Targeting Truncated Retinoid X Receptor-α by CF31 induces TNFα-dependent apoptosis

Abstract

A truncated version of retinoid X receptor-α, tRXRα, promotes cancer cell survival by activating the PI3K/AKT pathway. However, targeting the tRXRα-mediated survival pathway for cancer treatment remains to be explored. We report here our identification of a new natural product molecule, CF31, a xanthone isolated from Cratoxylum formosum ssp. Pruniflorum, and the biological evaluation of its regulation of the tRXRα-mediated PI3K/AKT pathway. CF31 binds RXRα and its binding results in inhibition of RXRα transactivation. Through RXRα mutational analysis and computational studies, we show that Arg316 of RXRα, known to form salt bridges with certain RXRα ligands such as 9-cis-retinoic acid (9-cis-RA), is not required for the antagonist effect of CF31, demonstrating a distinct binding mode. Evaluation of several CF31 analogs suggests that the antagonist effect is mainly attributed to an interference with Leu451 of helix H12 in RXRα. CF31 is a potent inhibitor of AKT activation in various cancer cell lines. When combined with TNFα, it suppresses TNFα activation of AKT by inhibiting TNFα-induced tRXRα interaction with the p85α regulatory subunit of PI3K. CF31 inhibition of TNFα activation of AKT also results in TNFα-dependent activation of caspase-8 and apoptosis. Together, our results demonstrate that CF31 is an effective converter of TNFα signaling from survival to death by targeting tRXRα in a unique mode and suggest that identification of a natural product that targets an RXR-mediated cell survival pathway that regulates PI3K/Akt may offer a new therapeutic strategy to kill cancer cells.

Introduction

Retinoid X receptor (RXR)α, a unique member of the nuclear receptor superfamily, regulates diverse biological processes, including growth, differentiation, apoptosis, and immune response, and ligands for RXRα show promise as therapeutic agents for many diseases such as cancer (1-4). Alterations in the expression and function of RXRα are implicated in the development of a number of diseases and cancer. Targeted disruption of RXRα gene leads to prostatic preneoplastic lesions (5) and skin abnormalities (6). Dysregulation of RXRα function by phosphorylation is associated with the development of human liver cancer (7) and colon cancer (8). Levels of RXRα protein are often reduced in cancer cells and tumor tissues (9-14), suggesting that diminished RXRα expression is associated with the development of certain malignancies. Numerous studies have demonstrated that proteolytic cleavage of RXRα protein is primarily responsible for its diminished levels in tumor cells and represents an important mechanism controlling the function and activities of RXRα (13-18). Recent studies showed that RXRα binding to PML/RARα is essential for the development of acute promeylocytic leukemia (19, 20), further demonstrating the oncogenic potential of this protein when it acts inappropriately.

Although significant progress has been made, how RXRα regulates cancer cell growth and how its ligands suppress tumorigenesis are still poorly understood. Like other nuclear receptors, RXRα interacts with DNA to regulate the transcription of target genes in a ligand-dependent manner (1-4). Aside from its role in DNA binding and transactivation, accumulating evidence indicates that RXRα also has extranuclear nongenomic actions (15, 21-24). RXRα resides in the cytoplasm at certain stages during development (25, 26). It migrates from the nucleus to the cytoplasm in response to differentiation (23), apoptosis (21), and inflammation (22, 24). Cytoplasmic localization of RXRα facilitates nuclear export of its heterodimerization partner Nur77, leading to Bcl-2 conversion and apoptosis (21, 27, 28).

We showed recently that proteolytic cleavage of RXRα results in production of truncated RXRα protein, tRXRα, which acquires new function that is different from the full-length RXRα (29). Unlike the full-length RXRα that resides in the nucleus, tRXRα is cytoplasmic and interacts with the p85α subunit of phosphatidylinositol-3-OH kinase (PI3K) in a tumor necrosis factor-α (TNFα)-dependent manner, leading to activation of the PI3K/AKT pathway, a major survival pathway important for uncontrolled growth of tumor and as drug resistance (29). Activation of the PI3K/AKT pathway by tRXRα contributes significantly to anchorage-independent growth of cancer cells in vitro and tumor growth in animals (29), offering an opportunity to suppress cancer cell growth by targeting the tRXRα-mediated PI3K/AKT pathway.

Epidemiological studies have shown that dietary phytochemicals provide beneficial effects for cancer prevention, and among them, xanthones are of great interest as cancer chemopreventive agents because of their potent anti-oxidative and anti-cancer activity (30-33). We previously purified a series of xanthones from a medicinal plant, Cratoxylum formosum ssp. pruniflorum, which belongs to the Clusiaceae family and is widely distributed in several Southeast Asian countries (34, 35). Here, we report our identification of one of the xanthones, CF31, which suppresses tRXRα-dependent AKT activation through its unique RXRα binding. CF31, when combined with TNFα inhibits TNFα-induced tRXRα-p85α interaction and AKT activation. Moreover, the CF31 and TNFα combination results in synergistic induction of TNFα-dependent caspase-8 activation and apoptosis in cancer cells. Thus, CF31 represents a new modulator of the tRXRα survival pathway and a potent converter of TNFα signaling from survival to death.

