Combination therapy with micellarized cyclopamine and temozolomide attenuate glioblastoma growth through Gli1 down-regulation

Abstract

Glioblastoma multiforme (GBM) is the most common and deadly brain cancer, characterized by its aggressive proliferation to adjacent tissue and high recurrence rate. We studied the efficacy and related mechanisms of the combination of cyclopamine (Cyp, a Sonic-hedgehog pathway (Shh) inhibitor) and temozolomide (TMZ, the clinically most used chemotherapeutic agent) in anti-GBM treatment. The micellarized Cyp (MCyp) showed better performance than Cyp solution in inhibiting GBM cells proliferation (3.77-fold against U87 MG cells and 3.28-fold against DBTRG-05MG cells) and clonogenity (1.35-fold against U87 MG cells and 2.17-fold against DBTRG-05MG cells), and preferred behavior of inhibiting cell invasion, colony formation through attenuated Gli1 expression. In addition, combination of MCyp and TMZ exhibited synergistic cytotoxicity, correlating with their ability in inducing apoptosis and eliminating neurospheres formation, and the combination of TMZ was accompanied with the enhanced blockage of Shh pathway. The optimal ratio of MCyp combined to TMZ was 1:20. So we proposed to use TMZ to kill tumor parenchyma and MCyp as the cancer stem cells inhibitor to resist tumor recurrence. These findings demonstrated that combination of TMZ with micellarized Cyp is a promising strategy for exerting different functions of drugs for tumor treatment.

Keywords: cyclopamine; glioma; hedgehog pathway; synergistic effect; temozolomide.

Figure 1

Size of MCyp determined by dynamic light scattering (A) and transmission electron microscope (B).

Figure 2

Survival rate of DBTRG-05MG cells (A) and U87 MG cells (B) cultured with Cyp solution, MCyp and the corresponding blank micelles for 48 h. Data are presented as the mean ± SEM (n = 6). Colony formation number of DBTRG-05MG cells (C) and U87 MG cells (D) after different concentrations of Cyp solution and MCyp treatment. Data are presented as the mean ± SEM (n = 3).

Figure 3

Representative images of the wound healing assay at 0 and 18 h for DBTRG-05MG (A) and U87MG (B) cells. The final concentration of Cyp was 5 μM.

Figure 4

Image of migration of DBTRG-05MG cells (A), U87 MG cells (B) after incubation with Cyp solution, MCyp and blank micelles for 30 h, respectively (magnification 10×). The final concentration of Cyp was 5 μM. Quantitative analysis of the relative percentage of DBTRG-05MG (C) and U87 MG cells (D) migrated compared to the control group. Data are presented as the mean ± SD (n = 17). Asterisks denote significant P < 0.001.

Figure 5

Image of invasion of DBTRG-05MG cells (A), U87 MG cells (B) after incubation with Cyp solution, MCyp and blank micelles for 30 h, respectively (magnification 10×). The final concentration of Cyp was 5 μM. Quantitative analysis of the relative percentage of DBTRG-05MG (C) and U87 MG cells (D) invaded compared to the control group. Data are presented as the mean ± SD (n = 22). Asterisks denote significant P < 0.001.

Figure 6

Survival rate of DBTRG-05 MG cells (A, B) and U87 MG cells (C, D) cultured with different concentrations of TMZ and fixed concentrations of Cyp solution (A, C) or MCyp (B, D) for 48 h. Data are presented as the mean ± SEM (n = 6).

Figure 7

Cytotoxicity study of free TMZ, MCyp and both drug combinations at various molar ratios after 72 h (A) and the corresponding CI vs Fa plot (B) of U87 MG cells. The concentration of the X-axis represents the molar concentration of TMZ, in addition to the MCyp group representing the Cyp concentration. Data are presented as the mean ± SD (n = 6).

Figure 8. Inverted microscope photographs on Day 0~4 of U87 MG tumor spheroids after exposed to different formulations

(A) The inhibitory effect on the growth of U87 MG spheroids after the application of drugs (B). Tumor volume of the fourth day compared to the initial volume of the spheroids (C). Data are presented as the mean ± SD (n = 6). 1/2(T+MCyp) and 1/2(T+Cyp) represent 1/2(TMZ+MCyp) and 1/2(TMZ+Cyp), respectively. (C)*P < 0.05; **P < 0.001.

Figure 9. The representative pictures of the DBTRG-05MG neurospheres after treated with of PBS, Cyp solution, MCyp, TMZ, TMZ+Cyp, TMZ+MCyp, and 1/2(TMZ+MCyp)

(A) Typical apoptosis profiles (B) and quantitative analysis (C) of the collected cells from the neurospheres after incubated with control (a1), MCyp (a2), TMZ (a3), TMZ+MCyp (a4), 1/2(TMZ+MCyp) (a5). LL, UL, LR and UR quadrant represents viable cells, necrotic cells, early and late apoptotic cells, respectively. Data are presented as the mean ± SD (n = 3).

Figure 10. Gli1 protein level of U87 MG cells after treatment of PBS (as control), Cyp solution, MCyp, TMZ, TMZ+Cyp, and TMZ+MCyp

The final concentration of the Cyp and TMZ are 30 μM and 600 μM, respectively. Data are presented as the mean ± SD (n = 3).