Bioactivity-guided Isolation of TRAIL-Resistance-Overcoming Activity Compounds From the Leaves of Murraya exotica

Abstract

Fractionation of the leaf extract from Murraya exotica led to the successful isolation of 12 compounds (1-12) with TRAIL-resistance-overcoming activity. Xanthinosin (1), 11α, 13-dihydroxanthinin (2), 11β, 13-dihydroxanthinosin (3), 4α, 11α, 13-trihydroxanthuminol (4), desacetylxanthanol (5), and lasidiol p-methoxybenzoate (6) were sesquiterpenes isolated from this plant for the first time, and 3 was isolated from natural sources for the first time. Among them, compounds 1 and 5 showed strong TRAIL-resistance-overcoming activity, but their mechanisms have already been revealed. Furthermore, dihydroxanthinin (2), 1, 5-dicaffeoylquinic acid (7), and (-) loliolide (8), which belong to different phytochemical groups, were investigated for their effects on increasing apoptosis induction to overcome TRAIL resistance using Western blot analysis. The results demonstrated that 2, 7, and 8 promoted TRAIL-induced apoptosis by increasing the expression of several proapoptotic markers, including cleaved caspases −3 and −8, and suppressing anti-apoptotic protein Bcl-2.

Introduction

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can induce apoptosis in cancer cells after complexing with death receptors ¹ . TRAIL has attracted great interest as an anticancer target because it selectively induces apoptosis in cancer cells while sparing normal cells ² . Unfortunately, over time, some cancers become resistant to TRAIL-apoptotic pathways such as gastric cancer, breast cancer, lung cancer, prostate cancer, and colon cancer ³ . We have already reported some compounds with TRAIL-resistance-overcoming activity^1,4; however, we are still searching for naturally derived compounds with TRAIL-resistance-overcoming activity.

Murraya exotica, an evergreen shrub in the Rutaceae family, has ethnomedical uses described in the Chinese Pharmacopeia, such as anti-stomachache, anti-rheumatic arthralgia, anti-toothache, anti-swelling, and analgesic ⁵ . M. exotica is reported to contain a variety of secondary metabolites, including alkaloids ⁶ , coumarins ⁷ , flavonoids ⁸ , and dipeptides ⁹ , having anti-implantation ¹⁰ , antioxidant ¹¹ , anti-inflammatory ¹² , and anti-proliferative activities ¹³ . However, the TRAIL-resistance-overcoming activity of M. exotica has not been explored.

Our screening results indicated that M. exotica extract has TRAIL-resistance-overcoming activity. In this study, we isolated 12 compounds (1-12) with TRAIL-resistance-overcoming activity from M. exotica and characterized them using one- and two-dimensional nuclear magnetic resonance (1D-, 2D-NMR) and electrospray ionization mass spectroscopy (ESI-MS). Finally, the candidates (compounds 2, 7, and 8) that were obtained in adequate amounts were studied for their effects on the TRAIL-induced apoptosis pathway by Western blot analysis.

Result and Discussion

Isolation and Structure Elucidation

Although there was a report of antimalarial and antipyretic activity of the methanolic extract of M. exotica ¹⁴ , the TRAIL-resistance-overcoming activity of M. exotica has not been studied. Plant extracts in our library were screened for TRAIL-resistance-overcoming activity and the M. exotica extract was identified as a hit sample (40% inhibition at 50 µg/mL). M. exotica leaves were extracted thrice with methanol (MeOH), passed through a Diaion HP-20 column, and partitioned with hexane, ethyl acetate (EtOAc), and butanol (BuOH) to afford their respective layers. The EtOAc layer was fractionated by column chromatography and preparative high-performance liquid chromatography (HPLC) together with the TRAIL-resistance-overcoming activity test to yield compounds 1 to 12 (Figure 1).

The isolated compounds were identified as xanthinosin (1) ⁴ , 11α, 13-dihydroxanthinin (2) ⁴ , 11β, 13-dihydroxanthinosin (3)^15,16, 4α, 11α, 13-trihydroxanthuminol (4) ⁴ , desacetylxanthanol (5) ⁴ , lasidiol p-methoxybenzoate (6) ⁴ , 1, 5-dicaffeoylquinic acid (7) ¹⁷ , (-)loliolide (8) ¹⁸ , caffeic acid (9) ¹⁹ , protocatechuic acid (10) ²⁰ , 4-hydroxybenzoic acid (11) ²¹ , and patuletin (12) ²² by comparing the obtained ¹H- and¹³C-NMR and ESI-MS data with that published in the literature (Supporting Information).

