For immunoblotting, an antibody against cleaved PARP (Asp214) was acquired from Cell Signaling Biotechnology (Beverly, MA, USA). Anti-P-gp (Calbiochem, San Diego, CA, USA), NF-κB p65 (F-6), SIRT1 (H-300), Sp1 (1C6), caspase-9 p35 (H-170), cytochrome c (A-8) and actin (C4) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. Nicotinamide and BAY11-7082 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

A549 or A549RT-eto cells, which were developed and kindly provided by the Laboratory of Biochemistry, Chulabhorn Research Institute, Thailand as described elsewhere (12), were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Gibco) at 37ºC in a humidified atmosphere of 5% CO₂ in air.

Fresh leaves of B. laetivirens (3 kg) were dried at 45ºC and ground into powder. The samples were extracted with MeOH. The extracts were partitioned between H₂O and CH₂Cl₂ and then assessed for cytotoxicity. A portion of the MeOH extract was chromatographed on Sephadex LH-20 gel (100 g; GE Healthcare, Piscataway, NJ, USA), and the fractions showing cytotoxicity to A549RT-eto cells were collected. The active fractions were then subjected to a silica gel (50 g; 60–230 mesh) column in hexane and eluted with MeOH:CH₂Cl₂ (5, 10 and 20%). The active fractions were again subjected to this column and eluted with MeOH:CH₂Cl₂ (0, 3, 5, 7.5 and 10%). The fractions showing a similar TLC profile were combined and then evaluated for cytotoxic activity. Isolation of the bioactive faction is diagrammed in Fig. 1, and the fraction, identified as F14, showed one single spot on TLC and the highest cytotoxic effect on A549RT-eto cells.

Cells were harvested and lysed with lysis buffer [150 mM NaCl, 1% NP-40, 50 mM Tris-HCl (pH 7.5)]containing 0.1 mM Na₂VO₃, 1 mM NaF and protease inhibitors (Sigma). For immunoblotting, proteins from whole cell lysates were resolved by 10 or 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes. Primary antibodies were used at 1:1,000 or 1:2,000 dilutions, and secondary antibodies conjugated with horseradish peroxidase were used at 1:2,000 dilutions in 5% non-fat dry milk. After the final washing, nitrocellulose membranes were exposed for an enhanced chemiluminescence assay using the LAS 4000 Mini (Fuji, Tokyo, Japan).

A549 or A549RT-eto cells (1×10⁶ cells/ml) were incubated with the F14 fraction or DMSO as control for 12 h, and the nuclear extracts were prepared. Cells were pelleted and resuspended in 0.4 ml hypotonic lysis buffer [20 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 0.2% Triton X-100 and 1 mM Na₂VO₃ plus protease inhibitors] and kept on ice for 20 min. After centrifugation at 14,000 × g for 5 min at 4ºC, the nuclear pellet was extracted with 0.1 ml hypertonic lysis buffer on ice for a further 20 min. After centrifugation at 14,000 × g for 5 min at 4ºC, the supernatants were diluted with 100 mM NaCl and incubated with 25 μl of agarose beads conjugated to a consensus NF-κB binding oligonucleotide (Santa Cruz Biotechnology) for 1 h at 4ºC. After 3 washes, sample buffer was added and boiled for 5 min. The binding of NF-κB (p65) protein to the oligonucleotide conjugated with agarose was detected by immunoblotting using an anti-p65 NF-κB antibody (Santa Cruz Biotechnology).

Cells were trypsinized and incubated overnight to achieve 60–70% confluency before siRNA transfection. SIRT1 siRNA (commercially pre-made at Bioneer Corporation, Daejeon, Korea; 100 nM), sense, 5′-ACU UUG CUG UAA CCC UGU A(dTdT)-3′; antisense, 5′-UAC AGG GUU ACA GCA AAG U(dTdT)-3′ or negative control siRNA (Bioneer) were mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cells were incubated with the transfection mixture for 6 h and then rinsed with RPMI-1640 medium containing 10% FBS. The cells were incubated for 48 h before harvesting.

