Meihua Jin

Stellettin B Induces G1 Arrest, Apoptosis and Autophagy in Human Non-small Cell Lung Cancer A549 Cells via Blocking PI3K/Akt/mTOR Pathway

Until now, there is not yet antitumor drug with dramatically improved efficacy on non-small cell lung cancer (NSCLC). Marine organisms are rich source of novel compounds with various activities. We isolated stellettin B (Stel B) from marine sponge Jaspis stellifera, and demonstrated that it induced G1 arrest, apoptosis and autophagy at low concentrations in human NSCLC A549 cells. G1 arrest by Stel B might be attributed to the reduction of cyclin D1 and enhancement of p27 expression. The apoptosis induction might be related to the cleavage of PARP and increase of ROS generation. Moreover, we demonstrated that Stel B induced autophagy in A549 cells by use of various assays including monodansylcadaverine (MDC) staining, transmission electron microscopy (TEM), tandem mRFP-GFP-LC3 fluorescence microscopy, and western blot detection of the autophagy markers of LC3B, p62 and Atg5. Meanwhile, Stel B inhibited the expression of PI3K-p110, and the phosphorylation of PDK1, Akt, mTOR, p70S6K as well as GSK-3β, suggesting the correlation of blocking PI3K/Akt/mTOR pathway with the above antitumor activities. Together, our findings indicate the antitumor potential of Stel B for NSCLC by targeting PI3K/Akt/mTOR pathway.

ZSTK474, a specific class I phosphatidylinositol 3-kinase inhibitor, induces G1 arrest and autophagy in human breast cancer MCF-7 cells

Multifaceted activities of class I phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 were investigated on human breast cancer cell MCF-7. ZSTK474 inhibited proliferation of MCF-7 cells potently. Flow cytometric analysis indicated that ZSTK474 induced cell cycle arrest at G1 phase, but no obvious apoptosis occurred. Western blot analysis suggested that blockade of PI3K/Akt/GSK-3β/cyclin D1/p-Rb pathway might contribute to the G1 arrest induced. Moreover, we demonstrated that ZSTK474 induced autophagy in MCF-7 cells by use of various assays including monodansylcadaverine (MDC) staining, transmission electron microscopy (TEM), tandem mRFP-GFP-LC3 fluorescence microscopy, and western blot detection of the autophagy protein markers of LC3B II, p62 and Atg 5. Inhibition of class I PI3K and the downstream mTOR might be involved in the autophagy-inducing effect. Combinational use of ZSTK474 and autophagy inhibitors enhanced cell viability, suggesting ZSTK474-induced autophagy might contribute to the antitumor activity. Our report supports the application of ZSTK474, which is being evaluated in clinical trials, for breast cancer therapy.

Anti-inflammatory activity of 6-hydroxy-2,7-dimethoxy-1,4-henanthraquinone from tuberous roots of Yam (Dioscorea batatas) through inhibition of prostaglandin D2 and leukotriene C4 production in mouse bone marrow-derived mast cells

Abstract6-Hydroxy-2,7-dimethoxy-1,4-phenanthraquinone (PAQ) isolated from the tuberous roots of Yam (Dioscorea batatas) inhibited cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) dependent prostaglandin D2 (PGD2) generation in mouse bone marrow-derived mast cells in a concentration-dependent manner with IC50 values of 0.08 μM and 0.27 μM, respectively. In the Western blotting with specific anti-COX-2 antibodies, the decrease of the quantity of PGD2 was accompanied by a decrease in the COX-2 protein level. But PAQ did not affect COX-1 protein level. In addition, this compound inhibited 5-lipoxygenase (5-LOX) dependent production of leukotriene C4 in a dose-dependent manner, with an IC50 of 0.032 μM. These results demonstrate that PAQ has a dual COX-2/5-LOX inhibitory activity. This compound also inhibited the degranulation reaction in a dose-dependent manner with an IC50 of 2.7 μM. Thus, these results suggest that PAQ may be useful in regulating mast cell-mediated inflammatory diseases.

