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Objective: Dracorhodin perchlorate (DP) was a synthetic analogue of the antimicrobial anthocyanin red pigment dracorhodin. It was reported that DP could induce apoptosis in human prostate cancer, human gastric tumor cells and human melanoma, but the cytotoxic effect of DP on human breast cancer was not investigated. This study would investigate whether DP was a candidate chemical of anti-human breast cancer.Methods: The MTT assay reflected the number of viable cells through measuring the activity of cellular enzymes. Phase contrast microscopy visualized cell morphology. Fluorescence microscopy detected nuclear fragmentation after Hoechst 33258 staining. Flowcytometric analysis of Annexin V-PI staining and Rodamine 123 staining was used to detect cell apoptosis and mitochondrial membrane potential (MMP). Real time PCR detected mRNA level. Western blot examined protein expression.Results: DP dose and time-dependently inhibited the growth of MCF-7 cells. DP inhibited MCF-7 cell growth through apoptosis. DP regulated the expression of Bcl-2 and Bax, which were mitochondrial pathway proteins, to decrease MMP, and DP promoted the transcription of Bax and inhibited Bcl-2. Apoptosis-inducing factor (AIF) and cytochrome c which localized in mitochondrial in physiological condition were released into cytoplasm when MMP was decreased. DP activated caspase-9, which was the downstream of mitochondrial pathway. Therefore DP decreased MMP to release AIF and cytochrome c into cytoplasm, further activating caspase 9, lastly led to apoptosis.Conclusion: Therefore DP was a candidate for anti-breast cancer, DP induced apoptosis of MCF-7 through mitochondrial pathway.

Recent studies indicate that mitochondrial pathways of apoptosis are potential chemotherapeutic target for the treatment of esophageal cancer. Azoxystrobin (AZOX), a methoxyacrylate derived from the naturally occurring strobilurins, is a known fungicide acting as a ubiquinol oxidation (Qo) inhibitor of mitochondrial respiratory complex III. In this study, the effects of AZOX on human esophageal squamous cell carcinoma KYSE-150 cells were examined and the underlying mechanisms were investigated. AZOX exhibited inhibitory effects on the proliferation of KYSE-150 cells with inhibitory concentration 50% (IC50) of 2.42 μg/ml by 48 h treatment. Flow cytometry assessment revealed that the inhibitory effect of AZOX on KYSE-150 cell proliferation occurred with cell cycle arrest at S phase and increased cell apoptosis in time-dependent and dose-dependent manners. Cleaved poly ADP ribose polymerase (PARP), caspase-3 and caspase-9 were increased significantly by AZOX. It is worth noted that the Bcl-2/Bax ratios were decreased because of the down-regulated Bcl-2 and up-regulated Bax expression level. Meanwhile, the cytochrome c release was increased by AZOX in KYSE-150 cells. AZOX-induced cytochrome c expression and caspase-3 activation was significantly blocked by Bax Channel Blocker. Intragastric administration of AZOX effectively decreased the tumor size generated by subcutaneous inoculation of KYSE-150 cells in nude mice. Consistently, decreased Bcl-2 expression, increased cytochrome c and PARP level, and activated caspase-3 and caspase-9 were observed in the tumor samples. These results indicate that AZOX can effectively induce esophageal cancer cell apoptosis through the mitochondrial pathways of apoptosis, suggesting AZOX or its derivatives may be developed as potential chemotherapeutic agents for the treatment of esophageal cancer.

