Curcumin-Loaded Poly(L-lactide-co-glycolide) Microbubble-Mediated Sono-photodynamic Therapy in Liver Cancer Cells.

Sono-photodynamic therapy (SPDT) activates the same photo-/sonosensitizer and exerts more marked antitumor effects than sonodynamic therapy or photodynamic therapy. We aimed to explore the utilization of curcumin (CUR)-loaded poly(L-lactide-co-glycolide) microbubble (MB)-mediated SPDT (CUR-PLGA-MB-SPDT) in HepG2 liver cancer cells. The cytotoxicity and intracellular accumulation of CUR were determined. We used 40 µM CUR as the photo-/sonosensitizer for 3 h. In a comparison of CUR-SDT or CUR-PDT, HepG2 cell viability decreased and apoptotic rate increased in CUR-SPDT. The CUR-PLGA MBs had round spheres with smooth surfaces and an average size of 3.7 µm. In CUR-PLGA MBs, drug entrapment efficiency and drug-loading capacity were 74.29 ± 2.60% and 17.14 ± 0.60%, respectively. CUR-loaded PLGA MBs (CUR-PLGA MBs) had good biocompatibility with normal L02 cells and were almost non-cytotoxic to HepG2 cells. Among CUR-SDT, CUR-PDT, CUR-SPDT or CUR-PLGA-MB-SDT, the cell CUR-PLGA-MB-SPDT had the lowest viability. Transmission electron microscopy revealed pyroptosis and apoptosis in the CUR-PLGA-MB-SPDT group; the potential mechanism was related to the mitochondrial membrane potential loss and increased production of intracellular reactive oxygen species. These findings suggested that CUR-PLGA-MB-SPDT may be a promising treatment for liver cancer.

Preparative separation of four isomers of synthetic anisodamine by HPLC and diastereomer crystallization.

Anisodamine (654-1), a well-known cholinergic antagonist, is marketed as synthetic anisodamine (mixture of four isomers, 654-2) in China. To preparative resolution and comparison of the bioactivities of the four isomers of synthetic anisodamine, current work explores an economic and effective separation method by using preparative high performance liquid chromatography (HPLC) and diastereomer crystallization. Their absolute configurations were established by single-crystal X-ray diffraction and circular dichroism method. The purities of each isomer were more than 95%. Among them, 654-2-A2 (6R, 2'S configuration) exhibited better effect on cabachol preconditioned small intestine tension more than 654-2 and other isomers. The direct separation method without using HPLC was tried as well, which was still on progress. This is the first report of the method for preparative separation of four isomers of synthetic anisodamine which could be used for large-scale production in industry.

Cucurbitacin B inhibits tumor angiogenesis by triggering the mitochondrial signaling pathway in endothelial cells.

Cucurbitacin B (CuB), the active component of a traditional Chinese herbal medicine, Pedicellus Melo, has been shown to exhibit antitumor and anti-inflammation effects, but its role in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism are unknown. Tumor angiogenesis is one of the hallmarks of the development in malignant neoplasias and metastasis. Effective targeting of tumor angiogenesis is a key area of interest for cancer therapy. Here, we demonstrated that CuB significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration, tubulogenesis in vitro, and blocked angiogenesis in chick embryo chorioallantoic membrane (CAM) assay in vivo. Furthermore, CuB induced HUVEC apoptosis and may induce apoptosis by triggering the mitochondrial apoptotic pathway. Finally, we found that CuB inhibiting angiogenesis was associated with inhibition of the activity of vascular endothelial growth factor receptor 2 (VEGFR2). Our investigations suggested that CuB was a potential drug candidate for angiogenesis related diseases.

FGF21 ameliorates the neurocontrol of blood pressure in the high fructose-drinking rats.

Fibroblast growth factor-21 (FGF21) is closely related to various metabolic and cardiovascular disorders. However, the direct targets and mechanisms linking FGF21 to blood pressure control and hypertension are still elusive. Here we demonstrated a novel regulatory function of FGF21 in the baroreflex afferent pathway (the nucleus tractus solitarii, NTS; nodose ganglion, NG). As the critical co-receptor of FGF21, ß-klotho (klb) significantly expressed on the NTS and NG. Furthermore, we evaluated the beneficial effects of chronic intraperitoneal infusion of recombinant human FGF21 (rhFGF21) on the dysregulated systolic blood pressure, cardiac parameters, baroreflex sensitivity (BRS) and hyperinsulinemia in the high fructose-drinking (HFD) rats. The BRS up-regulation is associated with Akt-eNOS-NO signaling activation in the NTS and NG induced by acute intravenous rhFGF21 administration in HFD and control rats. Moreover, the expressions of FGF21 receptors were aberrantly down-regulated in HFD rats. In addition, the up-regulated peroxisome proliferator-activated receptor-γ and -α (PPAR-γ/-α) in the NTS and NG in HFD rats were markedly reversed by chronic rhFGF21 infusion. Our study extends the work of the FGF21 actions on the neurocontrol of blood pressure regulations through baroreflex afferent pathway in HFD rats.

Bone morphogenetic protein-4 mediates cardiac hypertrophy, apoptosis, and fibrosis in experimentally pathological cardiac hypertrophy.