Compounds 1 to 6 were sesquiterpene lactones isolated from this plant for the first time. Although the isolation of 11β, 13-dihydroxanthinosin (3) from Tithonia diversifolia has been reported ²³ , our ¹H- and ¹³C-NMR data did not correspond well with the reported data. The published ¹H- and ¹³C-NMR data of 3 indicated that it can be obtained through the reduction of xanthatin; however, the stereochemistry at the C-11 position could not be confirmed ¹⁵ . Meanwhile, another study afforded 3 as a product of the cross metathesis of a xanthatin-intermediate and methyl vinyl ketone followed by 1,4- reduction, yielding 3 as the diastereomer of 11β,13-dihydro-tomentosin ¹⁶ . Therefore, this is the first isolation of 3 from natural sources. A one-dimensional Nuclear Overhauser Effect study of 3 at H-11 showed NOE correlation from H-11 [δ 2.23] to H-9β [δ 1.63] and H-8 [δ 4.25], with %NOE values of 5% and 0.5%, respectively (Figure 2), confirming the stereochemistry of 3 as 11β, 13-dihydroxanthinosin.

Figure 1.

Structures of compounds 1 to 12.

Figure 2.

Key NOE difference in 11β, 13-dihydroxanthinosin (3).

TRAIL-Resistance-Overcoming Activity of Isolated Compounds

All the isolated compounds (1-12) exhibited TRAIL-resistance-overcoming activity, but with different potencies. Most of the isolated sesquiterpene lactones showed strong TRAIL-resistance-overcoming activity, with AGS cell inhibition increases of 39%, 36%, 34%, 17%, 40%, and 24% observed for 1 (8 µM), 2 (20 µM), 3 (as a mixture, 40 µM), 4 (198 µM), 5 (10 µM), and 6 (13 µM), respectively. On the other hand, compounds 7 to 12 showed weaker TRAIL-resistance-overcoming activity, decreasing AGS cell viability by 19%, 24%, 23%, 36%, 20%, and 29% at concentrations of 200, 250, 280, 650, 1450, and 30 µM, respectively, when compared to treatment with the compound alone (no-TRAIL) (Figure 3).

Figure 3.

TRAIL-resistance-overcoming activity of isolated compounds (1-12) on AGS cells; DMSO (negative control), luteolin (positive control) (mean ± SD, n  =  3) (significance tests using paired t-test, *P < .05, **P < .01).

Although 1 and 5 showed strong TRAIL-resistance-overcoming activity, the effect of 1 on the expression of apoptotic proteins has already been revealed by Karmaka et al ⁴ and the structure of 5 is nearly identical to that of 1, which contains an α-methylene-γ-lactone moiety that can bind with cellular proteins and disrupt cell processes ²⁴ . Therefore, the effect of 11α, 13-dihydroxanthinin (2), 1, 5-dicaffeoylquinic acid (7), and (-)loliolide (8) on TRAIL-induced apoptosis was studied by Western blot analysis.

Effect of Compounds 2, 7, and 8 on the Expression of Apoptotic Proteins

The related apoptotic proteins were analyzed after overnight treatment of AGS cells with 2 (10, 20, and 40 µM), 7 (0.7, 1.2, and 1.6 mM), and 8 (102, 204, and 306 µM) with 100 ng/mL of TRAIL. The results showed that all the compounds induced apoptosis by increasing the expression of CHOP, resulting in DR5 upregulation (Figure 4A to C). CHOP is a transcription factor of the C/EBP family and is known to induce apoptosis through endoplasmic reticulum stress-mediated apoptotic molecules such as DR5 ²⁵ . In addition, 7 and 8 also induced the expression of p53, which is a proapoptotic protein that induces the expression of Aparf-1 and DR5 and suppresses the anti-apoptotic gene Bcl-2 ²⁶ . Accordingly, the p53 level in cells treated with 7 and 8 increased in a dose-dependent manner (Figure 4B and C), but p53 was not detected in cells treated with 2 (Figure 4A). Furthermore, the Bcl-2 level decreased in cells treated with compounds 2, 7, and 8 in a dose-dependent manner (Figure 4A to C).

Figure 4.

Western blot analysis of cleaved caspase-3, procaspase 8, cleaved caspase-8, DR5, CHOP, p53, Bcl-2, FLIP, and cleaved caspase-9 proteins in AGS cells after 24 h treatment using 2 (a), 7 (b), and 8 (c) with 100 ng/mL of TRAIL; β-actin was used as the internal control.

In the intrinsic pathway, the released cleaved caspase-8 activates Bid and transforms it into truncated Bid (tBid). Then, tBid translocates to the mitochondria and releases cytochrome c, which binds to Apaf-1 and forms apoptosome that subsequently activates the proteolytic cleaved caspase-9 ²⁷ . The released cleaved caspase-8 and −9 activate the transformation of procaspase-3 to active cleaved-caspase-3 in the final step. (-)Loliolide (8) induced apoptosis via both extrinsic and intrinsic pathways by increasing the levels of cleaved caspase-3, −8, and −9 and decreasing the level of anti-apoptotic FLIP in a dose-dependent manner (Figure 4C). Cleaved caspase-9 was not observed in cells treated with 2 (Figure 4A), whereas it was not concentration-dependent in cells treated with 7 (Figure 4B). Moreover, there was no obvious decrease in FLIP in cells treated with 2 and 7 (Figure 4A and B).

