| Summary: | 碩士 === 國立成功大學 === 藥理學研究所 === 105 === Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumors of the central nervous system. Although the standard treatment are well-development, the median survival rate is still very short within 12 to 15 months. The CD133 glioblastoma-derived stem cells (GSCs) are responsible for chemo-/radiation-resistance and cancer recurrence. Recently, targeting on GSCs becomes an important part of the therapeutic strategies. Imipramine (Imi), a tricyclic antidepressant drug (TCA), has been reported to inhibit glioma cells growth. However, the effectiveness and underlying mechanism by which Imi inhibits GSCs progression remains unknown. Previously, our laboratory isolated GSCs with high expression of tumor stem cells marker CD133 from the U87MG cell line. In this study, treatment with Imi significantly decreased the cell survival of GSCs in a concentration-dependent manner. Imi also showed a significant cytotoxicity in the TMZ-resistant and TMZ plus radiation-resistant U87 cells. In addition, a significant decrease in GSCs-derived neurosphere formation was observed in response to Imi treatment. Furthermore, we demonstrated that Imi increased the conversion of LC3-II within 48 h, increased p62 accumulation within 12 h and a significant increase in GFP-LC3 puncta formation within 48 h. Co-treatment with chloroquine, a lysosomal inhibitor, enhanced the Imi-induced cell death and Imi-induced expression of p62 and LC3-II in GSCs. In contrast, co-treatment with autophagy inhibitor 3-MA reversed the Imi-induced reduction in neurosphere formation. Similarly, the conversion of LC3-I to LC3-II and activation of caspase-3-induced by Imi were reversed by 3-MA. In addition, co-treatment with 3-MA attenuated the Imi-induced cell death. These results showed that Imi induced an impaired autophagic cell death. We further investigated whether the Imi-induced cell death was associated with apoptosis. We found that cleaved caspase-3 was detected after 48 h treatment with Imi. Combination of 3-MA and Z-DEVD-FMK could significantly reverse the effect of Imi on the survival of GSCs. Imi treatment significantly deceased the anti-apoptotic protein Bcl-2 expression, which further demonstrate the apoptosis was activated in response to Imi-induced cell death. We further investigated the anti-tumor activity of Imi in vivo. The results revealed that Imi significantly reduced the tumor growth, prolonged the survival time and prevented the body weight loss in the Imi-treated animals compared with vehicle group.
In summary, we have demonstrated Imi inhibited GSCs proliferation in vitro and in vivo, which was mediated by the induction of autophagic and apoptotic cell death. With the advantage of being able to cross the blood-brain barrier, we suggest Imi may be used to combine with the standard treatment for curing the GBM.
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