Profiles of and variations in aluminum species in PAC-MC used for the removal of blooming microalgae.

Polyaluminum chloride modified clay (PAC-MC) is a safe and efficient red tide control agent that has been studied and applied worldwide. Although it is well known that the distribution of hydrolytic aluminum species in PAC affects its flocculation, little is known about the influence of particulars aluminum species on the microalgae removal efficiency of PAC-MC; this lack of knowledge creates a bottleneck in the development of more efficient MCs based on aluminum salts. The ferron method was used in this study to quantitatively analyze the distributions of and variations in different hydrolytic aluminum species during the process of microalgae removal by PAC-MC. The results showed that Ala, which made up 5%-20% of the total aluminum, and Alp, which made up 15%-55% of the total aluminum, significantly affected microalgae removal, with Pearson's correlation coefficients of 0.83 and 0.89, respectively. Most of the aluminum in the PAC-MC sank rapidly into the sediments, but the rate and velocity of settlement were affected by the dose of modified clay. The optimal dose of PAC-MC for precipitating microalgae was determined based on its aluminum profile. These results provide guidance for the precise application of PAC-MC in the control of harmful algal blooms.

BI1071, a Novel Nur77 Modulator, Induces Apoptosis of Cancer Cells by Activating the Nur77-Bcl-2 Apoptotic Pathway.

Nur77 (also called TR3 or NGFI-B), an orphan member of the nuclear receptor superfamily, induces apoptosis by translocating to mitochondria where it interacts with Bcl-2 to convert Bcl-2 from an antiapoptotic to a pro-apoptotic molecule. Nur77 posttranslational modification such as phosphorylation has been shown to induce Nur77 translocation from the nucleus to mitochondria. However, small molecules that can bind directly to Nur77 to trigger its mitochondrial localization and Bcl-2 interaction remain to be explored. Here, we report our identification and characterization of DIM-C-pPhCF3 +MeSO3 - (BI1071), an oxidized product derived from indole-3-carbinol metabolite, as a modulator of the Nur77-Bcl-2 apoptotic pathway. BI1071 binds Nur77 with high affinity, promotes Nur77 mitochondrial targeting and interaction with Bcl-2, and effectively induces apoptosis of cancer cells in a Nur77- and Bcl-2-dependent manner. Studies with animal model showed that BI1071 potently inhibited the growth of tumor cells in animals through its induction of apoptosis. Our results identify BI1071 as a novel Nur77-binding modulator of the Nur77-Bcl-2 apoptotic pathway, which may serve as a promising lead for treating cancers with overexpression of Bcl-2.

Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity.

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs-s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs- with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs- showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs- activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs-, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs- in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.

Effects of modified clay used for the control of harmful algal blooms on Alexandrium pacificum cysts.

Cyst formation plays an important role in the resistance of dinoflagellates to adverse environments, and cyst germination is considered one of the causes of harmful algal blooms (HABs). Among the methods for mitigating HABs, modified clay (MC) is considered a promising strategy because of its high efficiency and low environmental impacts. The typical HAB species Alexandrium pacificum was focused on in this study to clarify the effects of MC on cyst formation and germination. The results showed that more than 90% of the vegetative cells were removed under the 0.6 g/L MC treatment. The vegetative cell density was monitored over 90 d and increased slightly to the peak at 10 d after the cell removal experiment, but persistent growth was not observed. The amount of cysts was maximal at 20 d after removal, however, most of the cysts were temporary cysts that subsequently disappeared. After 80 d, all the remaining cysts were resting ones. The total density of resting cysts was higher under MC concentrations of 0.2 and 0.4 g/L and lower under concentrations of 0.6, 0.8 and 1.0 g/L compared with that in the control. The total formation rate of resting cysts was 29.6% in the control group, and the lowest formation rate in the experimental groups was 15.5% at 0.6 g/L MC. The total germination rate of resting cyst decreased as the MC concentration increased, and approximately 68.0% of the resting cysts in the control group germinated successfully, whereas the addition of MC reduced the germination rate to as low as 12.4%. Our results indicated that the application of appropriate MC concentrations may provide an effective mitigation strategy for A. pacificum blooms because it does not leave more residual cysts, which can act as "seeds" for the initiation of HABs.

Controlling harmful algae blooms using aluminum-modified clay.

