Mitochondria-mediated apoptosis induced by acetogenins from Porcelia macrocarpa (Annonaceae) in K562 chronic myeloid...

Acetogenins from Annonaceae species are widely recognized for their potent cytotoxic and antitumor properties. Some acetogenin derivatives, such as bullatacin, have been reported to impair mitochondrial function, including inhibition of mitochondrial respiratory chain complex I. However, the cytotoxic effects of acetogenin derivatives isolated from Porcelia macrocarpa in leukemia cells remain poorly explored, as do the mitochondrial mechanisms underlying their biological activity.

The present study aimed to evaluate the cytotoxic potential of three acetogenins, namely (2S,3R,4R)-3‑hydroxy-4-methyl-2-(n‑eicos-11'-yn-19'-enyl)butanolide (1), (2S,3R,4R)-3‑hydroxy-4-methyl-2-(n‑eicos-11'-ynyl)butanolide (2), and (2S,3R,4R)-3‑hydroxy-4-methyl-2-(n‑eicos-11'-yn-19'-epoxy)butanolide (3), isolated from the seeds of Porcelia macrocarpa and to investigate the molecular mechanisms underlying the cytotoxic activity of the most active compound in K562 chronic myeloid leukemia cells.

A combined phytochemical, in silico, and In vitro experimental approach was employed to investigate the physicochemical and predicted pharmacokinetic properties, cytotoxic activity, and cellular responses associated with acetogenin exposure.

Acetogenins 1 - 3 were isolated and structurally characterized by NMR and HR-ESI-MS. In silico analyses were conducted to predict physicochemical properties, pharmacokinetic parameters, and toxicity profiles. Cytotoxicity was evaluated in Philadelphia chromosome positive K562 human chronic leukemia cells using MTT and trypan blue assays, while mitochondrial superoxide production, mitochondrial membrane potential, and cytosolic calcium levels were analyzed by flow cytometry. Apoptotic signaling was assessed by Western blot analysis of BAX, BCL-2, and caspase activation of caspase 9 and caspase 3.

In silico predictions indicated favorable gastrointestinal absorption and limited blood-brain barrier permeability for the evaluated acetogenins. In vitro assays demonstrated structure-dependent cytotoxicity, with acetogenins 1 and 3 significantly reducing leukemia cell viability, whereas compound 2 showed minimal activity. Mechanistic studies revealed that acetogenin 1 increased mitochondrial superoxide production, partially dissipated mitochondrial membrane potential, disrupted calcium homeostasis, and it was associated with modulation of the BAX/BCL-2 ratio and activation of caspase-9 and caspase-3, consistent with intrinsic apoptotic signaling.

These findings indicate that acetogenin 1 exposure is associated with mitochondrial alterations and intrinsic apoptotic signaling in K562 chronic myeloid leukemia cells and provide mechanistic insights into the cellular responses induced by acetogenins isolated from Porcelia macrocarpa.