Materials and Methods

Isolation of natural products

Compounds CF31, also called cochinchinone B or 1,3,6,7-tetrahydroxy-2-(3-methyl-2-butenyl)-5-(3,7-dimethyl-2,6-octadienyl)xanthone (36), CF13 (1,3,5-trihydroxy-4-Geranylxanthone), and CF26 (pruniflorone Q) were isolated from the stems of C. formosum ssp. pruniflorum as described (35).

Plasmids

The pGAL4-RXRα-LBD plasmid was obtained by inserting RXRα LBD sequence (amino acids 198–462) in-frame with the Gal4 DBD coding sequence in the pBIND vector (Promega).

Antibodies and regents

Antibodies for phospho-Akt(Ser473, D9E) and cleaved caspase-8(p43/p41) (Cell Signaling); Bax(6A7) (Sigma-Aldrich); p85α (Millipore); AKT1(C-20), actin, c-Myc(9E10), RXRα(ΔN197), RXRα(D20), and PARP(H-250) (Santa Cruz Biotechnology) were used. Caspase-3 and caspase-8 activity assay kits were from Biovision.

Stable transfections

GFP-RXRα/Δ80 was stably transfected into SW480, HepG2, MCF-7 cells to obtain SW480/RXRα/Δ80, HepG2/RXRα/Δ80 and MCF-7/RXRα/Δ80 respectively.

Ligand-binding assay

RXRα LBD was incubated with [³H]-9-cis-RA in the presence or absence of unlabeled 9-cis-RA or CF31. Bound [³H]-9-cis-RA in RXRα LBD protein was determined in a scintillation counter (29).

Transient transfection and reporter assays

CV-1 green monkey kidney cells were grown in DME medium supplemented with 10% fetal bovine serum (FBS). For CAT reporter assays, cells were seeded at 5 × 10⁴ cells/well in 24-well plates and transfected with 50 ng TREpal-tk-CAT reporter plasmid (37), 20 ng β-galactosidase expression vector (pCH 110; Amersham), 20 ng RXRα expression vectors using Lipofectamine 2000 (Invitrogen). Cells were then treated with CF31 at different dose for 20 hr. CAT activity was normalized with β-galactosidase activity. For luciferase reporter assay, 24 hr after transfected with pGL5 luciferase reporter vector (40 ng/well) and pGAL4-RXRα-LBD expression vector (40 ng/well), cells were incubated with varied concentrations of compounds for 12 hr. Luciferase activities were measured using the Dual-Luciferase Assay System kit (Promega).

Molecular docking and molecular dynamics (MD) simulations

AutoDock version 4.0 was used for the docking screening. The Lamarckian genetic algorithm was selected for ligand conformational searching. Docking parameters were as follows: grid box of 40 Å * 40 Å * 40 Å, xyz-coordinates of gridcenter: 49.192 63.991 -7.523, population size of 150, random starting position and conformation, translation step ranges of 2 Å, rotation step ranges of 50°, elitism of 1, mutation rate of 0.02, crossover rate of 0.8, local search rate of 0.06, and 2.5 million energy evaluations. MD simulations were adopted to sample possible configurations of each RXRα LBD complex using the AMBER (version 7.0) program (38). To set up each MD simulation, the electrostatic potentials of the ligand was computed by using the Gaussian 98 package at the HF/6-31G* level. Atom-centered partial charges were derived by using the Restrained Electrostatic potential method implemented in the AMBER package.

Immunofluorescence microscopy

HepG2 cells mounted on glass slides were permeabilized with PBS containing 0.1% Triton X-100 and 0.1 M glycine for 15 min, and blocked with 1% BSA in PBS for 30 min at room temperature. Cells were then incubated with primary antibody (anti-Bax (6A7) (1:500)) at 37°C for 1 hr and detected by anti-rabbit IgG conjugated with Cy3 (1:400) at room temperature for 30 min. Cells were co-stained with 4'6'-diamidino-2-phenylindole (DAPI) to visualize nuclei. The images were taken using LSM-510 confocal laser scanning microscope system (Carl Zeiss, Oberkochen, Germany).

Western blotting

Equal amounts of the lysates were electrophoresed on 8% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk in TBST [50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween 20] for 1hr, incubated with various primary antibodies for 2 hr and detected with either anti-rabbit (1:5000) or anti-mouse (1:5000) secondary antibodies for 1 hr, all of which were undertaken under room temperature. The final immunoreactive products were detected by using ECL system.

Caspase activity assay

Caspase activities were determined by a colorimetric assay based on the ability of caspase-3, -8 to change acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) and acetyl-Ile-Glu-Thr-Asp p-nitroanilide Ac-IETD-pNA) into a yellow formazan product [p-nitroaniline (pNA)], respectively. After treatment with CF31 and/or TNFα, lysates were prepared and assayed according to the manufacturer's protocol (Biovision Research Products).

RXRα siRNA

RXRα siRNA siGENOME SMARTpool (M-003443-02) and siRNA Non-specific Control IX (D-001206-09-05) were from DHARMACON, and they were transfected into cells by RNAiMAX reagent (Invitrogen).

