Hoi-Yeung Li

Mitosis-targeted anti-cancer therapies : where they stand.

The strategy of clinically targeting cancerous cells at their most vulnerable state during mitosis has instigated numerous studies into the mitotic cell death (MCD) pathway. As the hallmark of cancer revolves around cell-cycle deregulation, it is not surprising that antimitotic therapies are effective against the abnormal proliferation of transformed cells. Moreover, these antimitotic drugs are also highly selective and sensitive. Despite the robust rate of discovery and the development of mitosis-selective inhibitors, the unpredictable complexities of the human body’s response to these drugs still herald the biggest challenge towards clinical success. Undoubtedly, the need to bridge the gap between promising preclinical trials and effective translational bedside treatment prompts further investigations towards mapping out the mechanistic pathways of MCD, understanding how these drugs work as medicine in the body and more comprehensive target validations. In this review, current antimitotic agents are summarized with particular emphasis on the evaluation of their clinical efficacy as well as their limitations. In addition, we discuss the basis behind the lack of activity of these inhibitors in human trials and the potential and future directions of mitotic anticancer strategies.

Angiopoietin-like 4 Protein Elevates the Prosurvival Intracellular O2−:H2O2 Ratio and Confers Anoikis Resistance to Tumors

Cancer is a leading cause of death worldwide. Tumor cells exploit various signaling pathways to promote their growth and metastasis. To our knowledge, the role of angiopoietin-like 4 protein (ANGPTL4) in cancer remains undefined. Here, we found that elevated ANGPTL4 expression is widespread in tumors, and its suppression impairs tumor growth associated with enhanced apoptosis. Tumor-derived ANGPTL4 interacts with integrins to stimulate NADPH oxidase-dependent production of O(2)(-). A high ratio of O(2)(-):H(2)O(2) oxidizes/activates Src, triggering the PI3K/PKBα and ERK prosurvival pathways to confer anoikis resistance, thus promoting tumor growth. ANGPTL4 deficiency results in diminished O(2)(-) production and a reduced O(2)(-):H(2)O(2) ratio, creating a cellular environment conducive to apoptosis. ANGPTL4 is an important redox player in cancer and a potential therapeutic target.

Orally Active Peptidic Bradykinin B1 Receptor Antagonists Engineered from a Cyclotide Scaffold for Inflammatory Pain Treatment

Chronic pain is a universal health issue associated with numerous medical conditions, for example after severe burns or following major surgery. Compelling evidence suggests that bradykinin (BK) antagonists could be useful in treating chronic pain and inflammatory pain. Bradykinin and its homolog kallidin (lysyl-BK or KD), which are collectively known as kinins, participate in many pathophysiological insults. They are short-lived peptide mediators and the most potent endogenous pain inducers. Kinins are released during tissue injury or noxious stimulation and modulate pain through the activation of both the B1 and the B2 receptor, which are two G-protein-coupled receptors; the carboxypeptidase metabolites of kinins, des-Arg-BK and des-Arg-KD, activate the B1 receptor. [2b, 4] The B1 receptor stimulates the chronic phase of the inflammatory pain response, while the B2 receptor stimulates the acute phase owing to their differences in ligand dissociation, receptor desensitization, downregulation as well as internalization. Emerging evidence also suggests that the B1 receptor mediates various chronic pain responses through the activation of phospholipase C, thereby leading to the production of diacylglycerol and inositol triphosphate, which further activate protein kinase C and Ca mobilization. Numerous BK-antagonist peptides have been discovered from natural sources and structure–activity studies. Kinestatin isolated from frog skin and helokinestatin from lizard venom are examples of natural BK-antagonist peptides. Structure–activity studies have shown that removing the Cterminal Arg residue and concurrently replacing the penultimate residue Phe to Leu of bradykinin to des-Arg-[Leu]bradykinin or kallidin to des-Arg-[Leu]-kallidin (DALK) changes a bradykinin B2 receptor agonist to a B1 receptor antagonist (Figure 1). To increase potency and in vivo stability, several laboratories also developed BK antagonists

ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters

Vascular disruption induced by interactions between tumor-secreted permeability factors and adhesive proteins on endothelial cells facilitates metastasis. The role of tumor-secreted C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4) in vascular leakiness and metastasis is controversial because of the lack of understanding of how cANGPTL4 modulates vascular integrity. Here, we show that cANGPTL4 instigated the disruption of endothelial continuity by directly interacting with 3 novel binding partners, integrin α5β1, VE-cadherin, and claudin-5, in a temporally sequential manner, thus facilitating metastasis. We showed that cANGPTL4 binds and activates integrin α5β1-mediated Rac1/PAK signaling to weaken cell-cell contacts. cANGPTL4 subsequently associated with and declustered VE-cadherin and claudin-5, leading to endothelial disruption. Interfering with the formation of these cANGPTL4 complexes delayed vascular disruption. In vivo vascular permeability and metastatic assays performed using ANGPTL4-knockout and wild-type mice injected with either control or ANGPTL4-knockdown tumors confirmed that cANGPTL4 induced vascular leakiness and facilitated lung metastasis in mice. Thus, our findings elucidate how cANGPTL4 induces endothelial disruption. Our findings have direct implications for targeting cANGPTL4 to treat cancer and other vascular pathologies.

