Facile total synthesis of lysicamine and the anticancer activities of the RuII, RhIII, MnII and ZnII complexes of lysicamine.

Lysicamine is a natural oxoaporphine alkaloid, which isolated from traditional Chinese medicine (TCM) herbs and has been shown to possess cytotoxicity to hepatocarcinoma cell lines. Reports on its antitumor activity are scarce because lysicamine occurs in plants at a low content. In this work, we demonstrate a facile concise total synthesis of lysicamine from simple raw materials under mild reaction conditions, and the preparation of the Ru(II), Rh(III), Mn(II) and Zn(II) complexes 1-4 of lysicamine (LY). All the compounds were fully characterized by elemental analysis, IR, ESI-MS, 1H and 13C NMR, as well as single-crystal X-ray diffraction analysis. Compared with the free ligand LY, complexes 2 and 3 exhibited superior in vitro cytotoxicity against HepG2 and NCI-H460. Mechanistic studies indicated that 2 and 3 blocked the cell cycle in the S phase by decreasing of cyclins A2/B1/D1/E1, CDK 2/6, and PCNA levels and increasing levels of p21, p27, p53 and CDC25A proteins. In addition, 2 and 3 induced cell apoptosis via both the caspase-dependent mitochondrial pathway and the death receptor pathway. in vivo study showed that 2 inhibited HepG2 tumor growth at 1/3 maximum tolerated dose (MTD) and had a better safety profile than cisplatin.

Studies on antitumor mechanism of two planar platinum(II) complexes with 8-hydroxyquinoline: synthesis, characterization, cytotoxicity, cell cycle and apoptosis.

[Pt(Q)2] (1) and [Pt(MQ)2] (2) exhibited enhanced cytotoxicity against BEL-7404, Hep-G2, NCI-H460, T-24, A549 tumor cells but low cytotoxicity on normal HL-7702 cells. 1 and 2 could cause the cell cycle arrest in G2 and S phase, respectively. While pifithrin-α, a specific p53 inhibitor, induced cell cycle arrest in G1 phase. Although 1, 2 and pifithrin-α caused serious inhibition on p53, 1 and 2 significantly cause the loss of mitochondrial membrane potential and increase of the reactive oxygen species level, cytochrome c, apaf-1 and caspase-3/9 ratio in BEL-7404 cells. 1 and 2 may trigger the cell apoptosis through a mitochondrial dysfunction pathway whereas pifithrin-α does not. The interactions of 1 and 2 with DNA are most probably via an intercalation.

The decidual stromal cells-secreted CCL2 induces and maintains decidual leukocytes into Th2 bias in human early pregnancy.

The precise mechanism of characteristic Th2 predominance at maternal-fetal interface remains unresolved. In the present study, we investigated roles of the decidua-derived CCL2 in Th2 predominance at maternal-fetal interface. FCM shows that 55% CD56(+)CD16(-)CD3(-) decidual NK, 52% CD4(+) T cells and 75% CD14(+) monocytes express CCR2. Recombinant human CCL2 (rhCCL2) and the decidual stromal cells (DSCs)-derived supernatant can enhance proliferation and inhibit apoptosis of these decidual leukocytes (DLCs), and promote Th2 cytokines production, IL-4 and IL-10, with an increase in GATA-3 transcription. They also inhibit the secretion of Th1 cytokines, TNF-α and IFN-γ, with a decrease in T-bet transcription It is concluded that the secreted CCL2 by decidual stromal cells increases GATA-3 transcription and decreases T-bet transcription in the decidual leukocytes, which contributes to Th2 polarization at maternal-fetal interface. Furthermore, the Th2 cytokines, IL-4 and IL-10, rather than Th1 cytokines, was shown to increase CCL2 secretion of DSC.

Fusion of hC3d3 to hCGbeta enhances responsiveness in vitro of human peripheral immunocompetent cells upon the antigen primary challenge.