The performances of aluminum chloride modified clay (AC-MC), aluminum sulfate modified clay (AS-MC) and polyaluminum chloride modified clay (PAC-MC) in the removal of Aureococcus anophagefferens were compared, and the potential mechanisms were analyzed according to the dispersion medium, suspension pH and clay surface charges. The results showed that AC-MC and AS-MC had better efficiencies in removing A.anophagefferens than PAC-MC. The removal mechanisms of the three modified clays varied. At optimal coagulation conditions, the hydrolysates of AC and AS were mainly monomers, and they transformed into Al(OH)3(am) upon their addition to algae culture, with the primary mechanism being sweep flocculation. The PAC mainly hydrolyzed to the polyaluminum compounds, which remained stable when added to the algae culture, and the flocculation mainly occurred through polyaluminum compounds. The suspension pH significantly influenced the aluminum hydrolysate and affected the flocculation between the modified clay and algae cells.

Nitrostyrene Derivatives Act as RXRα Ligands to Inhibit TNFα Activation of NF-κB.

Retinoid X receptor alpha (RXRα) and its N-terminally truncated version, tRXRα, are widely implicated in cancer development and represent intriguing targets for cancer prevention and treatment. Successful manipulation of RXRα and tRXRα requires the identification of their modulators that could produce therapeutic effects. Here, we report that a class of nitrostyrene derivatives bind to RXRα by a unique mechanism, of which the nitro group of nitrostyrene derivatives and Cys432 of RXRα are required for binding. The binding results in the potent activation of Gal4-DBD-RXRα-LBD transactivation. However, the binding inhibits the transactivation of RXRα homodimer, which might be due to the distinct conformation of RXRα homodimer induced by these nitrostyrene derivatives. Two RXRα point mutants with Cys432 substituted with Tyr and Trp, respectively, could mimic the bindings of two nitrostyrene derivatives and have the ability of autotransactivation. In studying the functional consequences of the binding, we show that these nitrostyrene derivatives could potently inhibit the TNFα/NFκB signaling pathway in a tRXRα-dependent manner. tRXRα promotes TNFα-induced NF-κB activation through its interaction with TRAF2 and enhances TNFα-induced ubiquitination of RIP1, which is strongly inhibited by nitrostyrene derivatives. The inhibition of TNFα-induced NF-κB activation results in the synergistic effect of the combination of nitrostyrene derivatives and TNFα on the induction of cancer cell apoptosis. Together, our results show a new class of RXRα modulators that induce apoptosis of cancer cells through their unique binding mode and new mechanism of action.

Regulation of proteolytic cleavage of retinoid X receptor-α by GSK-3ß.

We recently reported that an N-terminally truncated retinoid X receptor-α (tRXRα) produced in cancer cells acts to promote cancer cell growth and survival through AKT activation. However, how RXRα is cleaved and how the cleavage is regulated in cancer cells remain undefined. In this study, we demonstrated that calpain II could cleave RXRα protein in vitro, generating two truncated RXRα products. The cleavage sites in RXRα were mapped by Edman N-terminal sequencing to Gly(90)↓Ser(91) and Lys(118)↓Val(119). Transfection of the resulting cleavage product RXRα/90, but not RXRα/118, resulted in activation of AKT in cancer cells, similar to the effect of tRXRα. In support of the role of calpain II in cancer cells, transfection of calpain II expression vector or its activation by ionomycin enhanced the production of tRXRα, whereas treatment of cells with calpain inhibitors reduced the levels of tRXRα. Co-immunoprecipitation assays also showed an interaction between calpain II and RXRα. In studying the regulation of tRXRα production, we observed that treatment of cells with lithium chloride or knockdown of glycogen synthase kinase-3ß (GSK-3ß) significantly increased the production of tRXRα. Conversely, overexpression of GSK-3ß reduced tRXRα expression. Furthermore, we found that the inhibitory effect of GSK-3ß on tRXRα production was due to its suppression of calpain II expression. Taken together, our data demonstrate that GSK-3ß plays an important role in regulating tRXRα production by calpain II in cancer cells, providing new insights into the development of new strategies and agents for the prevention and treatment of tRXRα-related cancers.

Relative impact of 3- and 5-hydroxyl groups of cytosporone B on cancer cell viability.

A novel and the shortest route, thus far, for preparing cytosporone B (Csn-B) is reported. Csn-B and two analogs were used to probe the importance of hydroxyl groups at the 3- and 5-positions of the Csn-B benzene ring in inhibiting the viability of human H460 lung cancer and LNCaP prostate cancer cells, inducing H460 cell apoptosis, and interacting with the NR4A1 (TR3) ligand-binding domain (LBD). These studies indicate that Csn-B and 5-Me-Csn-B, having a phenolic hydroxyl at the 3-position of their aromatic rings, had similar activities in inhibiting cancer cell viability and in inducing apoptosis, whereas 3,5-(Me)2-Csn-B was unable to do so. These results are in agreement with ligand-binding experiments showing that the interaction with the NR4A1 LBD required the presence of the 3-hydroxyl group.

