Effect of IFN-β on human glioma cell lines with temozolomide resistance

Abstract

Abstract. Human interferon-ß (IFN-ß) is known to exhibit pleiotropic biological activities including antitumor effects. On the other hand, temozolomide (TMZ), an oral bioavailable alkylating agent with excellent tolerability, has demonstrated efficacy and has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. More recent studies have indicated that there might be favorable therapeutic interactions between IFN-ß and TMZ, although the therapeutic advantages of such a combination have not yet been fully explored. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of IFN-ß and TMZ. The antitumor effect of and cell sensitivity to IFN-ß and TMZ and the synergistic potential of IFN-ß and TMZ in combination were evaluated in six malignant glioma cell lines. Correlations among the MGMT methylation status, quantitative level of MGMT mRNA, MGMT protein expression and the antitumor effect of these agents were also evaluated, since one of the most prominent resistance mechanisms to TMZ involves the DNA repair protein MGMT. The cell growth inhibitory effects of IFN-ß and TMZ on all tumor cell lines were observed in a dose-dependent manner, and the human malignant glioma-derived cell lines differed in their sensitivity to TMZ. The MGMT status, including promoter hypermethylation, quantitative mRNA expression and protein expression, was strongly correlated with TMZ sensitivity. A synergistic cell growth inhibitory effect and down-regulated MGMT mRNA levels were significantly observed when a clinically achievable CNS dose of IFN-ß was combined with TMZ, as compared to treatment with IFN-ß or TMZ alone in TMZ-resistant T98G cells. Furthermore, significant amounts of endogenous IFN-ß protein were detected in TMZ-treated T98G cells by ELISA. These results suggest that the clinical therapeutic efficacy of TMZ might be improved by a combination with IFN-ß in malignant gliomas unmethylated at the MGMT gene. The data provide an experimental basis for future strategies in TMZ chemotherapy, although further studies are needed to determine the detailed role of combined IFN-ß and TMZ chemotherapy in increasing tumor sensitivity.

Gene expression profiles predicting the response to IFN-β and a combination of temozolomide and IFN-β in malignant gliomas

“…Natsume et al suggested that a sensitizing effect between IFN-β and TMZ in TMZ-resistant glioma cells was possibly due to an attenuation of MGMT expression via induction of the protein p53 (10). Furthermore, we demonstrated that a significant synergistic antitumor effect and downregulated quantitative MGMT mRNA levels were observed when TMZ was combined with IFN-β at clinically relevant concentrations, as compared to treatment with TMZ or IFN-β alone in TMZ-resistant glioma cells, and that significant amounts of endogenous IFN-β protein were detected in the TMZ-treated cells by ELISA (12).…”

“…Our prior studies have shown that T98G, U-138MG and YH-13 had MGMT mRNA and protein expression, whereas A-172, AM-38, U-87MG and U-251 did not have them (3). Further, a synergistic antitumor effect of combination treatment with IFN-β and TMZ was observed in the TMZ-resistant glioma cells, T98G, due possibly to an attenuation of their MGMT mRNA levels (12). However, in the present study, other MGMT-positive cell lines, at least YH-13, did not exhibit a synergistic effect between IFN-β and TMZ, whereas the MGMT-negative cell line, A-172, displayed a synergistic effect with a combination of TMZ and IFN-β.…”

“…To assess the antitumor effect of a combination of TMZ and IFN-β, we investigated the following incubation conditions: 10 µM TMZ, 10 IU/ml IFN-β, or 10 µM TMZ and 10 IU/ml IFN-β. These conditions represent the relevant concentrations of TMZ and IFN-β (12). Briefly, cells were plated at 2x10 4 cells per well in 24-well, flat-bottomed plates and incubated with medium for 24 h. The cells were subsequently washed twice with medium and incubated further with fresh medium (control), or medium containing 10 µM TMZ, 10 IU/ml IFN-β, or 10 µM TMZ and 10 IU/ml IFN-β for 72 h. After exposure to the various conditions for 72 h, cells were detached by trypsinization and the numbers counted.…”

Gene expression profiling predicts response to temozolomide in malignant gliomas

“…Parts of the results obtained have recently been reported elsewhere (22,23) Table V. MGMT methylation status, quantitative of MGMT mRNA, and MGMT protein expression in seven malignant glioma cell lines.…”

“…To determine the protein levels of MGMT, Western blot analysis was employed as described previously (22,23). Briefly, soluble protein lysates of glioma cells were obtained employing lysis buffer (Medical & Biological Laboratories, Woburn, MA, USA) for 20 min on ice.…”