The proliferation, apoptosis, invasion of endothelial-like epithelial ovarian cancer cells induced by hypoxia

Abstract

Background: Epithelial ovarian cancer is one of the most malignant cancers in women because metastasis occurs in the most of patients by the time of diagnosis. Cancer cells have strong capacity to form angiogenesis or vasculogenic mimicry, which plays the major role in its malignant phenotype. Vasculogenic mimicry might contribute to the failure of the angiogenesis-targeted therapy strategies. Under the microenvironment of the tumor, hypoxia is the most common phenomena because of the vast energy and oxygen consuming. In the present study, the endothelial-like cells induced by hypoxia from SKOV-3 and ES-2 ovarian cancer cells were harvested to investigate the changes in their biological behaviors.

Methods: The endothelial-like cells from SKOV-3 and ES-2 cells were harvested by laser capture microdissection. The biological behaviors of the endothelial-like cells, including proliferation, cell cycle, apoptosis, invasion and telomerase activity were determined by MTT, FCM, Transwell chamber and TRAP-ELISA methods. HIF-1α is the most important factor for the behavior changes under hypoxic condition. Some other genes relative to biological behaviors are also changes following the changes of HIF-1α. In order to elucidate the underlying mechanisms for these changes by hypoxia, the relative genes expressions including HIF-1α, CyclinD1, Flk-1, VEGF, p53 and V-src were determined by real-time PCR.

Results: SKOV-3 and ES-2 cells were resistant to hypoxia by adoption of proliferation, apoptosis, differentiation and invasion. Combined with other studies, the more poorly cancer cells differentiate, the more strongly cells are resistant to hypoxia, the more possible to form vasculogenic mimicry. The changes in the expression of HIF-1α, and HIF-1α-dependent VEGF, Flk-1, Cyclin D1, and HIF-1α-independent p53 have been involved in this process.

Conclusions: HIF-1α took an important role in the behavioral changes of SKOV-3 and ES-2 cells by hypoxia. At the same time, other mechanisms were also involved in this process.

Figure 1

The morphology of the ELs from ovarian cancer induced by hypoxia and microdissected by LCM. The ovarian cancer cells were cultured in 3-dimisonal Matrigel system on EVA membrane under hypoxia for 7 d before harvest. The pictures were taken under the light microscope. A and B. The original and after microdissected by LCM of the single cell. C and D. The original and after microdissected by LCM of the grouped cells. Magnification X200. Arrow: The morphology of the cells after microdissection.

Figure 2

The proliferation, cell cycle, apoptosis, invasion of SKOV-3, ES-2 and HUVEC cells induced by hypoxia. The SKOV-3, ES-2 and HUVEC cells were cultured for 3 or 7 d in normoxia or hypoxia conditions before proliferation, cell cycle (S-phage), apopotosis and invasion detected by MTT, FCM (for cell cycle and apoptosis) and Transwell as shown in methods. A. The proliferation of three cells by MTT. B. The S-phase ratio in three cells by FCM. C. The apoptosis of three cells detected by FCM. D and E. The numbers of cells invasion through the membrane indicated by Transwell after incubated for 3 days (D) or 7 days (E). Data were shown in Mean ± S.D. from three separate experiments with the similar result. * and ** indicates P < 0.05 and P < 0.01 vs. Normoxia.

Figure 3

The genes expression in SKOV-3, ES-2, ELs from cancer cells and HUVEC induced by hypoxia. The SKOV-3, ES-2 and HUVEC cells were cultured for 7 d in normoxia or hypoxia conditions before harvested for the expression of HIF-1a, VEGF, Flk-1, CyclinD1, p53 and V-src genes detected by Real-time PCR. A. The genes expression in SKOV-3 and relative cells by Real-time PCR. B. The genes expression in ES-2 and relative cells by Real-time PCR. SKOV-3 EL: the endothelial-like cells induced from SKOV-3 cells; SKOV-3+Si: the SKOV-3 cells treated by Sirolimus under hypoxia; ES-2 EL: the endothelial-like cells induced from ES-2 cells; ES-2+Si: the ES-2 cells treated by Sirolimus under hypoxia; *, ^, and & indicates that P < 0.05 vs.HUVEC, SKOV-3 (or ES-2) and SKOV-3+Si (or ES-2+Si); **, ^^, and && indicates that P < 0.01 vs.HUVEC, SKOV-3 (or ES-2) and SKOV-3+Si (or ES-2+Si).