Novel Complex of PD-L1 Aptamer and Albumin Enhances Antitumor Efficacy In Vivo.

The PD-1/PD-L1 pathway blockade can generate a good clinical response by reducing immunosuppression and provoking durable antitumor immunity. In addition to antibodies, aptamers can also block the interaction between PD-1 and PD-L1. For the in vivo application, however, free aptamers are usually too small in size and quickly removed from blood via glomerular filtration. To avoid renal clearance of aptamer, we conjugated the PD-L1 aptamer to albumin to form a larger complex (BSA-Apt) and evaluated whether BSA-Apt would enhance the in vivo antitumor efficacy. The PD-L1 aptamer was thiol-modified and conjugated to the amino group of BSA via a SMCC linker. The average size of BSA-Apt was 11.65 nm, which was above the threshold for renal clearance. Functionally, BSA-Apt retained the capability of the PD-L1 aptamer to bind with PDL1-expressing tumor cells. Moreover, both the free aptamer and BSA-Apt augmented the PBMC-induced antitumor cytotoxicity in vitro. Furthermore, BSA-Apt generated a significantly stronger antitumor efficacy than the free PD-L1 aptamer in vivo without raising systemic toxicity. The results indicate that conjugating the PD-L1 aptamer to albumin may serve as a promising strategy to improve the in vivo functionality of the aptamer and that BSA-Apt may have application potential in cancer immunotherapy.

Novel Complex of PD-L1 Aptamer and Holliday Junction Enhances Antitumor Efficacy in Vivo.

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.

Novel Bispecific Aptamer Enhances Immune Cytotoxicity Against MUC1-Positive Tumor Cells by MUC1-CD16 Dual Targeting.

A promising strategy in cancer immunotherapy is the employment of a bispecific agent that can bind with both tumor markers and immunocytes for recruitment of lymphocytes to tumor sites and enhancement of anticancer immune reactions. Mucin1 (MUC1) is a tumor marker overexpressed in almost all adenocarcinomas, making it a potentially important therapeutic target. CD16 is expressed in several types of immunocytes, including NK cells, γδ-T cells, monocytes, and macrophages. In this study, we constructed the first bispecific aptamer (BBiApt) targeting both MUC1 and CD16. This aptamer consisted of two MUC1 aptamers and two CD16 aptamers linked together by three 60 nt DNA spacers. Compared with monovalent MUC1 or CD16 aptamers, BBiApt showed more potent avidity to both MUC1-positive tumor cells and CD16-positive immunocytes. Competition experiments indicated that BBiApt and monovalent aptamers bound to the same sites on the target cells. Moreover, BBiApt recruited more CD16-positive immunocytes around MUC1-positive tumor cells and enhanced the immune cytotoxicity against the tumor cells in vitro. The results suggest that, apart from bispecific antibodies, bispecific aptamers may also potentially serve as a novel strategy for targeted enhancement of antitumor immune reactions against MUC1-expressing malignancies.

Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-positive breast cancer cells in vitro.

BACKGROUND: Aptamer-based tumor targeted drug delivery system is a promising approach that may increase the efficacy of chemotherapy and reduce the related toxicity. HER2 protein is an attractive target for tumor-specific drug delivery because of its overexpression in multiple malignancies, including breast, gastric, ovarian, and lung cancers. METHODS: In this paper, we developed a new HER2 aptamer (HB5) by using systematic evolution of ligands by exponential enrichment technology (SELEX) and exploited its role as a targeting ligand for delivering doxorubicin (Dox) to breast cancer cells in vitro. RESULTS: The selected aptamer was an 86-nucleotide DNA molecule that bound to an epitope peptide of HER2 with a Kd of 18.9 nM. The aptamer also bound to the extracellular domain (ECD) of HER2 protein with a Kdof 316 nM, and had minimal cross reactivity to albumin or trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative breast cancer cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating Dox into the DNA structure of HB5. The Apt-Dox complex could selectively deliver Dox to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells in vitro. Moreover, Apt-Dox retained the cytotoxicity of Dox against HER2-positive breast cancer cells, but reduced the cytotoxicity to HER2-negative cells. CONCLUSIONS: The results suggest that the selected HER2 aptamer may have application potentials in targeted therapy against HER2-positive breast cancer cells.

