| Background and objective:Nasopharyngeal carcinoma (NPC) is a malignant tumor arising from the epithelial cells that cover the surface and line of nasopharynx. The etiology of NPC seems to follow a multi-factor process, in which EBV, ethnic background, and environmental carcinogens play an important role respectively. NPC is the most common head and neck tumor in Guangdong, South China, where the incidence peaks at50per100,000, but it is rare in the western world (1per100,000). NPC manifests high malignancy which in most cases invades adjacent regions and metastasizes to regional lymph nodes and distant organs.30to60percent of patients with NPC will eventually develop a distant metastasis. Although NPC tumors are sensitive to radiotherapy and chemotherapy, treatment failure is still high due to local recurrence and distant metastases, which are the key contributors to NPC mortality. However, the underlying cellular and molecular mechanisms of NPC metastasis and recurrence remain poorly understood. Therefore it is worth studying biological characteristics of NPC to facilitate better understanding of its initiation, progression and development.More insightful understanding of cancer stem cells may become a promising breakthrough to target therapeutic methods of special effectiveness benefiting NPC patients.The theory of cancer stem cell (CSC) was proposed recently by scientists, pointing out that cancer stem cells coming from organ stem cells or progenitor cells are both the source of tumorigenesis and the target organ of therapeutics. As a tiny minority of all tumor cells, CSC turns out to be the origin of the initiation, growth, and development of tumor. Even if the majority of tumor cells are killed, CSC repopulates the tumor in a comeback. As one of the crucial causes of tumor recurrence and metastasis, CSC possesses strong tumorigenicity and is able to survive chemotherapy and radiotherapy.In1996, Goodell et al described a side population (SP) of bone marrow-derived cells that were able to efflux Hoechst dye33342and were enriched in hematopoietic stem cells (HSCs). Since then, similar phenomena have been reported in a wide range of normal tissues and some solid tumors. SP cells express high levels of stem-associated genes, possess self-renewing and multipotent differentiation potential, suggesting that they have stem cell behaviors. Though SP cells have been identified in NPC cell line, molecular mechanism relating to metastasis and recurrence still remain elusive.The epithelial-mesenchymal transition (EMT) is a basic physiology and pathology phenomenon, involving in embryogenesis, tissue reconstruction and cancer metastasis. It is characterized as a switch from a polarized epithelial phenotype to a highly motile fibroblast or mesenchymal phenotype. EMT was first described by Garry Greenburg and Elisabeth Hay. During EMT, the links between epithelial cells and those between epithelial cell and extracellular matrix break down, and cells migrate to other locations in the body. EMT is critical to metazoan embryogenesis, chronic inflammation and fibrosis, and has been demonstrated to be a central mechanism in cancer invasiveness and metastasis. It has been reported that EMT generates cells with stem cell-like properties, and that EMT often occurs in cancer stem cells, which suggests that tumor progressions and metastases are sometimes caused by cancer cells that acquire stem cell characteristics. Since EMT plays an important role in tumor cell invasion and distants, thus it has become a hot spot in cancer research.Methods:The NPC CNE-2cells which were in logarithmic growth phase with Hoechst33342were dyed, comparing them with verapamil inhibitor, and then fluorescence positive NSP cells and fluorescence negative SP cells were collected When the purity requirement was met, the following experiments were operated:1) Colony formation assay.the SP and NSP cells were at once incubated in6-well plates at a density of500cells per well in RPMI-1640medium supplemented with10%FBS to do colony formation assay, comparing the difference of their colony formation ability.2) Dynamic observation of their morphological differences in the state of living cells. The fresh-sorted SP and NSP cells were seeded in RPMI-1640(with10%FBS) and UltraMEM (with5%FBS) in culture flasks for contrast. The cell morphologic changes were observed dynamically.3) Matrigel transwell invasion assay in vitro. Matrigel invasion chambers were used to compare the differences in the invasion and metastasis ability of the two cell subsets.4) Detection their expression differences in stem cell-associated genes and mesenchymal cell genes. Total-RNA of SP and NSP were extracted respectively, which were converted to cDNA afterwards. qRT-PCRs were carried out to detect their gene expression differences.Chemo-resistant ability of SP and NSP cells was examined on exposure to5-Fu, cis-diaminedichloroplatinum and adriamycin.5) Western Blot assay to test their expression differences in the epithelial cells cadherin (E-cadherin) and Vimentin. Cells were lysed and protein concentrations were determined. Then their expression differences in E-cadherin and Vimentin by western blotting were detected.6) Immunofluorescence staining assay to verify the above western blot results. Fresh-sorted cells were grown on sterile glass to do the immunofluorescence analysis of the expression of E-cadherin and Vimentin in SP and NSP cells in order to verify the western blotting results.7) Analysis of differences in the cell cycle of SP and NSP cells using flow cytometry. And compare the expression differences in cell-cycle related proteins by western blotting.8) NOD/SCID mice were used to determine the tumorigenicity in two-cell subsets in vivo which was one of the standard criteria for proving the cancer stem cell characterization.Results:A small population of SP cells exists in human NPC cell line CNE-2(16.2%-22.6%). Verapamil can specifically block the transporter function of the ATP-binding cassette (ABC) half transporter member2of G family protein, stop the fluorescent dye from effluxing out of SP cells, and decrease the proportion of SP cells to0.00%. The SP cells in CNE-2cell line share many characterizations with normal stem cells, possess the properties of cancer stem cells, and in addition, have more mesenchymal cell characteristics, such as unlimited cell growth, high colony formation ability (P<0.001), higher invasive potential (P<0.001), high expressing stem-related genes and mesenchymal cell-related genes; lower expression of E-cadherin and higher expression of Vimentin. The morphological changes of these SP cells are close to mesenchymal cells, showing fusiform. Chemo-resistant ability was higher in SP cells than that in NSP cells on exposure to5-Fu, cis-Diaminedi-chloroplatinum and Adriamycin. Cell cycle analysis showed that a larger percentage of SP cells were in phase S than in NSP cells. Cell cycle-related proteins like CDK2and Cycling Dl was upregulated in SP cells, while the cyclin-dependent kinase inhibitors including p21and p27were attenuated in SP cells. The lowest inoculation cell number was10,000for mice to form tumors with SP cells in contrast to NSP cells which could only form tumors with200,000cells in NOD/SCID mice. Tumor formation ability of SP cells is at least20times higher than that of NSP cells. Conclusion:Our results support that SP cells exist in human NPC CNE-2cell line. SP cells isolated from cell line CNE-2possess many stem cell properties and EMT characteristics, including high colony formation efficiency, strong invading ability as well as strong tumor formation ability in vivo in NOS/SCID mice. Furthermore, SP cells not only express stem cell-related genes but also express mesenchymal cell-related genes. Low level of E-cadherin and high level of Vimentin may be one of the mechanisms of NPC invading and metastasis. |
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