Fig. 6: Mahanine up-regulating LC3B through oxidative stress and inhibits cell growth and autophagy in primary cells isolated from patients with high-grade serous histology.

a Primary cancer cells were isolated from ascitic fluid of nine patients . Cells (1 × 10⁴ /well) were plated, treated with mahanine and processed as described in Fig. 3a. b Primary cells from three representative patients (5 × 10⁵ per well in 6-well plates) were treated with mahanine (near to IC₅₀ dose). Phase-contrast images showing the collapse of morphology in treated cells (scale bar: 100 μm). c Primary cells from three representative patients (PCAST-4, PCAST-14, and PCAST-17) were treated similarly for 24 h and processed to determine the percent autophagosome by FACS, which exhibited reduced autophagosome-formation up to ~ 70%. d Primary cells (1 × 10⁶/well) from these patients were treated with mahanine for 24 h. Cell lysates were electrophoresed and analyzed by western blot using anti-LC3B antibodies. e Model illustrating the possible mechanism of oxidative stress-induced enhanced LC3B leading to anoikis but inhibited autophagy. A pro-oxidant agent (mahanine) enhanced LC3B via oxidative stress, which induces cell detachment by modulating EMT markers and downregulating cell adhesion molecules like integrin-β3 and focal adhesion kinase (FAK), leading to form homeless cells. Enhance LC3B also induces the apoptotic molecules (caspase-3, caspase-9, Bax and PARP cleavage) and downregulate anti-apoptotic molecule (Bcl2), which induces detachment-induced cell death (Anoikis). Such oxidative stress induces autophagy-related molecules like Atg 3, 5, 7, 12, p62, Beclin1 and LC3B-II, and also inhibits the formation of ULK1 complex, which leads to inhibition of autophagy