Genistein attenuates vascular endothelial impairment in ovariectomized hyperhomocysteinemic rats.

Hyperhomocysteinemia (HHcy) is a well-known independent risk factor for vascular diseases in the general population. This study was to explore the effect of genistein (GST), a natural bioactive compound derived from legumes, on HHcy-induced vascular endothelial impairment in ovariectomized rats in vivo. Thirty-two adult female Wistar rats were assigned randomly into four groups (n = 8): (a) Con: control; (b) Met: 2.5% methionine diet; (c) OVX + Met: ovariectomy + 2.5% methionine diet; (d) OVX + Met + GST: ovariectomy + 2.5% methionine diet + supplementation with genistein. After 12 wk of different treatment, the rats' blood, toracic aortas and liver samples were collected for analysis. Results showed that high-methionine diet induced both elevation of plasma Hcy and endothelial dysfunction, and ovariectomy deteriorated these injuries. Significant improvement of both functional and morphological changes of vascular endothelium was observed in OVX + Met + GST group; meanwhile the plasma Hcy levels decreased remarkably. There were significant elevations of plasma ET-1 and liver MDA levels in ovariectomized HHcy rats, and supplementation with genistein could attenuate these changes. These results implied that genistein could lower the elevated Hcy levels, and prevent the development of endothelial impairment in ovariectomized HHcy rats. This finding may shed a novel light on the anti-atherogenic activities of genistein in HHcy patients.

A mutation in PHKG1 causes high drip loss and low meat quality in Chinese Ningdu yellow chickens.

With increasing societal development and the concurrent improvement in people's quality of life, meat consumption has gradually changed from a focus on "quantity" to "quality". Broiler production is increasingly used as a means to improve meat quality by altering various characteristics, especially its genetic factors. However, until now, little has been known about the genetic variants related to meat quality traits in Chinese purebred chicken populations. To better understand these genetic underpinnings, a total of 17 traits related to meat quality and carcass were measured in 325 Chinese Ningdu yellow chickens. We performed DNA sequencing to detect nucleotide mutations, after which we conducted association studies between PHKG1 gene polymorphisms and traits related to meat quality and carcass. Results indicated a large phenotypic variation in meat quality traits. More specifically, the single nucleotide polymorphism (SNP) rs15845448 was significantly associated with drip loss at 24 h (P = 8.04 × 10-6) and 48 h (P = 5.47 × 10-6), pH (P = 2.39 × 10-3), and meat color L* (P = 9.88 × 10-3). Moreover, the SNP rs15845448 reduced 24 h and 48 h drip loss by 3.62 and 5.97%, respectively. However, no significant associations were found between rs15845448 and carcass traits (P > 0.05). Furthermore, a haplotype block containing 2 adjacent SNPs (rs15845448 and rs15845450) was identified. This block displayed 4 distinct haplotypes that had significant association with drip loss at 24 h and 48 h, pH, and meat color L*. Collectively, these results provide new insights into the genetic basis of meat quality in Chinese Ningdu yellow chickens. Moreover, the significance of SNP rs15845448 could be incorporated into the selection programs involving this breed.

The effects of α-zearalanol and estradiol benzoate on expression of c-myc, c-fos and epidermal growth factor receptor mRNAs in breast tissues implanted into nude mice.

This study was designed to evaluate the effects of a novel phytoestrogen, α-zearalanol (α-ZAL), and estradiol benzoate (B-E2), on c-myc, c-fos, and EGFR expression in normal human breast tissues implanted into nude mice. A xenograft-model, pieces of normal human breast tissue implanted subcutaneously into 9-10-week-old athymic nude mice, was established. The mice were divided into five groups subjected to the following treatments: normal saline (Controls); α-ZAL at 1 and 5 mg/kg; and estradiol benzoate (B-E2) at 1 and 5 mg/kg. Treatment was given every other day, and human breast tissues were removed for experiments after treatment for 30 days. The expression of c-myc, c-fos, and EGFR mRNAs were determined by in situ hybridization. α-ZAL decreased expression of c-myc (p < 0.05). About 1 mg/kg α-ZAL increased EGFR expression (p < 0.05) and two dosage of α-ZAL increased c-fos expression (p < 0.01) compared with control. B-E2 significantly increased expression of c-myc, c-fos, and EGFR mRNAs expression compared with controls (p < 0.01). The extents of the increases in EGFRmRNA expression induced by α-ZAL and in c-fos mRNA by 5 mg/kg α-ZAL were lower than those induced by B-E2 (p < 0.01). These data suggest that the phytoestrogen α-ZAL may be safer than estrogen on breast.

