Glioblastoma multiforme (GBM) is the most malignant primary human brain cancer and cannot be cured by any therapy available today. Current therapy includes complex treatments of neurosurgical resection, radiotherapy, and chemotherapy, with extreme poor outcome. The typical life expectation for GBM patient is less than 1 year. The tumor localizes in the central nervous system and appears to be very solid to prevent its impermeability to large particles. A major challenge for GBM treatment is to efficiently control tumor growth by killing the cancer cells on the surface of the tumor more rapidly than replication of the inner tumor cells. Thus, high selectivity and safety of the treatment to avoid damage to the normal brain tissue, rapid and efficient cell killing, and inhibition of tumor cell replication may be all crucial for a successful treatment for GBM.
Most of GBM overexpressed epidermal growth factor receptor (EGFR). Therefore, drugs that specifically target EGFR and subsequently inhibit EGFR signaling in glioblastoma cells may have a therapeutic effect on GBM. Although approaches have been widely taken by researchers to search for ways to inhibit EGFR signaling in glioblastoma cells, no safe and efficient therapy for glioblastoma has been achieved.
Ginsenoside Rh2 is one of the characteristic components in red ginseng with potential bioactivity. Ginsenoside Rh2 and its derivatives have been reported of potentially therapeutic effects on ovarian cancers, pancreatic adenocarcinoma, prostate cancer, colorectal cancer, and breast cancer, with its antitumor effects through inhibition of tumor cell proliferation and induction of tumor cell apoptosis. Nevertheless, the exact molecular basis of the antitumor effect of ginsenoside Rh2 remains unclear. Moreover, the effect of ginsenoside Rh2 on glioblastoma has not been studied before.
Here, we show that ginsenoside Rh2 can substantially inhibit the growth of glioblastoma in a mouse model. Moreover, the inhibition of the tumor growth appears to result from combined effects on decreased tumor cell proliferation and increased tumor cell apoptosis. Further analyses suggest that ginsenoside Rh2 may have its antiglioblastoma effect through inhibition of the EGFR signaling pathways in tumor cells. In a lose-of-functionexperiment, recombinant EGFR was given together with ginsenoside Rh2 to the glioblastoma cells in vitro and in vivo, which completely blocked the antitumor effects of ginsenoside Rh2. Thus, our data not only reveal an antiglioblastoma effect of ginsenoside Rh2 but also demonstrate that this effect may function via inhibition of EGFR signaling in glioblastoma cells.
Materials and methods
Culture and label human glioblastoma cell line with a luciferase reporter
The human glioblastoma cell line A-172 (GC) established by Dr. Giard was cultured in 1:1 mixture of DMEM and Ham’s F12 medium supplemented with L-glutamine and 5 % FBS. To trace the glioblastoma cells in vivo, we infected the cells with a recombinant lentivirus expressing luciferase and green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter at MOI 100 and resulted in nearly 100 % infection efficiency based on green fluorescence. The reporter-carrying glioblastoma cells were termed GC-luc. Ginsenoside Rh2 (0.1 mg/ml) alone, or with recombinant EGFR (0.2 μg/ml) (Millipore, USA), was given to the cultured GC-luc for 24 h before analysis.
All mouse experiments were approved by the general principles contained in the Guide for the Care and Use of Laboratory Animals published by China Medical University. Ten-week-old male NOD/SCID mice were used for the experiments. Ginsenoside Rh2 (1 mg/kg body weight) was injected from the tail vein of the mice twice per week for 1 month, till the end of the experiment. Recombinant EGFR (2 μg/kg body weight) was stereotactically injected to the site of glioblastoma once per week, starting at the time of ginsenoside Rh2 administration, till the end of the experiment.
Ginsenoside Rh2 efficiently inhibited the growth of glioblastoma in vivo
The human glioblastoma cell line A-172 (GC) established by Dr. Giardwas used to generate a glioblastoma model to examine the effect of ginsenoside Rh2. To trace these glioblastoma cells in vivo, we infected the cells with a recombinant lentivirus expressing luciferase and GFP under the control of a CMV promoter. The reporter-carrying glioblastoma cells were termed GC-luc, which were stereotactically injected into the brains of male NOD/SCID mice 10 weeks of age as described (Fig. 1a). One month after, the animals were examined of tumor growth by luciferase assay, showing establishment of the brain tumor.
Shaoyi Li & Yu n G a o & Weining Ma & Wenchang Guo & Gang Zhou & Tianci Cheng & Yunhui Liu. Tumor Biol. (2014) 35:5593–5598