Ying-Li Wu

Adenanthin targets peroxiredoxin I and II to induce differentiation of leukemic cells

Peroxiredoxins (Prxs) are potential therapeutic targets for major diseases such as cancers. However, isotype-specific inhibitors remain to be developed. We report that adenanthin, a diterpenoid isolated from the leaves of Rabdosia adenantha, induces differentiation of acute promyelocytic leukemia (APL) cells. We show that adenanthin directly targets the conserved resolving cysteines of Prx I and Prx II and inhibits their peroxidase activities. Consequently, cellular H(2)O(2) is elevated, leading to the activation of extracellular signal-regulated kinases and increased transcription of CCAAT/enhancer-binding protein β, which contributes to adenanthin-induced differentiation. Adenanthin induces APL-like cell differentiation, represses tumor growth in vivo and prolongs the survival of mouse APL models that are sensitive and resistant to retinoic acid. Thus, adenanthin can serve as what is to our knowledge the first lead natural compound for the development of Prx I- and Prx II-targeted therapeutic agents, which may represent a promising approach to inducing differentiation of APL cells.

Role of HIF-1α in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells

Angiotensin-converting enzyme (ACE) enhances the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which contribute to the pathogenesis of hypoxic pulmonary hypertension (HPH). Previous reports have demonstrated that hypoxia upregulates ACE expression, but the underlying mechanism is unknown. Here, we found that ACE is persistently upregulated in PASMCs on the transcriptional level during hypoxia. Hypoxia-inducible factor 1alpha (HIF-1alpha), a key transcription factor activated during hypoxia, was able to upregulate ACE protein expression under normoxia, whereas knockdown of HIF-1alpha expression in PASMCs inhibited hypoxia-induced ACE upregulation. Furthermore, HIF-1alpha can bind and transactivate the ACE promoter directly. Therefore, we report that ACE is a novel target of HIF-1alpha. Recently, a homolog of ACE, ACE2, was reported to counterbalance the function of ACE. In contrast to ACE, we found that ACE2 mRNA and protein levels increased during the early stages of hypoxia and decreased to near-baseline levels at the later stages after HIF-1alpha accumulation. Thus HIF-1alpha inhibited ACE2 expression, and the accumulated ANG II catalyzed by ACE is a key mediator in the downregulation of ACE2 by HIF-1alpha. Moreover, a reduction of ACE2 expression in PASMCs by RNA interference was accompanied by significantly enhanced proliferation and migration during hypoxia. We conclude that ACE is directly regulated by HIF-1alpha, whereas ACE2 is regulated in a bidirectional way during hypoxia and may play a protective role during the development of HPH. In sum, these findings contribute to the understanding of the pathogenesis of HPH.

Pharicin B stabilizes retinoic acid receptor-α and presents synergistic differentiation induction with ATRA in myeloid leukemic cells

All-trans retinoic acid (ATRA), a natural ligand for the retinoic acid receptors (RARs), induces clinical remission in most acute promyelocytic leukemia (APL) patients through the induction of differentiation and/or eradication of leukemia-initiating cells. Here, we identify a novel natural ent-kaurene diterpenoid derived from Isodon pharicus leaves, called pharicin B, that can rapidly stabilize RAR-α protein in various acute myeloid leukemic (AML) cell lines and primary leukemic cells from AML patients, even in the presence of ATRA, which is known to induce the loss of RAR-α protein. Pharicin B also enhances ATRA-dependent the transcriptional activity of RAR-α protein in the promyelocytic leukemia-RARα-positive APL cell line NB4 cells. We also showed that pharicin B presents a synergistic or additive differentiation-enhancing effect when used in combination with ATRA in several AML cell lines and, especially, some primary leukemic cells from APL patients. In addition, pharicin B can overcome retinoid resistance in 2 of 3 NB4-derived ATRA-resistant subclones. These findings provide a good example for chemical biology-based investigations of pathophysiological and therapeutic significances of RAR-α and PML-RAR-α proteins. The effectiveness of the ATRA/pharicin B combination warrants further investigation on their use as a therapeutic strategy for AML patients.

Downregulation of ANP32B, a novel substrate of caspase-3, enhances caspase-3 activation and apoptosis induction in myeloid leukemic cells.

