Antitumor activity of gemcitabine against high-grade meningioma in vitro and in vivo

Abstract

Currently, there is no established therapeutic option for high-grade meningioma recurring after surgery and radiotherapy, and few chemotherapeutic agents are in development for the treatment of high-grade meningioma. Here in this study, we screened a panel of chemotherapeutic agents for their possible antitumor activity in high-grade meningioma and discovered that high-grade meningioma cells show a preferential sensitivity to antimetabolites, in particular, to gemcitabine. In vitro, gemcitabine inhibited the growth of high-grade meningioma cells effectively by inducing S-phase arrest and apoptotic cell death. In vivo, systemic gemcitabine chemotherapy suppressed not only tumor initiation but also inhibited the growth and achieved a long-term control of established tumors in xenograft models of high-grade meningioma. Histological analysis indicated that systemic gemcitabine blocks cell cycle progression and promotes apoptotic cell death in tumor cells in vivo. Together, our data demonstrate that gemcitabine exerts potent antitumor activity against high-grade meningioma through cytostatic and cytotoxic mechanisms. We therefore propose gemcitabine is a promising chemotherapeutic agent that warrants further investigation as a treatment option for high-grade meningioma.

Keywords: anaplastic meningioma; brain tumor; cancer; intracranial neoplasm; malignant meningioma.

Figure 1. Growth-inhibitory effects of chemotherapeutic agents on high-grade meningioma cells in vitro

High-grade meningioma cells (HKBMM and M-16-N) and IMR90 normal human fibroblasts were treated with the indicated chemotherapeutic agents for 3 days, and their viability relative to control (cells treated in the absence of the test drug) was determined. Values in the graphs represent means + SD from triplicate samples of a representative experiment repeated with similar results.

Figure 2. The gemcitabine sensitivity of cell lines derived from cancers for the treatment of which gemcitabine is indicated

Pancreatic cancer cells (PANC-1, PSN-1, BxPC-1 and AsPC-1), non-small cell lung cancer cells (A549 and H1299), ovarian cancer cells (SKOV-3 and RMG-1), and high-grade meningioma cells (HKBMM and M-16-N) were treated with the indicated concentrations of gemcitabine (GEM) for 3 days, and their viability relative to control (cells treated in the absence of gemcitabine) was determined. Values in the graphs represent means + SD from triplicate samples of a representative experiment repeated with similar results. Note that the data for HKBMM and M-16-N, which were included in this figure for comparison, are identical with those in Figure 1.

Figure 3. Anti-proliferative and pro-apoptotic activities of gemcitabine in high-grade meningioma cells

(A) HKBMM cells treated with the indicated concentrations of gemcitabine (GEM) for 24 h were subjected to cell cycle analysis by flow cytometry. Representative flow cytometry histograms are shown, with the percentage of cells in each cell cycle phase tabulated on the right. (B) Cells treated with the indicated concentrations of GEM for 3 days were subjected to cell death assay. The graphs show the percentage of dead cells, and values in the graphs represent means + SD from triplicate samples of a representative experiment repeated with similar results. *P < 0.05. (C) Cells treated with the indicated concentrations of GEM for 24 h were subjected to immunoblot analysis of cleaved caspase-3 and PARP expression. (D) HKBMM cells treated with the indicated concentrations of GEM for 3 days were cultured for another 1 week in the absence of GEM for colony formation assay. An image of a representative experiment (left panel) and the number of colonies (right graph) are shown. Values in the graph represent means + SD from triplicate samples of a representative experiment repeated with similar results. *P < 0.05.

Figure 4. Systemic gemcitabine administration inhibits tumor initiation and progression of high-grade meningioma and provides long-term control

(A) Mice (three for each group) implanted subcutaneously with 5 × 10⁵ viable HKBMM cells were treated, after randomization according to body weight, with intraperitoneal injection of control vehicle or gemcitabine (GEM, 20 mg/kg twice a week for 4 weeks) starting on the next day of implantation. Tumor volume (left) and the body weight of the mice (right) were measured at the indicated time points. Values in the graphs represent means + SD of each treatment group. (B) Mice (three for each group) were implanted subcutaneously with HKBMM cells (bilaterally, 1 × 10⁶ viable cells each) and, after randomization according to tumor volume and body weight, received intraperitoneal injection of vehicle alone, hydroxyurea (HU, 500 mg/kg/day for 15 days) or GEM (20 mg/kg twice a week for 4 weeks), which started at 4 weeks after implantation when the volume of the subcutaneous tumors reached ∼100 mm³. Tumor volume (left) and the body weight of the mice (right) measured at 4 (pre-treatment) and 12 (post-treatment) weeks after implantation are shown. Values in the graphs represent means + SD of each treatment group. *P < 0.05. (C) The volume of the tumors in the mice described in (B) was subsequently monitored at a weekly interval. Mice in the GEM treatment group were treated repeatedly with the same GEM regimen as indicated in the graph. Values in the graph represent means + SD of each treatment group. (D, E) Xenograft tumors (∼300 mm³) formed by subcutaneous (and bilateral) implantation of HKBMM cells were treated, after randomization according to tumor volume, with systemic administration of the control vehicle or GEM (20 mg/kg twice a week) for 2 weeks (two mice were treated for each group). On the next day of the final administration, the tumors were excised and subjected to immunofluorescence analysis of cleaved PARP (D) and phospho-histone H3 Ser10 (E) expression. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images (left, scale bars = 20 μm) and graphs indicating the number of positive cells per high-power field (right) are shown. Values in the graphs represent means + SD of three sections from tumors treated as indicated (two tumors from a mouse harboring the largest tumors in each treatment group were analyzed). *P < 0.05.