De-Hong Yu

Peptide-conjugated biodegradable nanoparticles as a carrier to target paclitaxel to tumor neovasculature

Antiangiogenic cancer therapy can be achieved through the targeted delivery of antiangiogenic agents to the endothelial cells of tumor neovasculature. In the present study, we developed a drug delivery system (DDS), nanoparticles conjugated with K237-(HTMYYHHYQHHL) peptides for tumor neovasculature targeting drug delivery. Paclitaxel, a chemotherapeutic agent with potent antiangiogenic activity, was used as a prototype drug. We synthesized the aldehyde poly(ethylene glycol)-poly(lactide) (aldehyde-PEG-PLA) block copolymer by ring opening polymerization. The nanoparticles loading paclitaxel (PTX-NP) were fabricated using the O/W emulsion and evaporation technique. K237 ligand, a peptide that can bind to the KDR receptors predominantly expressed on the surface of tumor neovasculature endothelial cells with high affinity and specificity and inhibit the VEGF-KDR angiogenic signal pathway, was conjugated to the aldehyde group of PEG chain using the N-terminal PEGylation technique. The K237 conjugated paclitaxel-loaded nanoparticles (K237-PTX-NP) had a hydrodynamic diameter of 150 nm. The K237 density on nanoparticle surface was 474 and the mean distance between two neighboring PEG chains linked to K237 peptide was 12 nm. The K237 conjugated nanoparticles could be significantly internalized by human umbilical vein endothelial cells (HUVEC) through the K237-KDR interaction, and this facilitated uptake led to the expected enhanced antiangiogenic activity shown by HUVEC proliferation, migration and tube formation compared to cells treated with the commercial formulation Taxol and PTX-NP. The long-circulating property and the K237 ligand of K237-PTX-NP warranted rapid, long-term, and accurate in vivo tumor neovasculature targeting, and thereafter the significant apoptosis of tumor neovasculature endothelial cells and necrosis of tumor tissues of MDA-MB-231 breast tumors implanted in female BLAB/c nude mice. This nanoparticulate DDS offers a new strategy for paclitaxel chemotherapy application and it could also be used to carry other chemotherapeutic drugs, genes, and proteins with antiangiogenic activity for antiangiogenic cancer therapy.

Dichloroacetate shifts the metabolism from glycolysis to glucose oxidation and exhibits synergistic growth inhibition with cisplatin in HeLa cells

The unique bioenergetic feature of cancer, aerobic glycolysis or the Warburg effect, is an attractive therapeutic target for cancer therapy. Reversing the glycolytic phenotype may trigger apoptosis in tumor cells. Recently, dichloroacetate (DCA) was proven to produce significant cytotoxic effects in certain tumor cells through this distinct mechanism. In this study, the effect of DCA on the metabolism of cervical cancer HeLa cells was explored and its synergistic growth inhibition with cisplatin was also evaluated. The intracellular changes in HeLa cells following DCA exposure were analyzed through cell viability, intracellular H2O2 and pH levels, mitochondrial membrane potential (MMP), expression of apoptotic proteins and Kv1.5 channel, and intracellular-free Ca2+ concentration ([Ca2+]i). For the evaluation of combination chemotherapy, HeLa cells were treated with a combination of DCA and cisplatin at various concentrations for 48 h. Cell viability was determined by CCK-8 assay and the synergy of the two agents was evaluated using the R index method. DCA shifted the metabolism of HeLa cells from aerobic glycolysis to glucose oxidation as shown by the increased intracellular H2O2 and pH levels. The change of the metabolism modality led to a drop in MMP and the increase of apoptotic proteins (caspase 3 and 9). The increased Kv1.5 expression and decreased [Ca2+]i established a positive feedback loop that resulted in reduced tonic inhibition of caspases. Combination chemotherapy of DCA and cisplatin exhibited a significant synergy in inhibiting the proliferation of HeLa cells. The specific apoptotic mechanism of DCA as distinguished from the cisplatin may be partly responsible for the synergy and further in vivo study on combination chemotherapy of the two agents in cervical cancer xenografts in mice is warranted.

