Bee Jen Tan

Perorally active nanomicellar formulation of quercetin in the treatment of lung cancer

Background Realizing the therapeutic benefits of quercetin is mostly hampered by its low water solubility and poor absorption. In light of the advantages of nanovehicles in the delivery of flavanoids, we aimed to deliver quercetin perorally with nanomicelles made from the diblock copolymer, polyethylene glycol (PEG)-derivatized phosphatidylethanolamine (PE). Methods Quercetin-loaded nanomicelles were prepared by using the film casting method, and were evaluated in terms of drug incorporation efficiency, micelle size, interaction with Caco-2 cells, and anticancer activity in the A549 lung cancer cell line and murine xenograft model. Results The incorporation efficiency into the nanomicelles was ≥88.9% when the content of quercetin was up to 4% w/w, with sizes of 15.4–18.5 nm and polydispersity indices of <0.250. Solubilization of quercetin by the nanomicelles increased its aqueous concentration by 110-fold. The quercetin nanomicelles were stable when tested in simulated gastric (pH 1.2) and intestinal (pH 7.4) fluids, and were non-toxic to the Caco-2 cells as reflected by reversible reduction in transepithelial electrical resistance and ≤25% lactose dehydrogenase release. The anticancer activity of quercetin could be significantly improved over the free drug through the nanomicellar formulation when tested using the A549 cancer cell line and murine xenograft model. The nanomicellar quercetin formulation was well tolerated by the tumor-bearing animals, with no significant weight loss observed at the end of the 10-week study period. Conclusion A stable PEG-PE nanomicellar formulation of quercetin was developed with enhanced peroral anticancer activity and no apparent toxicity to the intestinal epithelium.

Cloning and characterization of AWP1, a novel protein that associates with serine/threonine kinase PRK1 in vivo.

We describe the cloning and expression of cDNAs encoding a novel human protein of 208 amino acid residues with a predicted molecular mass of 22.6kDa and its mouse homologue. We name this protein as AWP1 (associated with PRK1). AWP1 is a ubiquitously expressed protein, and the Awp1 gene is switched on during early human and mouse development. When expressed in COS-1 cells, the Myc-tagged AWP1 has an apparent molecular mass higher than that deduced from its amino acid sequence. AWP1 possesses a conserved zf-A20 zinc finger domain at its N-terminal and a zf-AN1 zinc finger domain at its C-terminal. Co-immunoprecipitation experiments revealed that mouse AWP1 specifically interacts with a rat serine/threonine protein kinase PRK1 in vivo. Hence, AWP1 may play a regulatory role in mammalian signal transduction pathways.

Lipid-Based Nanoparticulate Systems for the Delivery of Anti-Cancer Drug Cocktails: Implications on Pharmacokinetics and Drug Toxicities

The use of drug cocktails has become a widely adopted strategy in clinical cancer therapy. Cytotoxic drug cocktails are often administered based on maximum tolerated dose (MTD) of each agent, with the belief of achieving maximum cell kill through tolerable toxicity level. Yet, MTD administration may not have fully captured the therapeutic synergism that exists among the individual agents in the drug cocktail, as the response to a cocktail regimen, that is, whether the effect is synergistic or not, could be highly sensitive to the concentration ratios of the individual drugs at the site of action. It is important to realize that the inherently different pharmacokinetic profiles of the individual agents could have significant influence on the response to an anti-cancer drug cocktail by dictating the amount of the individual agents reaching the tumor site and therefore the concentration ratios. Furthermore, the individual agents may have unfavorable pharmacokinetic interactions that add to the difficulty in determining the therapeutic and/or toxicological effects of the drug cocktail. In this review, we will focus on how lipid-based nanoparticulate systems could address the above issues associated with anti-cancer drug cocktails. Specifically, we will highlight the use of liposome systems as the means to control and coordinate the delivery of various anti-cancer drug cocktails, encompassing conventional chemotherapeutics, chemosensitizing agents and molecularly targeted agents.

