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Micheliolide derivative DMAMCL inhibits glioma cell growth in vitro and in vivo.

An Y, Guo W, Li L, Xu C, Yang D, Wang S, Lu Y, Zhang Q, Zhai J, Fan H, Qiu C, Qi J, Chen Y, Yuan S - PLoS ONE (2015)

Bottom Line: DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner.Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma.Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory Center, Chinese PLA Air Force General Hospital, Haidian, Beijing 100142, PR China.

ABSTRACT

Background: There is no highly effective chemotherapy for malignant gliomas to date. We found that dimethylaminomicheliolide (DMAMCL), a selective inhibitor of acute myeloid leukemia (AML) stem/progenitor cells, inhibited the growth of glioma cells.

Methods: The distribution of DMAMCL in brain was analyzed by an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) system. The anti-tumor evaluations of DMAMCL in vitro were performed by MTT, FACS and RT-PCR. In vivo, the mixture of C6 cells and matrigel was injected into caudatum, and the anti-tumor activity of DMAMCL was evaluated by tumor growth and rat survival. The toxicity of DMAMCL was evaluated by body weight, daily food intake, hematological or serum biochemical analyses, and histological appearance of tissues.

Results: The IC50 values of DMAMCL against the C6 and U-87MG cell lines in vitro were 27.18 ± 1.89 μM and 20.58 ± 1.61 μM, respectively. DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner. In a C6 rat tumor model, daily administration of DMAMCL for 21 days reduced the burden of C6 tumors by 60% to 88% compared to controls, and more than doubled the mean lifespan of tumor-bearing rats. Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma. Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity.

Conclusions: These results suggest that DMAMCL is highly promising for the treatment of glioma.

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Related in: MedlinePlus

DMAMCL-induced apoptosis.C6 and U-87MG cells were treated with DMAMCL. After 24 h, cells were collected for Annexin V and PI detection or total protein extraction. (A) Representative FACS results. (B) and (C) The percentage of cell apoptosis (the total of early and late apoptosis) for C6 and U-87MG cells, respectively. (D) and (E) Western blotting detection for Bax, Bcl-2 and β-actin protein expression for C6 and U-87MG cells, respectively. One-way ANOVAs followed by Tukey’s post-hoc tests were used and statistical significance is indicated by asterisks (*p < 0.01 compared to vehicle; **p < 0.01 compared to vehicle; ***p < 0.001 compared to vehicle).
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pone.0116202.g004: DMAMCL-induced apoptosis.C6 and U-87MG cells were treated with DMAMCL. After 24 h, cells were collected for Annexin V and PI detection or total protein extraction. (A) Representative FACS results. (B) and (C) The percentage of cell apoptosis (the total of early and late apoptosis) for C6 and U-87MG cells, respectively. (D) and (E) Western blotting detection for Bax, Bcl-2 and β-actin protein expression for C6 and U-87MG cells, respectively. One-way ANOVAs followed by Tukey’s post-hoc tests were used and statistical significance is indicated by asterisks (*p < 0.01 compared to vehicle; **p < 0.01 compared to vehicle; ***p < 0.001 compared to vehicle).

Mentions: We analyzed the ability of DMAMCL to induce apoptosis in C6 and U-87MG cells using flow cytometry and Western blots (Fig. 4). Dual-labeled fluorescence activated cell sorting (FACS) analysis (Annexin V and PI) was used to measure early and late apoptosis and necrosis of C6 and U-87MG cells. Annexin V detects early apoptosis, whereas cells positive for PI and Annexin V are in late apoptosis. By 24 hr post-treatment, DMAMCL significantly increased early and late apoptosis and necrosis of C6 and U-87MG cells in a dose-dependent manner (Fig. 4 A, B and C). There were few surviving C6 or U-87MG cells after treatment with DMAMCL at 240 μM. The Bcl-2 family of regulator proteins influence apoptosis and have been widely studied in glioma cells and the development of MG [24]. Hence, we investigated whether DMAMCL could reduce anti-apoptotic protein Bcl-2 expression and induce pro- apoptotic protein Bax expression. Western blots showed that DMAMCL at 60 μM and 120μM up-regulated Bax expression and down-regulated Bcl-2 expression (Fig. 4 D and E).


Micheliolide derivative DMAMCL inhibits glioma cell growth in vitro and in vivo.

