Demethoxycurcumin Suppresses Human Brain Glioblastoma Multiforme GBM 8401 Cell Xenograft Tumor in Nude Mice In Vivo

Abstract

Demethoxycurcumin (DMC), a derivate of curcumin, has been shown to induce apoptotic cell death in human glioblastoma multiforme GBM 8401 cells via cell cycle arrest and induction of cell apoptosis. However, there is no report showing DMC suppresses glioblastoma multiforme cells in vivo. In the present study, we investigated the effects of DMC on GBM8401 cells in vivo. At first, we established a luciferase-expressing stable clone named GBM 8401/luc2. Second, mice were inoculated subcutaneously with GBM 8401/luc2 cells to generate a xenograft tumor mice model. After inoculation, tumor volume reached 100-120 mm³, and all mice were randomly divided into three groups: Group I was treated with 110 µL phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 30 mg/kg of DMC, and Group III with 60 mg/kg of DMC. Mice from each group were given the oral treatment of DMC by gavage for 21 days. The body weight and tumor volume were recorded every 3 days. DMC significantly decreased the tumor volumes, and 60 mg/kg treatment showed a higher decrease in tumor volumes than that of 30 mg/kg, However, DMC did not affect the body weights. The photons emitted from mice tumors were detected with Xenogen IVIS imaging system, DMC at both doses decreased the total photon flux and 60 mg/kg treatment of DMC has low total photon flux than that of 30 mg/kg. The tumor volumes and weights in 60 mg/kg treatment of DMC were lower than that of 30 mg/kg. Immunohistochemical analysis was used to measure protein expression of tumors and results showed that DMC treatment led to lightly staining with anti-Bcl-2 and -XIAP and 60 mg/kg treatment of DMC has lighter staining with anti-Bcl-2 and -XIAP than that of 30 mg/kg. The higher dose (60 mg/kg) of DMC has higher signals of cleaved-caspase-3 than that of the lower dose (30 mg/kg). Furthermore, the hematoxylin and eosin (H&E) staining of liver tissues showed no significant difference between DMC-treated and control-groups. Overall, these observations showed that DMC suppressed tumor properties in vivo and DMC may be used against human glioblastoma multiforme in the future.

Keywords: demethoxycurcumin (DMC); glioblastoma multiforme; in vivo; nude mice; xenograft tumor.

Figure 1

Tumor growth was inhibited by DMC treatment. (A) An animal experiment flow chart was displayed. (B) Tumor volume was measured by caliper every three days and quantified. (C) Tumors were isolated and photographed from each group of mice on day 21. (D) Tumor weight was also measured and quantified. (a¹ p < 0.05 and a² p < 0.01 vs. control; b¹ p < 0.05 and b² p < 0.01 vs. DMC 30 mg/kg).

Figure 2

Luc2 signals from living tumor cells were suppressed by DMC treatment. (A) The representative BLI results from each group at different time points. (B) Quantification results of luc2 signal intensity of tumors. (a² < 0.01 vs. control; b¹ p < 0.05 and b² p < 0.01 vs. DMC 30 mg/kg).

Figure 3

Apoptosis-related proteins were modulated by DMC treatment. (A) The IHC staining images of Bcl-2 and XIAP were observed by microscope with 100 times magnification. (B) Quantification results of Bcl-2 and XIAP protein levels as compared to control. (C) The IHC staining images of cleaved caspase-3 and BAX were observed by microscope with 100 times magnification. (D) Quantification results of cleaved caspase-3 and BAX protein levels as compared to control. (a² < 0.01 vs. control; b¹ p < 0.05 vs. DMC 30 mg/kg).

Figure 4

No acute or delayed toxicity was found in DMC-treated glioblastoma-bearing mice. (A) Liver pathology photograph from the microscope with 100 times magnification. (B) Mouse body weight was measured and recorded every three days.