Journal of Stem Cells Research Development & Therapy Category: Medical Type: Research Article
Subcellular Localization of CD133 and Interleukin-6 Receptor (IL-6R) in Human Hepatoblastoma Cell-Line (HuH-6 Clone-5)
- Masumi Akita1*, Sachiko Matsumoto2, Noriko Murai2, Kumiko Komatsu2, Keiko Fujita3
- 1 Division Of Morphological Science, Biomedical Research Center, Saitama Medical University, 38 Moroyama, Iruma-gun, Saitama 350-0495, Japan
- 2 Division Of Morphological Science, Biomedical Research Center, Saitama Medical University, Saitama, Japan
- 3 Department Of Anatomy, Saitama Medical University, Saitama, Japan
*Corresponding Author:Masumi Akita
Division Of Morphological Science, Biomedical Research Center, Saitama Medical University, 38 Moroyama, Iruma-gun, Saitama 350-0495, Japan
Received Date: May 09, 2014 Accepted Date: Jun 17, 2014 Published Date: Jul 01, 2014
Recently, we reported that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia . We also reported the co-localization of CD133 with F-actin. Furthermore, an antibody against CD133 decreased cell migration, strongly suggesting that CD133 is involved in tumor cell migration . Co-localization of CD133 and IL-6Rwas reported in the lung cancer stem cells (CSCs) . There is evidence that IL-6/6R signaling acts as a critical factor for the growth and malignancy of cancer cells [20,21]. The aberrant production and increased secretion of IL-6 in cancer patients is profoundly linked to tumor progression and poor prognosis in many cancer types [22-24]. However, the role of IL-6R in cancer stem cells (CSCs) is not well defined.IL-6R expression is up regulated in CSCs [25-27]. Yi et al.  reported that the expression of IL-6R in the lung CSCs was markedly up regulated at both the gene and protein levels. The prominent distribution of IL-6R was confirmed on the cell surface of CSCs, but not on non-CSCs. They also reported the co-localization of CD133 and IL-6R. However, they did not reveal the subcellular localization of CD133 and IL-6R.
In the present study, we detected CD133 and IL-6R in the human hepatoblastoma cell-line (HuH-6 Clone-5). We demonstrated that CD133 and IL-6R were preferentially concentrated in the complex structure comprising filopodia and lamellipodia.
In the late stages of cultivation, CD133 was mainly localized to the plasma membrane. IL-6R was mainly localized at the dividing cells. Some dividing cells expressed co-localization of CD133 and IL-6R. Co-localization of CD133 and IL-6R may play an important role of cell division for stemness.
MATERIALS AND METHODS
The Aclar® plastic films for electron microscopy were sterilized by immersion in 70% ethanol for 20 min. The films were then rinsed twice in PBS and placed on the bottom of plastic culture dishes (35 mm).
Immunohistochemistry for light microscopy
Alexa Fluor 488-Fluoronanogold®-anti-rabbit Fab'(Nanoprobes, Inc., Yaphank, NY), Alexa Fluor 488-conjugated anti-rabbitFab' andAlexa Fluor 555-conjugated goat anti-mouse IgG (Molecular Probes, Inc., Eugene, OR) were also used for secondary antibodies. Diamidino-2-phenylindole (DAPI) staining was used to determine the number of nuclei.
Transmission Electron Microscopy (TEM)
Scanning Electron Microscopy (SEM)
Effect of CD133 antibody
Localization of CD133 and IL-6R by light microscopy
Time-dependent localization of CD133 and IL-6R
Co-localization of CD133 andIL6-R was partially observed. However, the expression of CD133 and IL-6R was heterogeneous (Figure 5).
Culture of late stages (7-day-old<)
CD133 was mainly localized to the plasma membrane. IL-6R was mainly localized at the dividing cells (Figure 6). Unequal distribution of CD133 was observed in some cells (Figure 7).
Effect of CD133 antibody
In the present study, we also demonstrated that the expression of CD133 and IL-6R was heterogeneous in different stages. In the late stages of cultivation, CD133 and IL-6R exhibited different patterns of subcellular localization. CD133 was mainly localized to the plasma membrane. Koyama-Nasu et al.  have shown that CD133 interacts with plakoglobin (also known as c-catenin), a desmosomal linker protein. They further demonstrate that knockdown of CD133 by RNA interference (RNAi) results in the down regulation of desmoglein-2, a desmosomal cadherin, and abrogate cell-cell adhesion and tumorigenicity of clear cell carcinoma of the ovary stem cells. In the present study, we confirmed the diminishing cell adhesion by decreasing desmosomal structure, after treatment with CD133 antibody. Present result obtained from CD133 antibody treatment also suggests that CD133-mediated regulation may be required for cell survival.
Co-localization of CD133 and IL-6R was demonstrated in the dividing cells. Some cells indicated an unequal distribution of CD133. Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials . Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates . Asymmetric cell division is a proposed mechanism to maintain CSCs. Lathia et al.  utilized glioma stem cells (GSCs) for two cell division modes. Through a symmetric cell division, two GSCs were generated. The other mode is to generate one GSC and one non-GSC through an asymmetric cell division. This division mode would contribute to increased cellular heterogeneity of the tumor, while still maintaining a GSC pool . Co-localization of CD133 and IL-6R may play an important role of cell division. Further studies are required to understand the relationship between symmetric cell division and CSCs.
IL-6R has been targeted for cancer treatments, resulting in cell growth inhibition or reduced angiogenesis [32-34]. Wang et al.  showed that knockdown of IL-6R diminished stemness and survival of glioma stem cells. Present study suggests that CD133 and IL-6R are targeted for cancer treatments, resulting in inhibition of cell proliferation and increasing apoptosis.
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Citation:Akita M, Matsumoto S, Murai N, Komatsu K, Fujita K (2014) Subcellular Localization of CD133 and Interleukin-6 Receptor (IL-6R) in Human Hepatoblastoma Cell-Line (HuH-6 Clone-5). J Stem Cell Res Dev Ther, 1: 001.
Copyright: © 2014 Masumi Akita, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.