Tubeimoside-I sensitizes temozolomide-resistant glioblastoma cells to chemotherapy by reducing MGMT expression and suppressing EGFR induced PI3K/Akt/mTOR/NF-κB-mediated signaling pathway

Qingfa Tang; Haihong Cao; Ni Tong; Yuanliang Liu; Wanyu Wang; Yuheng Zou; Lanyang Xu; Zhiyun Zeng; Wei Xu; Zhixin Yin; Wenjuan Ma; Qirui Wang

doi:10.1016/j.phymed.2022.154016

Tubeimoside-I sensitizes temozolomide-resistant glioblastoma cells to chemotherapy by reducing MGMT expression and suppressing EGFR induced PI3K/Akt/mTOR/NF-κB-mediated signaling pathway

Phytomedicine. 2022 May:99:154016. doi: 10.1016/j.phymed.2022.154016. Epub 2022 Feb 27.

Authors

Qingfa Tang¹, Haihong Cao², Ni Tong², Yuanliang Liu³, Wanyu Wang², Yuheng Zou², Lanyang Xu², Zhiyun Zeng⁴, Wei Xu⁴, Zhixin Yin⁴, Wenjuan Ma⁵, Qirui Wang⁶

Affiliations

¹ Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong 510282, China; School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China; Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, Guangzhou, 510515, China.
² Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong 510282, China; School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
³ Department of Pathology, Guangzhou First People's Hospital, Guangzhou, Guangdong 510180, China.
⁴ School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
⁵ Department of Intensive Care Unit, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, Guangdong, 510060, China. Electronic address: mawenj@sysucc.org.cn.
⁶ Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong 510282, China; School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China. Electronic address: wqrv@hotmail.com.

PMID: 35278900
DOI: 10.1016/j.phymed.2022.154016

Abstract

Background: Glioblastoma multiforme (GBM, World Health Organization [WHO] grade IV) is one of the malignant Central Nerve System (CNS) tumors with high incidence rate and poor prognosis. The use of alkylating agents, such as temozolomide (TMZ), has been the main method of cytotoxic therapy for glioma patients for decades. However, TMZ resistance may be one of the major reasons for treatment failure, so far. In searching for effective agents to reverse TMZ resistance, we found that Tubeimoside-I (TBMS1), a saponin from traditional Chinese medicine, Bolbostemma paniculatum (Maxim.) Franquet, showed activities of reversing TMZ resistance of GBM. However, the ability of TBMS1 enhancing the chemosensitivity of GBM has been rarely studied, and its underlying mechanisms remain unclear.

Purpose: This study purposes to reveal the synergistic effects and mechanism of TBMS1 and TMZ against TMZ-resistant GBM cells.

Methods: CCK8 assay was used to investigate the anti-proliferative effects on grade IV glioblastoma human T98G and U118 MG cells. Cell proliferation was determined by EdU assay and clonogenic assay after TMZ plus TBMS1 treatment. Apoptosis was analyzed by flow cytometry. DNA damage and DNA Double Strand Break (DSB) were assessed by cleaved Poly (ADP-ribose) polymerase (PARP), γH2AX Foci Assay and Comet Assay, respectively. Expression of proteins associated with apoptosis and DNA repair enzymes were measured by Western blot analysis. The prognostic significance of key proteins of the epidermal growth factor receptor (EGFR) induced PI3K/Akt/mTOR/NF-κB signaling pathway was analyzed using GEPIA (http://gepia.cancer-pku.cn) and validated by Western blotting.

Results: Here we demonstrated that TBMS1 sensitized TMZ-resistant T98G and U118 MG glioblastoma cells to chemotherapy and exhibited promotion of apoptosis and inhibition on cell viability, proliferation and clone formation. Coefficient of drug in interaction (CDI) values showed a notable synergistic effect between TBMS1 and TMZ. Moreover, we observed that combination of TBMS1 and TMZ induced apoptosis was accompanied by robust DSB, γH2AX Foci formation and increasing cleaved PARP, as well as the heightened ratio of Bax/Bcl-2, cleavages of caspase-3 and caspase-9. In addition, the synergistic anti-glioma effect between TBMS1 and TMZ was intimately related to the reduction of MGMT expression in TMZ-resistant GBM cells. Moreover, it was also associated with attenuated expression of EGFR, p-PI3K-p85, p-Akt (Ser473), p-mTOR (Ser2481) and p-NF-κB p65(Ser536), which implying deactivation of the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway.

Conclusion: We first demonstrated that synergistic effects of TBMS1 and TMZ induced apoptosis in GBM cells through reducing MGMT expression and inhibiting the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. This study provides a rationale for combined application of TMZ and TBMS1 as a potential chemotherapeutic treatment for MGMT⁺ GBM patients.

Keywords: EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway; Genotoxicant; Glioblastoma multiforme; MGMT; Temozolomide resistance; Tubeimoside-I.