The expression of p75(NTR) was only located in basal cells of normal laryngeal epithelia, consistent with the staining features of epithelial stem cells as evidenced by parallel staining of p63, a putative keratinocyte stem cell marker. p75(NTR) is differentially expressed in LSCC, although no significant relationship was found with many clinicopathologic factors, this expression and distribution may correlate to malignant transformation and tumor proliferation. Co-expression of p75(NTR) and CD133 was confirmed, showing the association of p75(NTR)-positive cells with cancer stem cells in Hep-2 cells.
Hypoxia renders tumor cells with reduced sensitivity and increased resistance to chemotherapeutic agents. One of the possible mechanisms underlying this unfavorable status is activation of the unfolded protein response (UPR) under hypoxic conditions, due to the upregulation of glucose-regulated protein 78 (GRP78) expression. GRP78, an endoplasmic reticulum chaperone protein and a key regulator of the UPR, has been reported to be overexpressed in various types of cancer. However, the role of GRP78 in regulating the cell growth and apoptosis of hypopharyngeal carcinoma cells, with regard to the severity of hypoxia, remains unclear. Therefore, the aim of the present study was to investigate whether, and under what circumstances, GRP78 is associated with hypoxia-induced chemoresistance in hypopharyngeal carcinoma. For this purpose, cells from the FaDu human hypopharyngeal carcinoma cell line were cultured under normoxic and hypoxic conditions for different time periods. No significant changes in GRP78 and C/EBP homology protein (CHOP) protein expression levels were revealed under moderately hypoxic conditions (oxygen concentration, 1%), but these levels were changed over time under severely hypoxic conditions (oxygen concentration, <0.02%). This indicated that severe hypoxia, rather than moderate hypoxia, leads to UPR activation in hypopharyngeal carcinoma cells. Knockdown of GRP78 with short hairpin RNA inhibited cell proliferation and promoted apoptosis under severely hypoxic conditions, even with cisplatin treatment, indicating that GRP78 confers FaDu cells resistant to chemotherapy in response to severe hypoxia. Furthermore, knockdown of GRP78 resulted in a significant increase in CHOP and Bax expression levels and a decrease in Bcl-2 expression levels with simultaneous increase in the levels of apoptosis under severely hypoxic conditions. It was concluded that severe hypoxia leads to UPR activation and elevation of GRP78 expression, promoting cell survival and inducing chemoresistance. Silencing of GRP78 may block the pro-survival arm of UPR, simultaneously promoting proapoptotic signaling through induction of CHOP. Downregulation of GRP78 may be a promising strategy for overcoming the resistance of hypopharyngeal cancer to chemotherapy.
Abstract. Short hairpin rna (shrna) targeting signal transducer and activator of transcription 3 (Stat3) potentiate the radiosensitivity of human laryngeal squamous carcinoma cells in vitro. in the present study, we investigated the inhibitory effect of Stat3 shrna plus radiotherapy on nude mouse laryngeal squamous cell carcinoma xenografts. the xenotransplanted tumors were treated with Stat3 shrna, with or without radiation, following a planned scheme. the inhibition rate for tumor growth was calculated and the tumor growth curve was plotted. in addition, the expression of p-Stat3, B cell lymphoma 2 (Bcl-2), p53, vascular endothelial growth factor (VEGF) protein and intratumoral microvessel density (mVd) was determined by immunohistochemistry. Flow cytometry was used to detect the rate of cell apoptosis. the results revealed that Stat3 shrna transfection plus radiotherapy significantly minimized tumor volume and increased the rate of tumor inhibition. p-Stat3 protein expression and intratumoral mVd were observed to be down-regulated, whereas apoptosis was increased. there was a positive correlation between the expression of p-Stat3 and Bcl-2, and also between the expression of p53 and VEGF, and MVD. These findings indicate that STAT3 shRNA potentiate the radiosensitivity of laryngeal carcinoma xenografts in vivo by regulating downstream signaling proteins in the Stat3 pathway.
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