Conclusion

Six natural sesquiterpenes (1-6) were isolated from M. exotica for the first time, and 3 was isolated from natural sources for the first time. All the compounds (1-12) possessed TRAIL-resistance-overcoming activity, but the strongest was in compounds 1 and 5. Western blot analysis demonstrated that compounds 2, 7, and 8 promoted TRAIL-induced apoptosis by increasing the expression of several proapoptotic markers and decreasing the expression of the anti-apoptotic protein Bcl-2.

Experimental

General Experimental Procedures

The analytical instruments and materials used in this study included a P-2200 polarimeter (JASCO) for optical rotations; an ECZ-600 spectrometer for NMR spectroscopy (solvent chemical shifts were used as the internal standard); a PU 980 (JASCO) pump and UV 970 (JASCO) for HPLC; an LCMS system including LCMS 2020 (Shimadzu) for ESI-MS; silica gel 60 F254 (0.25 mm, Merck) and silica gel 60 RP-18 F254S (0.25 mm, Merck) for analytical thin layer chromatography; silica gel 60 N (Kanto Chemical Co., Inc.) for column chromatography; and COSMOSIL AR-II (10.0  ×  250 mm, Nacalai Tesque), Inertsil-ODS-3 (10.0  ×  250 mm, GL sciences), and YMC-SIL06 (10  ×  250 mm, YMC Co., Ltd) for preparative HPLC.

Plant Material

Leaves of M. exotica (430 g) were collected from Bangladesh and deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan, under voucher specimen No. KKB301.

Extraction and Isolation

The dry leaf powder of M. exotica was macerated with MeOH (3  ×  800 mL) at room temperature. The crude extracts (18.3 g) were chromatographed on a Diaion HP-20 (5  ×  15 cm) to afford MeOH (9.1 g) and acetone extracts (3.8 g). Each extract was partitioned into n-hexane, EtOAc, BuOH, and water layers. The MeOH-EtOAc layer (2.4 g) was separated on a sequence of silica gel 60N and ODS columns to yield fractions 4A-4E. Fraction 4B (24.5 mg) was eluted on an Inertsil ODS-3 (10  ×  250 mm) column using 60% MeOH as the mobile phase to afford 1 (8.0 mg) and a mixture of 2 and 3 (2.6 mg). Fraction 1D (666.7 mg) was eluted on a silica gel column (3  ×  30 cm) using CHCl₃:MeOH (100:1-1:1) and 0.1% formic acid as eluents to afford fractions 11A-11I. Fraction 11F (25.6 mg) was separated by a Cosmosil AR-II (10  ×  250 mm) preparative HPLC column using 0.1% formic acid in 30% MeOH as the mobile phase to afford 9 (3.3 mg) and 10 (4.1 mg). Fractions 11E (107.4 mg) and 1E (866.3 mg) were eluted using a Sephadex® LH-20 (3  ×  25 cm) column with MeOH as the eluent to yield 12 (2.0 mg) and 7 (59.6 mg), respectively. Fraction 25D (236.6 mg) was eluted on a Cosmosil AR-II (10  ×  250 mm) column using 0.1% formic acid in 50% MeOH as the mobile phase to afford 4 (34.5 mg), 11 (7.1 mg), and 8 (6.8 mg). Fraction 25C (90.1 mg) was eluted on a preparative normal-phase HPLC column (YMC-SIL06, 10  ×  250 mm) using n-hexane:EtOAc as the mobile phase to afford 5 (7.0 mg) and 2 (9.5 mg). The acetone-hexane layer extract (2.2 g) was eluted on a silica gel 60N column chromatography (4  ×  20 cm) using n-hexane:EtoAc:MeOH (50:1:0-0:1:1) as the eluent and washed with 0.1% trifluoroacetic acid in MeOH to afford fractions 36A-36AJ. Fraction 36G (318.3 mg) was fractionated on a silica gel column (2  ×  20 cm) using CHCl₃:MeOH (1:0-0:1) as the eluent to afford fractions 37A-37L. Fraction 37C (81.3 mg) was eluted on an ODS column (2  ×  20 cm) with 90% acetonitrile as the mobile phase to yield 6 (19.4 mg).

The structures of the isolated compounds were elucidated based on ¹H- and ¹³C-NMR, heteronuclear multiple quantum coherence and heteronuclear multiple bond coherence spectral analysis, and ESI-MS data, as well as by comparison with published data. The physical and spectroscopic data of isolated compounds 1 to 12 are provided in the Supporting Information.

Bioassay

The TRAIL-resistance-overcoming activity of the isolated compounds was determined according to our previously published method ⁴ . The results in the bar graph are expressed as mean ± SD together with significance tests. Western blot analysis of compounds 2, 7, and 8 was performed as described in our previous report ⁴ with slight modifications. All primary antibodies used in this study were prepared at a 1:1000 ratio in Can get Signal® Solution 1 (Toyobo Co., Ltd), while the secondary antibodies were prepared at 1:2000 in Can get Signal® Solution 2 (Toyobo Co., Ltd).

Supporting Information

NMR spectra and TRAIL-resistance-overcoming activity of isolated compounds (PDF).