Contraceptive vaccines based on hCGbeta have not met clinical application because of poor immunogenicity. In the present study, the eukaryotic expression vectors pCI-gs-signal-6His-hCGbeta and pCI-gs-signal-6His-hCGbeta-hC3d3 were constructed, and transfected into CHO cells with aid of Lipofectaine 2000 reagent to gain the secretory recombinant protein. Isolated B cells from human peripheral blood, combined B cells with T cells, and PBMC were treated in vitro, respectively, with 1 nM, 10 nM, 100 nM hCGbeta, hCGbeta-hC3d3 or PWM for 12 days. Immunoglobulin (Ig) and anti-hCG antibody levels in the supernatant were measured by an indirect enzyme-linked immunosorbent assay (ELISA). The expressions of CD80/CD86 on B cells, and CD154/CD25 on T cells, were analyzed by flow cytometry (FCM), and IL-2 production was assayed by ELISA. It was found that the Ig levels in the B-cell supernatants, the combined B with T cells, and PBMC treated with 100 nM hCGbeta-C3d3 fusion protein were 4-fold, 10-fold and 10.9-fold more, respectively, than that of hCGbeta. The anti-hCG antibody could be produced in the combined B cells with T cells, as well as PBMC challenged with 100 nM hCGbeta-C3d3, but no anti-hCG antibody was produced in the challenge with hCGbeta. The hCGbeta-hC3d3 fusion protein enhanced the expression of CD80 and CD86 on B cells, especially CD86 (P<0.05), and significantly increased the expression of CD154 and CD25 molecules on T cells compared to that of hCGbeta (P<0.05). The hCGbeta-hC3d3 promoted human PBMC producing more IL-2 than hCGbeta. These findings indicate that the fusion of hC3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may contribute to a more efficient humoral immune response, and might provide a potential application of protein vaccine strategies in humans in the future.

[Gene conjugation of molecular adjuvant C3d3 to hCGbeta enhanced the anti-hCGbeta humoral immune response via upregulation of CXCR4 expression on splenic B cells in DNA vaccination].

To investigate mechanism of the anti-hCGbeta humoral immune responsive enhancement by gene conjugation of the molecular adjuvant C3d3 to hCGbeta in DNA immunization, BALB/c mice were inoculated intramuscularly with pCMV4-hCGbeta-C3d3, pCMV4-hCGbeta and pCMV4, respectively. The titers of anti-hCGbeta IgG/IgA antibody in serum were determined by indirect ELISA. The IgG/IgA ASCs levels were evaluated by ELISPOT. The expressions of chemokine receptors on B cells were analyzed by RT-PCR, and CXCR4 expression was analyzed respectively by RT-PCR and FCM. The expression of CXCL12 in spleen was investigated respectively by RT-PCR and ELISA. We found that anti-hCGbeta IgG antibody titer in serum after pCMV4-hCGbeta-C3d3 immunization was significantly higher than that of pCMV4-hCGbeta immunization. But the anti-hCGbeta IgA antibody titers appeared no difference between the two immunized groups. The level of IgG ASCs in spleen of pCMV4-hCGbeta-C3d3 immunization was significantly higher than that of pCMV4-hCGbeta immunization,but the levels of IgA ASCs appeared no difference between the two groups. The expression of CXCR4 on B cell increased after pCMV4-hCGbeta-C3d3 immunization, and significantly higher than that of pCMV4-hCGbeta immunization. The rate of CXCR4+ cell was correlated to the numbers of the ASC (r = 0.966, P < 0.05). CXCL12 expression increased after pCMV4-hCGbeta and pCMV4-hCGbeta-C3d3 immunization, and appeared no difference between the two groups. These results above indicate that gene fusion of the molecular adjuvant C3d3 to hCGbeta can significantly enhance the antigen-specific antibody response, and the level of IgG ASCs in spleen via upregulation of CXCR4 expression on splenic B cells in DNA vaccination.

Regulation of C-C motif chemokine ligand 2 and its receptor in human decidual stromal cells by pregnancy-associated hormones in early gestation.

