An oriental medicinal plant derived from the bark of the magnolia tree shows promise as a treatment for prostate cancer. In traditional Asian medicine, extracts of magnolia have been used for pain relief and as a treatment for anxiety and depression, and the plant is marketed widely as an alternative medicine. Honokoi (HNK) is a bioactive natural product obtained from magnolia and research has shown that it has a range of potentially therapeutic effects on the body, including anti-tumor, promotion of neural growth, inhibition of platelet aggregation, anti-inflammatory, antioxidant, and others that have attracted interest in it as a drug candidate. Honokiol has shown activity against a number of solid tumor types including breast, prostate, gastric, and ovarian cancers. In order to demonstrate activity of honokiol against prostate cancer, investigators from the University of Pittsburgh and Kagoshima University in Japan exposed human and mouse prostate cancer cells to pharmacologic doses of HNK and observed the cells for evidence of autophagy (cellular self-degradation), cell viability, and reactive oxygen species that would indicate apoptosis (programmed cell death). They assessed autophagy by transmission electron microscopy, immunofluorescence, and immunoblotting for the presence of LC3BII, a protein marker for autophagy, and determined cell viability by trypan blue assay, and apoptosis by DNA fragmentation and Annexin V/propidium iodide assay. EPR was performed on a Bruker X-band benchtop EPR spectrometer as one of several methods to identify reactive oxygen species in the cells treated with honokiol. EPR signals in the human cells were measured after four hours of treatment with honokiol or a control DMSO solution. The treated cells had a significantly higher EPR signal than the DMSO-treated cells. The results were confirmed by fluorescence. Researchers further characterized the nature of the anticancer effects by investigating how other treatments of the cells changed the results. They found that induction of autophagy by honokiol was cytoprotective through the use of a chemical inhibitor of autophagy, leading to increased apoptosis. Knocking out a protein called ATG5, which is critical to the autophagy process, also increased the rate of apoptosis. On the other hand, antioxidants reversed some, but not all, effects of honokiol on the cancer cells. Increases in LC3BII were partially suppressed in the presence of the antioxidant. However, rates of apoptosis induced by honokiol were not significantly affected by treatment with antioxidants. Previous studies of honokiol’s anticancer effects had not established its ability to induce autophagy. The use of multiple methods, including transmission electron microscopy, western blotting for LC3BII, and microscopic visualization allowed investigators to establish a clear effect of autophagy induction, suggesting that this effect can be exploited for development of cancer therapy. The researchers were also able to establish that induction of autophagy by honokiol was partly dependent on generation of reactive oxygen species. However, inhibition of autophagy in the presence of antioxidants did not increase apoptosis as expected. That could be due masking of an apoptotic effect of honokiol which would have been inhibited in the presence of antioxidants.