Targeted Treatment of Colon Cancer with Aptamer-Guided Albumin Nanoparticles Loaded with Docetaxel.

PURPOSE: Chemotherapy of colon cancer needs improvement to mitigate the severe adverse effects (AEs) associated with the cytotoxic drugs. The aim of this study is to develop a novel targeted drug delivery system (TDDS) with practical application potential for colon cancer treatment. METHODS: The TDDS was built by loading docetaxel (DTX) in albumin nanoparticles (NPs) that were functionalized with nucleolin-targeted aptamers (AS1411). RESULTS: The TDDS (Apt-NPs-DTX) had an average size of 62 nm and was negatively charged with a zeta potential of -31.2 mV. DTX was released from the albumin NP with a typical sustained release profile. Aptamer-guided NPs were preferentially ingested by nucleolin-expressing CT26 colon cancer cells vs the control cells. In vitro cytotoxicity study showed that Apt-NPs-DTX significantly enhanced the killing of CT26 colon cancer cells. Importantly, compared with non-targeted drug delivery, Apt-NPs-DTX treatment significantly improved antitumor efficacy and prolonged the survival of CT26-bearing mice, without raising systemic toxicity. CONCLUSION: The results suggest that Apt-NPs-DTX has potential in the targeted treatment of colon cancer.

Targeted Therapy of Colon Cancer by Aptamer-Guided Holliday Junctions Loaded with Doxorubicin.

PURPOSE: Chemotherapy is the primary treatment for advanced colon cancer, but its efficacy is often limited by severe toxicities. Targeted therapy in the form of selectively drug delivery system (SDDS) is an important strategy to reduce adverse effects. Here, we aim to design a novel SDDS with potential for practical application using biocompatible components and scalable production process, for targeted delivery of doxorubicin (Dox) to colon cancer cells. METHODS: The SDDS was made of a self-assembled DNA nano-cross (Holliday junction, or HJ) functionalized by four AS1411 aptamers (Apt-HJ) and loaded with Dox. RESULTS: Apt-HJ had an average size of 12.45 nm and a zeta potential of -11.6 mV. Compared with the monovalent AS1411 aptamer, the quadrivalent Apt-HJ showed stronger binding to target cancer cells (CT26). A complex of Apt-HJ and doxorubicin (Apt-HJ-Dox) was formed by intercalating Dox into the DNA structure of Apt-HJ, with each complex carrying approximately 17 Dox molecules. Confocal microscopy revealed that Apt-HJ-Dox selectively delivered Dox into CT26 colon cancer cells but not the control cells. Moreover, Apt-HJ-Dox achieved targeted killing of CT26 cancer cells in vitro and reduced the damage to control cells. Importantly, compared with free Dox, Apt-HJ-Dox significantly enhanced the antitumor efficacy in vivo without boosting the adverse effects. CONCLUSION: These results suggest that Apt-HJ-Dox has application potential in targeted treatment of colon cancer.

Targeted distribution of bacterial magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 in healthy Sprague-Dawley rats.

Bacterial magnetosomes (BMs) have drawn great interest as novel magnetic targeted carriers and have been widely used as carriers for enzymes, nucleic acids and antibodies experimentally. However, BMs have rarely been employed as drug carriers in vivo mainly due to the unclear biocompatibility and pharmacokinetics of BMs. In this study, we provided a unique effective method for purification and sterilization of BMs. The BMs obtained exhibited sterility, high purity, narrowed size-distribution, uniformity in morphology, intact membrane and abundant amino groups in BMs membrane. To elucidate the in vivo body distribution of BMs and if BMs displayed any rejection by animals when they are delivered into the vascular system, the BMs were injected into the sublingual vena of Sprague-Dawley (SD) rats and the tissue distribution of BMs in dejecta, urine, serum and main organs was examined. A target distribution of BMs in SD rats was observed. After injected into the sublingual vena, BMs were only found in livers and there was no obvious evidence to indicate the existence of BMs in the dejecta and urine within 72 h following the intravenous administration.

