Chloropupukeananin and Pestalofone C Regulate Autophagy through AMPK and Glycolytic Pathway

Abstract

Chloropupukeananin (RN56‐6) and Pestalofone C (RN56‐49), isolated from the culture of the plant endophytic fungus Pestalotiopsis fici, have been shown cytotoxic, anti‐HIV, and antimicrobial activities. However, the underlying mechanism of their regulatory roles in autophagy remains unknown. In the present study, we revealed that both compounds increased the formation of autophagosome and enhanced autophagic flux. While RN56‐6 upregulated the expression of HK2, one of the key rate‐limiting enzymes of glycolysis, the inhibition of glycolysis chemically attenuated RN56‐6‐induced autophagy. On the contrary, RN56‐49 downregulated the expression of HK2, while the suppression of glycolysis promoted RN56‐49‐dependent autophagic flux. Moreover, the knockdown of AMPKβ1, a scaffolding subunit of AMPK, decreased autophagy induced by these two compounds. Collectively, these findings revealed that RN56‐6 and RN56‐49 regulated autophagic process through AMPK and glycolytic pathway.

Different Role of Raptor and Rictor in Regulating Rasfonin‐Induced Autophagy and Apoptosis in Renal Carcinoma Cells

“…The membrane will be incubated in milk at the room temperature upon 1 h. Western blot was performed by using appropriate primary antibodies and horseradish peroxidase-conjugated suitable secondary antibodies, followed by detection with enhanced chemiluminescence (Pierce Chemical Rockford, IL, USA). [16,46] Cell Viability Assay (MTS) For MTS assay, cells culturing in 96-well plates (8,000 cells per well) with 100 μL complete culture media was carried out. After incubation overnight, cells were replaced with Phenol red free complete medium which was added with either drug-free or rasfonin (6 μM), or Z-V-FMK (20 μM) and Nec-1 (30 μM).…”

“…Cells were cultured for indicated period and the cell viability was detected at 492 nm by CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega). [16,28] Flow-Cytometry Assay ACHN cells were challenged with the mentioned compounds in figure legends, then, trypsinized and harvested (keeping all floating cells), washed with cold PBS buffer, followed by incubation with fluorescein isothiocyanate-labeled annexin V (FITC) and propidium iodide (PI) as instructed in the Annexin-V-FITC Apoptosis Detection Kit (BioVision Inc., Milpitas, CA, USA, K101-100) and analyzed by flow cytometry (FACSAria, Becton Dickinson, Franklin Lakes, NJ, USA). While PIpositive staining was considered to be necrotic, the cells with annexin V-positive and PI-negative staining were calculated as apoptotic.…”

“…Ultimately, transmission electron microscope (JEM-1230, Akishima, Tokyo, Japan) was employed to observe the images of the thin sections. [16,46]…”

“…[15] Macroautophagy (hereafter referred to as autophagy) is a catabolic process, which is conserved in eukaryotic cell and involved in the degradation of cytoplasmic contents as well as organelles. [16] Autophagy continually happens in all eukaryotic cells at basal level and can be activated with variety of stimulations, such as nutrient deprivation, oxidative stress exposure and drug challenge. [17 -19] Autophagy has been demonstrated to manage cell growth, survival, differentiation, development and protein homeostasis, while it also plays a critical role in maintaining a balance between the synthesis, degradation, and subsequent recycling of cellular products.…”

“…Macroautophagy (hereafter referred to as autophagy) is a catabolic process, which is conserved in eukaryotic cell and involved in the degradation of cytoplasmic contents as well as organelles [16] . Autophagy continually happens in all eukaryotic cells at basal level and can be activated with variety of stimulations, such as nutrient deprivation, oxidative stress exposure and drug challenge [17–19] .…”