Co-immunoprecipitation and immunoblotting assays

Cells extracts were cleared by incubation with the Protein A/G plus Agarose beads and then incubated with appropriate antibody and 30μl of Protein A/G plus Agarose beads overnight at 4°C. Immunoreactive products were detected by chemiluminescence with an enhanced chemiluminescence system (ECL) (Amersham) .

Colony formation assay

HeLa cells were seeded in 6-well plate (500 cells/well) for 5 days, treated with CF31 in 0.5% serum medium for 3 days, and fixed with 4% paraformaldehyde. Colonies were stained with 0.1% crystal violet.

Statistical analyses

Data were analyzed using an analysis of variance or Student's test and were presented as the mean SEM.

Results

CF31 binds to RXRα

To identify new agents for regulating the tRXRα survival pathway, we screened a natural product library prepared from Chinese Herbal Medicine for compounds capable of regulating RXRα transcriptional function. For this purpose, transient transfection assays using a CAT reporter containing TREpal capable of binding to bind to RXRα homodimer (37) were used. Compounds were evaluated for their effect on TREpal activity in the absence or presence of 9-cis-retinoic acid (RA), the natural RXR ligand known to induce the formation of RXRα homodimer and its activation of TREpal (37). Among compounds that were identified to modulate RXRα activity, CF31 (Fig. 1A), which was prepared from Cratoxylum formosum ssp. Pruniflorum (35), potently inhibited 9-cis-RA-induced TREpal-reporter activity in a dose dependent manner (Fig. 1B). CF31 at 10 μM showed a similar inhibitory effect when compared to BI1003 (1 μM), a known RXRα antagonist (39). To further study the inhibitory effect of CF31, we cloned the ligand-binding domain (LBD) of RXRα as a Gal4 fusion, and used the resulting Gal4-RXRα/LBD chimera and Gal4 reporter system to evaluate the inhibitory effect of CF31. Gal4-RXRα/LBD strongly activated the Gal4 reporter in the presence of 9-cis-RA, which was inhibited by 1 μM UVI3003, another RXRα antagonist (40) (Fig. 1C). Similar to UVI3003, treatment of cells with CF31 resulted in inhibition of 9-cis-RA-induced reporter activity in a CF31 dose dependent manner. Since UVI3003 was derived from a potent and selective RXRα agonist CD3254 (40), the effect of CF31 on CD3254-induced RXRα activity was also evaluated. CD3254 strongly induced Gal4-RXRα/LBD activity, which was comparable to that induced by 9-cis-RA. Interestingly, CD3254-induced reporter activity was similarly inhibited by UVI3003 and CF31 (Fig. 1C). We next determined whether CF31 could bind to RXRα. Thus, a ligand competition assay with [³H]9-cis-RA binding to RXRα LBD was used. While unlabeled 9-cis-RA was able to efficiently displace [³H]9-cis-RA from binding to RXRα LBD with an IC₅₀ of 7.6 nM, CF31 displaced [³H]9-cis-RA with an IC₅₀ of 9.6 μM (Fig. 1D). Together, our data demonstrate that CF31 acts as a RXRα antagonist by binding to the RXRα LBD.

Figure 1. CF31 binds to RXRα in a unique mode.

(A) Structure of CF31.

(B) Inhibitory effect of CF31 on RXRα transcriptional activity. CV-1 cells transfected with TREpal-tk-CAT and RXRα expression vector were treated with the indicated concentrations of CF31 in the presence of 10^-7 M 9-cis-RA. Reporter activities were measured and normalized. For comparison, the effect of BI-1003 (1 μM) was shown. One of five similar experiments is shown. Bars represent means ± SEM.

(C) Inhibition of Gal4-RXRα-LBD activity by CF31. CV-1 cells transfected with pGL5 luciferase reporter vector and pGAL4-RXRα-LBD expression vector (40 ng/well) were incubated with or without 9-cis-RA (10^-7 M) or CD3254 (10^-7 M) in the presence or absence of the indicated concentrations of CF31 or UVI3003 for another 12 h. Luciferase activities were measured using the Dual-Luciferase Assay System kit. One of three similar experiments is shown. Bars represent means ± SEM.

(D) CF31 binding to RXRα in vitro. RXRα LBD protein was incubated with [³H] 9-cis-RA in the presence or absence of CF31 or unlabeled 9-cis-RA. Bound [³H] 9-cis-RA was quantitated by liquid scintillation counting.

Arg316 is not required for the antagonist effect of CF31

Molecular conformation analyses showed there was a large degree of structural overlapping between CF31 and LG100754 (Fig. 2A), an antagonist of RARα/RXRα heterodimer (41). This suggested that CF31 might bind to the antagonist form of the RXRα LBD. However, CF31, unlike many natural and synthetic RXRα ligands, lacks a carboxylate moiety known to form salt bridges with Arg316 in the L-shaped RXRα ligand-binding pocket (LBP) (41-43). To understand how CF31 binds RXRα, a docking study using the antagonist structure of the RXRα LBD in complex with LG100754 (41) was conducted. CF31 docked well with a good score to the RXRα antagonist conformation. Superposing the docked configuration of CF31 and the crystal structure of LG100754 showed that their scaffold orientations were highly conserved (Fig. 2A). An interesting difference, however, is that while the carboxylate group of LG100754 made a strong salt bridge with Arg316 in helix H5, CF31 could not form such a bridge (Fig. 2B).