Relaxing effects of phytoestrogen alpha-zearalanol on rat thoracic aorta rings in vitro.

The aim of this research is to investigate the vasorelaxing effects and mechanisms involved in the phytoestrogen alpha-zearalanol (alpha-ZAL) in rat thoracic aortas rings. Intact or endothelium denuded rat thoracic aortas rings were put in individual organ chamber to observe the endothelium-dependent or independent vasorelaxing effects of alpha-ZAL (10(-10)-10(-5) M). The thoracic aortas rings were pre-contracted with phenylephrine. The relaxing effects of alpha-ZAL were observed and the influence of N(omega)-nitro-L-arginine methylester (L-NAME, NOS inhibitor), methylene blue (MB, guanylate cyclase inhibitor), charybdotoxin (ChTX, Ca(2+)-activated K+ channel blocker), glibenclamide (ATP-sensitive K+ channel blocker), (-) BayK8644 (L-type Ca2+ channel agonist) and ICI182,780 (estrogen receptor antagonist) were pre-incubated with alpha-ZAL, respectively, to explore the possible mechanisms involved in this vasorelaxation. Furthermore, the Phospho-eNOS expression and cGMP level in the aortas tissue were detected by Western blot and radioimmunity, respectively; the NO level in perfusate was assaied by chromatometry. Our result showed that alpha-ZAL (10(-10)-10(-5) M) induced both endothelium-dependent and -independent relaxation of rat thoracic aortas rings. The vasorelaxing effects of alpha-ZAL were dose-dependent whether the endothelium was intact or not. In endothelium-intact aortas rings, alpha-ZAL-induced vasorelaxation might be inhibited by L-NAME, MB, charybdotoxin, glibenclamide and (-) BayK8644, but not ICI182,780. (-) BayK8644 could also inhibit alpha-ZAL-induced vasorelaxation in endothelium-denuded aortas rings.10(-7)-10(-5) M alpha-ZAL might induce the Phospho-eNOS expression in thoracic aorta tissue, increase the NO level in perfusate and cGMP content in thoracic aorta tissue. Meanwhile, L-NAME might decrease both NO and its downstream cGMP level. Methylene blue might decrease the level of cGMP. These results suggest that alpha-ZAL induces a partly endothelium-dependent relaxation of rat thoracic aortas rings; the possible mechanisms involved in this rapid vasorelaxation include activation of eNOS/NO/cGMP pathway, opening of VSMCs ATP-sensitive and Ca(2+)-activated K+ channels through secretion of EDHF from endothelium. Furthermore, this relaxation also appears to be mediated by both direct and indirect inhibition of voltage-dependent Ca2+ channel of VSMCs, while it is not concerned with activation of estrogen receptor.

[Inhibitory effect of low molecular weight heparin on the secretion of vascular endothelial growth factor by tumor cells in vitro].

