Abstract
Objective: This study was designed to analyze the combinatorial chemotherapeutic effect of temozolomide (TMZ), the most common drug in glioblastoma treatment and a purified carbohydrate (Fr-II) from the edible mushroom Pleurotus florida, on human glioblastoma cell lines.Methods: Fr-II was purified by size-exclusion chromatography and characterised by different mass spectroscopy analysis. Human glioblastoma cells were treated with TMZ, Fr-II, and combination of TMZ and Fr-II. Cell cytotoxicity was measured by MTT assay, cell cycle phase distribution was determined by cell cycle analysis and followed by the relative p53 protein expression was analyzed by western blot analysis.Results: Chemical analysis of Fr-II confirmed the glycosidically linked two units of glucose with terminally attached mannitol with mass of 506 Da. Fr-II treatment exhibited cytotoxicity in both the cell lines in a dose-dependent manner with most effective dose at 200µg/ml. When Fr-II (200µg/ml) was combined with a dose range of TMZ it showed a more cellular cytotoxicity compared to the cytotoxicity of TMZ alone with most oppressive combinatorial dose at 400µM (TMZ)+200µg/ml (Fr-II). In compliance, with the above results, both cell lines showed a 10% increase in no. of cells (p<0.05) in G2/M phase indicating an arrest of cell cycle and increased p53 protein expression (p<0.05) at the combinatorial dose than TMZ alone at 400µM, but Fr-II alone didn’t show any cell cycle arrest nor did it show increased p53 expression.Conclusion: Therefore it confirms that Fr-II synergizes with TMZ to significantly intensify its anti-proliferative properties, thereby emerging as an effective element for combinatorial treatment of glioblastoma.
Highlights
Glioblastoma multiforme (GBM) is the most prevalent and aggressive form of primary brain tumors which demonstrates a high proliferation rate, an aggressive growth pattern and is mostly resistant to chemotherapy [1]
Human glioblastoma cell line U373 was a kind donation from Dr Chitra Mondal, Indian Institute of Chemical Biology (Kolkata, India) and U87MG was purchased from National Centre for Cell Science (Pune, India)
U373 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and U87MG in Eagle’s Minimal Essential Medium (MEM)supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin and grown in 5% CO2 and 37 °C conditions
Summary
Glioblastoma multiforme (GBM) is the most prevalent and aggressive form of primary brain tumors which demonstrates a high proliferation rate, an aggressive growth pattern and is mostly resistant to chemotherapy [1]. The infiltrative growth of glioma cells and their resistance to standard therapy have limited successful treatment, as a result of which the median survival of patients with GBM is approximately 12–15 mo [2]. One of the most promising drugs for the treatment of brain tumor is Temozolomide (TMZ), an oral alkylating agent belonging to imidazotertrazines, which exhibits anti-cancer properties against malignancies [4]. According to Uzzaman et al, the average survival of patients with glioblastoma after TMZ treatment is 22 mo, which has not rendered the drug entirely satisfactory for therapy [12]
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