Figure 2. Analysis of CF31 RXRα binding.

(A) Comparison of the docked conformation of CF31 (grey and red) to the crystal structure of LG100754 (Green) (PDB code: 3A9E).

(B) The interactions of LG100754 (yellow) and CF31 (green) respectively with the RXRα LBP. Only residues closer than 4.2 Å to the ligand are shown (blue). Salt bridge is shown as dotted green line. The secondary structure of the RXRα-LBD from structure of PDB 3A9E was used and specific residues of RXRα are labeled.

(C) Role of Arg316 mutation. CV-1 cells transiently transfected with 40 ng of pGL5-Luc reporter vector and 40 ng of Gal4-RXRα-LBD or Gal4-RXRα-LBD/R316A were treated with or without 9-cis-RA (10^-7 M) or CD3254 (10^-7 M) in the presence or absence of the indicated concentrations of CF31 or UVI3003. One of three similar experiments is shown. Bars represent means ± SEM.

(D) The interactions of 9-cis-RA (blue, PDB 1FBY)) and CD3254 (black, PDB 3FUG) respectively with Arg316 of RXRα LBP. Comparison of ligand-residue interaction spectrum for 9-cis-RA and CF31. Binding free energies were computed using the MM-PB/SA method, including each residue in the RXRα LBP (from aa 260 to 450) and the overall binding free energy given by the MM-PB/SA method for 9-cis-RA and CF31.

To study the requirement of Arg316 in CF31 binding, Arg316 was replaced with Glu, and the resulting mutant RXRα/R316E was evaluated for its activation by 9-cis-RA and CD3254. As shown in Fig. 2C, mutation of Arg316 completely abolished the effect of 9-cis-RA on activating the Gal4 reporter. In contrast, CD3254 could still activate the RXRα/R316E mutant. Thus, while Arg316 is capable of establishing an ionic interaction with the carboxylate group of 9-cis-RA (43), it fails to form salt bridges with CD3254 (40). Consistently, our computational analysis showed that 9-cis-RA formed two shorter hydrogen bonds (2.098 Å, 2.423 Å) with Arg316 than those formed with CD3254 (2.901 Å, 2.553 Å). Therefore, Arg316 plays a more important role in the binding of 9-cis-RA than that of CD3254. We then studied whether CF31 could inhibit CD3254-induced RXRα/R316E activity. When RXRα/R316E was cotransfected with the Gal4 reporter, CD3254-induced reporter activity was potently inhibited by CF31 in a dose dependent manner (Fig. 2C), demonstrating that Arg316 was not required for CF31 binding.

To identify which amino acid residues in the RXRα LBP were key to CF31 binding, we used the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PB/SA) method (44) to evaluate binding free energy of CF31 in complex with the RXRα LBP. For comparison, 9-cis-RA was also studied. Our examination of the interaction spectrum of CF31 with amino acid residues in the RXRα LBP with that of 9-cis-RA revealed that Arg316 was indeed mainly responsible for the binding of 9-cis-RA but not CF31 (Fig. 2E)(Supplemental Table 1). However, CF31 made considerable contacts with several amino acid residues in the RXRα LBP, including Val265 and Ile268 in H3, Ile310 and Phe313 in H5, and V342, I345, F346 in H7, and Leu436 in H11 (Fig. 2E). Thus, the nonpolar van der Waals interactions of CF31 with the hydrophobic residues in the RXRα LBP may serve to stabilize the binding of CF31, revealing its distinct mode of RXRα binding.

In an attempt to gain more understanding of the antagonist effect of CF31, we evaluated two additional xanthones isolated from Cratoxylum formosum ssp. Pruniflorum, CF13 and CF26 (Fig. 3A), for their antagonist effect (Fig. 3B). While CF13 had no inhibitory effect of 9-cis-RA-induced RXRα transactivation, the inhibitory effect of CF26 was even stronger than that of CF31 (Fig. 3B). Modeling studies suggested that a potential steric hindrance between side chain of Leu541 in H12 and substituent at position 3 of ring A of these compounds might mediate their antagonist effect (Fig. 3C). The size of the substituent correlated well with their antagonist effect, with 3-methyl-2-butenyl in CF26 being the most effective.

Figure 3. Antagonist effect of CF31 compounds of RXRα transactivation.

(A) Structure of compounds CF13, CF31, and CF26. The red moiety of each compound is predicted to mediate its antagonistic activity.

(B) Antagonist effect of CF13, CF31, and CF26. CV-1 cells transfected with TREpal-tk-CAT and RXRα expression vector were treated with or without 10^-7 M 9-cis-RA in the absence or presence of 10 μM CF13, CF26, or CF31. Reporter activities were measured and normalized. One of three similar experiments is shown. Bars represent means ± SEM.

