The dual PI3K/mTOR inhibitor dactolisib elicits anti-tumor activity in vitro and in vivo

Abstract

Glioblastomas (GBMs) are among the most malignant of all human tumors and have poor prognosis. The current standard of care (SOC) includes maximal surgical tumor resection followed by adjuvant temozolomide (TMZ) and concomitant radiotherapy (RT). However, even with this treatment, the 5-year survival rate is less than 10%, and thus, follow-up treatment is required to improve efficacy. In GBMs as well as many other solid cancers, PI3K/mTOR signaling is overactivated. Therefore, multiple tumor-based PI3K inhibitors have been studied in various cancers. In the current study, we investigated the effect of the dual PI3K/mTOR inhibitor dactolisib on TMZ+RT treatment in three human GBM cell lines and a orthotopic xenograft model. Dactolisib alone induced cytotoxicity and pro-apoptotic effects, which act as antitumor factors. Combined with SOC treatment, dactolisib inhibited cell viability, induced enhanced pro-apoptotic effect, and attenuated migration/invasion in all three cell lines, thereby enhancing the SOC therapeutic effect. Protein microarray analysis showed that A172 cells treated with TMZ+RT+dactolisib had higher p27 and lower Bcl-2 expression than other groups. Moreover, in the xenograft model, oral dactolisib combined with TMZ+RT inhibited tumor growth and prolonged survival. Thus, SOC combined with dactolisib shows potent anti-tumor activity and has promising potential for solid tumor treatment.

Keywords: chemotherapy; dactolisib (NVP-BEZ235); dual PI3K/mTOR inhibitor; glioblastoma; radiotherapy.

Figure 1. Dactolisib treatment enhances the reduction in GBM cell viability by TMZ+RT

(A) Cell viability in A172, SHG44, and T98G GBM cells treated with 25–200 μM TMZ for 24 h. (B) Cell viability in A172, SHG44, and T98G GBM cells treated with 10–80 nM dactolisib. Cell viability was assessed after 48 h of drug exposure. *P < 0.05 vs. control. (C) Cell viability in A172, SHG44, and T98G GBM cells treated with 20 nM dactolisib for 24, 72, and 48 h. (D) Cell viability in A172 cells exposed to TMZ+RT or TMZ+RT+dactolisib for 48 h. *P < 0.05 vs. control. DMSO served as a vehicle control. Cell viabilities were assessed by CCK-8. Treatments included TMZ (100 μM), RT (2 Gy), and dactolisib (20 nM).

Figure 2. Dactolisib enhances the inhibitory effect of TMZ+RT on migration of glioma cells

(A) Micrographs showing cell migration of A172, SHG44, and T98G cells exposed to dactolisib, TMZ+dactolisib, or TMZ+RT+dactolisib for 24 hours. (B) Quantitative data of cell migration as shown in panel (A) Data are from three independent experiments. *P < 0.05 vs. control. Treatments included TMZ (100 μM), RT (2 Gy), and dactolisib (20 nM).

Figure 3. Dactolisib enhances the inhibitory effect of TMZ+RT on invasion of glioma cells

(A) Micrographs showing cell invasion of A172, SHG44, and T98G cells exposed to dactolisib, TMZ+dactolisib, or TMZ+RT+dactolisib for 24 hours. (B) Quantitative data of cell invasion as shown in panel (A). Data are from three independent experiments. *p < 0.05 from control. Treatments included TMZ (100 μM), RT (2 Gy), and dactolisib (20 nM).

Figure 4. Dactolisib enhances the pro-apoptotic effect of TMZ+RT in glioma cells

(A) Representative flow-cytometric results of the effects of dactolisib, RT, RT+TMZ, and TMZ+RT+dactolisib in A172 cells. Cells were treated for 24 hours. (B) Histograms showing the percentage of apoptotic cells in A172, SHG44, and T98G cells treated with dactolisib, RT, RT+TMZ, and TMZ+RT+dactolisib. Data are from three independent experiments. Treatments included TMZ (100 μM), RT (2 Gy), and dactolisib (20 nM).

Figure 5. Treatment with TMZ, RT, and dactolisib induces cell-cycle arrest in glioma cells

(A) Representative flow-cytometric results showing the percentages of cells in G1, S, or G2-M phase in T98G, SHG44, and A172 cells exposed to dactolisib, RT+TMZ, or TMZ+RT+dactolisib for 24 hours. (B) Histograms showing the percentages of glioma cells in G1/G0, S, and G2/M phases. Treatments included TMZ (100 μM), RT (2 Gy), and dactolisib (20 nM).

Figure 6. Cell-cycle arrest in glioma cells treated with TMZ, RT, and dactolisib in different time point

(A) Representative flow-cytometric results showing the percentages of cells in G1, S, or G2-M phase in A172 cells after the indicated treatments for 12, 24, and 48 h. (B) Histograms showing the percentages of glioma cells in G1/G0, S, and G2/M phases. Treatments included TMZ (100 μM), RT (2 Gy), and dactolisib (20 nM).

Figure 7. MGMT expression and dactolisib intervention on PI3K/mTOR pathway

(A) O6-Methylguanine DNA methyltransferase (MGMT) expression was determined by western blot analysis. Data were obtained from three independent experiments. MGMT levels were reduced by dactolisib in T98G cells. (B) All three cell lines were treated with TMZ (100 μM), dactolisib (20 nM), RT (2 Gy), or the indicated combination for 24 h. Total protein level and phosphorylation status of AKT, Bcl-2, and p27 were determined by western blot analysis. β-Actin served as a loading control.

Figure 8. Dactolisib inhibits tumor growth in vivo and prolongs survival of orthotopic glioblastoma-bearing rats

(A) Axial 3D fast spoiled gradient-echo images. Tumor volumes were significantly different among 3 groups on days 10 and 14. (B) Kaplan–Meier method was used to assess animal survival time. The survival time of rats in the RT+TMZ+dactolisib group was significantly longer than other groups.