Sequential treatment of phenethyl isothiocyanate increases sensitivity of Temozolomide resistant glioblastoma cells by decreasing expression of MGMT via NF-κB pathway

Abstract

Background: Existence of acquired or intrinsic resistance to Temozolomide (TMD) remains a point of concern in treating glioblastoma (GBM). Here we established mechanism by which Phenethyl isothiocyanate (PEITC) reverses TMD resistance in T98G cell lines both in vitro and in vivo.

Methods: For the study TMD-resistant cell lines were generated by stepwise exposing the parental cell lines (U87 and U373) to TMD. The 50% inhibitory concentration (IC₅₀) values were established. MTT assay was done for cell survival studies, apoptosis assay by FITC Annexin V/PI staining, luciferase reporter assay for NF-κB transcription activity, cell colony survival and cell invasion assay, protein expression by western blot was done. For in vivo studies nude mouse model of GBM was established, TUNEL assay was done for apoptosis in tumor specimens.

Results: We established that T98G, U87-R and U373-R showed higher NF-κB activity and exhibited higher IC₅₀ of TMD with significantly increased MGMT expression compared to untreated cells. Next, we found that PEITC suppressed proliferation of resistant GBM cells, inhibited NF-κB activity, decreased expression of MGMT and reversed the resistance in U373-R, U87-R and T98G cells. Exposure to PEITC followed by sequential treatment of TMD produced synergistic effect. In U373-R grafted xenografts mouse model PEITC suppressed cell growth and enhanced cell death.

Conclusion: Altogether, the present research established that combination of PEITC with TMD could enhance its clinical efficacy in resistant GBM by suppressing MGMT via inhibiting NF-κB activity.

Keywords: MGMT; Temozolomide; glioblastoma; resistance.

Figure 1

Expression of MGMT in GBM cell lines. The expression of MGMT is found to be positively expressed in T98G cells, whereas it is negatively expressed in other selected cell lines.

Figure 2

Temozolamide resistance in GBM cell lines (U373, U373-R, U87 and U87-R). A. Drug sensitivity assay for IC₅₀ values of U373, U87 parental and resistance i.e U373-R and U87-R cell lines. B. NF-κB transcription activity in both U373-R and U87-R cell lines was significantly activated compared to their parental cell lines (**P < 0.01 compared to respective parental cell lines). C. Expression of MGMT in Temozolamide resistant cell lines showed increased expression in resistance cells.

Figure 3

Anti proliferative effect of PEITC on Temozolamide resistant GBM cells. A. PEITC inhibits cell viability in T98G, U373-R and U87-R cell line in a dose dependent manner. (^aP < 0.05, ^*aP < 0.01, ^**aP < 0.001 compared to dose of 10 μM). B. A significant decrease in number of colonies (^**P < 0.01) for concentration 10 and 20 μM was observed for U373-R and U87-R, whereas in T98G cells, concentration of 20 μM resulted in significant decrease compared to control (^**P < 0.01, ^*P < 0.05 compared to untreated cells).

Figure 4

Results of IC₅₀ values for PEITC for T98G, U373-R and U87-R cell lines were 50.4, 50.1 and 56.4 μM respectively.

Figure 5

PEITC inhibits the levels of MGMT via NF-κB pathway in all the three TMD resistant cell lines. A. Luciferase assay showed that treatment of PEITC significantly decreased NF-κB transcriptional activity. B. The treatment of PEITC suppressed levels of MGMT in all the three resistant cell lines with increasing concentrations.

Figure 6

Treatment of PEITC reversed the resistance against Temozolomide. A. The invasive capacity reduced significantly in cells receiving pretreatment of PEITC at 10 and 20 μM combined with TMD 270 μM. B. The outcomes of FACS analysis showed that PEITC enhanced the TMD mediated apoptosis significantly (P < 0.01) at dose of 20 μM combined with 270 μM of TMD. C. The caspase 3/7 activity increased significantly in all the three cell lines exposed to PEITC at 10 and 20 μM combined with TMD 270 μM.

Figure 7

The in vivo studies using GBM tumor mouse model confirmed PEITC increased the sensitivity against Temozolamide. A. The tumor volume decreased significantly after 25 days of treatment compared to control (P < 0.01). B. The combined treatment of PEITC with TMD decreased the expression of MGMT, MMP-2, MMP-9, p65 and Ki-67 whereas showed expression of caspase 3. C. The TUNEL assay demonstrated increase in number of TUNEL positive cells indicating xenograft apoptosis.