(C) Superposition of the docked binding modes of compounds CF13 (blue), CF31 (green), and CF26 (black) with RXRα and their interactions with the residue Leu451 of Helix 12.

CF31 inhibits AKT activation

In the first step to study the effect of CF31 on tRXRα-dependent AKT survival pathway, we determined whether CF31 could regulate AKT activation. Figure 4A showed that CF31 dose-dependently inhibited the activation of AKT in HeLa cells, revealed by its inhibition of the expression of phospho-AKT but not total AKT using Western blotting. Time course analysis showed that the inhibition of AKT activation by CF31 was observed when cells were treated by 10 μM CF31 for as short as 8 hr (Fig. 4B). The inhibition of AKT activation by CF31 also closely correlated with apoptosis induction as shown by enhanced cleavage of PARP protein (Figs 4A and B).

Figure 4. Inhibition of AKT activation and cells survival by CF31.

(A) Dose dependent effect of CF31 on AKT activation and apoptosis induction. HeLa cells were treated with CF31 (1, 10, 20 μM) for 12 hr and analyzed by immunoblotting.

(B) Time-course analysis. HeLa cells were treated with CF31 (10μM) for 8, 16, 24 hr and analyzed by immunoblotting.

(C) CF31 inhibits clonogenic survival of HeLa cells. Cells grown in 6-well plates for 5 days were treated with CF31 (50 μM) or K-80003 (50 μM) or Sulindac (50 μM) for 3 days.

(D) RXRα dependent enhancement of the apoptotic effect of camptothecine by CF31. HepG2 cells transfected with control or RXRα siRNA were treated with CF31 (5 μM) and/or camptothecine (10 μM) for 9 hr, and analyzed by immunoblotting using anti-PARP and D20 anti-RXRα antibody.

The ability of CF31 to inhibit AKT activation prompted us to examine the effect of CF31 on the survival of HeLa cells by colonogenic survival assays. Treatment of cells with CF31 for 3 days completely inhibited colony formation of HeLa cells, which was as effective as K-80003, a Sulindac analog that inhibits AKT activation through its binding to tRXRα (29). In contrast, Sulindac showed little effect under the same conditions (Fig. 4C). We also determined whether CF31 inhibition of AKT activation could enhance the apoptotic response of cancer cells to chemotherapeutics. To this end, we examined the death effect of the topoisomerase-I inhibitor camptothecine (CPT), which has shown significant anti-tumor activity across a broad spectrum of human tumors (45), in the presence or absence of CF31 in HepG2 liver cancer cells. Immunoblotting showed that PARP cleavage induced by CPT was significantly enhanced when cells were cotreated with CF31, while CF31 alone had undetectable effect on PARP cleavage under the conditions used (Fig. 4D). When cells were transfected with RXRα siRNA, the enhancing effect of CF31 was impaired. Thus, CF31 may sensitize cancer cells to the apoptotic effect of certain anti-cancer drugs through its inhibition of AKT activation.

Antagonist effect of CF31 on TNFα activation of AKT

TNFα, a multifunctional cytokine, can activate AKT in a tRXRα-dependent manner in certain cancer cells (29). In HeLa cells, TNFα treatment enhanced AKT activation, while CF31 and K-80003 showed comparable effect on inhibiting AKT activation. Both compounds were much more effective than Sulindac when used at 10 μM (Fig. 5A). We then examined whether CF31 could inhibit TNFα activation of AKT. Treatment of HeLa cells with TNFα for 30 min strongly activated AKT, which was inhibited by either CF31 or K-80003 in a dose dependent manner (Fig. 5B). Both compounds at 20 μM completely inhibited the effect of TNFα on activating AKT. CF31 could also inhibit basal and TNFα-induced AKT activation in other cancer cell lines, including A549 lung cancer and HepG2 liver cancer cells (Fig. 5C). All these cancer cell lines expressed high levels of tRXRα, as revealed by immunoblotting using ΔN197 anti-RXRα antibody recognizing the LBD of RXRα (29) (Fig. 5C).

Figure 5. CF31 inhibits TNFα-induced AKT activation.

(A) Inhibition of basal AKT activation and induction of apoptosis by CF31. HeLa cells were treated with CF31 (10 μM) or K-80003 (10 μM) or Sulindac (10 μM) for 24 hr and analyzed by immunoblotting.

(B) Inhibition of TNFα-induced AKT activation by CF31. HeLa cells were treated with CF31 (5, 10, 20 μM) or K-80003 (10, 20 μM) for 12 hr before exposed to TNFα (10 ng/ml) for 30 min and analyzed by immunoblotting.

(C) Inhibition of AKT activation by CF31. Cells were starved overnight and pretreated with CF31 (10 μM) for 12 hr before exposed to TNFα (10 ng/ml) for 30 min. AKT activation was analyzed by immunoblotting. Expression of RXRα and tRXRα was analyzed by immunoblotting using ΔN197 anti-RXRα antibody.

(D) Inhibition of AKT activation by RXRα siRNA. A549 cells transfected with control or RXRα siRNA for 24 hr were treated with CF31 (10 μM) for 12hr before exposed to TNFα(10 ng/ml) for 30 min. AKT activation was analyzed by immunoblotting. Reduction of RXRα level was examined by using D20 anti-RXRα antibody.

