Daocheng Wu

The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular carcinoma cells

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.

Biofunctionalization of fluorescent-magnetic-bifunctional nanospheres and their applications

Hydrazide-containing bifunctional nanospheres were covalently coupled on the surface with IgG, avidin, and biotin, to generate novel fluorescent-magnetic-biotargeting trifunctional nanospheres, which can be used in a number of biomedical applications, including visual sorting and manipulation of apoptotic cells as demonstrated here.

Apogossypolone, a novel inhibitor of antiapoptotic Bcl-2 family proteins, induces autophagy of PC-3 and LNCaP prostate cancer cells in vitro.

Limited treatment options are available for aggressive prostate cancer. Gossypol has been reported to have a potent anticancer activity in many types of cancer. It can increase the sensitivity of cancer cells to alkylating agents, diminish multidrug resistance and decrease metastasis. Whether or not it can induce autophagy in cancer cells has not yet been determined. Here we investigated the antiproliferative activity of apogossypolone (ApoG2) and (-)-gossypol on the human prostate cancer cell line PC3 and LNCaP in vitro. Exposure of PC-3 and LNCaP cells to ApoG2 resulted in several specific features characteristic of autophagy, including the appearance of membranous vacuoles in the cytoplasm and formation of acidic vesicular organelles. Expression of autophagy-associated LC3-II and beclin-1 increased in both cell lines after treatment. Inhibition of autophagy with 3-methyladenine promoted apoptosis of both cell types. Taken together, these data demonstrated that induction of autophagy could represent a defense mechanism against apoptosis in human prostate cancer cells.

Temperature-Triggered Enzyme Immobilization and Release Based on Cross-Linked Gelatin Nanoparticles

A glucoamylase-immobilized system based on cross-linked gelatin nanoparticles (CLGNs) was prepared by coacervation method. This system exhibited characteristics of temperature-triggered phase transition, which could be used for enzyme immobilization and release. Their morphology and size distribution were examined by transmission electron microscopy and dynamic light scattering particle size analyzer. Their temperature-triggered glucoamylase immobilization and release features were also further investigated under different temperatures. Results showed that the CLGNs were regularly spherical with diameters of 155±5 nm. The loading efficiencies of glucoamylase immobilized by entrapment and adsorption methods were 59.9% and 24.7%, respectively. The immobilized enzyme was released when the system temperature was above 40°C and performed high activity similar to free enzyme due to the optimum temperature range for glucoamylase. On the other hand, there was no enzyme release that could be found when the system temperature was below 40°C. The efficiency of temperature-triggered release was as high as 99.3% for adsorption method, while the release of enzyme from the entrapment method was not detected. These results indicate that CLGNs are promising matrix for temperature-triggered glucoamylase immobilization and release by adsorption immobilization method.

Preparation of rhodamine B fluorescent poly(methacrylic acid) coated gelatin nanoparticles

Poly(methacrylic acid) (PMAA)-coated gelatin nanoparticles encapsulated with fluorescent dye rhodamine B were prepared by the coacervation method with the aim to retard the release of rhodamine B from the gelatin matrix. With sodium sulfate as coacervation reagent for gelatin, a kind of biopolymer with excellent biocompatibility, the formed gelatin nanoparticles were crosslinked by formaldehyde followed by the polymerization of methacrylic acid coating. The fluorescent poly(methacrylic acid) coated gelatin (FPMAAG) nanoparticles had a uniform spherical shape and a size distribution of 60 ± 5 nm. Infrared spectral analysis confirmed the formation of PMAA coating on the gelatin nanoparticles. Based on UV-Vis spectra, the loading efficiency of rhodamine B for the FPMAAG nanoparticles was 0.26 µg per mg nanoparticles. The encapsulated rhodamine B could sustain for two weeks. Favorable fluorescence property and fluorescence imaging of cells confirmed that the FPMAAG nanoparticles have promising biochemical, bioanalytical, and biomedical applications.

Inhibition of proliferation of prostate cancer cell line, PC-3, in vitro and in vivo using (-)-gossypol.

We investigated the antiproliferative activity of (-)-gossypol on the human prostate cancer cell line PC3 in vitro and in vivo to elucidate its potential molecular mechanisms. Cell growth and viability were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell apoptosis was detected by flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and electron microscopy. Expression of proliferating cell nuclear antigen (PCNA), Bcl-2, CD31, caspase-3 and caspase-8 in tumour tissue was determined by immunohistochemistry. The drug concentration that yielded 50% cell inhibition (IC(50) value) was 4.74 microg mL(-1). In the PC-3 tumour xenograft study, (-)-gossypol (> 5 mg kg(-1)) given once a day for 7 days significantly inhibited tumour growth in a dose-dependent manner. Immunohistochemical analysis revealed that (-)-gossypol enhanced caspase-3 and caspase-8 expression and decreased the expression of PCNA, Bcl-2 and CD31 in tumour tissues. It suggested that cell apoptosis and inhibition of angiogenesis might contribute to the anticancer action of (-)-gossypol.

