RESUMO
Tumor recurrence and metastasis of nasopharyngeal cancer (NPC) often result in the failure of treatment due to chemoradioresistance. Cancer stem cells (CSCs) have been observed to drive tumor initiation and tumor chemoradioresistance. Therefore, the poor prognosis of advanced NPC is likely to result from the failure to kill CSCs. Sphere formation may be used as an experimental method to enrich potential CSC subpopulations. At present, there are few reports on NPC tumorspheres. The present study focused on examining the cancer stem-like properties of NPC tumorspheres from NPC cell lines. Western blot analysis revealed that NPC tumorspheres had a higher expression of stem cell markers Nanog homeobox and SRY-box 2, compared with parental cells. It was additionally verified that NPC tumorspheres contained a high aldehyde dehydrogenase (ALDH) enzymatic activity compared with parental cells. ALDH+ cells were amplified by 9- to 10-fold in tumorspheres, compared with parental cells (1.8 vs. 16.9%). The tumorsphere cells exhibited an increased half maximal inhibitory concentration value of >10-fold with cisplatin compared with the control parental cells. Compared with the parental cells, the percentage of side population cells in the tumorsphere cell population increased significantly (10.3 vs. 2.3%; P<0.05). NPC tumorsphere cells demonstrated enhanced resistance to radiation. Further investigation verified that salinomycin inhibited NPC CSCs by selectively targeting its stem cells. Altogether, the data revealed that NPC tumorspheres contain cancer stem-like populations with increased chemoradioresistance. It was suggested that the serum-free culture of NPC cells may provide an appropriate model for researching the sensitivity of CSCs to therapeutic agents. It was additionally revealed that salinomycin is an efficient inhibitor of NPC CSCs, supporting the hypothesis that salinomycin may eliminate CSCs and imply a need for further clinical evaluation.
RESUMO
Radiotherapy is the primary and most important treatment for nasopharyngeal carcinoma (NPC). Cancer stem-like cells (CSCs) have been shown to be resistant to radiation. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene has been suggested to play a role in stem cell self-renewal. In the present study, we sorted PTEN-/+ cells using a flow cytometer. The clone formation assay showed that PTEN- cells were more radioresistant than PTEN+ NPC cells. We found that PTEN- cells demonstrated a significant increase in tumorsphere formation and CSCs markers compared with PTEN+ cells. Silencing the expression of PTEN with siRNA resulted in increased expression of p-AKT, active ß-catenin and Nanog. siPTEN cells irradiated showed more radioresistant and DNA damage than parental cells. We also confirmed that down-regulation of ß-catenin expression with shRNA resulted in a reduced percentage of side population cells and expression of Nanog. shß-catenin cells significantly decreased survivin expression at 4 Gy irradiation in PTEN- cells compared with PTEN+ cells. In siPTEN cells, ß-catenin staining shifted from the cytoplasmic membrane to the nucleus. Furthermore, immunofluorescence showed that following irradiation of PTEN- cells, at 4 Gy, active ß-catenin was mainly found in the nucleus. Immunohistochemistry analysis also demonstrated that the PTEN-/p-AKT+/ß-catenin+/Nanog+ axis may indicate poor prognosis and radioresistance in clinical NPC specimens. Thus, our findings strongly suggest that PTEN- cells have CSCs properties that are resistant to radiation in NPC. PTEN exerts these effects through the downstream effector PI3K/AKT/ß-catenin/Nanog axis which depends on nuclear ß-catenin accumulation.
