Nutrient starvation induces apoptosis and autophagy in C6 glioma stem-like cells

Abstract

Glioblastoma is a severe cancer with extremely poor survival. Its treatment typically involves a combination of surgery, chemotherapy, and radiation therapy. However, glioma stem-like cells (GSCs)-a subpopulation of tumor-propagating glioblastoma cells—cause post-treatment recurrence and are a major factor in the poor prognosis of the disease. GSCs have higher proliferation than non-GSCs and are more resistant to invasive chemotherapy and radiotherapy. In this study, we subjected GSCs to nutrient starvation (deprived of glucose, glutamine, and calcium) to determine whether cell death can be triggered as a potential strategy to improve treatment outcomes. Flow cytometry revealed that 35.1%, 96.1%, and 99.9% of starved GSCs underwent apoptosis on days 1, 3, and 5, respectively, along with nearly 100% autophagy on all three days. Western blots detected cleaved caspase-3 (an apoptosis marker) and phospho-beclin 1, LC 3B-I, LC 3B-II (autophagy markers) in C6 GSCs after nutrient starvation for 1, 3, 4, and 5 days. Transmission electron microscopic observation of GSC ultrastructure after starvation treatment revealed that compared with control GSCs, starved cells had more pyknotic nuclei, membrane bleb, swollen endoplasmic reticulum, degenerative mitochondria, lipid droplets, and microvilli loss. Thus, nutrient starvation stresses cells by increasing free radicals. Cell stress opens more channels between mitochondria and endoplasmic reticulum. This study demonstrated that nutrient starvation decreases proliferation by approximately 81%, while increasing apoptosis (99.9%) and autophagy (94.6%) in C6 GSCs by the fifth day. Nutrient starvation of GSCs may, therefore, be an effective therapeutic strategy that can trigger apoptotic and autophagic metabolic reprogramming in cancer cells.