The acidic leucine-rich nuclear phosphoprotein 32 (ANP32)B has been reported to regulate gene expression by acting as a histone chaperone or modulate messenger RNA trafficking by serving as a HuR ligand. However, its exact cellular functions are poorly understood. By utilizing a proteomics-based approach, in this work, we identify that the human ANP32B protein is cleaved during apoptosis induction by NSC606985, a novel camptothecin analog. Further investigation shows that various apoptosis inducers cause a decrease of full-length ANP32B in multiple cell lines with a concomitant increase of an approximately 17 kDa fragment. The proteolytic cleavage of ANP32B is inhibited by a specific caspase-3 inhibitor Z-DEVD-fmk, and it cannot be seen in NSC606985-induced death of caspase-3-deficient MCF-7 cells. In vitro caspase cleavage assay and mutagenesis experiment reveal that ANP32B is a direct substrate of caspase-3 and it is primarily cleaved at the sequence of Ala-Glu-Val-Asp, after Asp-163. Additionally, the reduced expression of endogenous ANP32B by specific small interfering RNA enhances caspase-3 activation and apoptosis induction by NSC606985 and etoposide. These results suggest that ANP32B is a novel substrate for caspase-3 and acts as a negative regulator for apoptosis, the mechanism of which remains to be explored.

Protein Kinase C-δ mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta (DeltaPKC-delta). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the DeltaPKC-delta, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that DeltaPKC-delta mediated the down-regulation of hnRNP K protein during apoptosis: PKC-delta inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-delta-deficient apoptotic KG1a cells; conditional induction of DeltaPKC-delta in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of DeltaPKC-delta. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-delta down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

Important Role of SUMOylation of Spliceosome Factors in Prostate Cancer Cells

Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins of SUMO. Furthermore, we demonstrated that K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation.

Pharicin A, a novel natural ent-kaurene diterpenoid, induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function.

In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon xerophilus, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content, and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization, and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.

Alantolactone induces apoptosis in chronic myelogenous leukemia sensitive or resistant to imatinib through NF-κB inhibition and Bcr/Abl protein deletion

Alantolactone, an allergenic sesquiterpene lactone, has recently been found to have significant antitumor effects on malignant tumor cells. Here, we investigated the potential effect of alantolactone on Bcr/Abl+ imatinib-sensitive and -resistant cells. Alantolactone treatment resulted in obvious apoptosis in both imatinib-sensitive and -resistant K562 cells, as shown by the increase in Annexin V-positive cells, caspase-3 activation, poly(ADP-ribose) polymerase-1 (PARP-1) cleavage and mitochondrial membrane potential collapse. Alantolactone significantly inhibited NF-κB-dependent reporter gene activity, decreased the DNA-binding activity of NF-ОκB, and blocked TNF-α-induced IκBα phosphorylation. Of interest, the oncogenic Bcr/Abl fusion protein but not its mRNA levels were quickly reduced upon alantolactone exposure in imatinib-sensitive and -resistant K562 cells. Bcr/Abl knockdown enhanced the apoptosis driven by alantolactone. Bcr/Abl protein reduction could not be reversed by the addition of proteasome or caspase-3 inhibitors. The overexpression of p65 inhibited alantolactone-induced apoptosis, whereas p65 or Bcr/Abl silencing enhanced its apoptotic-inducing effect. Furthermore, Bcr/Abl-transfected 32D cells showed more sensitivity to alantolactone than vector-transfected control cells, and the Bcr/Abl protein was depleted, as observed in K562 cells. Finally, alantolactone-induced apoptosis was also observed in primary CD34+ CML leukemic cells. Collectively, these findings suggest that alantolactone is a promising potent agent to fight against CML cells via the inhibition of the NF-κB signaling pathway and depletion of the Bcr/Abl protein.

Targeting peroxiredoxins against leukemia

Peroxiredoxins (Prx), a family of small non-seleno peroxidases, are important regulators for cellular reactive oxygen species (ROS), which contribute to many signaling pathways and pathogenesis of diseases. Targeting redox homeostasis is being developed as a promising therapeutic strategy for many diseases such as cancers. This mini-review attempts to focus on our recent discoveries on adenanthin as the first natural molecule to specifically target the resolving cysteines of Prx I and Prx II and thus inhibit their peroxidase activities, and its role in differentiation induction in vitro and in vivo of acute myeloid leukemic cells.

Proteomic Identification of Common SCF Ubiquitin Ligase FBXO6-Interacting Glycoproteins in Three kinds of Cells

FBOX6 ubiquitin ligase complex is involved in the endoplasmic reticulum-associated degradation pathway by mediating the ubiquitination of glycoproteins. FBXO6 interacts with the chitobiose in unfolded N-glycoprotein, pointing glycoproteins toward E2 for ubiquitination. Although the glycoprotein-recognizing mechanism of FBXO6 is well documented, its bona fide interacting glycoproteins are largely unknown. Here we utilized a protein purification approach combined with LC-MS to systematically identify the FBXO6-interacting glycoproteins. Following identification of 39 proteins that specifically interact with FBXO6 in all three different cell lines, 293T, HeLa and Jurkat cells, we compared the protein complex organization between wild-type FBXO6 and its mutant, which fails to recognize glycoproteins. Combining these databases, 29 highly confident glycoproteins that interact with FBXO6 in an N-glycan dependent manner are identified. Our data provide valuable information for the discovery of the interacting glycoproteins of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other FBAs (F-box associated regions) containing F-box proteins.