TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells but not in normal cells, hence TRAIL has recently emerged as a novel anti-cancer agent. Adult T-cell Leukemia/Lymphoma (ATLL) is a neoplasm of T-lymphocyte origin etiologically associated with HTLV-I, and is known to be resistant to standard anti-cancer therapies. In a previous study, we showed that although most ATLL cells express TRAIL-death receptors DR4 and/or DR5, they are resistant to TRAIL. To overcome this resistance, we tried to find natural materials that increase the sensitivity to TRAIL in the present study. Here, we report for the first time that dihydroflavonol from a plant increases TRAIL sensitivity by enhancing death receptor expression.

Materials and methods: We used a TRAIL-resistant ATLL cell line, KOB, for screening, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. The cell surface expression of DR4, DR5, and decoy-receptors DcR1 and DcR2 was examined using flow cytometry (FCM). Cell viability was assessed by MTS assay after incubation with various combinations of natural extracts and TRAIL for 48 hours. Apoptosis was evaluated by Annexin V binding and propidium iodide (PI) staining. We also analyzed caspase−8, − 9 and −3 activation and pro- and anti-apoptotic proteins by Western blot.

Results and Discussion: We screened more than 500 natural products. Among them, the most striking synergism with TRAIL was observed in a dihydroflavonol that was extracted from the Blumea Balsamifera, a family of chrysanthemum from Thailand. It was a methyldihydroquercetin (molecular weight, 318), and we named it BB-1. The viability of KOB cells treated with either TRAIL or BB-1 was 98% and 97%, respectively, and decreased to 60% with concurrent treatment. Interestingly, sequential treatment, pre-treatment with BB-1 followed by TRAIL, further augmented the synergism, and the viability decreased to 38%. Using Annexin V and PI, we confirmed that these are the results of apoptosis. Although it has been reported that anti-apoptotic proteins FLIP, Bcl-2, Bcl-xL and XIAP play key roles in TRAIL resistance, we did not find any change in these factors in the BB-1-treated cells. Instead, we found that BB-1 increased DR5 expression. Subsequent activation of caspase−8, −9 and −3 was observed in a time-dependent manner in the sequentially treated cells, but not in cells treated with BB-1 or TRAIL alone. More importantly, BB1 did not induce DR5 expression or enhance TRAIL sensitivity in PBMCs. Thus, BB-1 augmented TRAIL-induced apoptosis by up-regulating DR5 expression without any cytotoxic activity on normal PBMCs. These results suggest that combined treatment with BB-1 and TRAIL can be a new strategy for cancer immunotherapy against TRAIL-resistant cells.

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