A Novel Fluoride Anion Modified Gelatin Nanogel System

PURPOSE Controlled drug release, especially tumor-targeted drug release, remains a great challenge. Here, we prepare a novel fluoride anion-modified gelatin nanogel system and investigate its characteristics of ultrasound-triggered drug release. METHODS Adriamycin gelatin nanogel modified with fluoride anion (ADM-GNMF) was prepared by a modified co-precipitation method with fluoride anion and sodium sulfate. The loading and encapsulation efficiency of the anti-neoplastic agent adriamycin (ADM) were measured by high performance liquid chromatography (HPLC). The size and shape of ADM-GNMF were determined by electron microscopy and photo-correlation spectroscopy. The size distribution and drug release efficiency of ADM-GNMF, before and after sonication, were measured by two designed measuring devices that consisted of either a submicron particle size analyzer and an ultrasound generator as well as an ultrasound generator, automatic sampler, and HPLC. RESULTS The ADM-GNMF was stable in solution with an average diameter of 46+/-12 nm; the encapsulation and loading efficiency of adriamycin were 87.2% and 6.38%, respectively. The ultrasound-triggered drug release and size change were most efficient at a frequency of 20 kHz, power density of 0.4w/cm2, and a 1~2 min duration. Under this ultrasound-triggered condition, 51.5% of drug in ADM-GNMF was released within 1~2 min, while the size of ADM-GNMF changed from 46 +/- 12 nm to 1212 +/- 35 nm within 1~2 min of sonication and restored to its previous size in 2~3 min after the ultrasound stopped. In contrast, 8.2% of drug in ADM-GNMF was released within 2~3 min without sonication, and only negligible size changes were found. CONCLUSIONS The ADM-GNMF system efficiently released the encompassed drug in response to ultrasound, offering a novel and promising controlled drug release system for targeted therapy for cancer or other diseases.

High drug-loading nanomedicines: progress, current status, and prospects

Drug molecules transformed into nanoparticles or endowed with nanostructures with or without the aid of carrier materials are referred to as “nanomedicines” and can overcome some inherent drawbacks of free drugs, such as poor water solubility, high drug dosage, and short drug half-life in vivo. However, most of the existing nanomedicines possess the drawback of low drug-loading (generally less than 10%) associated with more carrier materials. For intravenous administration, the extensive use of carrier materials might cause systemic toxicity and impose an extra burden of degradation, metabolism, and excretion of the materials for patients. Therefore, on the premise of guaranteeing therapeutic effect and function, reducing or avoiding the use of carrier materials is a promising alternative approach to solve these problems. Recently, high drug-loading nanomedicines, which have a drug-loading content higher than 10%, are attracting increasing interest. According to the fabrication strategies of nanomedicines, high drug-loading nanomedicines are divided into four main classes: nanomedicines with inert porous material as carrier, nanomedicines with drug as part of carrier, carrier-free nanomedicines, and nanomedicines following niche and complex strategies. To date, most of the existing high drug-loading nanomedicines belong to the first class, and few research studies have focused on other classes. In this review, we investigate the research status of high drug-loading nanomedicines and discuss the features of their fabrication strategies and optimum proposal in detail. We also point out deficiencies and developing direction of high drug-loading nanomedicines. We envision that high drug-loading nanomedicines will occupy an important position in the field of drug-delivery systems, and hope that novel perspectives will be proposed for the development of high drug-loading nanomedicines.

Incorporating fluorescent dyes into monodisperse melamine–formaldehyde resin microspheres via an organic sol–gel process: a pre-polymer doping strategy

An organic sol-gel process was developed to incorporate fluorescent dyes into monodisperse melamine-formaldehyde (MF) resin microspheres. Various organic fluorescent dyes have been successfully incorporated by this process, and monodisperse fluorescent MF microspheres were prepared. Fluorescence-encoded microsphere arrays with dozens of sets were obtained by quantitatively incorporating several dyes at different doping concentrations. The characteristics and incorporating mechanism of these microspheres and their dyes were investigated by scanning electron microscopy, Malvern particle analysis, fluorescence spectroscopy, laser scanning confocal microscopy (LSCM), and flow cytometric analyses. Resonance energy transfer (RET) interactions of the doped dyes were investigated by steady-state and time-resolved fluorescence spectroscopy. The dye-incorporated microspheres were stable, and no leakage or deformation was found. With the occurrence of the RET effect among multi-doped dyes, prepared microspheres exhibited single excited, doping ratio-related emission signatures. The prepared dye-doped microspheres were coated with silica shells, which provided favorable surface properties for bioconjugate applications. This process of incorporating organic dyes could also be used to coat particles with dye-doped fluorescent MF shells. Multi-shell structured composite microspheres with fluorescent shells were also prepared by alternately repeating silica and MF coatings.

Apogossypolone, derivative of gossypol, mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage

Gossypol is a polyphenolic aldehyde that is produced in the cotton plant. Since long it has been reported to possess antiproliferative activity against a variety of cancer cell lines as well as tumor regression in animal models. However, the toxicity of gossypol does not permit it to be an effective antitumor agent. One of the derivatives of gossypol to show promising results is apogossypolone. For example, it has been shown to specifically target tumor growth in hepatocellular carcinoma xenograft in nude mice without causing any damage to normal tissue. Using human peripheral lymphocytes, in this paper we show that both gossypol and its semi-synthetic derivative apogossypolone cause oxidative DNA breakage in these cells through the mobilization of endogenous copper ions. Such cellular DNA breakage is inhibited by copper specific chelator but nor by iron or zinc chelating agents. Similar results are obtained with isolated nuclei indicating that chromatin bound copper is mobilized in this reaction. Further, apogossypolone showed enhanced DNA breakage and increased oxidative stress in whole lymphocytes as compared with gossypol indicating that this is possibly the result of greater permeability of apogossypolone. It is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be subject to greater electron transfer between copper ions and gossypol/apogossypolone to generate reactive oxygen species responsible for DNA cleavage. This may account for the preferential cytotoxicity of apogossypolone towards tumor cells.