(E) Transfection of SW480 cells with GFP-RXRα/Δ80 sensitizes their response to CF31. SW480 and SW480/RXRα/Δ80 were starved overnight and pretreated with CF31 (10 μM) for 12 hr before exposed to TNFα(10 ng/ml) for 30 min. AKT activation and RXRα expression were analyzed by immunoblotting. Expression of endogenous and transfected GFP-RXRα/Δ80 was examined by using ΔN197 anti-RXRα antibody.

(F) CF31 inhibits tRXRα-p85α interaction. A549 cells treated with CF31 (10 μM) for 12 hr before exposed to TNFα(10 ng/ml) for 30 min were analyzed for RXRα-p85α interaction by co-immunoprecipitation using ΔN197 antibody.

To study the role of tRXRα in CF31 action, we first assessed the effect of RXRα siRNA transfection. Transfection of RXRα siRNAs in A549 cells, which reduced levels of both the full-length RXRα and tRXRα (29), abolished the inhibitory effect of CF31 on AKT activation either in the absence or presence of TNFα (Fig. 5D). To complement this study, we determined whether overexpression of tRXRα could enhance the inhibitory effect of CF31. For this purpose, we stably expressed GFP-RXRα/Δ80 (29), a GFP-tagged RXRα mutant lacking 80 N-terminal amino acid residues, in SW480 colon cancer cells that expressed little tRXRα protein (Fig. 5E), SW480 cells exhibited low basal AKT activation, and their AKT activation was insensitive to regulation by TNFα and (Fig. 5E). However, stable expression of GFP-RXRα/Δ80 enhanced not only their basal AKT activation but also their response to TNFα induction of AKT activation (Fig. 5E). Moreover, the sensitivity of SW480 cells to CF31 was restored, as it could effectively inhibit TNFα-induced AKT activation in SW480/RXRα/Δ80 cells. Thus, tRXRα expression is involved in AKT activation by TNFα and is essential for its inhibition by CF31.

Previously, we reported that tRXRα binding to p85α was essential for the activation of the PI3K/AKT pathway (29). Thus, we determined whether CF31 inhibition of tRXRα-dependent AKT activation could be attributed to its interference with tRXRα interaction with p85α. Consistent with our previous observation, TNFα induced interaction of tRXRα with p85α, as revealed by co-immunoprecipitation using the ΔN197 anti-RXRα antibody (Fig. 5F). However, when cells were co-treated with CF31, TNFα-induced tRXRα-p85α interaction was largely abolished. Thus, CF31 may modulate AKT activation through its ability to inhibit the interaction of tRXRα with p85α.

CF31 induces tRXRα-dependent apoptosis

To study whether CF31 inhibition of AKT activation could result in apoptosis, we first examined the effect of CF31 on activation of pro-apoptotic Bcl-2 family member Bax in HepG2 cells in the presence of TNFα by immunostaining using a Bax conformation-sensitive antibody Bax/6A7 that recognizes active Bax protein (46). As shown in Figure 6A, immunostaining with Bax/6A7 antibody demonstrated that activated Bax protein was undetectable in control cells. However, when cells were treated with CF31, a strong immunostaining was observed, suggesting the activation of Bax by CF31. The apoptotic effect of CF31 in the presence of TNFα was also illustrated by its induction of PARP cleavage in HepG2 and HeLa cells (Fig. 6B). Induction of PARP cleavage by TNFα/CF31 combination treatment was associated with reduction of AKT activation, suggesting that the inhibition of AKT activity plays a role in their induction of apoptosis. In support of the role of AKT inhibition by the combination treatment, transfection of the constitutive-active AKT (CA-AKT) expression vector prevented its induction of PARP cleavage (Fig. 6C).

Figure 6. CF31 induces RXRα-dependent apoptosis.

(A) CF31 activation of Bax. HepG2 cells treated with CF31 (10 μM/ 12hr) were immunostained with Bax/6A7 antibody and DAPI. About 40% CF31-treated cells showed Bax staining.

(B) CF31 induction of PARP cleavage. Cells were treated with CF31 (10 μM) for 12 hr before exposed to TNFα (10 ng/ml) for 30 min and analyzed by immunoblotting.

(C) Regulation of CF31/TNFα-induced PARP cleavage by AKT activation. HepG2 cells transfected with CA-AKT expression vector were treated with TNFα (10 ng/ml) and/or CF31 (10 μM) for 4 hr and analyzed by immunoblotting.

(D) Inhibition of CF31-induced PARP cleavage by RXRα siRNA. HeLa cells transfected with control or RXRα siRNA were treated with CF31 (10 μM) for 12 hr and analyzed by immunoblotting.

(E) RXRα/Δ80 expression sensitizes A549 cells to apoptotic effect of CF31. A549 cells were transfected with Myc-RXRα/Δ80, treated with CF31 (10 μM) for 12 hr and analyzed by immunoblotting.