Stellettin B induces apoptosis in human chronic myeloid leukemia cells via targeting PI3K and Stat5

Novel agents are still urgently expected for therapy of chronic myeloid leukemia (CML). The in vitro anti-leukemia activity of Stellettin B (Stel B), a triterpenoid we isolated from marine sponge Jaspis stellifera, on human CML K562 and KU812 cells was recently investigated. Stel B inhibited K562 and KU812 cell proliferation with IC50 as 0.035 μM and 0.95 μM respectively. While no obvious cell cycle arrest was observed, apoptosis was induced in K562 cells after Stel B treatment. The Stel B-induced apoptosis might be in mitochondrial pathway, with increase of Bad and Bax, decrease of Bcl-2 and activation of caspase-9. In addition, dose-dependent increase of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (MMP) occurred. Meanwhile, Stel B inhibited phosphorylation of Stat5, expression of 4 PI3K catalytic isoforms, and phosphorylation of the downstream effectors including PDK1 and Akt, suggesting that inhibition against Stat5 and PI3K might be involved in the apoptosis-inducing effect. Combination of Stel B with Imatinib with ratio as IC50 Stel B: 5×IC50 Imatinib led to synergistic effect. Stel B might become a promising candidate for CML therapy alone or together with Imatinib.

Roburic Acid Suppresses NO and IL-6 Production via Targeting NF-κB and MAPK Pathway in RAW264.7 Cells

In the present study, we investigated the anti-inflammatory effect of roburic acid on production of nitric oxide (NO) and interlukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. We found that roburic acid reduced production of NO and IL-6, and the expression of inducible nitric oxide synthases (iNOS). Meanwhile, phosphorylation of inhibitor of κBα (IκBα) and IκB kinase α/β (IKKα/β), as well as translocation of nuclear factor-κB (NF-κB) to the nucleus, was suppressed by roburic acid treatment. In addition, phosphorylation of mitogen-activated protein kinase (MAPKs) including p38 and c-Jun-NH2-terminal kinase (JNK) was inhibited. Roburic acid exhibited inhibitory activities on production of NO and IL-6 via blocking IKK/IκB/NF-κB and MAPKs pathway, suggesting the potential application as a drug candidate for therapy of inflammatory diseases.

Discovery of two Bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental Fire-bellied toad, Bombina orientalis

Amphibian skin secretions are known to contain numerous peptides with a large array of biological activities. Bombinins are a group of amphibian‐derived peptides with broad spectrum antimicrobial activities that have been only identified from the ancient toad species, Bombina. In this study, we described the identification and characterization of a novel bombinin precursor which encoded a bombinin‐like peptide (BLP‐7) and a novel bombinin H‐type peptide (named as Bombinin H‐BO) from the skin secretion of Oriental fire‐bellied toad, Bombina orientalis. The primary structures of both mature peptides were determined by combinations of molecular cloning of peptide precursor‐encoding cDNAs and mass spectrometry techniques. Secondary structure prediction revealed that both peptides had cationic amphipathic α‐helical structural features. The synthetic replicate of BLP‐7 displayed more potent antimicrobial activity than Bombinin H‐BO against Gram‐positive and Gram‐negative bacteria and yeast. Also, in vitro antitumour assay showed that both peptides possessed obvious antiproliferative activity on three human hepatoma cells (Hep G2/SK‐HEP‐1/Huh7) at the non‐toxic doses. These results indicate the peptide family of bombinins could be a potential source of drug candidates for anti‐infection and anticancer therapy.

Antiproliferative effect of ZSTK474 alone or in combination with chemotherapeutic drugs on HL60 and HL60/ADR cells