Nanoparticle-mediated drug delivery to tumor neovasculature to combat P-gp expressing multidrug resistant cancer

Anticancer drug resistance is a common intractable obstacle in clinical cancer chemotherapy. Here, we hypothesize that antiangiogenic cancer therapy through the targeted delivery of antiangiogenic agents to the tumor endothelial cells (EC), not the resistant cancer cells, may have the potential of combating multidrug resistant cancer. The K237 peptide-conjugated paclitaxel loaded nanoparticles (K237-PTX-NP), which can target KDR receptors highly expressed in the tumor vasculature, were fabricated for this investigation and the human colorectal adenocarcinoma HCT-15 with naturally expressed P-gp on the cell surface was adopted as the resistant tumor model. The human umbilical vein endothelial cells (HUVEC, a classical cell model mimicking tumor EC) were much more sensitive, in the cytotoxicity and apoptosis test, to K237-PTX-NP than Taxol and non-targeted PTX-NP. The enhanced antiangiogenic feature of K237-PTX-NP can be ascribed to the active internalization mediated by the interaction of K237 and KDR specifically highly expressed on the HUVEC, and the significantly extended intracellular drug retention. The tumor vessel targeting of K237-PTX-NP led to increased nanoparticle accumulation in HCT-15 tumors, and more importantly, induced significant apoptosis of tumor vascular EC and necrosis of tumor tissues. Low dose paclitaxel formulated in K237-PTX-NP (1 mg/kg) achieved significant anticancer efficacy of inhibiting the growth of HCT-15 tumors, but the same efficacy could be only obtained with 8 fold dose paclitaxel (8 mg/kg) in Taxol plus XR9576, a potent P-gp inhibitor. The anticancer efficacy of K237-PTX-NP was well related with the improved antiangiogenic effect shown in the dramatically decreased intratumoral microvessel density and pronouncedly increased apoptotic tumor cells, and such approach did not lead to obvious toxicity in the mice. These results suggest that the nanoparticles targeting drug to tumor neovasculature may be a promising strategy for the treatment of multidrug resistant cancer.

The use of nanoparticulate delivery systems in metronomic chemotherapy

Metronomic chemotherapy aiming at inhibiting tumor angiogenesis with conventional chemotherapeutics is a promising strategy for antiangiogenic cancer therapy. However, current metronomic chemotherapy mainly focuses on free small-molecule drugs, without any effort to achieve tumor-specific biodistribution, which may lead to long-term toxicity concerns. Metronomic chemotherapy using nanoparticulate drug delivery system (DDS) offers significant upside to reduce off-target side effects, decrease accumulated dose, and enhance the efficacy of tumor vessel targeting without compromising antitumor efficacy; but there has been a lack of thorough experimental data describing the targeted metronomic chemotherapy. Here, we develop a new nanoparticulate DDS, SP5.2 peptide conjugated, Flt-1 (VEGFR-1) targeted nanoparticles for docetaxel (SP5.2-DTX-NP), as a model for the investigation of targeted metronomic chemotherapy with respect to both antitumor efficacy and toxicity. The results demonstrate that metronomic SP5.2-DTX-NP exerts antitumor activity mainly through the antiangiogenic effect of docetaxel, which is specifically delivered into the tumor vascular endothelial cells through the nanoparticle internalization mediated by the interaction of SP5.2 and over-expressed Flt-1 receptors on tumor vessels. Moreover, the antitumor efficacy of targeted metronomic chemotherapy is better than that of the treatment with the DDS given in the maximum tolerated dose (MTD) regimen, which is shown in significantly prolonged mice survival and minimal drug-associated toxicity (bone marrow suppression, hematological toxicity, and mucosal injury of small intestine). The present research reveals and highlights the significance of targeted metronomic therapy with nanoparticulate DDS in antiangiogenic cancer therapy.

