Gymnema sylvestre Extract Restores the Autophagic Pathway in Human Glioblastoma Cells U87Mg

SIMPLE SUMMARY: The treatment of GBM is extremely difficult and complicated by the heterogeneous nature of neoplastic cells. The problems inherent in treating any central nervous system tumour are due to the anatomical complexity and the limited repair mechanisms of the surrounding unaffected tissues. The choice of the most suitable treatment for GBM depends on several factors: the location of the disease, the extent, and the nature of the tumour. The limit of this choice is mainly due to the degree of complexity of the disease and to the mechanisms of drug resistance that the neoplasm develops during the treatment. Herbal medicines and their derived phytocompounds are increasingly recognised as useful complementary treatments for cancer. Numerous clinical studies have reported the beneficial effects of herbal medicines on survival, immune modulation, and quality of life of cancer patients when used in combination with conventional therapies. In this study, we investigated all the mechanisms that control tumour cell growth after induction with Gymnema sylvestre (GS) extract and the key proteins that regulate these mechanisms in glioblastoma cells. The study is of great translational interest because the natural substances used could be proposed as natural adjuvant drugs for the treatment of glioblastoma, and therefore could act by modulating new molecular targets for the control of brain tumour cell growth. ABSTRACT: Glioblastoma is a brain tumour, characterised by recurrent or innate resistance to conventional chemoradiotherapy. Novel natural molecules and phyto-extracts have been proposed as adjuvants to sensitise the response to Temozolomide (TMZ). In this study, we investigated the effect of GS extract on human glioblastoma cells U87Mg. According to the IC50-values, GS extract displayed a significant cytotoxicity. This was confirmed by cell growth inhibition and alteration in metabolic activity evaluated by cell count and MTT assay. GS induced reduction in Pro-caspase 9, 3, but not PARP cleavage nor DNA fragmentation. Thus, in GS-induced cytotoxicity, cell death is not associated with apoptosis. In this context, short-term treatment of U87Mg cells with GS extract (1 mg/mL) reduced the phosphorylation levels of mTOR and of its downstream target P70 S6 kinase, highlighting the role of GS extract into autophagy induction. The activation of autophagic flux by GS extract was confirmed by Western blot analysis, which revealed the reduction in p62 and the concomitant increase in LC3B II/I ratio. Immunofluorescence evidenced the accumulation of LC3B puncta in U87Mg cells pretreated with autophagy inhibitor Bafilomycin A1. Furthermore, as main key regulators of type II programmed cell death, p53, p21 and CDK4 were also investigated and were inhibited by GS treatment. In conclusion, GS extract could be considered as an autophagy inducer in glioblastoma cells U87Mg.