Targeting multiple pathways in gliomas with stem cell and viral delivered S-TRAIL and Temozolomide

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells. However, its short half-life, poor delivery, and TRAIL-resistant tumor cells have diminished its clinical efficacy. In this study, we explored whether novel delivery methods will represent new and effective ways to treat gliomas and if adjuvant therapy with the chemotherapeutic agent temozolomide would enhance the cytotoxic properties of TRAIL in glioma lines resistant to TRAIL monotherapy. We have engineered adeno-associated virus (AAV) vectors encoding recombinant secreted TRAIL (S-TRAIL) and bioluminescent-fluorescent marker fusion proteins and show that AAV-delivered S-TRAIL leads to varying degrees of killing in multiple glioma lines, which correspond with caspase-3/7 activation. In vivo, dual bioluminescent imaging revealed efficient delivery of therapeutic AAV vectors directly into the tumor mass, which induced marked attenuation of tumor progression. Treatment of glioma cells with the chemotherapeutic agent temozolomide alone lead to a significant accumulation of cells in G(2)-M phase, activated the cell cycle checkpoint protein Chk1, and increased death receptor expression in a time-dependent manner. Furthermore, combined treatment with AAV-S-TRAIL or neural stem cell-S-TRAIL and temozolomide induced cell killing and markedly up-regulated proapoptotic proteins in glioma cells least sensitive to TRAIL. This study elucidates novel means of delivering S-TRAIL to gliomas and suggests combination of clinically relevant temozolomide and S-TRAIL may represent a new therapeutic option with increased potency for glioblastoma patients.

Figure 1. AAV-mediated expression of bi-modal fusion proteins and S-TRAIL

(A) Schematic representation of recombinant AAV-2 vectors encoding fluorescent-bioluminescent fusion marker and S-TRAIL. (B) TRAIL-resistant 293T cell lines were transduced with AAV-Gluc-DsRed2 or AAV-S-TRAIL (MOI 1 000) and 48 h post-infection, cells were imaged using fluorescence microscopy. (C) Representative bioluminescent images and summary graphs of 293T cell lines transduced with increasing concentrations of AAV-Gluc-DsRed2 or AAV-S-TRAIL (500-20 000 MOI). After 24 h, cells were incubated with coelenterazine and bioluminescent imaging was used to determine Gluc expression. (D) 293T cells (4×10⁵) were transduced with control (Gluc-DsRed2) or S-TRAIL and 24 h post-infection secreted TRAIL concentration per mL of media was determined by enzyme-linked immunosorbent assay (ELISA). Maginification-10× (B).

Figure 2. S-TRAIL induced apoptosis and caspase upregulation in human glioma cells in vitro

Gli36-EGFRvIII (A,B), U87 (C,D), and U251 (E,F) human glioma cells were transduced with S-TRAIL or Gluc-DsRed2 at increasing MOI (1 000-10 000) and 24 h post-transduction both cell viability (A,C,E) and caspase activation (B,D,F) were determined using bioluminescent-based assays. *p<0.05 vs. control.

Figure 3. Real-time assessment of AAV transgene expression and effects of S-TRAIL on human glioma xenograft progression in vivo

(A) Flow cytometry analysis of Gli36-EGFRvIII human glioma cells transduced with LV-GFP-Fluc. Gli36-EGFRvIII glioma cells expressing GFP-Fluc were implanted subcutaneously into nude mice and 48 and 96 h post-implantation, 1×10⁹ particles of S-TRAIL or Gluc-DsRed2 was injected directly into the tumor mass (n=4 tumors/group, Day 1). Viral transgene expression and glioma progression were subsequently monitored by dual bioluminescent imaging. (B) 10 days after delivery of AAV-Gluc-DsRed2, mice were injected with coelenterazine and imaged for viral transgene expression. Forty-eight hours later, mice were injected with d-luciferin to visualize tumor mass. Representative images are shown to demonstrate co-localization of viral expression and glioma mass. (C) Representative images and summary data demonstrating the effects of AAV-S-TRAIL on tumor progression 1, 4, 7, and 12 days after AAV-S-TRAIL or AAV-Gluc-DsRed2 injection. (D) Seven days post-AAV-S-TRAIL or AAV-Gluc-DsRed2 injection, tumors were removed, sectioned, and stained for caspase activation. *p<0.05 vs. control.

Figure 4. TMZ induces cell cycle checkpoint arrest and upregulates death receptors on glioma cells

(A-F) Summary data of Gli36-EGFRvIII (A-C) and U251 (D-F) human glioma cells treated with TMZ (100 μM) for 24 (B,E), 72 h (C,F), or left untreated (A,D). Cells were pulsed for 1 h with bromodeoxyuridine, collected, stained, and cell cycle progression was analyzed by FACS. Data is expressed as percentage of cells in subG1, G1, S, or G2/M (G-I) Representative scatter plots of untreated Gli36 cells (G) and cells treated for 24 (H) or 72 h (I) with TMZ. (J) Western blot analysis demonstrating the effects of TMZ on the levels of total Chk1 and phosphorylated (p)Chk1. (K) Western blot analysis showing the expression of death receptor DR4 in U251 cells treated with TMZ for 24 or 72 h. All data is normalized to control cells (untreated). *p<0.05 vs. untreated cells.

Figure 5. Synergistic cell killing by TMZ and S-TRAIL in human glioma cell lines

Human glioma cell lines Gli36-EGFRvIII (A,B)and U251 (C,D) were treated with increasing concentrations of TMZ alone (0-1000 μM), control virus (AAV-Gluc-DsRed2: 4 000 MOI), S-TRAIL alone (AAV-S-TRAIL: 0-4 000 MOI; NSC-S-TRAIL: 0-600 ng/mL), or a combination of TMZ and S-TRAIL. Eighteen h later, cell viability was determined by luciferase-based assay. All data is normalized to control cells (TMZ 0 μM, AAV-Gluc-DsRed2 or NSC-S-TRAIL 0 ng/mL). *p<0.05 vs. control. †p<0.05 vs. S-TRAIL alone.

Figure 6. TMZ pre-treatment enhances the synergistic effects of TMZ and S-TRAIL in TRAIL-resistant glioma cells

Summary data showing the viability of U251 glioma cells following concomitant TMZ and S-TRAIL treatment, or following 24 h of TMZ sensitization. Cells were left untreated (ns) or sensitized (s) with TMZ for 24 h. Both sensitized and untreated cells were subsequently treated or re-treated with TMZ alone, control virus (AAV-Gluc-DsRed2: 4 000 MOI), S-TRAIL alone (Low: AAV-S-TRAIL=1 000 MOI, NSC-S-TRAIL=200 ng/mL; High: AAV-S-TRAIL=4 000 MOI, NSC-S-TRAIL=600 ng/mL), or a combination of TMZ and S-TRAIL for an additional 18 h at which time cell viability (A,B) or caspase 3/7 activation (C) were determined. Representative images and summary data of western blot analysis showing the expression of cleaved PARP (D) and immunocytochemical analysis of cleaved caspase-3 expression (E) in U251 cells following treatment with TMZ, NSC-S-TRAIL, concomitant TMZ and S-TRAIL, or TMZ and S-TRAIL following 24 h of TMZ sensitization. All data is normalized to control cells (TMZ 0 μM, AAV-Gluc-DsRed2 or S-TRAIL 0 ng/mL). *p<0.05 vs. S-TRAIL/TMZ no pretreatment.