BACKGROUND: Decidua is in close contact with the fetal trophoblasts, and involved in immune relationship of mother to fetus. However, the roles of decidua and decidual stromal cells (DSC) in materno-fetal immune regulation remain to be elucidated. In the present study, the expression and regulation of chemokines and their receptors in decidua and DSCs were investigated. METHODS AND RESULTS: The transcription of 18 chemokine receptors in human first-trimester decidual tissue and DSC were first analysed by RT-PCR. Among these receptors, C-C motif chemokine receptor-2 (CCR2) was highly transcribed. It was demonstrated by RT-PCR and immunostaining that both CCR2 and its major ligand, C-C motif chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1), were expressed in decidua and DSC. We then detected CCL2 in the supernatant of primary cultures of DSC by enzyme-linked immunosorbent assay. It was shown that DSC secreted CCL2 spontaneously and continuously over 72 h (21.72 +/- 2.34 ng/ml), and the CCR2 antagonist RS102895 and an inhibitor of the map kinase kinase/mitogen-activated protein kinase (ERK/MAPK) signal pathway decreased significantly the CCL2 secretion of DSC (both P < 0.05). We further studied effects of the pregnancy-associated hormones, estrogen, progesterone or HCG on CCL2 secretion by DSC. CCL2 secretion by DSC was up-regulated by estrogen, progesterone or HCG. CONCLUSIONS: CCR2 and CCL2 are co-expressed by human first-trimester DSC and decidual tissue. CCL2 is secreted in an autocrine manner through the ERK/MAPK pathway, and is up-regulated by the pregnancy-associated hormones, estrogen, progesterone and HCG, which suggests that CCL2 may play an important role at materno-fetal interface.

[Diagnosis and treatment of biotinidase deficiency-clinical study of six patients].

OBJECTIVE: To investigate the clinical and neurodevelopmental profiles of patients with biotinidase deficiency and to determine the efficacy of current therapy with respect to outcome. METHODS: Six patients aged from 3 months to 14 years with biotinidase deficiency were confirmed by urinary organic acid analysis with gas chromatography/mass spectrometry (GC/MS) and biotinidase assay on dried blood spots. Biotin was supplemented individually (10-40 mg/d). Their clinical features, laboratory findings, and treatment regimen were reviewed. RESULTS: All the 6 patients presented with some extent of neurological abnormalities and dermatological lesions. Cases 1 - 3 had poor feeding, vomiting, seizures, mental retardation, and lethargy onset from their early infancy, with varied degree of anemia, ketosis, acidosis, and hypoglycemia. Case 2 exhibited eczema and dermatitis from his age of 7 months. Case 4 displayed motor deficit and ataxia after 6 months of age, and generalized pustular psoriasis when he was 8 months old. Cases 5 and 6 gradually showed muscle weakness and paraplegia at the age of 7 years and 5 years, respectively. Inflammatory demyelination changes of cervical cord were evident on magnetic resonance imaging in these two patients. Case 6 had progressive optic atrophy, eczema and alopecia. Remarkable elevations of urinary lactate, pyruvate, 3-OH-propionate, methylcitrate, propionylglycine, 3-OH-isovalerate, 3-methylcrontonylglycine were confirmed in cases 1, 2, 3 and 5. Slight increase of urinary lactate, pyruvate, and 3-methylcrontonylglycine was observed in cases 4 and 6. Biotinidase activities assayed on dried blood spots from all the patients were below 0.1 pmol/(min.3 mm) Biotin supplementation for all the patients, except for case 3 who was not treated, resulted in pronounced and rapid clinical and biochemical improvement. Cases 4 and 6 had residual neurological damage comprising ataxia and motor handicap of legs, due to prolonged disease course. CONCLUSIONS: Biotinidase deficiency intensively impairs nervous system and skin in the affected patients. Urinary organic acid analysis and blood biotinidase assay are crucial to the diagnosis. Early diagnosis and biotin supplementation can contribute significantly to the improvement of prognosis.