Protein–protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four‐and‐a‐half LIM‐only protein 3 (FHL3) is a type of LIM‐only protein that contains four tandemly repeated LIM motifs with an N‐terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two‐hybrid assay. Furthermore, site‐directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site‐directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two‐fusion fluorescence resonance energy transfer (FRET), in the determination of protein–protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi‐fluorescent microscope equipped with an FRET specific filter set. J. Cell. Biochem. 80:293–303, 2001.

Interaction of hCLIM1, an enigma family protein, with α‐actinin 2

Enigma proteins are proteins that possess a PDZ domain at the amino terminal and one to three LIM domains at the carboxyl terminal. They are cytoplasmic proteins that are involved with the cytoskeleton and signal transduction pathway. By virtue of the two protein interacting domains, they are capable of protein‐protein interactions. Here we report a study on a human Enigma protein hCLIM1, in particular. Our study describes the interaction of the human 36kDa carboxyl terminal LIM domain protein (hCLIM1), the human homologue of CLP36 in rat, with α‐actinin 2, the skeletal muscle isoform of α‐actinin. hCLIM1 protein was shown to interact with α‐actinin 2 by yeast two‐hybrid screening and immunochemical analyses. Yeast two‐hybrid analyses also demonstrated that the LIM domain of hCLIM1 binds to the EF‐hand region of α‐actinin 2, defining a new mode of LIM domain interactions. Immunofluorescent study demonstrates that hCLIM1 colocalizes with α‐actinin at the Z‐disks in human myocardium. Taken together, our experimental results suggest that hCLIM1is a novel cytoskeletal protein and may act as an adapter that brings other proteins to the cytoskeleton. J. Cell. Biochem. 78:558–565, 2000.

Survivin withdrawal by nuclear export failure as a physiological switch to commit cells to apoptosis.

Apoptosis is a tightly controlled process regulated by many signaling pathways; however, the mechanisms and cellular events that decide whether a cell lives or dies remain poorly understood. Here we showed that when a cell is under apoptotic stress, the prosurvival protein Survivin redistributes from the cytoplasm to the nucleus, thus acting as a physiological switch to commit the cell to apoptosis. The nuclear relocalization of Survivin is a result of inefficient assembly of functional RanGTP–CRM1–Survivin export complex due to apoptotic RanGTP gradient collapse. Subsequently, Survivin undergoes ubiquitination, which not only physically prevents its diffusion back to the cytoplasm but also facilitates its degradation. Together, this spatial and functional regulation of Survivin abolishes its cytoprotective effect toward the apoptotic executors and thus commits a cell to apoptosis. Our data indicate that the withdrawal of Survivin is a novel and active physiological regulatory mechanism that tilts the survival balance and promotes the progression of apoptosis.

Translocation of a human focal adhesion LIM‐only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle.

Characterization of tissue‐specific LIM domain protein (FHL1C) which is an alternatively spliced isoform of a human LIM‐only protein (FHL1)

We have cloned and characterized another alternatively spliced isoform of the human four‐and‐a‐half LIM domain protein 1 (FHL1), designated FHL1C. FHL1C contains a single zinc finger and two tandem repeats of LIM domains at the N‐terminus followed by a putative RBP‐J binding region at the C‐terminus. FHL1C shares the same N‐terminal two‐and‐a‐half LIM domains with FHL1 but different C‐terminal protein sequences. Due to the absence of the exon 4 in FHL1C, there is a frame‐shift in the 3′ coding region. Sequence analysis indicated that FHL1C is the human homolog of murine KyoT2. The Northern blot and RT‐PCR results revealed that FHL1 is widely expressed in human tissues, including skeletal muscle and heart at a high level, albeit as a relatively low abundance transcript in brain, placenta, lung, liver, kidney, pancreas, and testis. In contrast, FHL1C is specifically expressed in testis, skeletal muscle, and heart at a relatively low level compared with FHL1. The expression of FHL1C transcripts was also seen in aorta, left atrium, left, and right ventricles of human heart at low level. Immunoblot analysis using affinity‐purified anti‐FHL1C antipeptide antibodies confirmed a 20 kDa protein of FHL1C in human skeletal muscle and heart. Unlike FHL1B, which is another FHL1 isoform recently reported by our group and localized predominantly in the nucleus [Lee et al., 1999 ], FHL1C is localized both in the nucleus and cytoplasm of mammalian cell. J. Cell. Biochem. 82: 1–10, 2001.

Apoptotic histone modification inhibits nuclear transport by regulating RCC1

A number of signalling pathways have been identified that regulate apoptosis, but the mechanism that initiates apoptosis remains incompletely understood. We have found that the nuclear RanGTP level is diminished during the early stages of apoptosis, which correlates with immobilization of RCC1 on the chromosomes. Furthermore, the expression of phosphomimetic histone H2B or caspase-activated Mst1 immobilizes RCC1 and causes reduction of nuclear RanGTP levels, which leads to inactivation of the nuclear transport machinery. As a consequence, nuclear localization signal (NLS)-containing proteins, including NF-κB–p65, remain bound to importins α and β in the cytoplasm. Knocking down Mst1 allows resumption of nuclear transport and the nuclear entry of NF-κB–p65, which have important roles in rescuing cells from apoptosis. Therefore, we propose that RCC1 reads the histone code created by caspase-activated Mst1 to initiate apoptosis by reducing the level of RanGTP in the nucleus.