Abstract
Brucea javanica oil emulsion (BJOE), the petroleum ether extract of B. javanica emulsified by phospholipid, is widely used in China as an anticancer agent. The extracts from B. javanica induce cancer cell death by various mechanisms; however, it is not known whether these mechanisms involve autophagy, which is an important process in cancer development and treatment. Thus, the current study aimed to investigate whether BJOE modulates autophagy in HCT116 human colon cancer cells and whether modulation of autophagy is an anticancer mechanism of BJOE. Immunoblotting was employed to analyze the protein expression levels of microtubule-associated protein light-chain 3 (LC3), a specific protein marker of autophagy, in HCT116 cancer cells following exposure to BJOE. The apoptosis rate of the HCT116 cancer cells was detected by performing an Annexin V-fluorescein isothiocyanate/propidium iodide assay. According to the effect of BJOE administration on autophagy in the HCT116 cancer cells (induction or suppression), a functionally opposite agent (autophagy suppressor or inducer) was applied to counteract this effect, and the apoptosis rate of the cancer cells was detected again. The role of autophagy (pro-survival or pro-death) was demonstrated by comparing the rates of apoptotic cancer cells prior to and following the counteraction. The results revealed that BJOE suppressed the protein expression levels of LC3, including the LC3-I and LC3-II forms, and induced apoptosis in the HCT116 cancer cells with a high level of basal LC3. The apoptosis-inducing activity of BJOE was significantly attenuated when autophagy was induced by the administration of trehalose, an autophagy inducer. The data indicates that autophagy inhibition is involved in BJOE-induced cancer cell death, and that this inhibition may be a potential anticancer mechanism of BJOE.
Highlights
Nature's abundant botanical, animal and mineral resources have provided a large supply of materials for anticancer discovery, with >60% of anticancer agents being either natural products or based thereon [1]
The aim of the current study was to investigate whether B. javanica oil emulsion (BJOE) modulates autophagy in HCT116 human colon cancer cells and whether the modulation of autophagy is a mechanism by which Brucea javanica oil emulsion (BJOE) kills cancer cells
The Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) Apoptosis Detection kit was obtained from BestBio (Shanghai, China), and the trehalose was purchased from Difco, BD Biosciences (Franklin Lakes, NJ, USA)
Summary
Nature's abundant botanical, animal and mineral resources have provided a large supply of materials for anticancer discovery, with >60% of anticancer agents being either natural products or based thereon [1]. The effect of B. javanica or its extracts on autophagy in cancer cells has yet to be reported. Microtubule‐associated protein light chain 3 (LC3) is the key factor in the formation of autophagosomes and includes two forms, cytosolic LC3‐I and membrane‐bound LC3 ‐II, which are produced in the process of autophagy. LC3‐II serves as a good indicator of autophagosome formation, and the detection of LC3 conversion (LC3‐I to LC3‐II) by immunoblot analysis is widely used to monitor autophagy [12]. Beclin‐1 is a key protein involved in the initiation of autophagosome formation and closure, and has been indicated to be involved in tumor development. Bim [ known as B-cell lymphoma 2 (Bcl 2)‐like protein 11] has recently been identified to inhibit autophagy by binding to Beclin‐1 independent of its proapoptotic function, acting as a novel molecular link between autophagy and apoptosis. BimEL and BimL interact with Beclin‐1, only a weak interaction appears to exist between BimS and Beclin‐1 [14]
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