OBJECTIVE: To investigate whether low molecular weight heparin (LMWH) may suppress the expression and secretion of vascular endothelial growth factor (VEGF) from tumor cells in vitro and inhibit the VEGF-induced proliferation of human tumor vascular endothelial cells. METHODS: Human lung cancer cell line A549, human liver cancer cell line HepG2, human colon carcinoma cell lines HCT116 and HCT8 were used in this study. The expression levels of VEGF and TNF-alpha (tumor necrosis factor-alpha) in the tumor cells with or without pretreatment of LMWH/heparin were measured by standard sandwich ELISA technique. The VEGF mRNA level of HepG2 cells cultured with or without LMWH/heparin was determined by RT-PCR and real time PCR. Human umbilical vein endothelial cells (HUVEC) were cultured in tissue culture medium (TCM) with or without LMWH/heparin for 3 days. Then non-radioactive cell proliferation assay (MTS) kit and cell cycle assay by flow cytometry were performed to measure the proliferation of HUVEC. RESULTS: The VEGF levels in the control, LMWH, and heparin groups of the pulmonary adenocarcinoma cell line A549 were (1045.89 +/- 165.30) pg/ml, (782.45 +/- 67.17) pg/ml and (916.54 +/- 71.25) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups of the colon adenocarcinoma cell line HCT116 were (955.76 +/- 51.14) pg/ml, (822.89 +/- 142.39) pg/ml and (951.77 +/- 188.22) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the colon adenocarcinoma cell line HCT8 were (1290.62 +/- 41.23) pg/ml, (1063.34 +/- 63.82) pg/ml and (1257.14 +/- 11.40) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the liver cancer cell line HepG2 were (1083.00 +/- 134.35) pg/ml, (758.00 +/- 84.85) pg/ml and (874.00 +/- 22.62) pg/ml, respectively. The VEGF expression levels in the above mentioned cell lines cultured in TCM were significantly reduced in the LMWH-treated groups compared with that of the control group (P < 0.05). But the level of TNF-alpha in TCM-cultured cells was unaffected by LMWH. The VEGF mRNA was reduced in the LMWH-treated HepG2 cell line. Moreover, TCM exhibited stimulating effect on proliferation of HUVEC and the effect was significantly impaired by LMWH treatment. Flow cytometric analysis revealed that LMWH treatment arrested HUVECs at the G1 phase of cell cycle. CONCLUSION: LMWH can suppress the expression and secretion of VEGF by tumor cell lines and therefore have a potential inhibiting effect on angiogenesis induced by VEGF.

Selection of a novel DNA aptamer against OFA/iLRP for targeted delivery of doxorubicin to AML cells.

The standard treatment for most acute myeloid leukemia (AML) is chemotherapy, which is often associated with severe adverse effects. One strategy to reduce the adverse effects is targeted therapy that can selectively deliver anticancer drugs to tumor cells. Immature laminin receptor protein (OFA/iLRP) is a potential target for AML treatment, because it is over-expressed on the surface of AML cells but under-expressed in normal tissue. In this study, we developed the first aptamer for OFA/iLRP and explored its potential as a targeting ligand for delivery of doxorubicin (Dox) to AML cells in vitro. The selected aptamer (AB3) was a 59-base DNA oligonucleotides. It bound to OFA/iLRP structure with a Kd of 101 nM and had minimal cross-reactivity to albumin, trypsin, or ovalbumin. Moreover, AB3 could bind to OFA/iLRP-positive AML cells but not the OFA/iLRP-negative control cells. An aptamer-doxorubicin (Apt-Dox) complex was formed by intercalating doxorubicin into the DNA structure of AB3. Apt-Dox selectively delivered Dox to OFA/iLRP-positive AML cells but notably decreased the drug intake by OFA/iLRP-negative control cells. In addition, cytotoxicity study revealed that Apt-Dox efficaciously destroyed the OFA/iLRP-positive AML cells, but significantly reduced the damage to control cells. The results indicate that the OFA/iLRP aptamer AB3 may have application potential in targeted therapy against AML.

Carbon nanotubes conjugated to tumor lysate protein enhance the efficacy of an antitumor immunotherapy.

The biomedical applications of carbon nanotubes (CNTs) have attracted deep interest in recent years. Antitumor immunotherapy has the potential to improve the prognosis of cancer treatment but the efficacy of current immunotherapy generally needs further improvement. Multi-walled CNTs conjugated to tumor lysate protein are investigated as to whether they would enhance the efficacy of an immunotherapy employing a tumor-cell vaccine in a mouse model bearing the H22 liver cancer. The tumor cure rate is found to be markedly improved by CNTs conjugated to tumor lysate protein. The cellular antitumor immune reaction is also enhanced. Moreover, the observed antitumor immune response is relatively specific against the tumor intended for treatment. These findings suggest that CNTs may have a prospective role in the development of new antitumor immunotherapies.

Reactive oxygen species mediate oxidized low-density lipoprotein-induced endothelin-1 gene expression via extracellular signal-regulated kinase in vascular endothelial cells.