Antagonist effect of triptolide on AKT activation by truncated retinoid X receptor-alpha.

BACKGROUND: Retinoid X receptor-alpha (RXRα) is a key member of the nuclear receptor superfamily. We recently demonstrated that proteolytic cleavage of RXRα resulted in production of a truncated product, tRXRα, which promotes cancer cell survival by activating phosphatidylinositol-3-OH kinase (PI3K)/AKT pathway. However, how the tRXRα-mediated signaling pathway in cancer cells is regulated remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: We screened a natural product library for tRXRα targeting leads and identified that triptolide, an active component isolated from traditional Chinese herb Trypterygium wilfordii Hook F, could modulate tRXRα-mediated cancer cell survival pathway in vitro and in animals. Our results reveal that triptolide strongly induces cancer cell apoptosis dependent on intracellular tRXRα expression levels, demonstrating that tRXRα serves as an important intracellular target of triptolide. We show that triptolide selectively induces tRXRα degradation and inhibits tRXRα-dependent AKT activity without affecting the full-length RXRα. Interestingly, such effects of triptolide are due to its activation of p38. Although triptolide also activates Erk1/2 and MAPK pathways, the effects of triptolide on tRXRα degradation and AKT activity are only reversed by p38 siRNA and p38 inhibitor. In addition, the p38 inhibitor potently inhibits tRXRα interaction with p85α leading to AKT inactivation. Our results demonstrate an interesting novel signaling interplay between p38 and AKT through tRXRα mediation. We finally show that targeting tRXRα by triptolide strongly activates TNFα death signaling and enhances the anticancer activity of other chemotherapies. CONCLUSIONS/SIGNIFICANCE: Our results identify triptolide as a new xenobiotic regulator of the tRXRα-dependent survival pathway and provide new insight into the mechanism by which triptolide acts to induce apoptosis of cancer cells. Triptolide represents one of the most promising therapeutic leads of natural products of traditional Chinese medicine with unfortunate side-effects. Our findings will offer new strategies to develop improved triptolide analogs for cancer therapy.

Regulation of Nur77 expression by ß-catenin and its mitogenic effect in colon cancer cells.

The orphan nuclear receptor Nur77 is an immediate-early response gene whose expression is rapidly induced by various extracellular stimuli. The aims of this study were to study the role of Nur77 expression in the growth and survival of colon cancer cells and the mechanism by which Nur77 expression was regulated. We showed that levels of Nur77 were elevated in a majority of human colon tumors (9/12) compared to their nontumorous tissues and that Nur77 expression could be strongly induced by different colonic carcinogens including deoxycholic acid (DCA). DCA-induced Nur77 expression resulted in up-regulation of antiapoptotic BRE and angiogenic VEGF, and it enhanced the growth, colony formation, and migration of colon cancer cells. In studying the mechanism by which Nur77 was regulated in colon cancer cells, we found that ß-catenin was involved in induction of Nur77 expression through its activation of the transcriptional activity of AP-1 (c-Fos/c-Jun) that bound to and transactivated the Nur77 promoter. Together, our results demonstrate that Nur77 acts to promote the growth and survival of colon cancer cells and serves as an important mediator of the Wnt/ß-catenin and AP-1 signaling pathways.

NSAID sulindac and its analog bind RXRalpha and inhibit RXRalpha-dependent AKT signaling.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their anticancer effects through cyclooxygenase-2 (COX-2)-dependent and independent mechanisms. Here, we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-alpha (RXRalpha). We identified an N-terminally truncated RXRalpha (tRXRalpha) in several cancer cell lines and primary tumors, which interacted with the p85alpha subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-alpha (TNFalpha) promoted tRXRalpha interaction with the p85alpha, activating PI3K/AKT signaling. When combined with TNFalpha, Sulindac inhibited TNFalpha-induced tRXRalpha/p85alpha interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRalpha but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRalpha-dependent AKT activation and tRXRalpha tumor growth in animals.

Oncogenic potential of retinoic acid receptor-gamma in hepatocellular carcinoma.

