Tumor-derived hepatocyte growth factor is associated with poor prognosis of patients with glioma and influences the chemosensitivity of glioma cell line to cisplatin in vitro

Abstract

Background: We examined the association of tumor-derived hepatocyte growth factor (HGF) with the clinicopathological features of gliomas and investigated the effect of HGF inhibition on the biological behavior of tumor cells in vitro in order to determine whether HGF is a valuable prognostic predictor for glioma patients.

Methods: Seventy-six cases of glioma were collected. The tumor-derived HGF expression, cell proliferation index (PI) and intratumoral microvessels were evaluated by immunohistochemistry. Correlation between immunostaining and clinicopathological parameters, as well as the follow-up data of patients, was analyzed statistically. U87MG glioma cells were transfected with short interference (si)-RNA for HGF, and the cell viability, migratory ability and chemosensitivity to cisplatin were evaluated in vitro.

Results: Both high HGF expression in tumor cells (59.2%, 45/76) and high PI were significantly associated with high-grade glioma and increased microvessels in tumors (P < 0.05). However, only histological grading (P = 0.004) and high-expression of HGF (P = 0.008) emerged as independent prognostic factors for the overall survival of glioma patients. The tumor-derived HGF mRNA and protein expressions were significantly decreased in vitro after transfection of HGF siRNA. HGF siRNA inhibited the cell growth and reduced cell migratory ability. Moreover, HGF siRNA transfection enhanced the chemosensitivity of U87MG glioma cells to cisplatin.

Conclusion: This study indicated that there was significant correlation among tumor cell-derived HGF, cell proliferation and microvessel proliferation in gliomas. HGF might influence tumor progression by modulating the cell growth, migration and chemoresistance to drugs. Increased expression of HGF may be a valuable predictor for prognostic evaluation of glioma patients.

Figure 1

Immunohistochemical staining of glioma tissues. a Strong and diffuse expression of HGF was found in high-grade glioma; b positive staining of HGF was shown focally and weakly in low-grade gliomas. c The extent of Ki-67-positive signal represented the proliferation of gliomas. Most high-grade gliomas had a high proliferation index (PI). d Intratumoral microvessels of gliomas were highlighted by staining endothelial cells for anti-CD34. Any brown-staining endothelial cell or cell cluster that was clearly separated from the adjacent microvessels was considered as a single, countable microvessel. (a-c immunohistochemical staining with original magnification, ×400; d immunohistochemical staining × 200).

Figure 2

Kaplan-Meier survival analysis in 76 patients with gliomas. a Kaplan-Meier curve showing the patients with high-grade tumor have a lower survival rate than those with low-grade tumor. b Patients with a tumor with high HGF expression had a lower survival rates than those with low HGF expression tumors. c A significant difference in survival rates was found between patients with a high proliferation index and low index in their tumor. d There was no significant difference in survival rates between the patients with or without tumor recurrence.

Figure 3

HGF siRNA inhibited tumor cell-derived HGF expression. a Immunofluorescence assay showed that tumor cell-derived HGF protein expression was significantly decreased in U87MG glioma cells with HGF siRNA transfection (bottom) when compared with control siRNA transfected cells (middle) and mock (top) (immunofluorescence staining with original magnification, ×400). Western blot assay (b) and RT-PCR (c) exhibited that HGF protein and mRNA expression levels were decreased in the cells with HGF siRNA transfection.

Figure 4

The effect of HGF inhibition on cell growth and migratory abilities of glioma cells. a MTT assays were performed in the U87MG glioma cell line. Cells were cultured in a 96-well plate and transfected with HGF siRNA or control siRNA for 24, 48 and 72 h points with an average of five independent experiments in each group (*P <0.05 versus control cells; one-way ANOVA). b Optical microscopic images of in vitro wound healing at 0, 12, 24 and 48 h after the creation of wounds. HGF siRNA-transfected cells displayed significantly slower wound closure at all time points compared with control siRNA-transfected cells.

Figure 5

HGF siRNA-induced enhancement of chemosensitivity to cisplatin. Sensitivity curve of the U87MG cell line toward cisplatin via MTT assay. Growth inhibition of HGF siRNA-transfected cells was observed after exposure to different concentrations of cisplatin for 24 h (*P < 0.05 versus control cells, one-way ANOVA).