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) promotes expression and secretion of endothelin-1 (ET-1), however, the precise mechanism involved is unclear. This study was designed to identify the regulatory mechanism of oxLDL-induced ET-1 expression in endothelial cells. METHODS: ET-1 mRNA expression, secretion and promoter activity were evaluated by reverse transcriptase-PCR (RT-PCR), enzyme immunometric and luciferase assays, respectively. RESULTS: oxLDL (35 microg/ml) significantly enhanced reactive oxygen species (ROS), mRNA expression, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), all of which were nullified by the antioxidant N-acetyl cysteine (NAC). oxLDL stimulated the extracellular signal-regulated kinase (ERK) phosphorylation in HUVECs, which was blocked by NAC and the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD98059. NAC and PD98059 stopped oxLDL-elicited increase in mRNA expression, secretion and promoter activity of ET-1. Fusion plasmids with decreasing length of 5'-flanking sequence of ET-1 from -566 bpLuc to -250 bpLuc displayed increased luciferase activity after 24 h of oxLDL treatment. Interestingly, fusion plasmid from -233 and -185 bpLuc significantly reduced the luciferase activity in control and oxLDL-treated HUVECs. In addition, transfection of the reporter construct -250Luc, which contains a 2 bp mutation at activator protein-1 site, abolished both basal and oxLDL-stimulated ET-1 promoter activities. CONCLUSION: Collectively, our data favor the notion that oxLDL stimulates ERK phosphorylation via ROS accumulation, which in turn stimulates vascular endothelial transcriptional factor activator protein-1 and ET-1 expression as well as secretion.

Preparation and anti-tumor efficiency evaluation of doxorubicin-loaded bacterial magnetosomes: magnetic nanoparticles as drug carriers isolated from Magnetospirillum gryphiswaldense.

Bacterial magnetosomes (BMs) are commonly used as vehicles for certain enzymes, nucleic acids and antibodies, although they have never been considered drug carriers. To evaluate the clinical potential of BMs extracted from Magnetospirillum gryphiswaldense in cancer therapy, doxorubicin (DOX) was loaded onto the purified BMs at a ratio of 0.87 +/- 0.08 mg/mg using glutaraldehyde. The DOX-coupled BMs (DBMs) and BMs exhibited uniform sizes and morphology evaluated by TEM. The diameters of DBMs and BMs obtained by AFM were 71.02 +/- 6.73 and 34.93 +/- 8.24 nm, respectively. The DBMs released DOX slowly into serum and maintained at least 80% stability following 48 h of incubation. In vitro cytotoxic tests showed that the DBMs were cytotoxic to HL60 and EMT-6 cells, manifested as inhibition of cell proliferation and suppression in c-myc expression, consistent with DOX. These observations depicted in vitro antitumor property of DBMs similar to DOX. The approach of coupling DOX to magnetosomes may have clinical potential in anti-tumor drug delivery.

alpha-Zearalanol attenuates oxLDL-induced ET-1 gene expression, ET-1 secretion and redox-sensitive intracellular signaling activation in human umbilical vein endothelial cells.

alpha-Zearalanol (alpha-ZAL), a phytochemical with both antioxidant and estrogen-like properties, has been shown to retard progression of atherosclerosis and regulate cardiovascular function in part through suppression of endothelin-1 (ET-1) secretion. However, the precise nature behind alpha-ZAL-elicited inhibition on ET-1 cascade is not largely known. Oxidized low density lipoprotein (oxLDL) plays a critical role in the expression and secretion of ET-1 as well as the onset and progression of atherosclerosis through accumulation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase stress signaling cascade. Therefore, this study was designed to examine the effect of alpha-ZAL on oxLDL-induced extracellular signal-regulated kinase (ERK) phosphorylation, ROS generation, activation of the transcriptional factor activator protein-1 (AP-1), expression, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVEC). ROS generation was monitored using 2,7-dichlorofluorescin fluorescence. ET-1 expression and promoter activity were evaluated by RT-PCR and luciferase assays, respectively. oxLDL (35 microg/ml) significantly enhanced ERK phosphorylation, ROS generation, AP-1 activity, mRNA expression, secretion and promoter activity of ET-1 in HUVECs, all of which were abrogated by alpha-ZAL and the antioxidant N-acetyl-l-cysteine. Collectively, these data favor the notion that alpha-ZAL antagonizes oxLDL-induced upregulation of ET-1 gene expression and secretion via suppression of oxLDL-induced ROS accumulation, ERK phosphorylation, and activation of the endothelial transcriptional factor AP-1.