An Y, Guo W, Li L, Xu C, Yang D, Wang S, Lu Y, Zhang Q, Zhai J, Fan H, Qiu C, Qi J, Chen Y, Yuan S - PLoS ONE (2015)

DMAMCL-induced apoptosis.C6 and U-87MG cells were treated with DMAMCL. After 24 h, cells were collected for Annexin V and PI detection or total protein extraction. (A) Representative FACS results. (B) and (C) The percentage of cell apoptosis (the total of early and late apoptosis) for C6 and U-87MG cells, respectively. (D) and (E) Western blotting detection for Bax, Bcl-2 and β-actin protein expression for C6 and U-87MG cells, respectively. One-way ANOVAs followed by Tukey’s post-hoc tests were used and statistical significance is indicated by asterisks (*p < 0.01 compared to vehicle; **p < 0.01 compared to vehicle; ***p < 0.001 compared to vehicle).
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pone.0116202.g004: DMAMCL-induced apoptosis.C6 and U-87MG cells were treated with DMAMCL. After 24 h, cells were collected for Annexin V and PI detection or total protein extraction. (A) Representative FACS results. (B) and (C) The percentage of cell apoptosis (the total of early and late apoptosis) for C6 and U-87MG cells, respectively. (D) and (E) Western blotting detection for Bax, Bcl-2 and β-actin protein expression for C6 and U-87MG cells, respectively. One-way ANOVAs followed by Tukey’s post-hoc tests were used and statistical significance is indicated by asterisks (*p < 0.01 compared to vehicle; **p < 0.01 compared to vehicle; ***p < 0.001 compared to vehicle).
Mentions: We analyzed the ability of DMAMCL to induce apoptosis in C6 and U-87MG cells using flow cytometry and Western blots (Fig. 4). Dual-labeled fluorescence activated cell sorting (FACS) analysis (Annexin V and PI) was used to measure early and late apoptosis and necrosis of C6 and U-87MG cells. Annexin V detects early apoptosis, whereas cells positive for PI and Annexin V are in late apoptosis. By 24 hr post-treatment, DMAMCL significantly increased early and late apoptosis and necrosis of C6 and U-87MG cells in a dose-dependent manner (Fig. 4 A, B and C). There were few surviving C6 or U-87MG cells after treatment with DMAMCL at 240 μM. The Bcl-2 family of regulator proteins influence apoptosis and have been widely studied in glioma cells and the development of MG [24]. Hence, we investigated whether DMAMCL could reduce anti-apoptotic protein Bcl-2 expression and induce pro- apoptotic protein Bax expression. Western blots showed that DMAMCL at 60 μM and 120μM up-regulated Bax expression and down-regulated Bcl-2 expression (Fig. 4 D and E).

Bottom Line: DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner.Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma.Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory Center, Chinese PLA Air Force General Hospital, Haidian, Beijing 100142, PR China.

ABSTRACT

Background: There is no highly effective chemotherapy for malignant gliomas to date. We found that dimethylaminomicheliolide (DMAMCL), a selective inhibitor of acute myeloid leukemia (AML) stem/progenitor cells, inhibited the growth of glioma cells.

Methods: The distribution of DMAMCL in brain was analyzed by an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) system. The anti-tumor evaluations of DMAMCL in vitro were performed by MTT, FACS and RT-PCR. In vivo, the mixture of C6 cells and matrigel was injected into caudatum, and the anti-tumor activity of DMAMCL was evaluated by tumor growth and rat survival. The toxicity of DMAMCL was evaluated by body weight, daily food intake, hematological or serum biochemical analyses, and histological appearance of tissues.

Results: The IC50 values of DMAMCL against the C6 and U-87MG cell lines in vitro were 27.18 ± 1.89 μM and 20.58 ± 1.61 μM, respectively. DAMMCL down-regulated the anti-apoptosis gene Bcl-2 and increased apoptosis in C6 and U-87MG cells in a dose-dependent manner. In a C6 rat tumor model, daily administration of DMAMCL for 21 days reduced the burden of C6 tumors by 60% to 88% compared to controls, and more than doubled the mean lifespan of tumor-bearing rats. Distribution analysis showed that the DMAMCL concentration was higher in the brain than in plasma. Evaluations for toxicity revealed that oral administration of DMAMCL at 200 or 300 mg/kg once a day for 21 days did not result in toxicity.

Conclusions: These results suggest that DMAMCL is highly promising for the treatment of glioma.

Show MeSH
Related in: MedlinePlus