Novel derivative of benzofuran induces cell death mostly by G2/M cell cycle arrest through p53-dependent pathway but partially by inhibition of NF-kappaB

Abstract

The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor kappaB (NF-kappaB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-kappaB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-kappaB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells.

FIGURE 1.

Effect of Benfur on cell death. The structural formula (5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydro-1-benzofuran-3-carboxylic acid, designated IUPAC name) (inset) and chemical characterization by NMR data for benzofuran lignan (Benfur) are provided (A). Jurkat, U-937, HT29, and MCF-7 cells (10,000/well) were treated with different concentrations of Benfur for 24 and 48 h in triplicate. Cell viability was determined by MTT assay and indicated in percentage of cell viability. Error bars indicate ± S.D. B, different cells were treated with 100 nm Benfur for 24 h, and the level of apoptosis was measured by incubating cells with FITC-PARP (cleaved fragment) and detected in FACS. Percentage of apoptotic populations are represented as the M2-gated population (C). Untreated controls showed an M2 population (Apoptotic) between 4–8% (figures are not included here). Jurkat and U-937 cells were treated with different concentrations of Benfur for 48 h in triplicate. The culture supernatant was assayed for LDH and indicated in percentage above untreated cells, considering the 1% Triton X-100-treated cells as 100% (D). Jurkat and T-cells (isolated from fresh human blood and purified by nylon wool column) were incubated with 100 nm Benfur for 24 h. Cell viability was measured by MTT assay and indicated as inhibition of cell viability in percentage (E).

FIGURE 2.

Effect of Benfur on PARP cleavage, caspase activity, cell cycle stages, and amounts of cytoplasmic cytochrome c, Bax, and Bcl-2. Jurkat cells were treated with 100 and 500 nm Benfur for the indicated time period, and PARP cleavage was determined using FACS analysis as described under “Experimental Procedures.” Percentage apoptotic populations are represented as the M2-gated population (A). Jurkat and U-937 cells were treated with different concentrations of Benfur for 48 h, and then PARP was detected from whole cell extracts by Western blot (B). Jurkat cells were treated with indicated concentrations of the compound for 24 h and harvested in lysis buffer. Cellular lysates were incubated with caspase 3 substrate (Ac-DVED-pNA) (C1) or caspase 8 substrate (Ac-ITED-pNA) (C2), and absorbance was recorded at 405 nm. Results are presented as percentage of caspase activities considering 100% for untreated cells. Oleandrin (OL) (100 ng/ml) was used to treat the cells as a positive control. Jurkat and U-937 cells were treated with 100 nm Benfur for variable times, and the amounts of cytochrome c, Bax, and Bcl-2 were measured from cytoplasmic extracts by Western blot (D). Jurkat (5 × 10⁵) cells were treated with different concentrations of Benfur for 24 h and after staining with PI, cell cycle distribution was analyzed using a flow cytometer. The data indicate the percentage of live cell population in each phase of the cell cycle (E1). All experiments were performed in duplicate and showed similar results. The percentage apoptotic population is depicted in the graph (E2). Jurkat cells were treated with 100 and 500 nm Benfur for 24, 48, and 72 h in triplicate, and the percentage live population in the cell cycle phases (G1, S, and G2/M) were analyzed by flow cytometry (F). U-937 (5 × 10⁵) cells were treated with different concentrations of the Benfur for 24 h and after staining with PI, cell cycle distribution was analyzed using a flow cytometer. Data indicate the percentage of live cell population in each phase of the cell cycle (G1). All experiments were performed in duplicate and gave similar results. The percentage apoptotic population is depicted in the graph (G2).

FIGURE 3.

Effect of Benfur on SA-LPS-induced activation of NF-κB and nuclear translocation of p65. Jurkat and U-937 cells were treated with different concentrations of Benfur for 12 h and then stimulated with 100 ng/ml SA-LPS for 1 h. Nuclear extracts were prepared and used for NF-κB DNA binding (A). Jurkat and U-937 cells were transiently transfected with an NF-κB containing plasmid linked to the SEAP gene. After 12 h, cells were treated with different concentrations of Benfur for 12 h and then stimulated with 100 ng/ml SA-LPS for 6 h. Cell culture-conditioned medium was harvested and assayed for SEAP activity as described in the Clontech Great EscApe SEAP protocol. Results are expressed as fold activity over the nontransfected control value. Transfection efficiency was analyzed with pEGFP-N1 (Clontech) control vector (B). Jurkat and U-937 cells, pretreated with Benfur (100 nm) for 12 h were stimulated with 100 ng/ml SA-LPS for different times. Cytoplasmic extracts were used to measure the amount of IκBα, and the same blot was reprobed for p65 and tubulin. Nuclear extracts were used to measure p65 by Western blot (C). Jurkat and U-937 cells were transfected with p65 and NF-κB-SEAP reporter constructs for 3 h with Lipofectamine. Cells were cultured for 12 h and then treated with different concentrations of Benfur for 12 h. Nuclear extracts were used to measured NF-κB DNA binding (D). Jurkat and U-937 cells (vector, p65-transfected, or IκBα-DN (dominant negative IκBα) transfected for 3 h and then cultured for 12 h) were treated without or with 100 nm Benfur for 24 h. Cell viability was assayed by MTT assay and indicated as inhibition of cell viability in percentage (E). Jurkat cells and T-cells (isolated from fresh human blood by Ficoll-hypaque followed by nylon wool column methods) were treated with 100 nm Benfur for different times. NF-κB was assayed from nuclear extracts (F).

FIGURE 4.

Effect of Benfur on the amounts of p53, p21, p27, cyclin B, and retinoblastoma. U-937 and Jurkat cells were treated with different concentrations of Benfur for 12 h. The p53 DNA binding was detected from nuclear extracts by gel shift assay (A). The amounts of p21, p27, cyclin B, phospho-Rb, and Rb were measured from 100 μg of whole cell extracts by Western blot (B). All those blots were reprobed for tubulin. U-937 and MCF-7 cells were treated with Benfur (100 nm) for 12 h, and cells were used to measure p21 by immunofluorescence microscope (C). HCT116 and p53 stably transfected [HCT116 (p53^+/+)] cells were treated with different concentrations of Benfur for 48 h. The amount of p53 was measured by Western blot (D), and cell viability was assayed by MTT assay and indicated as inhibition of cell viability in percentage (E).

FIGURE 5.

Effect of Benfur on the amount of MDM2 and Sp1 DNA binding. U-937 and Jurkat cells were treated with Benfur (100 nm) for different times. The amounts of MDM2 and actin were detected by RT-PCR followed by PCR from total RNA (A). The amounts of MDM2 and p53 were measured from whole cell extracts by Western blot (B). The DNA binding to Sp1 and Oct1 was detected from nuclear extracts by gel shift assay (C). Nuclear extracts from Jurkat cells were incubated with different concentrations of Benfur for 2 h, and Sp1 DNA binding was carried out by gel shift assay (D). Docking interaction of Benfur with Sp1 transcription factor (PDB ID: 1SP1) was performed with the AutoDock 4.0 program (E).