Jin-Zhang Zeng

Regulation of Nur77 expression by β-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 β‐catenin was involved in induction of Nur77 expression through its activation of the tran‐scriptional 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/β‐catenin and AP‐1 signaling pathways.—Wu, H., Lin, Y., Li, W., Sun, Z., Gao, W., Zhang, H., Xie, L., Jiang, F., Qin, B., Yan, T., Chen, L., Zhao, Y., Cao, X., Wu, Y., Lin, B., Zhou, H., Wong, A.S.‐T., Zhang, X.‐K., Zeng, J.‐Z. Regulation of Nur77 expression by β‐catenin and its mitogenic effect in colon cancer cells. FASEB J. 25, 192–205 (2011). www.fasebj.org

NSAID Sulindac and Its Analog Bind RXRα and Inhibit RXRα-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-γ 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.

Inhibition of β-catenin signaling by nongenomic action of orphan nuclear receptor Nur77.

Dysregulation of β-catenin turnover due to mutations of its regulatory proteins including adenomatous polyposis coli (APC) and p53 is implicated in the pathogenesis of cancer. Thus, intensive effort is being made to search for alternative approaches to reduce abnormally activated β-catenin in cancer cells. Nur77, an orphan member of the nuclear receptor superfamily, has a role in the growth and apoptosis of cancer cells. Here, we reported that Nur77 could inhibit transcriptional activity of β-catenin by inducing β-catenin degradation via proteasomal degradation pathway that is glycogen synthase kinase 3β and Siah-1 independent. Nur77 induction of β-catenin degradation required both the N-terminal region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for β-catenin binding. Nur77/ΔDBD, a Nur77 mutant lacking its DNA-binding domain, resided in the cytoplasm, interacted with β-catenin, and induced β-catenin degradation, demonstrating that Nur77-mediated β-catenin degradation was independent of its DNA binding and transactivation, and might occur in the cytoplasm. In addition, we reported our identification of two digitalis-like compounds (DLCs), H-9 and ATE-i2-b4, which potently induced Nur77 expression and β-catenin degradation in SW620 colon cancer cells expressing mutant APC protein in vitro and in animals. DLC-induced Nur77 protein was mainly found in the cytoplasm, and inhibition of Nur77 nuclear export by the CRM1-dependent nuclear export inhibitor leptomycin B or Jun N-terminal kinase inhibitor prevented the effect of DLC on inducing β-catenin degradation. Together, our results demonstrate that β-catenin can be degraded by cytoplasmic Nur77 through their interaction and identify H-9 and ATE-i2-b4 as potent activators of the Nur77-mediated pathway for β-catenin degradation.

Targeting Truncated Retinoid X Receptor-α by CF31 Induces TNF-α–Dependent Apoptosis

A truncated version of retinoid X receptor-α, tRXR-α, promotes cancer cell survival by activating the phosphoinositide 3-kinase (PI3K)/AKT pathway. However, targeting the tRXR-α-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 biologic evaluation of its regulation of the tRXR-α-mediated PI3K/AKT pathway. CF31 binds RXR-α and its binding results in inhibition of RXR-α transactivation. Through RXR-α mutational analysis and computational studies, we show that Arg316 of RXR-α, known to form salt bridges with certain RXR-α ligands, such as 9-cis-retinoic acid (9-cis-RA), is not required for the antagonist effect of CF31, showing 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 RXR-α. CF31 is a potent inhibitor of AKT activation in various cancer cell lines. When combined with TNF-α, it suppresses TNF-α activation of AKT by inhibiting TNF-α-induced tRXR-α interaction with the p85α regulatory subunit of PI3K. CF31 inhibition of TNF-α activation of AKT also results in TNF-α-dependent activation of caspase-8 and apoptosis. Together, our results show that CF31 is an effective converter of TNF-α signaling from survival to death by targeting tRXR-α 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.

