Cytotoxic Effects of Arsenite in Combination With Gamabufotalin Against Human Glioblastoma Cell Lines

Abstract

Glioblastoma is a fatal primary malignant brain tumor, and the 5-year survival rate of treated glioblastoma patients still remains <5%. Considering the sustained development of metastasis, tumor recurrence, and drug resistance, there is an urgent need for the novel therapeutic approaches to combat glioblastoma. Trivalent arsenic derivative (arsenite, AsIII) with remarkable clinical efficacy in leukemia has been shown to exert cytocidal effect against glioblastoma cells. Gamabufotalin, an active bufadienolide compound, also shows selective cytocidal effect against glioblastoma cells, and has been suggested to serve as a promising adjuvant therapeutic agent to potentiate therapeutic effect of conventional anticancer drugs. In order to gain novel insight into therapeutic approaches against glioblastoma, the cytotoxicity of AsIII and gamabufotalin was explored in the human glioblastoma cell lines U-87 and U-251. In comparison with U-251 cells, U-87 cells were highly susceptible to the two drugs, alone or in combination. More importantly, clinically achieved concentrations of AsIII combined with gamabufotalin exhibited synergistic cytotoxicity against U-87 cells, whereas showed much less cytotoxicity to human normal peripheral blood mononuclear cells. G2/M cell cycle arrest was induced by each single drug, and further augmented by their combination in U-87 cells. Downregulation of the expression levels of cdc25C, Cyclin B1, cdc2, and survivin was observed in U-87 cells treated with the combined regimen and occurred in parallel with G2/M arrest. Concomitantly, lactate dehydrogenase leakage was also observed. Intriguingly, SB203580, a specific inhibitor of p38 MAPK, intensified the cytotoxicity of the combined regimen in U-87 cells, whereas wortmannin, a potent autophagy inhibitor, significantly rescued the cells. Collectively, G2/M arrest, necrosis and autophagy appeared to cooperatively contribute to the synergistic cytotoxicity of AsIII and gamabufotalin. Given that p38 MAPK serves an essential role in promoting glioblastoma cell survival, developing a possible strategy composed of AsIII, gamabufotalin, and a p38 MAPK inhibitor may provide novel insight into approaches designed to combat glioblastoma.

Cytotoxic Effects of Hellebrigenin and Arenobufagin Against Human Breast Cancer Cells

“…In fact, Dong et al have demonstrated that bufadienolide compounds such as gamabufotalin and bufalin sensitize human breast cancer cells to TRAIL-induced apoptosis (52). We also recently suggested that gamabufotalin could serve as a promising adjuvant therapeutic agent to potentiate therapeutic effect of arsenite in glioblastoma cells (15). The studies on cytocidal effects of conventional anticancer drugs in combination of Helle or Areno are ongoing in our laboratory.…”

“…Bufadienolides are another kind of important effective constituents of cinobufacini, and we also demonstrated that active bufadienolide compounds such as gamabufotalin, hellebrigenin (Helle) and arenobufagin (Areno) exhibited selective cytocidal effects against intractable cancer cells (12)(13)(14). Furthermore, we recently demonstrated that clinically achieved concentrations of trivalent arsenic derivative (As III ) combined with gamabufotalin exhibited synergistic cytotoxicity against glioblastoma cell line U-87, whereas showed much less cytotoxicity to human normal peripheral blood mononuclear cells (PBMCs) (15). These findings thus provide fundamental insight into the mechanisms underlying the anti-inflammatory and anticancer activity of cinobufacini.…”

“…After treatment of MCF-7 cells with various concentrations of Helle (10, 30 and 100 nM) for 48 h, cell cycle analysis was performed using a FACS Canto ™ flow cytometer (Becton Dickinson, CA, USA) according to the methods reported previously (14,15,30). Briefly, cells were washed twice with cold PBS, fixed with 1% paraformaldehyde/PBS on ice for 30 min, washed twice again with cold PBS, permeabilized in 70% (v/ v) cold ethanol and kept at -20°C for at least 4 h. The cell were then incubated with 0.25% Triton-X 100 for 5 min on ice.…”

“…Protein concentrations of the supernatant were determined according to Bradford's method using the Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc.), according to the manufacturer's instructions, using BSA as the standard. Western blot analysis was carried out according to the method previously described (14,15). Protein bands were detected using the following primary antibodies: Mouse anti-human b-actin (cat.…”

“…Moreover, survivin is highly expressed in most human cancer cells and implicated in cell cycle transitions (12,14,26). Besides, induction of autophagic cell death has emerged as a critical mechanism underlying cytocidal effect of various anticancer drugs (12,14,15). Although previous studies have demonstrated that the cytotoxicity of some bufadienolide compounds such as Helle and Areno are attributed to the induction of either of apoptosis/necrosis, cell cycle arrest and autophagy in hepatoma and glioblastoma cells (12,27,28), whether and how these biological phenomena contribute to the cytocidal effects of Helle and Areno in human breast cancer cells remain to be seen.…”