Glioma is the most common malignant tumor among all intracranial malignancies  and is a complex tumor lesion that is characterized by invasiveness and a high recurrence rate. Despite the availability of treatments, such as early surgical resection, radiation, or chemotherapy, the prognosis of patients with glioma is poor. In addition to the poor therapeutic effect of chemotherapy, early radiotherapy, and late adjuvant therapy on malignant glioma, these treatments also have toxic side effects. Therefore, identifying new therapeutic agents to improve the prognosis of patients with malignant glioma is critical [12, 13]. To overcome the limitations of the existing treatment methods for malignant glioma, many new treatment methods such as molecular targeted therapy, immunotherapy, gene therapy, stem cell therapy, and nanotechnology have begun to appear in preclinical and clinical research. In recent years, traditional Chinese medicine has shown significant value in the treatment of human glioma. Bi  found that Paris polyphylla saponin reduced the viability of U251 cells by inhibiting the expression of ARA1 and ARA3, leading to inhibited phosphorylation of Akt and p44/42 MAPK and induced apoptosis and cell cycle arrest. Liu  showed that berberine inhibited the proliferation of glioma cells by interfering with wild-type and mutant p53.
OA is an effective anti-tumor drug that inhibits glioma cells with a variety of malignant phenotypes. Studies have shown that OA and its derivatives have no cytotoxicity to normal human cells  and it inhibits tumor development in various malignant tumors through multiple methods and pathways. In vivo experiments showed that OA significantly reduced the size and quality of tumors, and in vitro experiments revealed that OA has a significant inhibitory effect on cell migration, cell viability and proliferation ability [17, 18]. OA has a sensitizing effect on radiotherapy of C6 cell–bearing rats . The pathway by which OA inhibits cell processes varies in different tumor cells, and its anti-tumor effect is related to the inhibition of intracellular signaling pathways such as STAT3, VEGF, MAPK, ERK, JNK, Akt, mTOR, and NF-κB pathways. In this study, molecular docking technology showed that the binding energies of OA and IKK-β, MAPK3, and MAPK4 proteins were all less than − 5 kcal /mol, indicating that OA had a good binding activity with the three proteins. We speculate that OA may inhibit glioma proliferation by downregulating IKK-β, MAPK3, and MAPK4 proteins.
To further study the effect of OA on glioma, we performed in vitro experiments. CCK-8 and MTT assays revealed that the proliferation inhibition rate of cells increased in response to OA in a dose-dependent manner. Colony formation assays showed that the number of U251 cells decreased significantly after OA treatment. Together these results indicated that OA inhibits the proliferation of U251 cells. Invasion is a critical biological feature of malignant tumors. It is the process by which tumor cells penetrate the blood vessel wall and enter the circulatory system. Transwell assays showed that the number of invaded U251 cells treated with OA was significantly reduced compared with the control group, suggesting that OA inhibits the invasion of U251 cells. Apoptosis is a process in which cells undergo programmed death through gene regulation. Many tumor suppressor genes inhibit tumor development through inducing apoptosis, and apoptosis induction is one of the main mechanisms of current tumor treatments. Flow cytometry showed that the apoptosis rate of cells treated with OA was significantly increased, indicating OA has a promoting effect on cell apoptosis. Together these results show that OA inhibits the proliferation and invasion of glioma U251 cells and promotes glioma cell apoptosis, thereby exhibiting anti-cancer effects in glioma.
The IKK and MAPK pathways play an essential role in the proliferation, migration, and invasion of glioma cells. The overexpression of IKK-β is closely related to the occurrence of a variety of malignant tumors, and inhibition of IKK-β in tumor cells results in specific anti-tumor effects [20–22]. Recent studies found a close relationship between malignant tumors and chronic inflammation. IKK-β activation helps promote the occurrence of cancer and inflammatory lesions, and inflammation can inhibit cell apoptosis through the IKK-β pathway, thus enabling the development malignant tumors. The specific inhibition of IKK-β provides a new strategy and approach for the prevention and targeted treatment of inflammation-associated tumors. Activation of the IKK/NF-κB pathway induces tumor cells to proliferate and migrate and inhibits apoptosis . Wang et al. showed that TGFβ1 regulates TNF-κB activity induced by TNF-α in glioma cells through the PP1A/IKK-β pathway and promotes the progression and development of gliomas .
The MAPK pathway participates in the regulation of cancer cells through roles in proliferation, differentiation, and apoptosis, and other critical biological responses [25, 26]. Recent studies showed that the MAPK signaling pathway has a series of active metastases in glioma and other malignant tumor tissues, and MAPK directly mediates the anti-tumor effects of a variety of drugs. One study showed that miR-326 inhibits the malignant biological behavior of glioma cells through the MAPK signaling pathway . MAPK3 and MAPK4 are members of the MAPK pathway. Yang et al. showed that miR-127-3p inhibits the proliferation of U251 glioma cells by down-regulating the target gene MAPK4 . Some researchers also reported that a new MAPK3 and MAPK4 double-molecule inhibitor PCC0208017 reduced glioma cell migration, glioma cell invasion, and angiogenesis and may be a promising lead compound for the treatment of glioma . Studies have confirmed that CYP17A1 gene silencing significantly inhibits the invasiveness of glioma cells, and CYP17A1 gene silencing promotes the apoptosis of glioma cells . Recent studies have shown that PTGS2 can not only promote the proliferation rate of glioma cells, but also improve radiation tolerance . The studies described above showed that the CYP17A1/IKK-β/PTGS2/MAPK3/MAPK4 signaling pathway plays an essential role in the development of glioma. In this study, our western blot analysis showed that the protein expression levels of CYP17A1, IKK-β, PTGS2, MAPK3, and MAPK4 were suppressed in glioma cells treated with OA. We speculate that OA may inhibit IKK-β, MAPK3, and MAPK4 protein expression by directly binding to the proteins. In this study, in vitro experiments observed that OA has inhibitory effects on the proliferation, cloning and invasion of glioma cells, and can promote their apoptosis. Furthermore, through transcriptome sequencing, it was found that the significantly differentially expressed genes are mainly involved in processes such as inflammation, metabolism, immunity, and regulation of cell growth. In the GO function enrichment analysis, information about cellular biological processes, defense response, cellular response to type interferon, basement membrane, endoplasmic reticulum, extracellular matrix, cytokine activity, cytokine receptor binding, transcription factor activity and transcription activator activity, etc. There are many genes with significant differences in activity. It can be seen from KEGG results that oleanolic acid may activate MAPK pathway through TNF pathway, thus inhibiting the proliferation of human neural U251 cells.