For the first time, we propose the role of TSN in the inhibition of the viability and metastasis of glioma cells through the promotion of induced apoptosis and inhibition of the PI3K/Akt/mTOR pathway

For the first time, we propose the role of TSN in the inhibition of the viability and metastasis of glioma cells through the promotion of induced apoptosis and inhibition of the PI3K/Akt/mTOR pathway. First, the CCK-8 assay showed the dose- and time-dependent cytotoxicity of TSN on glioma cells (U87MG, LN18, U251, LN229); furthermore, it showed the limited toxicity of TSN on normal human astrocytes. pathways as demonstrated Rabbit Polyclonal to Doublecortin (phospho-Ser376) by western blotting analysis. In addition, the effects of TSN on glioma cell viability, apoptosis, cell cycle arrest, migration, and invasion were reversed by 740Y-P, a PI3K activator. Finally, the mouse xenograft model Sabinene confirmed the suppressive effect of TSN on tumor growth in vivo. Conclusion Our results suggest that TSN is usually a promising chemotherapeutic drug for patients with glioma. strong class=”kwd-title” Keywords: Toosendanin, Glioma, Apoptosis, Proliferation, PI3K, Akt, mTOR pathway Introduction Gliomas are tumors originating from brain glial cells such as astrocytes, oligodendrocytes, and ependymal cells. Among its different types, glioblastomas (GBM) are the most Sabinene common intracranial tumor with the highest degree of malignancy, accounting for approximately half of the incidence, i.e., 3C4 per 100,000 [1]. The classic Stupp protocol is the most common and effective treatment for GBM. This protocol includes total or subtotal resection within the scope of safety and postoperative conventional radiotherapy plus adjuvant chemotherapy using temozolomide [2, 3]. In addition, other treatments include tumor treating fields, targeted medication, immunotherapy, and traditional Chinese medicine [4, 5]. However, the high degree of malignancy, rapid growth, and high recurrence rate of GBM has resulted in an extremely low survival rate despite completion of active treatment. A study found ?13% 5-year survival rate and ?20.5?months survival time in patients with GBM [6]. Therefore, relevant preclinical studies are warranted to determine an effective and safe treatment for GBM. Toosendanin (TSN) is usually a tetracyclic triterpenoid derived from the Chuan neem seed or blast. It is mainly used as an ascaris repellent; additionally, it has biological activities including anti-botulism, neuromuscular junction inhibition, and central nervous and respiratory system effects [7]. A large number of studies have shown the efficacy of natural products in cancer treatment, particularly, in cancer drug Sabinene research [8C10]. Among them, TSN has recently been shown to possess anti-tumor activity in many tumor cells including gastric, lung, pancreatic, colorectal, liver, and breast malignancy cells [11C17]. However, studies regarding the major pharmacological and molecular mechanisms of TSN on glioma cells remain lacking. Therefore, this study aimed to explore the anti-tumor effects of TSN on glioma cell lines (U87MG and LN18) in vivo and in vitro and potentially elucidate the underlying mechanisms. Methods Reagents TSN (purity of approximately 99%) was purchased from Chroma Biotechnology Company (Chengdu, China). TSN was dissolved in dimethyl sulfoxide (DMSO), which was diluted with intact cell culture medium to achieve the concentration required for the experiment. The final concentration of DMSO was ?0.1% to avoid side effects. Dulbeccos altered Eagles medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher (Waltham, MA, USA). Penicillin/streptomycin (100) and phosphate-buffered saline (PBS) were purchased from Shanghai Basal Media Technologies Co., Ltd. (Shanghai, China). Cell counting Kit-8 (CCK-8), BCA Protein Assay Kit, Annexin V-FITC Apoptosis Detection Kit, Hoechst Staining Kit, Cell Cycle and Apoptosis Analysis Kit, electrochemiluminescence (ECL) kit, and Tris-buffered saline with Tween-20 (TBS-T) were purchased from Beyotime Biotechnology Company (Shanghai, China). 740Y-P was purchased from Sabinene TargetMol (Shanghai, China). Primary antibodies against cleaved caspase-3, Bax, cyclin D1, p21, matrix metalloproteinase-2 (MMP2), phosphoinositol 3-kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt (p-Akt) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against PARP-1, phosphorylated PI3K (p-PI3K), mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), matrix metalloproteinase-9 (MMP9), Bcl-2, cleaved caspase-9, p27, CDK-4, and CDK-6 were purchased from Abcam (Cambridge, MA, UK). Secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) were obtained from Cell Signaling.