Total RNA was extracted from the cells using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. Three micrograms of total RNA was converted to cDNA using SuperScript II reverse transcriptase (Invitrogen), and PCR was performed using the following specific primers: human MDR1, sense 5′-CCC ATC ATT GCA ATA GCA GG-3′ and antisense 5′-GTT CAA ACT TCT GCT CCT GA-3′; MRP2, sense 5′-ACA GAG GCT GGT GGC AAC C-3′ and antisense 5′-ACC ATT ACC TTG TCA CTG TCC-3′; BCRP, sense 5′-GAT CAC AGT CTT CAA GGA GAT C-3′ and antisense 5′-CAG TCC CAG TAC GAC TGT GAC A-3′. The cDNAs of each sample were diluted, and PCR was run at the optimized cycle number. β-actin mRNA was measured as an internal standard. After amplification, the products were subjected to electrophoresis on 2.0% agarose gel and detected by ethidium bromide staining

A549RT-eto cells were transfected with hMDR1-luciferase or pGL3 empty vector as a control luciferase vector. To normalize transfection efficiency, a pGK-β-gal vector that expresses β-galactosidase from a phosphoglucokinase promoter was included in the transfection mixture. F14 extract was added to the transfected cells at 12 h before harvesting. At 48 h post-transfection, cells were washed with cold PBS and lysed in lysis solution [25 mM Tris (pH 7.8), 2 mM EDTA, 2 mM DTT, 10% glycerol and 1% Triton X-100]. Luciferase activity was measured with a luminometer by using a luciferase kit (Promega, Madison, WI, USA).

SIRT1 activity from A549, A549RT-eto or A549RT-eto cells treated with F14 extract was measured using the SIRT1 direct fluorescence kit provided by Cayman (Ann Arbor, MI, USA) according to the manufacturer’s recommendations. Briefly, 25 μl of assay buffer was added to the cell supernatant. Subsequently, 15 μl of substrate solution (containing a final concentration of 125 μM peptides and 3 mM NAD⁺) was added, and the sample mixture was incubated for 45 min before addition of stop solution. Fluorescence (an excitation wavelength 360 nm and an emission wavelength 460 nm) was measured using the Victor3 microplate reader (Perkin-Elmer, Waltham, MA, USA).

MTT assays were performed for the measurement of cell survival as previously described (13). Dye solution containing tetrazolium was added to the cells in a 96-well plate and incubated for 2 h. The absorbance of the formazan produced by living cells was measured at 570 nm. The relative percentage of cell survival was calculated by the following formula: % Cell survival = (OD_T/OD_C), where OD_T is the mean absorbance of the treated cells and OD_C is the mean absorbance of the control cells.

Data are presented as means ± standard deviation (SD). The Student’s t-test was used for statistical analysis, with P-value <0.05 defined as indicative of statistical significance.

We previously observed that human A549 lung cancer cells resistant to etoposide (A549RT-eto) exhibit upregulation of Stat1 and HDAC4, leading to the enhancement of P-gp protein levels (unpublished data). Moreover, recent studies have shown that NF-κB and SIRT1 are also involved in multidrug resistance (MDR). We thus examined whether NF-κB and SIRT1 protein levels are upregulated in A549RT-eto cells in addition to upregulation of Stat1 and HDAC4. We found that A549RT-eto cells showed higher levels of NF-κB and SIRT1 proteins when compared to these levels in the A549 parental cells (Fig. 2A). We next wondered whether the elevated protein levels of NF-κB in A549RT-eto cells indicate higher activity of NF-κB compared to A549 cells. To address this question, we prepared nuclear extracts from A549 and A549RT-eto cells. Since the active NF-κB protein is translocated into the nucleus and binds to its binding site, we examined whether p65 NF-κB protein found at higher levels in A659RT-eto cells can bind to NF-κB oligonucleotides conjugated with agarose. We found that more active p65 proteins were detected in the oligonucleotide mixtures from A549RT-eto cells than those from A549 cells (Fig. 2B). With the same intent, we also measured SIRT1 activity as we observed enhanced protein levels of SIRT1 in A549RT-eto cells when compared to these levels in A549 cells. We found that A549RT-eto cells showed higher activity of SIRT1 than that in A549 cells using a SIRT1 activity kit (Fig. 2C).

On the basis of our previous study to screen extracts from medicinal plant sources exhibiting the the strongest cytotoxic activity against A549RT-eto cells (13), we chose B. laetivirens. We, thus, purified and collected an active pool from B. laetivirens, and then named the active fraction as F14 extract (Fig. 1). We first optimized the concentration and treatment time of the F14 extract, and found that 20 μg/ml of F14 extract significantly induced the cell death of A549RT-eto cells at 24 h post-treatment but not at 12 h post-treatment (Fig. 3A). However, when we examined the protein levels of NF-κB, SIRT1 and P-gp in the A549RT-eto cells treated with F14 (20 μg/ml) at 12 h post-treatment, we found that F14 treatment drastically reduced the expression levels of NF-κB, SIRT1 and P-gp (Fig. 3B). In addition, we detected reduced pro-caspase-9 levels, and enhanced cytochrome c levels in the same lysates of A549RT-eto cells, indicating induction of intrinsic apoptosis (Fig. 3A). Furthermore, when we treated A549RT-eto cells with the F14 extract at 0.5 and 5 μg/ml for 12 h, we found that the F14 extract at 5 μg/ml was sufficient to reduce not only expression levels of NF-κB, SIRT1 and P-gp but also activities of NF-κB and SIRT1 (Fig. 3C and D).