Selection of a novel CD19 aptamer for targeted delivery of doxorubicin to lymphoma cells.

CD19 is overexpressed in most human B cell malignancies and considered an important tumor marker for diagnosis and treatment. Aptamers are oligonucleotides that may potentially serve as tumor-homing ligand for targeted cancer therapy with excellent affinity and specificity. In this study, we selected a novel CD19 aptamer (LC1) that was a 59-nucleotide single strand DNA. The aptamer could bind to recombinant CD19 protein with a Kd of 85.4 nM, and had minimal cross reactivity to bovine serum albumin (BSA) or ovalbumin (OVA). Moreover, the aptamer was found capable of binding with the CD19-positive lymphoma cells (Ramos and Raji), but not the CD19-negative cell lines (Jurkat and NB4). An aptamer-doxorubicin complex (Apt-Dox) was also formulated, and selectively delivered doxorubicin to CD19-positive lymphoma cells in vitro. The results indicate that aptamer LC1 can recognize CD19-positive tumor cells and may potentially function as a CD19-targeting ligand.

In vitro and in vivo antitumor effects of doxorubicin loaded with bacterial magnetosomes (DBMs) on H22 cells: the magnetic bio-nanoparticles as drug carriers.

Hepatocellular carcinoma (HCC) is the most common form of cancer although effective therapeutic strategy especially targeted therapy is lacking. We recently employed bacterial magnetosomes (BMs) as the magnetic-targeted drug carrier and found an antitumor effect of doxorubicin (DOX)-loaded BMs (DBMs) in EMT-6 and HL60 cell lines. The aim of this study was to evaluate the in vitro and in vivo anti-neoplastic effects of DBMs on hepatic cancer. DBMs, DOX and BMs displayed tumor suppression rates of 86.8%, 78.6% and 4.3%, respectively, in H22 cell-bearing mice. The mortality rates following administration of DBMs, DOX and BMs were 20%, 80% and 0%, respectively. Pathological examination of hearts and tumors revealed that both DBMs and DOX effectively inhibited tumor growth although DBMs displayed a much lower cardiac toxicity compared with DOX. The DBMs were cytotoxic to H22 cells manifested as inhibition of cell proliferation and c-myc expression, consistent with DOX. The IC(50) of DOX, DBMs and BMs in target cells were 5.309 +/- 0.010, 4.652 +/- 0.256 and 22.106 +/- 3.330 microg/ml, respectively. Our data revealed both in vitro and in vivo antitumor property of DBMs similar to that of DOX. More importantly, the adverse cardiac toxicity was significantly reduced in DBMs compared with DOX. Collectively, our study suggests the therapeutic potential of DBMs in target-therapy against liver cancer.

Cardiac nitric oxide synthases are elevated in dietary copper deficiency.

Dietary copper (Cu) deficiency leads to cardiac morphological and functional defects suggestive of heart failure. However, simultaneous cytoprotective events also appear to occur. The molecular mechanisms responsible for this complex alteration of cardiac function by Cu deficiency have not been elucidated. Because prior work has implicated altered nitric oxide (NO) metabolism in this altered function, we have examined this pathway in further detail. Male Sprague-Dawley rats were fed diets that were either Cu adequate (6 mg Cu/kg diet) or Cu deficient (<0.5 mg Cu/kg diet) for 5 weeks. Endothelial NO synthase (NOS) and inducible NOS (iNOS) protein expressions, as measured by Western blot analysis, were 58% and 40% higher, respectively, in Cu-deficient than in Cu-adequate rat hearts. Cardiac NOS activity, as measured by conversion of (3)H-arginine to (3)H-citrulline, was 130% higher in Cu-deficient than in Cu-adequate rats. NFkappaB is a known transcription factor for iNOS. Activation of NFkappaB, determined by an ELISA for the p65 subunit, was found to be 33% higher in Cu-deficient than in Cu-adequate rats. Coupled with prior evidence of elevated cardiac nitrate/nitrite production in Cu-deficient rats, these data suggest multiple pathways for enhanced NO production that may contribute to altered cardiac function under dietary Cu deficiency.