Retinoic acid receptors (RAR; alpha, beta, and gamma), members of the nuclear receptor superfamily, mediate the pleiotropic effects of the vitamin A metabolite retinoic acid (RA) and derivatives (retinoids) in normal and cancer cells. Abnormal expression and function of RARs are often involved in the growth and development of cancer. However, the underlying molecular mechanisms remain largely elusive. Here, we report that levels of RARgamma were significantly elevated in tumor tissues from a majority of human hepatocellular carcinoma (HCC) and in HCC cell lines. Overexpression of RARgamma promoted colony formation by HCC cells in vitro and the growth of HCC xenografts in animals. In HepG2 cells, transfection of RARgamma enhanced, whereas downregulation of RARgamma expression by siRNA approach impaired, the effect of RA on inducing the expression of alpha-fetoprotein, a protein marker of hepatocarcinogenesis. In studying the possible mechanism by which overexpression of RARgamma contributed to liver cancer cell growth and transformation, we observed that RARgamma resided mainly in the cytoplasm of HCC cells, interacting with the p85alpha regulatory subunit of phosphatidylinositol 3-kinase (PI3K). The interaction between RARgamma and p85alpha resulted in activation of Akt and NF-kappaB, critical regulators of the growth and survival of cancer cells. Together, our results show that overexpression of RARgamma plays a role in the growth of HCC cells through nongenomic activation of the PI3K/Akt and NF-kappaB signaling pathways.

Retinoid X receptor-alpha mediates (R )-flurbiprofen's effect on the levels of Alzheimer's beta-amyloid.

Alzheimer's disease (AD) is characterized by the formation of extracellular senile plaques in the brain, whose major component is a small peptide called beta-amyloid (Abeta). Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) has been found beneficial for AD and several reports suggest that NSAIDs reduce the generation of Abeta, especially the more amyloidogenic form Abeta42. However, the exact mechanism underlying NSAIDs' effect on AD risk remains largely inconclusive and all clinical trials using NSAIDs for AD treatment show negative results so far. Recent studies have shown that some NSAIDs can bind to certain nuclear receptors, suggesting that nuclear receptors may be involved in NSAID's effect on AD risk. Here we find that (R)-flurbiprofen, the R-enantiomer of the racemate NSAID flurbiprofen, can significantly reduce Abeta secretion, but at the same time, increases the level of intracellular Abeta. In addition, we find that a nuclear receptor, retinoid X receptor alpha (RXRalpha), can regulate Abeta generation and that down-regulation of RXRalpha significantly increases Abeta secretion. We also show that (R)-flurbiprofen can interfere with the interaction between RXRalpha and 9-cis-retinoid acid, and that 9-cis-retinoid acid decreases (R)-flurbiprofen's reduction of Abeta secretion. Moreover, the modulation effect of (R)-flurbiprofen on Abeta is abolished upon RXRalpha down-regulation. Together, these results suggest that RXRalpha can regulate Abeta generation and is also required for (R)-flurbiprofen-mediated Abeta generation.

[Effects of different phosphorus substrates on the growth and phosphatase activity of Skeletonema costatum and Prorocentrum donghaiense].

The effects NaH2PO4, adenosine disodium triphosphate (ATP), glucose 6-phosphate (G-6-P) and sodium beta-glycerophosphate (G-P) on the growth and phosphatase activity of Skeletonema costatum and Prorocentrum donghaiense were studied. The results showed that both species could utilize both dissolved inorganic phosphate (DIP) and dissolved organic phosphorus (DOP), and DOP had more effects on the growth of two species than DIP. For S. costatum, after 8 days, the cell abundances of the four treatments (NaH2PO4, ATP, G-6-P and G-P) were 48 x 10(4), 73 x 10(4), 63 x 10(4) and 54 x 10(4) cells/mL, respectively; For P. donghaiense, after 10 days, the cell abundances of the four treatments were 8.7 x 10(4), 15.5 x 10(4), 12.4 x 10(4) and 9.5 x 10(4) cells/mL, respectively. On the first 3-4 days, the phosphatase activity of all treatments of the two species showed a decreasing trend, but different changes were observed for the different phosphorus substrate treatments in latter days. For the NaH2PO4 treatment, both the AP and AcP activity of two species increased from the fifth day onwards. For S. costatum, the AP activity of the ATP and G-6-P treatment groups showed no obvious changes and AcP activity had a slight increase from the fifth day to the eighth day, while the activity of G-P treatment had highest phosphatase activity which increased from the fifth day on. At the end of the experiment, the AP activity of the three DOP treatment groups (ATP, G-6-P and G-P) was 0.004 x 10(-5), 0.014 x 10(-5) and 0.029 x 10(-5) U/cell, respectively, and the AcP activity was 0.006 x 10(-5), 0.011 x 10(-5) and 0.018 x 10(-5) U/cell, respectively. For P. donghaiense, both the AP and AcP activity of the three DOP treatments had similar trends, i.e., ATP < G-6-P < G-P. Under the same nutrient conditions, S. costatum had a much higher phosphatase activity and could absorb P from the environment much faster than P. donghaiense.

