Abstract
Xihuang pill (XHP), a traditional Chinese herbal formula, has long been used as an effective agent against multiple tumors. The aim of this study is to evaluate the effects of XHP on the growth inhibition and apoptosis in glioblastoma U-87 MG cells. Gas chromatography-mass spectrometry (GC-MS) was performed for constituent analysis of XHP. Cell viability, cell cycle arrest, generation of reactive oxygen species (ROS), and apoptosis were measured by CCK-8 assay, PI/RNase staining, DCFH-DA assay, TUNEL assay, Annexin V-FITC/PI double staining, and JC-1 assay, respectively. The role of XHP in the regulation of Akt/mTOR/FOXO1 interaction was clarified by using Western Blotting (WB), immunofluorescence (IF), pharmacological inhibitor or antioxidant, and siRNA silencing. The results suggested that XHP could inhibit U-87 MG cells growth and arrest cells in S-phase cell cycle significantly and that the generation of ROS, collapse of mitochondrial membrane potential, enhancement of Bax/Bcl-xL ratio, and reduction of the precursor forms of caspase-9 and caspase-3 caused by XHP prompted that a ROS-mediated mitochondria-dependent apoptosis was possibly involved. Furthermore, XHP affected the Akt/mTOR/FOXO1 pathway via inhibiting the phosphorylation of Akt, mTOR, and FOXO1 and increasing both prototype and nuclear translocation of FOXO1. Inhibition of Akt, mTOR, and FOXO1 by specific inhibitors or siRNA could interpose the apoptotic induction. In conclusion, we demonstrate for the first time that XHP may regulate glioblastoma U-87 MG cell apoptosis via ROS-mediated Akt/mTOR/FOXO1 pathway.
Highlights
Gliomas, a type of highly heterogeneous tumor, have been the leading lethal causes in primary central nerve system (CNS) tumors
The experimental research showed that its ability to induce apoptosis in cancer cells or cancer stem cells (CSCs) was possibly attributed to the mitochondrial-related B-cell lymphoma- (Bcl-) 2 regulation [10, 11], extracellular signalregulated kinase (ERK)/mitogen-activated protein kinase (MAPK) [12], or Wnt [13] signaling pathways, as well as cell cycle arrest [14, 15]
We investigated the effects of Xihuang pill (XHP) on human U-87 MG glioblastoma cell growth and its underlying mechanisms related to reactive oxygen species (ROS)-mediated Akt/Mammalian target of rapamycin (mTOR)/FOXO1 pathway, so as to provide some experimental data to the further research and development on antiglioblastoma application of XHP
Summary
A type of highly heterogeneous tumor, have been the leading lethal causes in primary central nerve system (CNS) tumors. According to the 2016 World Health Organization (WHO) classification of CNS tumors, gliomas in adults are mainly included astrocytoma (Grades I-IV), oligodendroglioma (Grades II-III), oligoastrocytoma (Grades IIIII), and glioblastoma multiforme (GBM) (Grade IV) [1, 2]. Xihuang pill (XHP, called Xihuang Wan), a formula composed by four traditional Chinese medicines, Olibanum, Myrrh, Moschus, and Calculus bovis, has been used as a complementary and alternative medicine for tumor treatment in China since 18th century [4]. XHP has been proved effectively on various solid tumors including breast cancer [5], cervical cancer [6], glioma [7], colorectal cancer [8], and non-Hodgkin’s lymphoma [9]. XHP exhibited the capability of converting tumor immunosuppressive microenvironment by reducing proportion of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and Treg cells [19], or by downregulating expression of cytokines such as interleukin- (IL-) 6, IL-10, and transforming growth factor(TGF-) β [20] or by increasing IL-2, Interferon- (IFN-) γ, and the ratio of CD4+/CD8+ T cells [21, 22]
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