Baicalin suppresses proliferation, migration, and invasion in human glioblastoma cells via Ca2+-dependent pathway

Yihao Zhu,1,* Jiang Fang,2,* Handong Wang,1 Maoxing Fei,3 Ting Tang,1 Kaichao Liu,4 Wenhao Niu,2 Yali Zhou1 1Department of Neurosurgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, Jiangsu 210002, China; 2Department of Neurosurgery, Jinling Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210002, China; 3Department of Neurosurgery, Jinling Hospital, Nanjing Medical University, Nanjing, Jiangsu 210002, China; 4Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China *These authors contributed equally to this work Objective: Baicalin, a kind of flavonoid extracted from the dry root of Scutellaria, possesses potent anticancer bioactivities in various tumor cell lines. Accumulating evidences show that baicalin induces autophagy and apoptosis to suppress the cancer growth. Moreover, the antineoplastic role of baicalin in human glioblastoma cells remains to be uncovered.Methods: Both U87 and U251 human glioblastoma cell lines were employed in the present study. Cell viability was tested by Cell Counting Kit-8 and colony-forming assay; Flow cytometry was employed to analyze cell apoptosis, cell cycle, and Ca2+ content. Cell immunofluorescence assays were used for analyzing terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), light chain 3 beta (LC3B), 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanineiodide (JC-1), and Ca2+ content. The protein levels were tested by Western blot. The SPSS software was used for statistical analysis.Results: Baicalin suppressed the proliferation, migration, and invasion ability of human glioblastoma cells in a dose-dependent manner. Baicalin induced the loss of mitochondrial membrane potential and led to mitochondrial apoptosis. The maturation of microtubule-associated protein 1A/1B-LC3B indicated the activation of autophagy potentially through PI3K/Akt/mTOR pathway, and inhibition of autophagy by 3-methyladenine decreased the apoptotic cell ratio. Besides, baicalin increased the intercellular Ca2+ content; meanwhile, chelation of free Ca2+ by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid inhibited both apoptotic and autophagy. Finally, baicalin suppressed tumor growth in vivo.Conclusion: Our observations suggest that baicalin exerts cytotoxic effects on human glioblastoma cells by the autophagy-related apoptosis through Ca2+ movement to the cytosol. Furthermore, baicalin has the potential as a candidate for the treatment of glioblastoma. Keywords: baicalin, glioblastoma, autophagy, mitochondrial apoptosis, PI3K/Akt/mTOR pathway, Ca2+-dependent pathway