Isolation of Various Flavonoids with TRAIL Resistance-Overcoming Activity from Blumea lacera

Eighteen compounds, including fourteen flavonoids (1–14), one steroid (15), two fatty acids (16,17), and one nitrogen-containing compound (18), were isolated from the methanol extract of the whole Blumea lacera plant collected in Thailand. Compounds 1–11 and 15–17 exhibited tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance-overcoming activity. Among them, bonanzin (2) and cirsilineol (7) had particularly strong TRAIL resistance-overcoming activity, where the IC50 values against the human gastric adenocarcinoma cell line AGS in the presence of TRAIL (100 ng/mL) were 10.7 μM and 5.9 μM, respectively.


Introduction
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to the death receptor and selectively induces apoptosis in cancer cells. Therefore, TRAIL is considered an attractive therapeutic target for cancer treatment. However, various types of cancer cells, including gastric, prostate, and breast cancers, are resistant to TRAIL-induced apoptosis [1]. Thus, compounds that can overcome this resistance may lead to the development of new anticancer drugs.
Blumea lacera is an annual herb in the Asteraceae family that is distributed in Southeast Asia, Australia, China, India, and tropical Africa. This plant has been used as traditional medicine by exploiting its antipyretic, antioxidant, and stimulant [2] properties, and is reported to have antiviral [3], anti-leukemic [3], and cytotoxic activities against several cancer cell lines [4,5]. Several compounds such as nicotifiorin [5], mauritianin [5], and rotundioic acid [6] have been isolated from Blumea lacera, whereas the bioactive natural compounds with TRAIL resistance-overcoming activity have not been explored in this plant.
We previously reported some naturally derived compounds with TRAIL resistanceovercoming activity [7,8]. In this study, we describe the isolation of eighteen compounds from the whole plant of Blumea lacera and report their TRAIL resistance-overcoming activity.

Plant Material
Whole plants of Blumea lacera (Asteraceae) were collected from Thailand and identified by the late Thaworn Kowithayakorn. A voucher specimen (KKP649) was deposited in the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan.

Assay of Cell Viability (TRAIL Resistance-Overcoming Activity Assay)
Cell viability was assessed using a fluorometric microculture cytotoxicity assay [9] in the presence and absence of TRAIL using TRAIL-resistant AGS cells. Cells were seeded in 96-well culture plates (1 × 10 4 cells per well) in 200 µL of medium containing 10% FBS and 1% PS. After 24 h of incubation, samples with or without TRAIL (100 ng/mL) were added to each well. Dimethyl sulfoxide (DMSO, 0.1%) was used as the negative control. Luteolin (17.5 µM) was used as a positive control. After another 24 h of incubation, the cells were washed with phosphate-buffered saline (PBS), and 200 µL PBS containing fluorescein diacetate (10 µg/mL) was added to each well. The plates were incubated at 37 • C for 1 h, and fluorescence at 538 nm with excitation at 485 nm was measured using a Fluoroskan Ascent (Thermo Fisher Scientific, Waltham, USA). IC 50 values were calculated using TRAIL (0 ng/mL), and the survival rate was 100%. For example, the IC 50 value of 2 with TRAIL was determined based on the viability at 7 µM (85.9%) and 13 µM (33.9%).

TRAIL Resistance-Overcoming Activity of Isolated Compounds
The ability of the isolated compounds to overcome TRAIL resistance was examined using the human gastric adenocarcinoma cell line AGS . Compounds 1-7 and 15-17, along with two mixtures of compounds (8/9 and 10/11), exhibited TRAIL resistance-overcoming activity. Most of the isolated flavonoids exhibited TRAIL resistance-overcoming activity ( Figure 2). Bonanzin (2) and cirsilineol (7) had particularly strong TRAIL resistanceovercoming activity, with IC 50 values of 10.7 µM and 5.9 µM in the presence of TRAIL (100 ng/mL), whereas compounds 2 and 7 had IC 50 values of 26.1 µM and 20.7 µM without TRAIL, respectively ( Figure 2). There have been no reports on the TRAIL resistanceovercoming activity of these compounds [27]. Patulitrin (13), a 7-O-glucoside of patuletin, did not exhibit strong TRAIL resistance-overcoming activity, but was cytotoxic against AGS cells, with an IC 50 value of 165 µM. However, the other two glucosides of the flavonoids (12 and 14) exhibited no bioactivity at 200 µM, where it is proposed that the presence of glucose at C-3 prevents these compounds from interacting with the target. Further research is necessary to clarify these interactions.

TRAIL Resistance-Overcoming Activity of Isolated Compounds
The ability of the isolated compounds to overcome TRAIL resistance was examined using the human gastric adenocarcinoma cell line AGS. Compounds 1-7 and 15-17, along with two mixtures of compounds (8/9 and 10/11), exhibited TRAIL resistance-overcoming activity. Most of the isolated flavonoids exhibited TRAIL resistance-overcoming activity ( Figure 2). Bonanzin (2) and cirsilineol (7) had particularly strong TRAIL resistance-overcoming activity, with IC50 values of 10.7 μM and 5.9 μM in the presence of TRAIL (100 ng/mL), whereas compounds 2 and 7 had IC50 values of 26.1 μM and 20.7 μM without TRAIL, respectively ( Figure 2). There have been no reports on the TRAIL resistance-overcoming activity of these compounds [27]. Patulitrin (13), a 7-O-glucoside of patuletin, did not exhibit strong TRAIL resistance-overcoming activity, but was cytotoxic against AGS cells, with an IC50 value of 165 μM. However, the other two glucosides of the flavonoids (12 and 14) exhibited no bioactivity at 200 μM, where it is proposed that the presence of glucose at C-3 prevents these compounds from interacting with the target. Further research is necessary to clarify these interactions.

Conclusions
Eighteen compounds, including fourteen flavonoids, were isolated from the MeOH extract of Blumea lacera and its partitions, guided by the TRAIL resistance-overcoming activity. Compounds 1-7 and 15-17, along with two mixtures of compounds (8/9 and 10/11), exhibited TRAIL resistance-overcoming activity, whereas compound 13 was cytotoxic against AGS cells without TRAIL resistance-overcoming activity. Although the compounds have the same flavonoid skeleton, the strength of the TRAIL resistance-overcoming activity depends on the substituents. Among the isolated flavonoids, bonanzin (2) and cirsilineol (7) exhibited strong TRAIL resistance-overcoming activity, with IC 50 values of 10.7 µM and 5.9 µM, respectively.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding authors upon reasonable request.