Identifying the key factor mediating pathological cardiac hypertrophy is critically important for developing the strategy to protect against heart failure. Bone morphogenetic protein-4 (BMP4) is a mechanosensitive and proinflammatory gene. In this study, we investigated the role of BMP4 in cardiac hypertrophy, apoptosis, and fibrosis in experimentally pathological cardiac hypertrophy. The in vivo pathological cardiac hypertrophy models were induced by pressure-overload and angiotensin (Ang) II constant infusion in mice, and the in vitro model was induced by Ang II exposure to cultured cardiomyocytes. The expression of BMP4 increased in pressure overload, Ang II constant infusion-induced pathological cardiac hypertrophy, but not in swimming exercise-induced physiological cardiac hypertrophy in mice. BMP4 expression also increased in Ang II-induced cardiomyocyte hypertrophy in vitro. In turn, BMP4 induced cardiomyocyte hypertrophy, apoptosis, and cardiac fibrosis, and these pathological consequences were inhibited by the treatment with BMP4 inhibitors noggin and DMH1. Moreover, Ang II-induced cardiomyocyte hypertrophy was inhibited by BMP4 inhibitors. The underlying mechanism that BMP4-induced cardiomyocyte hypertrophy and apoptosis was through increasing NADPH oxidase 4 expression and reactive oxygen species-dependent pathways. Lentivirus-mediated overexpression of BMP4 recapitulated hypertrophy and apoptosis in cultured cardiomyocytes. BMP4 inhibitor DMH1 inhibited pressure overload-induced cardiac hypertrophy in mice in vivo. The plasma BMP4 level of heart failure patients was increased compared with that of subjects without heart failure. In summary, we conclude that BMP4 is a mediator and novel therapeutic target for pathological cardiac hypertrophy.

Large T-antigen up-regulates Kv4.3 K⁺ channels through Sp1, and Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through activation of calcium/calmodulin-dependent protein kinase II.

Down-regulation of Kv4.3 K⁺ channels commonly occurs in multiple diseases, but the understanding of the regulation of Kv4.3 K⁺ channels and the role of Kv4.3 K⁺ channels in pathological conditions are limited. HEK (human embryonic kidney)-293T cells are derived from HEK-293 cells which are transformed by expression of the large T-antigen. In the present study, by comparing HEK-293 and HEK-293T cells, we find that HEK-293T cells express more Kv4.3 K⁺ channels and more transcription factor Sp1 (specificity protein 1) than HEK-293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K⁺ channel expression in HEK-293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K⁺ expression in HEK-293 cells. Since the ultimate determinant of the phenotype difference between HEK-293 and HEK-293T cells is the large T-antigen, we conclude that the large T-antigen up-regulates Kv4.3 K⁺ channel expression through an increase in Sp1. In both HEK-293 and HEK-293T cells, inhibition of Kv4.3 K⁺ channels with 4-AP (4-aminopyridine) or Kv4.3 small interfering RNA induces cell apoptosis and necrosis, which are completely rescued by the specific CaMKII (calcium/calmodulin-dependent protein kinase II) inhibitor KN-93, suggesting that Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through CaMKII activation. In summary, we establish: (i) the HEK-293 and HEK-293T cell model for Kv4.3 K⁺ channel study; (ii) that large T-antigen up-regulates Kv4.3 K⁺ channels through increasing Sp1 levels; and (iii) that Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through activating CaMKII. The present study provides deep insights into the mechanism of the regulation of Kv4.3 K⁺ channels and the role of Kv4.3 K⁺ channels in cell death.

HIV Tat protein inhibits hERG K+ channels: a potential mechanism of HIV infection induced LQTs.

HIV-infected patients have a high prevalence of long QT syndrome (LQTs). hERG K(+) channel encoded by human ether-a-go-go related gene contributes to IKr K(+) currents responsible for the repolarization of cardiomyocytes. Inhibition of hERG K(+) channels leads to LQTs. HIV Tat protein, the virus transactivator protein, plays a pivotal role in AIDS. The aim of the present study is to examine the effects of HIV Tat protein on hERG K(+) channels stably expressed in HEK293 cells. The hERG K(+) currents were recorded by whole-cell patch-clamp technique and the hERG channel expression was measured by real-time PCR and Western blot techniques. HIV Tat protein at 200 ng/ml concentration showed no acute effect on hERG currents, but HIV Tat protein (200 ng/ml) incubation for 24 h significantly inhibited hERG currents. In HIV Tat incubated cells, the inactivation and the recovery time from inactivation of hERG channels were significantly changed. HIV Tat protein incubation (200 ng/ml) for 24h had no effect on the hERG mRNA expression, but dose-dependently inhibited hERG protein expression. The MTT assay showed that HIV Tat protein at 50 ng/ml and 200 ng/ml had no effect on the cell viability. HIV Tat protein increased reactive oxygen species (ROS) generation and the inhibition of hERG channel protein expression by HIV Tat protein was prevented by antioxidant tempol. HIV Tat protein in vivo treatment reduced IKr currents and prolonged action potential duration of guinea pig cardiomyocytes. We conclude that HIV Tat protein inhibits hERG K(+) currents through the inhibition of hERG protein expression, which might be the potential mechanism of HIV infection induced LQTs.