Since not only the MDR1 gene but also other genes such as MRP2 and BCRP are known to be involved in drug resistance (14,15), we examined levels of transcripts related to MDR1 such as the MDR1, MRP2 and BCRP genes in A549RT-eto cells during F14 treatment. In addition, we aimed to ascertain whether the decrease in P-gp protein levels is regulated at the transcriptional level by F14 treatment. Cells were thus treated with the F14 extract (5 μg/ml) for 12 h, and total RNAs were isolated. After cDNA synthesis, MDR1, MRP2 and BCRP genes were amplified in A549RT-eto cells treated with F14. We found that A549RT-eto drastically reduced levels of MDR1 transcripts and slightly reduced levels of BCRP in a dose-dependent manner, but not MRP2 (Fig. 4A). Furthermore, we examined MDR1 transcriptional activity in the cells using a MDR1-promoter luciferase reporter vector (7). As shown in Fig. 4B, F14 treatment induced a drastic decrease in MDR1-mediated luciferase activity in A549RT-eto cells (Fig. 4B), indicating that the F14 extract reduced MDR1 transcriptional activity. On the basis of these results, we suggest that the F14 extract inhibited MDR1 transcription, resulting in a decrease in P-gp protein levels, which eventually sensitized A549RT-eto cells to apoptosis.

Since we observed enhanced NF-κB protein levels and activity in A549RT-eto cells, we explored whether NF-κB is involved in resistance to etoposide in A549 cells through upregulation of P-gp. We treated A549RT-eto cells with BAY11-7082 (BAY; 10 μM), an inhibitor of NF-κB, and examined cell viability with MTT assays. BAY treatment alone did not cause inhibition of cell growth in A549RT-eto cells while the F14 extract (1 μg/ml) inhibited ~50% cell growth at 24 h post-treatment (Fig. 5A). Furthermore, we found that the combined treatment with the F14 extract and BAY accelerated F14 extract-mediated apoptosis in A549RT-eto cells (Fig. 5A), which was confirmed by observation of cleaved PARP and pro-caspase-9 (Fig. 5B). Moreover, when we examined protein levels of NF-κB, SIRT1 and P-gp after treatment with Bay alone, F14 extract alone, or Bay plus F14 extract, we found that BAY treatment alone decreased the expression levels of NF-κB and P-gp but did not influence protein levels of SIRT1 in the cells (Fig. 5B). F14 treatment alone drastically diminished the protein levels of NF-κB, SIRT1 and P-gp in the A549RT-eto cells (Figs. 3A and 5B). We also observed that the combined treatment more significantly reduced protein levels of NF-κB, SIRT1 and P-gp (Fig. 5B). These results suggest that NF-κB is involved in MDR in A549 cells by upregulation of P-gp, resulting in resistance to etoposide.

We observed the enhanced levels of SIRT1 in A549RT-eto cells (Fig. 2A) while F14 treatment reduced SIRT1 as well as P-gp expression levels (Fig. 3B). We aimed to ascertain whether downregulation of the SIRT1 protein level was attributed to a decrease in P-gp expression, leading to F14 fraction-induced apoptosis. Thus, we introduced SIRT1 siRNA to suppress SIRT1 levels in A549RT-eto cells. We first optimized the SIRT1 siRNA concentration (100 nM; data not shown). We then treated A549RT-eto cells with F14 under suppression of SIRT1 using its siRNA and examined cell viability with MTT assays. Importantly, we found that suppression of SIRT1 did not accelerate F14-induced apoptosis of A549RT-eto cells (Fig. 6A) as noted in the combined treatment with Bay and F14 (Fig. 5). We also observed that inhibition of SIRT1 activity with nicotinamide (NAM; 500 μM) did not accelerate F14 extract-induced apoptosis of A549RT-eto cells (Fig. 6C). Furthermore, neither suppression of SIRT1 protein levels nor inhibition of SIRT1 activity significantly affected P-gp expression (Fig. 6B and D). However, F14 treatment (1 μg/ml) decreased expression of P-gp irrespective of SIRT1 protein levels and activity (Fig. 6B and D). These results suggest that the elevated levels of SIRT1 were not related to the upregulation of P-gp in A549RT-eto cells.