ABSTRACT
OBJECTIVES: This study was designed to investigate the activation of the unfolded protein response (UPR) in tumor associated endothelial cells (TECs) and its association with chemoresistance during acidic pH stress. MATERIALS AND METHODS: Endothelial cells from human oral squamous cell carcinomas (OSCC) were excised by laser capture microdissection (LCM) followed by analysis of UPR markers (Grp78, ATF4 and CHOP) using quantitative PCR. Grp78 expression was also determined by immunostaining. Acidic stress was induced in primary human dermal microvascular endothelial cells (HDMECs) by treatment with conditioned medium (CM) from tumor cells grown under hypoxic conditions or by adjusting medium pH to 6.4 or 7.0 using lactic acid or hydrochloric acid (HCl). HDMEC resistance to the anti-angiogenic drug Sunitinib was assessed with SRB assay. RESULTS: UPR markers, Grp78, ATF4 and CHOP were significantly upregulated in TECs from OSCC compared to HDMECs. HDMECs cultured in acidic CM (pH 6.0-6.4) showed increased expression of the UPR markers. However, severe acidosis led to marked cell death in HDMECs. Alternatively, HDMECs were able to adapt when exposed to chronic acidosis at pH 7.0 for 7 days, with concomittant increase in Grp78 expression. Chronic acidosis also confers drug resistance to HDMECs against Sunitinib. Knockdown of Grp78 using shRNA resensitizes HDMECs to drug treatment. CONCLUSIONS: UPR induction in ECs under acidic pH conditions is related to chemoresistance and may contribute to therapeutic failures in response to chemotherapy. Targeting Grp78, the key component of the UPR pathway, may provide a promising approach to overcome ECs resistance in cancer therapy.
Subject(s)
Acidosis/pathology , Dermis/pathology , Drug Resistance, Neoplasm , Endothelium, Vascular/pathology , Heat-Shock Proteins/metabolism , Mouth Neoplasms/pathology , Unfolded Protein Response/drug effects , Acidosis/drug therapy , Acidosis/metabolism , Angiogenesis Inhibitors/pharmacology , Apoptosis , Blotting, Western , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Cycle , Cell Proliferation , Cells, Cultured , Dermis/drug effects , Dermis/metabolism , Endoplasmic Reticulum Chaperone BiP , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fluorescent Antibody Technique , Heat-Shock Proteins/genetics , Humans , Hydrogen-Ion Concentration , Immunoenzyme Techniques , Laser Capture Microdissection , Mouth Neoplasms/drug therapy , Mouth Neoplasms/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Loco-regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl-2 expression in lymphatic endothelial cells. METHODS: Endothelial cells were selectively retrieved from paraffin-embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT-PCR was used to evaluate Bcl-2 expression in tumor-associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)-C on the expression of Bcl-2 in primary human lymphatic endothelial cells. RESULTS: We observed that Bcl-2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage-matched tumors without metastasis. VEGF-C induced Bcl-2 expression in lymphatic endothelial cells via VEGFR-3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF-C and induce Bcl-2 in lymphatic endothelial cells. CONCLUSIONS: Collectively, this work unveiled a mechanism for the induction of Bcl-2 in lymphatic endothelial cells and suggested that endothelial cell Bcl-2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.