EMAP-II sensitize U87MG and glioma stem-like cells to temozolomide via induction of autophagy-mediated cell death and G2/M arrest

Abstract

Despite the fact that temozolomide (TMZ) has been widely accepted as the key chemotherapeutic agent to prolong the survival of patients with glioblastoma, failure and recurrence cases can still be observed in clinics. Glioma stem-like cells (GSCs) are thought to be responsible for the drug resistance. In this study, we investigate whether endothelial monocyte-activating polypeptide-II (EMAP-II), a pro-inflammatory cytokine, can enhance TMZ cytotoxicity on U87MG and GSCs or not. As described in prior research, GSCs have been isolated from U87MG and maintained in the serum-free DMEM/F12 medium containing EGF, b-FGF, and B27. TMZ and/or EMAP-II administration were performed for 72 h, respectively. The results showed that TMZ combined with EMAP-II inhibit the proliferation of U87MG and GSCs by a larger measure than TMZ single treatment by decreasing the IC₅₀. EMAP-II also enhanced TMZ-induced autophagy-mediated cell death and G2/M arrest. Moreover, we found that EMAP-II functioned a targeted suppression on mTOR, which may involve in the anti-neoplasm mechanism. The results suggest that EMAP-II could be considered as a combined chemotherapeutic agent against glioblastoma by sensitizing U87MG and GSCs to TMZ.

Keywords: EMAP-II; G2/M arrest; autophagy; glioma stem cells; temozolomide.

Figure 1.

EMAP-II enhanced cytotoxic effect of TMZ on U87MG and GSCs (A) U87MG cells were cultured in the DMEM, containing 10% FBS in flasks. After sorted by FACS, the CD133-positive cells were collected and maintained in the DMEM/F12, containing 2% B27 supplements, 20ng/ml EGF, and bFGF. Suspended cells became spheres after 48 h culture. (B) GSCs expressing the stemness biomarkers of CD133 and Nestin by immunofluorescence. (C) Both U87MG and GSCs were treated with TMZ alone or a combination of TMZ and EMAP-II with assigned concentration for 72 h respectively, IC₅₀ of TMZ calculated from dose-inhibition response curves was remarkably decreased on both U87MG and GSCs. (D) Cells were treated with 100μM TMZ and/or 0.5 nM EMAP-II for 72 h respectively and cell viability was then determined. EMAP-II enhanced the TMZ-induced cytotoxic effect on U87MG and GSCs.

Figure 2.

EMAP-II increased TMZ-induced autophagy (A) U87MG was treated with either TMZ or EMAP-II and both TMZ and EMAP-II for 72 h respectively. Expression and distribution of LC3B II were determined by immunofluorescence staining, all images were taken with exactly the same settings. Compared to the control, cells treated with either EMAP-II or TMZ both showed an obvious higher level of LC3BII expression, and cells treated with both EMAP-II and TMZ showed even higher LC3BII staining. (scale bar = 20μM). (B-D) U87MG was treated with either TMZ or EMAP-II and both TMZ and EMAP-II for 72 h. TMZ induced a higher LC3BII/LC3BIlevel than EMAP-II; Combination of TMZ and EMAP-II showed a significant elevation on LC3BII/LC3BIthan either TMZ or EMAP-II; P62/SQSTM1 was obviously inhibited by combination of TMZ and EMAP-II;(E-G) GSCs was treated with either TMZ or EMAP-II and both TMZ and EMAP-II for 72 h. The combination of TMZ and EMAP-II showed a significant elevation on LC3BII/LC3BIthan either TMZ or EMAP-II; P62/SQSTM1 was obviously inhibited by the combination of TMZ and EMAP-II; Values present means ± SD (n = 3, each). *p < 0.05, **p < 0.01, or ***p < 0.001.

Figure 3.

Effect on the cell progression. After U87MG and GSCs were treated with either TMZ or EMAP-II and combination of TMZ with EMAP-II for 72 h, the cell population in different phase were determined by flow cytometry. (A-B) U87MG treated with TMZ showed an increased proportion of the G2/M population, combined TMZ and EMAP-II induced a higher G2/M accumulation than TMZ. (C-D) GSCs treated TMZ did not show an increased proportion of the G2/M population. only combined TMZ and EMAP-II induced a higher G2/M accumulation. The values present means ± SD (n = 4, each). *p < 0.05

Figure 4.

Effect on PI3K/AKT/mTOR pathway. U87MG and GSCs were treated with either TMZ or EMAP-II and combination of TMZ with EMAP-II for 72 h. (A-D) Western Blot showed relative expressions of PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR, combination of TMZ with EMAP-II enhanced inhibition of p-mTOR/mTOR on U87MG; (E-H) Western Blot showed relative expressions of PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR, combination of TMZ with EMAP-II enhanced inhibition of p-mTOR/mTOR on GSCs. The values present means ± SD (n = 4, each). *p < 0.05, **p < 0.01, or ***p < 0.001.