Antiangiogenic Triterpenes Isolated from Chinese Herbal Medicine Actinidia chinensis Planch

Actinidia chinensis Planch. is a famous Chinese herbal medicine to treat many diseases such as cancers. Triterpenes, polyphenols and anthraquinones are normally considered as the main constituents for its effects. In this study, eleven known triterpenes were isolated from the root of Actinidia chinensis., and were examined for its antiangiogenic activities. Their structures were elucidated by comprehensive spectroscopic methods, including IR, UV, HR-ESI-MS, and 1D and 2D NMR techniques. The eleven compounds are following: 2α,3α,19-trihydroxyurs-12-en-28-oic acid (1), 2α,3β-dihydroxyurs-12-en-28-oic acid (2), 2α,3α,23-trihydroxyurs-12-en-28-oic acid (3), asiatic acid (4), ursolic acid (5), 2α,3β,19,24-tetrahydroxyurs-12-en-28-oic acid (6), 2α,3β,19-trihydroxyolean-12-en-28-oic acid (7), 2α,3α,24-trihydroxyolean-12-en-28-oic acid (8), oleanolic acid (9), 3β-O-acetyloleanolic acid (10), 2α,23-dihydroxylmicromeric acid (11). All these compounds were evaluated with respect to their antiangiogenic activities utilizing the assays of human umbilical vein endothelial cells (HUVEC) proliferation and tube formation and Ursolic acid (used as control) and compounds 2, 3, 4, and 8 exhibited significant, dose-dependently, antiangiogenic activity in the tested concentration range. Our findings suggest that antitumor action of Actinidia chinensis Planch. is partly via inhibiting tumor angiogenesis by triterpenes, and compounds 2, 3, 4, and 8 as the novel potential antiangiogenic agents are worthy of further translational research.

Secolignans with antiangiogenic activities from Peperomia dindygulensis.

Two new secolignans, peperomins G and H (1 and 2, resp.), were isolated from the whole plant of Peperomia dindygulensis, together with five known secolignans, peperomin A (3), peperomin E (4), peperomin B (5), 2,3‐trans‐2‐methyl‐3‐{(3‐hydroxy‐4,5‐dimethoxyphenyl)[5‐methoxy‐3,4‐(methylenedioxy)phenyl]methyl}butyrolactone (6), 2,3‐cis‐2‐(hydroxymethyl)‐3‐{bis[5‐methoxy‐3,4‐(methylenedioxy)phenyl]methyl}butyrolactone (7). Their structures and configurations were elucidated by spectroscopic methods including 2D‐NMR techniques. Antiangiogenic effects of all compounds were evaluated using human umbilical vein endothelial cells (HUVEC) proliferation and tube‐formation tests, with compounds 4 and 5 being active in the bioassay. Compounds 4 and 5 induced obvious cell toxicity to HUVEC with IC50 values of 1.64±0.19 and 8.44±0.4 μM, respectively. Compounds 4 and 5 also exhibited significant HUVEC tube formation‐inhibiting activity with IC50 values of 3.13±0.09 and 6.24±0.12 μM, respectively.

Determination of Raddeanin A in rat plasma by liquid chromatography-tandem mass spectrometry: Application to a pharmacokinetic study.

A simple, rapid and sensitive LC-MS/MS analysis method was developed and validated for the determination of Raddeanin A (RA) in rat plasma. Protein precipitation with three volumes of methanol as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Thermo Syncronis C18 column with the mobile phase of methanol-water (containing 5mM ammonium formate, pH 2.2) (85:15, v/v). RA and glycyrrhetinic acid (internal standard) were monitored under negative electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of RA and IS were 2.1 min and 3.5 min, respectively. The limit of detection was 5 ng/mL and the linear range was 50-50,000 ng/mL. The intra-day and inter-day precision was 1.87-2.94% and 3.25-5.36%, and the intra-day and inter-day accuracy ranged from 5.9% to 10.5% and 5.6% to 11.1%, respectively. The absolute recovery was above 90.3%. The method has been successfully translated to the pharmacokinetic study of RA in rats after intravenous and intraperitoneal administration (0.75 mg/kg).

IF7-Conjugated Nanoparticles Target Annexin 1 of Tumor Vasculature against P-gp Mediated Multidrug Resistance.