(F) RXRα/Δ80 expression sensitizes MCF-7 cells to apoptotic effect of CF31. MCF-7 and MCF-7 cells transfected with GFP-RXRα/Δ80 were starved overnight and pretreated with CF31 (10 μM) for 12 hr. PARP cleavage were analyzed by immunoblotting.

We also determined the role of RXRα in CF31-induced apoptosis by transfecting control and RXRαsiRNAin HeLa cells. Comparison of the effect of CF31 in cells transfected with control siRNA or RXRα siRNA demonstrated that transfection of RXRα siRNA impaired the ability of CF31 to induce PARP cleavage and to inhibit AKT activation (Fig. 6D). To address the role of tRXRα, we examined the effect of CF31 on PARP cleavage in cells stably expressing RXRα/Δ80. In A549 cells, CF31 showed little effect on inducing PARP cleavage (Fig. 6E). However, it significantly induced PARP cleavage in cells stably expressing RXRα/Δ80 tagged with Myc epitope, Myc-RXRα/Δ80. Similar results were obtained in MCF-7 breast cancer cells stably expressing GFP-RXRα/Δ80 (Fig. 6F). Thus, the expression of tRXRα plays a role in the apoptosis induction by CF31.

CF31 activates TNFα-induced extrinsic apoptotic pathway

TNFα is capable of inducing opposing biological activities such as cell survival and death, and its killing effect in cancer cells are often antagonized by its survival function that is mainly mediated by activation of the NF-κB and PI3K/AKT pathways (47, 48). Our observation that both inhibition of AKT activation and induction of apoptosis by CF31 occurred in the presence of TNFα prompted us to study whether CF31 could convert TNFα from a survival to a killing molecule. Thus, caspase-3 activation was examined in HeLa cells treated with CF31 or TNFα alone or their combination. Treatment of cells with either CF31 or TNFα alone had little effect on caspase-3 activation, whereas the combination treatment resulted in strong caspase-3 activation (Fig. 7A). The combination treatment also strongly induced the activation of caspase-8, the downstream mediator of the TNFα-dependent apoptotic pathway (47, 48). Caspase-8 activation by CF31/TNFα combination was also revealed by immunoblotting showing strong induction of cleaved caspase-8 products, p43/p41, an indication of caspase-8 activation (Fig. 7B). The induction of caspase-8 activation and PARP cleavage by the CF31/TNFα combination was partially impaired by transfection of RXRα siRNA (Fig. 7B), demonstrating a role of RXRα.

Figure 7. Activation of extrinsic apoptotic pathway by CF31.

(A) Induction of caspase-3 and caspase-8 activity by CF31/TNFα combination. HeLa cells were treated with CF31 (10 μM) and/or TNFα (10 ng/ml) for 4 hr, and analyzed by caspase-3 and caspase-8 activity assay kit.

(B) Inhibition of CF31/TNFα-induced caspases-8 cleavage by RXRαsiRNA. HeLa cells transfected with control or RXRα siRNA were treated with TNFα (10 ng/ml) and CF31 (10 μM) and analyzed by immunoblotting.

(C, D) Inhibition of CF31/TNFα-induced PARP cleavage by caspase-8 inhibitor and siRNA. HeLa cells transfected with control or caspase-8 siRNA (C) or pretreated with Z-IETD-fmk (40 μM) (D) for 1hr were treated with TNFα (10 ng/ml)and CF31 (10 μM), then analyzed by immunoblotting.

Induction of PARP cleavage by CF31/TNFα combination treatment was closely correlated with caspase-8 activation (Fig. 7B), suggesting an activation of TNFα-dependent apoptotic pathway by CF31. To further address the role of caspase-8 activation in apoptosis induction by CF31, we examined the effect of caspase-8 siRNA transfection. Transfection of HeLa cells with caspase-8 siRNA reduced the level of p43/p41 and PARP cleavage (Fig. 7C). In addition, PARP cleavage induced by the CF31/TNFα combination treatment was largely inhibited in cells treated with the caspase-8 inhibitor Z-IETD-fm (Fig. 7D). These results demonstrated that apoptosis induction by the CF31/TNFα combination treatment was largely mediated by their activation of TNFα-mediated extrinsic apoptotic pathway, suggesting the role of CF31 in shifting the TNFα signaling from the survival to death.

Discussion

Traditional medicinal plants and dietary factors provide a fertile ground for modern drug development, with some compounds, such as paclitaxel, etoposide, camptothecin, and vincristine, being successfully used as anti-cancer drugs (49). Xanthones, a class of three-membered heterocyclic ring compounds mainly found as secondary metabolites in higher plants and microorganisms, exert very diverse biological profiles, including antihypertensive, antioxidative, antithrombotic and anticancer activity, depending on their diverse structures (30-33). A large number of naturally occurring and synthetic xanthones such as psorospermin (50), dimethylxanthesone-4-acetic acid (DMXAA)(51), and α-mangostin (52) have shown potent anti-cancer activities. However, the biological targets of xanthone compounds remain elusive (30-33). Cratoxylum is a small genus distributed in Southeast Asia with some of its species used medicinally, and xanthones are the most characteristic biologically active components of this genus (31, 35, 52, 53). We have previously identified several bioactive xanthones from the stems of Cratoxylum formosum ssp. Pruniflorum (34, 35). We report here that CF31, one of the xanthones that we isolated, acts as a potent negative regulator of the tRXRα-mediated PI3K/AKT survival signaling pathway by binding to RXRα, thus identifying CF31 as a new lead for a class of anti-cancer agents targeting this newly identified cancer survival pathway.

