Long-term temozolomide treatment induces marked amino metabolism modifications and an increase in TMZ sensitivity in Hs683 oligodendroglioma cells

Abstract

Gliomas account for more than 50% of all primary brain tumors. The worst prognosis is associated with gliomas of astrocytic origin, whereas gliomas with an oligodendroglial origin offer higher sensitivity to chemotherapy, especially when oligodendroglioma cells display 1p19q deletions. Temozolomide (TMZ) provides therapeutic benefits and is commonly used with radiotherapy in highly malignant astrocytic tumors, including glioblastomas. The actual benefits of TMZ during long-term treatment in oligodendroglioma patients have not yet been clearly defined. In this study, we have investigated the effects of such a long-term TMZ treatment in the unique Hs683 oligodendroglioma model. We have observed increased TMZ sensitivity of Hs683 orthotopic tumors that were previously treated in vitro with months of progressive exposure to increasing TMZ concentrations before being xenografted into the brains of immunocompromised mice. Whole-genome and proteomic analyses have revealed that this increased TMZ sensitivity of Hs683 oligodendroglioma cells previously treated for long periods with TMZ can be explained, at least partly, by a TMZ-induced p38-dependant dormancy state, which in turn resulted in changes in amino acid metabolism balance, in growth delay, and in a decrease in Hs683 oligodendroglioma cell-invasive properties. Thus, long-term TMZ treatment seems beneficial in this Hs683 oligodendroglioma model, which revealed itself unable to develop resistance against TMZ.

Figure 1

(A) Morphologic illustration (hematoxylin-eosin staining, x 40) of a TMZ-S Hs683 oligodendroglioma xenograft in the brain of an immunocompromised mouse after having orthotopically injected 105 TMZ-S Hs683 tumor cells 17 days earlier. T indicates tumor; BP, brain parenchyma. (B) Survival of immunocompromised mice with brain xenografts of TMZ-S (black circles) versus TMZ-LTT (black squares) Hs683 oligodendroglioma cells. Mice have been orthotopically grafted into their brains with 10⁵ Hs683 tumor cells on day 0 (D0). Hs683 oligodendroglioma cell populations that have been left untreated with TMZ are labeled as TMZ-S Hs683 cells, whereas Hs683 oligodendroglioma cells that have been able to grow in vitro over months in the presence of 1 mM TMZ have been labeled TMZ-LTT Hs683 cells (see Materials and Methods). (C) Survival of immunocompromised mice with brain xenografts of TMZ-S Hs683 cells treated (open circles) or not (black circles) with TMZ compared to TMZ-LTT Hs683 cells treated (open squares) or not (black squares) with TMZ. The TMZ treatment (80 mg/kg per os) was given three times a week (Monday, Wednesday, Friday) during three consecutive weeks, with treatment starting at D5.

Figure 2

(A) mRNA expression (RT-PCR analyses) of vimentin, cytokeratin 20 (CK20), CD133, MELK, and CD44 in Hs683 oligodendroglioma cell populations that have been left untreated (TMZ-SHs683 cells) or that have been able to growin vitro over months in the presence of 1mM TMZ (TMZ-LTT Hs683 cells; see Materials and Methods). HT-29 colon cancer cells have been used as an internal control. Actin RT-PCR was used as a quality control for the reverse transcription and the PCR. (B) Quantitative determination of MGMT mRNA expression (quantitative RT-PCR) in TMZ-S versus TMZ-LTT Hs683 oligodendroglioma cells. Wild-type U373 and T98G astroglioma cells [11,18,21] have been chosen as internal references.

Figure 3

Western blot analysis of BCAT1 and PSAT1 expression in Hs683 oligodendroglioma cell populations that have been left untreated (TMZ-S Hs683 cells) or that have been able to grow in vitro over months in the presence of 1 mM TMZ(TMZ-LTT Hs683 cells; see Materials and Methods), and in TMZ-S Hs683 cells treated with 100 µM TMZ for 72 hours. Tubulin was used as quality and loading control.

Figure 4

(A) PERK activation by Grp78 under stress condition lead to the phosphorylation of eIF2α, which activates the translation of the transcription factor ATF4, which in turn regulates gene expression in response to environmental stresses; p38 MAPK activation induces growth arrest and tumor cell dormancy through the activation of PERK and also through the suppression of ERK signaling. (B) Western blot analyses of Grp78, Grp94, GADD34, ATF4, and phospho-p38 MAPK expression in TMZ-S versus TMZ-LTT Hs683 cells and in TMZ-S Hs683 cells treated with 100 µM TMZ for 72 hours (see the legend to Figure 3). Tubulin was used as a quality and loading control.

Figure 5

(A) Quantitative determination of the percentage of mitotic cells during 72 hours in Hs683 oligodendroglioma cell populations that have been left untreated (TMZ-S Hs683 cells) or that have been able to grow in vitro over months in the presence of 1 mM TMZ (TMZ-LTT Hs683 cells; see Materials and Methods). (B) Flow cytometry analysis of double-stained (Annexin V and PI) TMZ-S and TMZ-LTT Hs683 cells. Apoptotic cells include annexin V⁺/PI^- (early apoptosis) and annexin V⁺/PI⁺ (late apoptosis), whereas necrotic cells are annexin V^-/PI⁺ and normal cells annexin V^-/PI^-. (C) Characterization of the invasiveness of TMZ-S and TMZ-LTT Hs683 cells cultured for 24 hours in Matrigel-coated Boyden chambers. Data are illustrated in A to C as mean ± SEM values, and all experiments have been carried out in triplicate.