Derivation of a retinoid X receptor scaffold from peroxisome proliferator-activated receptor gamma ligand 1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene.

PPARgamma agonist DIM-Ph-4-CF(3), a template for RXRalpha agonist (E)-3-[5-di(1-methyl-1H-indol-3-yl)methyl-2-thienyl] acrylic acid: DIM-Ph-CF(3) is reported to inhibit cancer growth independent of PPARgamma and to interact with NR4A1. As both receptors dimerize with RXR, and natural PPARgamma ligands activate RXR, DIM-Ph-4-CF(3) was investigated as an RXR ligand. It displaces 9-cis-retinoic acid from RXRalpha but does not activate RXRalpha. Structure-based direct design led to an RXRalpha agonist.1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene (DIM-Ph-4-CF(3)) is reported to inhibit cancer cell growth and to act as a transcriptional agonist of peroxisome proliferator-activated receptor gamma (PPARgamma) and nuclear receptor 4A subfamily member 1 (NR4A1). In addition, DIM-Ph-4-CF(3) exerts anticancer effects independent of these receptors because PPARgamma antagonists do not block its inhibition of cell growth, and the small pocket in the NR4A1 crystal structure suggests no ligand can bind. Because PPARgamma and NR4A1 heterodimerize with retinoid X receptor (RXR), and several PPARgamma ligands transcriptionally activate RXR, DIM-Ph-4-CF(3) was investigated as an RXR ligand. DIM-Ph-4-CF(3) displaces 9-cis-retinoic acid from RXRalpha but does not transactivate RXRalpha. Structure-based design using DIM-Ph-4-CF(3) as a template led to the RXRalpha transcriptional agonist (E)-3-[5-di(1-methyl-1H-indol-3-yl)methyl-2-thienyl]acrylic acid. Its docked pose in the RXRalpha ligand binding domain suggests that binding is stabilized by interactions of its carboxylate group with arginine 316, its indoles with cysteines 269 and 432, and its 1-methyl groups with hydrophobic residues lining the binding pocket. As is expected of a selective activator of RXRalpha, but not of RARs and PPARgamma, this RXRalpha agonist, unlike DIM-Ph-4-CF(3), does not appreciably decrease cancer cell growth or induce apoptosis at pharmacologically relevant concentrations.

Modulation of orphan nuclear receptor Nur77-mediated apoptotic pathway by acetylshikonin and analogues.

Shikonin derivatives, which are the active components of the medicinal plant Lithospermum erythrorhizon, exhibit many biological effects including apoptosis induction through undefined mechanisms. We recently discovered that orphan nuclear receptor Nur77 migrates from the nucleus to the mitochondria, where it binds to Bcl-2 to induce apoptosis. Here, we report that certain shikonin derivatives could modulate the Nur77/Bcl-2 apoptotic pathway by increasing levels of Nur77 protein and promoting its mitochondrial targeting in cancer cells. Structural modification of acetylshikonin resulted in the identification of a derivative 5,8-diacetoxyl-6-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones (SK07) that exhibited improved efficacy and specificity in activating the pathway. Unlike other Nur77 modulators, shikonins increased the levels of Nur77 protein through their posttranscriptional regulation. The apoptotic effect of SK07 was impaired in Nur77 knockout cells and suppressed by cotreatment with leptomycin B that inhibited Nur77 cytoplasmic localization. Furthermore, SK07 induced apoptosis in cells expressing the COOH-terminal half of Nur77 protein but not its NH(2)-terminal region. Our data also showed that SK07-induced apoptosis was associated with a Bcl-2 conformational change and Bax activation. Together, our results show that certain shikonin derivatives act as modulators of the Nur77-mediated apoptotic pathway and identify a new shikonin-based lead that targets Nur77 for apoptosis induction.

A short Nur77-derived peptide converts Bcl-2 from a protector to a killer.

