, AQP4-IgG causes astrocyte cytotoxicity, initially by complement activation and then by a cascade of inflammatory events such as granulocyte infiltration and then macrophage infiltration, then causes further disruption of the blood-brain barrier, oligodendrocyte death, and ultimately neuronal cell death [27]. Moreover, tumor altered immune status by secreting cytokines, such as interleukin 6 [28], leading to patients more susceptible to immune attack.…”

The Biological Effects of Interleukin-6 and Their Clinical Applications in Autoimmune Diseases and Cancers

“…Signifi cant elevation of serum IL-6 is found in breast cancer patients when compared with healthy controls [55,56]. Breast cancer cells may secrete IL-6 to support the proliferation of cancer, and reduction of proliferation of cancer is observed after IL-6 knockdown [57]. In another study about breast cancer, the expression of IL-6 is associated with the expression of estrogen receptor [58].…”

A homeobox protein, NKX6.1, up-regulates interleukin-6 expression for cell growth in basal-like breast cancer cells

“…Our results showed that NKX6.1 controls tumor formation and cell growth. The knockdown system [22,23]. The qRT-PCR analysis showed that NKX6.1 expression was increased as a result of IL-6 knockdown (Fig.…”

“…We searched genes involving cell proliferation based on gene ontology, and obtained candidate genes ( Supplementary Table S4). Among these candidates, this study focused on IL6, the gene encoding the IL-6 protein, because secretion of IL-6 is high in basal-like breast cancer, where it exhibits a paracrine effect to positively regulate cell growth [22,23,28]. We measured IL6 mRNA levels in NKX6.1 knockdown and knockout cells by qRT-PCR.…”

“…Interleukin-6 (IL-6) is a cytokine that plays a role in various biological activities, including cell growth and differentiation. In basal-like breast cancer, the level of IL-6 secretion is high, and the IL-6/Janus kinase (JAK) 2/signal transducer and activator of the transcription 3 (STAT3) pathway is active in regulating cell growth [22,23]. In this pathway, IL-6 binds to IL-6 receptor and activates JAK2 tyrosine kinase.…”