High-Throughput Multiparameter Analysis of Individual Mitochondria

Mitochondria are one of the most important organelles responsible for cellular energy metabolism and apoptosis regulation. However, single-mitochondrion analysis is challenging, because of their small sizes and the low content of organelle constituents. Here, we report the development of a sensitive and versatile platform for high-throughput multiparameter analysis of individual mitochondria. Employing specific fluorescent staining with a laboratory-built high-sensitivity flow cytometer (HSFCM), we demonstrate the simultaneous detection of side scatter, cardiolipin, and mitochondria DNA (mtDNA) of a single mitochondrion. Simultaneous measurements of side scatter, porin, and cytochrome c of individual mitochondria are reported for the first time. Correlation analysis among multiple attributes on an organelle-by-organelle basis could provide a more definitive assessment of the purity, structure integrity, and apoptosis-related proteins of isolated mitochondria than bulk measurement. This work represents a significant advancement in single-mitochondrion analysis. We believe that the HSFCM holds great potential for studying apoptotic signal transduction pathways at the single-mitochondrion level.

A Unique Cytoplasmic Localization of Retinoic Acid Receptor-γ and Its Regulations

Recent evidence suggests that extranuclear action of retinoid receptors is involved in mediating the pleiotropic effects of retinoids. However, whether they reside in the cytoplasm remains elusive. Here, we showed that retinoic acid receptor-γ (RARγ) was cytoplasmic in confluent cells, or when cells were released from serum depletion or treated with growth factors. In studying the regulation of RARγ subcellular localization, we observed that ectopically overexpressed RARγ was mainly cytoplasmic irrespective of serum concentration and cell density. The cytoplasmic retention of RARγ was inhibited by ligand retinoic acid (RA). In addition, coexpression of retinoid X receptor-α (RXRα) resulted in nuclear localization of RARγ through their heterodimerization. Mutagenesis studies revealed that a C-terminal fragment of RXRα potently prevents RA-induced RARγ nuclear localization and transcriptional function. Furthermore, our results showed that the cytoplasmic retention of RARγ was due to the presence of its unique N-terminal A/B domain, which was subject to regulation by p38 MAPK-mediated phosphorylation. Deletion or mutation of the N-terminal A/B domain largely impaired its cytoplasmic localization. Together, our data demonstrate that the subcellular localization of RARγ is regulated by complex interactions among ligand binding, receptor phosphorylation, and receptor dimerizations.

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.

Identification of Mitochondria-Targeting Anticancer Compounds by an in Vitro Strategy

Mitochondria play a pivotal role in determining the point-of-no-return of the apoptotic process. Therefore, anticancer drugs that directly target mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. In this study, we report the development of an in vitro strategy to quickly identify the therapeutic agents that induce apoptosis via directly affecting mitochondria. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (Δψ(m)) fluorescence of individual mitochondria using a laboratory-built high-sensitivity flow cytometer. The feasibility of this method was tested with eight widely used anticarcinogens. Dose-dependent Δψ(m) losses were observed for paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, and triptolide, but not for cisplatin or actinomycin D, which agrees well with their mechanisms of apoptosis induction reported in the literature. The as-developed method offers an effective approach to identify mitochondria-targeting anticancer compounds.

Venezuelines A–G, new phenoxazine-based alkaloids and aminophenols from Streptomyces venezuelae and the regulation of gene target Nur77

Five new phenoxazine-based alkaloids venezuelines A-E (1-5) and two new aminophenols venezuelines F-G (6-7), as well as three known analogues exfoliazone, chandrananimycin D and carboxyexfoliazone were isolated from the fermentation broth of the marine-derived bacterium Streptomyces venezuelae. The structures of new compounds were determined on the basis of extensive spectroscopic analysis. The cytotoxic activity of these compounds against a panel of tumor cell lines were tested, while the regulation of gene target Nur77 of 2 and exfoliazone (8) were evaluated.