Multidrug resistance is the main cause of clinical chemotherapeutic failure. Antiangiogenic cancer therapy with nanomedicine that allows the targeted delivery of antiangiogenic agents to tumor endothelial cells may contribute to innovative strategies for treating multidrug-resistant cancers. In this study, we developed a new nanodrug delivery system (nano-DDS), with improved antiangiogenic efficacy against multidrug resistant human breast cancer MCF-7/ADR cells. Here, the IF7 ligand was a peptide designed to bind the annexin 1 (Anxa 1), a highly specific marker of the tumor vasculature surface, with high affinity and specificity. IF7-conjugated Anxa 1-targeting nanoparticles containing paclitaxel (IF7-PTX-NP) allowed controlled drug release and displayed favorable prolonged circulation in vivo. IF7-PTX-NP was significantly internalized by human umbilical vein endothelial cells (HUVEC) through the IF7-Anxa 1 interaction, and this facilitated uptake enhanced the expected antiangiogenic activity of inhibiting HUVEC proliferation, migration, and tube formation in a Matrigel plug relative to those of Taxol and PTX-NP. As IF7-PTX-NP targeted the tumor vessels, more nanoparticles accumulated in MCF-7/ADR tumors, and more importantly, induced significant apoptosis of the tumor vascular endothelial cells and necrosis of the tumor tissues. Low dose paclitaxel (1 mg/kg) formulated in IF7-PTX-NP showed significant anticancer efficacy, delaying the growth of MCF-7/ADR tumors. The same efficacy was only obtained with an 8-fold dose of paclitaxel (8 mg/kg) as Taxol plus XR9576, a potent P-gp inhibitor. The anticancer efficacy of IF7-PTX-NP was strongly associated with the improved antiangiogenic effect, evident as a dramatic reduction in the tumor microvessel density and pronounced increase in apoptotic tumor cells, with no obvious toxicity to the mice. This nano-DDS, which targets the tumor neovasculature, offers a promising strategy for the treatment of multidrug-resistant cancer.

Influence of E3 region on conditionally replicative adenovirus mediated cytotoxicity in hepatocellular carcinoma cells.

Virotherapy employing conditionally replicative adenovirus (CRAd) represents a novel targeted strategy for the hepatocellular carcinoma (HCC) treatment. In this study, we explored the potential influence of E3 region, which encodes several TRAIL-inhibiting proteins (E3-6.7K, E3-10.4K/14.5K and E3-14.7K), on CRAd mediated cytotoxicity to HCC cells. Two E1B-55 kDa-deleted CRAds containing E3 region (Ad.ΔE1B) or no E3 region (Ad.ΔE1B.ΔE3) were fabricated. Ad.ΔE1B.ΔE3 exhibited higher cytocidal potency than Ad.ΔE1B in all tested HCC cells (Hep3B, BEL-7404, BEL-7402, HuH7, PLC/PRF/5 and HepG2), suggesting that Ad.ΔE1B.ΔE3 mediated cytotoxicity was partly attributed to the absence of E3 region encoding TRAIL-inhibiting proteins. In representative Hep3B cells, Ad.ΔE1B.ΔE3 led to more drop of mitochondrial membrane potential (MMP) and much lower ATP level than Ad.ΔE1B. Moreover, Ad.ΔE1B.ΔE3 induced early apoptotic cells and the late apoptotic/ necrotic cells for three and four times more than those infected by Ad.ΔE1B. The cytotoxicity to all TRAIL endogenously expressing HCC cells and MMP drop of Hep3B cells induced by Ad.ΔE1B.ΔE3 but not Ad.ΔE1B could be significantly inhibited by z-vad-fmk, a pan caspase inhibitor, suggesting that the endogenous TRAIL-mediated apoptotic pathway may be implicated in the cytocidal potency of Ad.ΔE1B.ΔE3 on HCC cells although other unknown mechanisms may be also involved. Our findings provided the first evidence that CRAd without E3 region might be a smart choice for the virotherapy of HCC.