Bcl-2 can be converted into a proapoptotic molecule by nuclear receptor Nur77. However, the development of Bcl-2 converters as anticancer therapeutics has not been explored. Here we report the identification of a Nur77-derived Bcl-2-converting peptide with 9 amino acids (NuBCP-9) and its enantiomer, which induce apoptosis of cancer cells in vitro and in animals. The apoptotic effect of NuBCPs and their activation of Bax are not inhibited but rather potentiated by Bcl-2. NuBCP-9 and its enantiomer bind to the Bcl-2 loop, which shares the characteristics of structurally adaptable regions with many cancer-associated and signaling proteins. NuBCP-9s act as molecular switches to dislodge the Bcl-2 BH4 domain, exposing its BH3 domain, which in turn blocks the activity of antiapoptotic Bcl-X(L).

Hypericum sampsonii induces apoptosis and nuclear export of retinoid X receptor-alpha.

Natural products derived from plants provide a rich source for development of new anticancer drugs. Recent studies suggest that modulation of subcellular localization of retinoid X receptor-alpha (RXRalpha) represents a potential approach for inducing cancer cell apoptosis. In this study, we screened a herbal library for inducing translocation of RXRalpha from the nucleus to the cytoplasm. Our results revealed that the extract of Hypericum sampsonii, a member of the genus Hypericum, had remarkable effect on RXRalpha subcellular localization in various cancer cells. Treatment of NIH-H460 human lung cancer cells with H. sampsonii extract resulted in relocalization of RXRalpha from the nucleus to the cytoplasm. Cytoplasmic RXRalpha induced by H. sampsonii was associated with mitochondria, accompanied with cytochrome c release and apoptosis. H. sampsonii extract effectively inhibited the growth of various cancer cell lines, including NIH-H460 lung cancer, MGC-803 stomach cancer and SMMC7721 liver cancer cells. The growth inhibitory effect of H. sampsonii extract depended on levels of RXRalpha, as it failed to inhibit the growth of CV-1 cells lacking detectable RXRalpha, whereas transfection of RXRalpha into CV-1 cells restored its apoptotic response to H. sampsonii. Furthermore, the apoptotic effect of H. sampsonii was significantly enhanced when RXRalpha was overexpressed in NIH-H460 cells. Together, our results demonstrate that H. sampsonii contains ingredient(s) that induce apoptosis of cancer cells by modulating subcellular localization of RXRalpha.

Retinoid X receptor regulates Nur77/TR3-dependent apoptosis [corrected] by modulating its nuclear export and mitochondrial targeting.

Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways by acting as a ubiquitous heterodimerization partner of many nuclear receptors, including the orphan receptor Nur77 (also known as TR3 [corrected] or NGFI-B), which translocates from the nucleus to mitochondria, where it interacts with Bcl-2 to induce apoptosis. Here, we report that RXRalpha is required for nuclear export and mitochondrial targeting of Nur77 through their unique heterodimerization that is mediated by dimerization interfaces located in their DNA-binding domain. The effects of RXRalpha are attributed to a putative nuclear export sequence (NES) present in its carboxyl-terminal region. RXRalpha ligands suppress NES activity by inducing RXRalpha homodimerization or altering RXRalpha/Nur77 heterodimerization. The RXRalpha NES is also silenced by RXRalpha heterodimerization with retinoic acid receptor or vitamin D receptor. Consistently, we were able to show that the mitochondrial targeting of the RXRalpha/Nur77 heterodimer and its induction of apoptosis are potently inhibited by RXR ligands. Together, our results reveal a novel nongenotropic function of RXRalpha and its involvement in the regulation of the Nur77-dependent apoptotic pathway [corrected]

Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3.

The Bcl-2 family proteins are key regulators of apoptosis in human diseases and cancers. Though known to block apoptosis, Bcl-2 promotes cell death through an undefined mechanism. Here, we show that Bcl-2 interacts with orphan nuclear receptor Nur77 (also known as TR3), which is required for cancer cell apoptosis induced by many antineoplastic agents. The interaction is mediated by the N-terminal loop region of Bcl-2 and is required for Nur77 mitochondrial localization and apoptosis. Nur77 binding induces a Bcl-2 conformational change that exposes its BH3 domain, resulting in conversion of Bcl-2 from a protector to a killer. These findings establish the coupling of Nur77 nuclear receptor with the Bcl-2 apoptotic machinery and demonstrate that Bcl-2 can manifest opposing phenotypes, induced by interactions with proteins such as Nur77, suggesting novel strategies for regulating apoptosis in cancer and other diseases.