Functionalized carbon nanomaterials: exploring the interactions with Caco-2 cells for potential oral drug delivery

Although carbon nanomaterials (CNMs) have been increasingly studied for their biomedical applications, there is limited research on these novel materials for oral drug delivery. As such, this study aimed to explore the potential of CNMs in oral drug delivery, and the objectives were to evaluate CNM cytotoxicity and their abilities to modulate paracellular transport and the P-glycoprotein (P-gp) efflux pump. Three types of functionalized CNMs were studied, including polyhydroxy small-gap fullerenes (OH-fullerenes), carboxylic acid functionalized single-walled carbon nanotubes (f SWCNT-COOH) and poly(ethylene glycol) functionalized single-walled carbon nanotubes (f SWCNT-PEG), using the well-established Caco-2 cell monolayer to represent the intestinal epithelium. All three CNMs had minimum cytotoxicity on Caco-2 cells, as demonstrated through lactose dehydrogenase release and 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Of the three CNMs, f SWCNT-COOH significantly reduced transepithelial electrical resistance and enhanced transport of Lucifer Yellow across the Caco-2 monolayer. Confocal fluorescence microscopy showed that f SWCNT-COOH treated cells had the highest perturbation in the distribution of ZO-1, a protein marker of tight junction, suggesting that f SWCNT-COOH could enhance paracellular permeability via disruption of tight junctions. This modulating effect of f SWCNT-COOH can be reversed over time. Furthermore, cellular accumulation of the P-gp substrate, rhodamine-123, was significantly increased in cells treated with f SWCNT-COOH, suggestive of P-gp inhibition. Of note, f SWCNT-PEG could increase rhodamine-123 accumulation without modifying the tight junction. Collectively, these results suggest that the functionalized CNMs could be useful as modulators for oral drug delivery, and the differential effects on the intestinal epithelium imparted by different types of CNMs would create unique opportunities for drug-specific oral delivery applications.

Liposome co-encapsulation of synergistic combination of irinotecan and doxorubicin for the treatment of intraperitoneally grown ovarian tumor xenograft.

Liposome co-encapsulation of synergistic anti-cancer drug combination is an emerging area that has demonstrated therapeutic benefit in clinical trials. Remote loading of two or more drugs into a single liposome constitutes a new challenge that calls for a re-examination of drug loading strategies to allow the loading of the drug combination efficiently and with high drug content. In this study, the Mn2+ gradient coupled with A23187 ionophore was applied in the sequential co-encapsulation of doxorubicin and irinotecan, as this drug loading method is capable of remotely loading drugs by apparently two different mechanisms, namely, coordination complexation and pH gradient. Doxorubicin and irinotecan could be co-encapsulated into liposomes in a wide range of drug-to-drug ratio, with encapsulation efficiencies of >80%. The total encapsulated drug content was non-linearly correlated with increases in the intraliposomal Mn2+ concentration, with a maximum total drug-to-lipid molar ratio of 0.8:1 achieved with 600mM Mn2+. This high encapsulated drug content did not affect the stability of the co-encapsulated liposomes upon storage for six months. Regardless of the encapsulated drug amount, the liposomes did not exhibit the fiber bundle precipitate morphology but rather an undefined structural organization in the aqueous core. The co-encapsulated liposome formulation was further tested in an intraperitoneally grown, human ovarian tumor xenograft model, and was shown to significantly improve the survival of the tumor-bearing animals. The improvement in therapeutic efficacy was possibly due to the increase in systemic drug exposure, with the maintenance of the synergistic molar drug ratio of 1:1 in circulation.