While chemotherapy remains to be one of the main approaches in the clinical treatment of acute myeloid leukemia (AML), multidrug resistance (MDR) has become a serious problem which limits the therapeutic efficacy. The important roles of the PI3K/Akt pathway in modulating cell proliferation and MDR suggest that PI3K inhibitor might be effective for treatment of AML. In the present study, the antiproliferative effects of PI3K inhibitor ZSTK474 on AML cell HL60 and the adriamycin (ADR)-resistant HL60/ADR cells were investigated. Our data indicated that ZSTK474 exhibited potent antiproliferative activity, induced G1 cell cycle arrest, but no obvious apoptosis in both cell lines. Moreover, ZSTK474 affected the protein levels of cell-cycle-related molecules including increased p27, decreased cyclin D1 and phosphorylated Rb in dose-dependent manner. The proteins downstream of PI3K including phosphorylated PDK1, Akt and GSK-3β were reduced in a dose-dependent manner after ZSTK474 treatment. ZSTK474 reversed ADR resistance, increased the intracellular accumulation of ADR, and reduced the expression and function of multidrug resistance (MDR) proteins including both P-gp and MRP1 in HL60/ADR cells. The combination of ZSTK474 and chemotherapeutic drugs cytarabine or vincristine led to a synergistic effect in HL60 and HL60/ADR cells. In conclusion, ZSTK474 showed potent antiproliferative effect on HL60 and HL60/ADR cells; combination with cytarabine or vincristine resulted in synergistic effect. Our results suggest ZSTK474 has the potential to be applied in the treatment of AML patients, while further evidences particularly those about in vivo efficacy are needed.

1,6- O , O -Diacetylbritannilactone Inhibits Eotaxin-1 and ALOX15 Expression Through Inactivation of STAT6 in A549 Cells

Abstract1,6-O,O-Diacetylbritannilactone (OODBL), a plant sesquiterpene lactone, was previously reported to show multiple pharmacological effects such as anti-cancer and anti-inflammatory activities. In this study, we investigated the anti-inflammatory effect of OODBL on interleukin (IL)-4-induced signal transducer and activator of transcription 6 (STAT6) signaling pathway in human lung A549 cells. We found that OODBL dramatically inhibited IL-4-induced messenger RNA (mRNA) expression of eotaxin-1 and arachidonate 15-lipoxygenase-1 (ALOX15) in a dose-dependent manner. To clarify the action mechanism of OODBL, we examined the effect of OODBL on activation of STAT6. OODBL decreased both STAT6 phosphorylation and reporter gene activity. Furthermore, OODBL suppressed phosphorylation of Janus Kinase 3 (JAK3) without affecting JAK1. Taken together, OODBL abolished IL-4-induced eotaxin-1 and ALOX15 mRNA expressions by repressing the activation of STAT6 and JAK3. These results suggest that OODBL has a potential therapeutic efficacy on inflammatory diseases especially allergic airway disease as a lead compound.

1,6-O,O-Diacetylbritannilactone suppresses activation of mast cell and airway hyper-responsiveness

Abstract Mast cells play critical roles in allergic disorders such as atopic dermatitis and allergic asthma. The aim of this study was to investigate the anti-inflammatory and anti-asthmatic activities of 1,6-O,O-diacetylbritannilactone (OODBL) isolated from Inula japonica Thunb. (I. japonica) in a murine asthma model and bone marrow-derived mast cells (BMMCs). In an ovalbumin-induced asthma model, OODBL administration attenuated the airway hyper-responsiveness induced by aerosolized methacholine and serum IgE level in asthmatic mice. In vitro system, we found that OODBL reduced leukotriene C4 production and degranulation through the suppression of cytosolic phospholipase A2 phosphorylation and phospholipase Cγ-mediated Ca2+ influx in IgE/antigen-stimulated BMMCs. Taken together, OODBL may have therapeutic potential in the treatment of allergic diseases such as asthma.

Britanin attenuates ovalbumin-induced airway inflammation in a murine asthma model

We previously demonstrated the alleviation of ovalbumin (OVA)-induced airway inflammation by Inulae flos. In the present study, the effects of britanin, a sesquiterpene compound isolated from Inulae flos, were evaluated in an in vivo animal model for anti-asthma activity through observation of airway hyperresponsiveness (AHR), eosinophil recruitment, Th2 cytokine and IgE levels, and lung histopathology. Britanin administration effectively reduced AHR induced by aerosolized methacholine, airway eosinophilia, Th2 cytokines in bronchoalveolar lavage fluids and the supernatant of cultured splenocytes compared with OVA-induced mice. Histological studies showed that increased inflammatory cell infiltration and mucus secretion were reduced by britanin administration. Thus, britanin may have therapeutic potential for treating allergic asthma.