CF31 binds to RXRα in a unique mode and acts as a RXRα antagonist. The LBP of RXR is highly restrictive to flexible and elongated ligands. The published crystal structures of RXRα bound to natural or synthetic ligands show that a carboxylate group in these ligands forms salt bridges with basic residue Arg316 at the end of the L-shaped RXRα LBP to establish anchoring ionic interaction for stabilization (41-43). However, CF31 lacks such a carboxylate moiety (Fig. 1A) and is therefore incapable of interacting with Arg316. This was supported by our mutagenesis study, which showed that Arg316 was not required for its antagonist effect (Fig. 2C). Although Arg316 was not required for CF31 binding, our MM-PB/SA analysis suggested that the binding of CF31 was stabilized by its extensive van der Waals interactions with several hydrophobic residues in the RXRα (Fig. 2E), thus revealing a distinct binding mode for CF31. It is noteworthy that mutation of Arg316, which completely impaired the transactivation function of 9-cis-RA, did not show much effect on CD3254 (Fig. 2C), implying that CD3254 binding to RXRα does not require its ionic interaction with Arg316. Very recently, crystal structure studies showed that begelovin, a RXRα agonist lacking the carboxylate moiety, bound to RXRα (54) in a mode similar to CF31. Thus, the LBP of RXRα is more flexible than expected to mediate diverse activities of compounds with different structural features.

CF31 effectively inhibited constitutive and inducible AKT activation and cell survival in several cancer cell lines (Figs. 4, 5). It was much more effective than Sulindac, an NSAID that was previously reported to inhibit tRXRα-mediated AKT activation (29), and was comparable to K-80003, an improved Sulindac analog (29), on inhibiting AKT activation (Fig. 5A and data not shown). The inhibitory effect of CF31 on AKT activation occurred at concentrations under that CF31 could bind to RXRα, suggesting that it achieved its inhibitory effect on AKT activation by RXRα binding. In support of this conclusion, we showed that knocking down RXRα expression by RXRα siRNA impaired its inhibitory effects on basal and TNFα-induced AKT activation (Fig. 5D), while overexpression of tRXRα resulted in an enhancement (Fig. 5E).

Another unique property of CF31 is its ability to convert TNFα from a survival molecule to a killer of cancer cells. TNFα is a multifunctional cytokine that plays roles in diverse cellular events such as cell survival and death (47, 48, 55, 56). The apoptotic effect of TNFα is mediated by caspase-8-dependent apoptotic pathway, whereas its survival function involves activation of PI3K/AKT and NF-κB pathways. Because the death effect of TNFα is antagonized or suppressed by its abnormally elevated survival function in cancer cells, TNFα often acts a survival instead of killer in the cells (47, 48, 55, 56). Since TNFα is produced by malignant or host cells in the tumor microenvironment but not in normal cells, there has been tremendous interest in developing strategies to change a tumor-promoting microenvironment to a tumor-inhibiting state by shifting TNFα signaling from survival to death (47, 48, 57-59). Our previous discovery that tRXRα mediates AKT activation by TNFα provides an opportunity to convert TNFα's function from survival to death in cancer cells by targeting tRXRα (29). Our current study provides several lines of evidence that CF31 could act as such a converter through its ability to suppress tRXRα-mediated AKT activation by TNFα. First, we found that CF31 could potently induce PARP cleavage in cancer cells when used together with TNFα (Fig. 6). Moreover, combination of CF31 and TNFα led to a synergistic effect on PARP cleavage (Fig. 6C) and caspase activation (Fig. 7A), demonstrating that the apoptotic effect of CF31 requires the TNFα signaling pathway. Secondly, induction of PARP cleavage by the combination treatment in cancer cells was associated with their inhibition of AKT activation (Fig. 6B), which was also tRXRα dependent (Figs. 6D-F). In contrast, transfection of constitutive-active AKT abolished the inducing effect of CF31/TNFα combination on PARP cleavage (Fig. 6C). Thus, inhibition of AKT activation was essential for their induction of apoptosis. Thirdly, induction of caspase-3 activation and PARP cleavage by the CF31/TNFα combination was mediated by activation of caspase-8 (Fig. 7), a critical mediator of TNFα-induced intrinsic apoptotic pathway. Thus, CF31 can serve to relieve the anti-apoptotic function of AKT, leading to activation of TNFα-dependent apoptosis. Together, our results identify CF31 as a new converter of TNFα survival signaling in cancer cells. With its ability to shift TNFα signaling from survival to death by its unique RXRα binding, CF31 represents a promising lead for a class of RXRα modulators that selectively induce apoptosis of cancer cells. Because of the proven natural product drug discovery track record, identification of a natural product that targets an RXR-mediated cell survival pathway that regulates PI3K/Akt may offer a new therapeutic strategy to kill cancer cells.