The PKCα-D294G Mutant Found in Pituitary and Thyroid Tumors Fails to Transduce Extracellular Signals

Protein kinase C (PKC) is a key regulator of cell proliferation, differentiation, and apoptosis and is one of the drug targets of anticancer therapy. Recently, a single point mutation (D294G) in PKCalpha has been found in pituitary and thyroid tumors with more invasive phenotype. Although the PKCalpha-D294G mutant is implicated in the progression of endocrine tumors, no apparent biochemical/cell biological abnormalities underlying tumorigenesis with this mutant have been found. We report here that the PKCalpha-D294G mutant is unable to bind to cellular membranes tightly despite the fact that it translocates to the membrane as efficiently as the wild-type PKCalpha upon treatment of phorbol ester. The impaired membrane binding is associated with this mutants inability to transduce several antitumorigenic signals as it fails to mediate phorbol ester-stimulated translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS), to activate mitogen-activated protein kinase and to augment melatonin-stimulated neurite outgrowth. Thus, the PKCalpha-D294G is a loss-of-function mutation. We propose that the wild-type PKCalpha may play important antitumorigenic roles in the progression of endocrine tumors. Therefore, developing selective activators instead of inhibitors of PKCalpha might provide effective pharmacological interventions for the treatment of certain endocrine tumors.

Role of oxidative stress, endoplasmic reticulum stress and ERK activation in triptolide-induced apoptosis

Since its isolation from Tripterygium wilfordii in 1972, triptolide has been shown to possess potent anticancer activity against a variety of cancers, and has entered phase I clinical trial. It is a diterpenoid triepoxide that acts through multiple molecular targets and signaling pathways. The mitogen-activated protein kinases are well known for their modulation of cell survival and proliferation. In particular, the ERK pathway has a dual role in cell proliferation and cell death. Thus far, data on the effect of triptolide on ERK signaling remain limited. In our current study, we have shown for the first time that ERK activation rather than inhibition occurred in a dose- and time-dependent manner following triptolide treatment in MDA-MB-231 breast cancer cells. ERK activation was crucial in mediating triptolide-induced caspase-dependent apoptosis. Tritpolide-induced ERK activation modulated the expression of the Bcl-2 protein family member Bax but was not involved in the downregulation of Bcl-xL expression. Signals acted upstream of ERK activation included generation of reactive oxygen species (ROS) and endoplasmic reticulum stress predominantly via the PERK‑eIF2α pathway, as the MEK inhibitor U0126 did not inhibit the phosphorylation of PERK and eIF2α or the generation of ROS.

The Last 10 Amino Acid Residues beyond the Hydrophobic Motif Are Critical for the Catalytic Competence and Function of Protein Kinase Cα

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ∼60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

Synthesis and Heterocyclizations of 3,4-Dihydroquinazolin-2-yl Guanidine in the Search of New Anticancer Agents

The cyclocondensations of 3,4-dihydroquinazolin-2-yl guanidine with a variety of electrophilic reagents viz. aldehydes, ketones, triethyl orthoformate, diethyl ethoxymethylenemalonate, carbon disulfide and trichloroacetonitrile were found to afford 1,3,5-triazino[2,1-b]quinazolines. However, some unexpected reactions were also observed. The structural properties such as tautomerism and hinderance to conformational rotation were also investigated. The results of biological testing suggested that the 1,3,5-triazino[2,1-b]quinazoline nucleus could be a new promising scaffold for the development of potential anticancer agents.

Signaling via a novel integral plasma membrane pool of a serine/threonine protein kinase PRK1 in mammalian cells

Mammalian serine/threonine protein kinases, except for TGF‐β receptor kinase family, are intracellular proteins. PRK1/PKN is a member of the protein kinase C superfamily of serine/threonine kinases and is one of the first identified effectors for RhoA GTPase. However, the role of PRK1 in mediating signaling downstream of activated RhoA is largely unknown. Here, we present evidence that identifies a novel plasma membrane pool of PRK1. This integral membrane form of PRK1 is catalytically active. The phosphorylation of serine377 of PRK1 is required for its integration into membranes. This integration is essential for PRK1 to function as a Rho effector as only the integral plasma membrane PRK1 is able to initiate RhoA‐mediated and ligand‐dependent transcriptional activation of the androgen receptor in human epithelial cells and to mediate RhoA‐induced neurite retraction in mouse neuronal cells. These results indicate that RhoA signals via the integral membrane pool of its effectors in its immediate vicinity at the plasma membrane, thus establishing a new paradigm in mammalian cell signaling.