To address the role of the transforming growth factor beta (TGFβ)-Smad3 signaling pathway in dendrite growth and associated synaptogenesis, we used small inhibitory RNA to knockdown the Smad3 gene in either cultured neurons and or primary astrocytes. We found that TGFβ1 treatment of primary neurons increased dendrite extensions and the number of synapsin-1-positive synapses. When Smad3 was knockdown in primary neurons, dendrite growth was inhibited and the number of synapsin-1-positive synapses reduced even with TGFβ1 treatment. When astrocyte-conditioned medium (ACM), collected from TGFβ1-treated astrocytes (TGFβ1-stimulated ACM), was added to cultured neurons, dendritic growth was inhibited and the number of synapsin-1-positive puncta reduced. When TGFβ1-stimulated ACM was collected from astrocytes with Smad3 knocked down, this conditioned media promoted the growth of dendrites and the number of synapsin-1-positive puncta in cultured neurons. We further found that TGFβ1 signaling through Smad3 increased the expression of chondroitin sulfate proteoglycans, neurocan, and phosphacan in ACM. Application of chondroitinase ABC to the TGFβ1-stimulated ACM reversed its inhibitory effects on the dendrite growth and the number of synapsin-1-positive puncta. On the other hand, we found that TGFβ1 treatment caused a facilitation of Smad3 phosphorylation and translocation to the nucleus induced by status epilepticus (SE) in wild-type (Smad3(+/+)) mice, and this treatment also caused a promotion of γ-aminobutyric acid-ergic synaptogenesis impaired by SE in Smad3(+/+) as well as in Smad3(-/-) mice, but more dramatic promotion in Smad3(+/+) mice. Thus, we provide evidence for the first time that TGFβ-Smad3 signaling pathways within neuron and astrocyte differentially regulate dendrite growth and synaptogenesis, and this pathway may be involved in the pathogenesis of some central nervous system diseases, such as epilepsy.

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A herbal decoction (Danggui Buxue Tong, DBT) is a traditional Chinese medicine formula comprising Danggui (Radix Angelicae) and Huang Qi (Radix Astragali), which has been used for promotion of “blood production” for centuries and have shown favorable effects on thrombocytopenia. Previously, we have showed that it has significant effects on promoting hematopoiesis and thrombopoiesis (Yang M et al. J Ethnopharmacol, 2009). Many chemical components have been identified in DBT. Among them is the polysaccharides fraction of Angelica sinensis and Radix Astragali. Our study has demonstrated that Angelica Polysaccharide (APS) promotes hematopoiesis and platelet recovery in a mouse model (Liu et al, BMC Complement Altern Med, 2010). In this present study, we further assess the effect of Astragalus Polysaccharide (ASPS) on hematopoiesis and thrombopoiesis in-vivo and in vitro. A myelosuppression mouse model (4-Gy-irradiated) was treated with ASPS (10 mg/kg/day) and TPO (1 mg/kg/day). Peripheral blood cells from ASPS, TPO and vehicle-treated samples were counted on days 0, 7, 14 and 21. Using the colony-forming unit (CFU) assays, we determine the effects of ASPS on the hematopoietic stem/progenitor cells and megakaryocytic lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were also conducted in HL-60 cells and megakaryocytic cell line M-07e. The effects of ASPS on cells treated with Ly294002, a PI3K inhibitor and the effect of ASPS on the phosphorylation of AKT were further studied. Results showed that ASPS, like TPO, significantly enhanced the recovery of WBC and platelet count, and bone marrow CFU-GM and CFU-MK formation (n=8) in this model. Morphological examination of bone marrows also showed that ASPS treatment significantly increased the recovery of hematopoietic stem/progenitor cells and megakaryocytic series. We further analyzed the in vitro effect of ASPS on CFU-MK and CFU-GM formation. ASPS (50-100 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK (p=0.05, n=4), and CFU-GM formation (p=0.04, n=4). However, ASPS alone (1-500 ug/ml) did not show significant effect on CFU-MK and CFU-GM proliferation (n=4). Moreover, the anti-apoptotic effect of ASPS in HL-60 and M-07e cells were also demonstrated by using Annexin-V, Caspase-3, and JC-1 assays. Addition of Ly294002 alone increased the percentage of cells undergoing apoptosis. However, additional of ASPS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of ASPS significantly increased the phosphorylation of AKT. Here, we showed that ASPS, the polysaccharide from the root of Radix Astragali, has hematopoietic and thrombopoietic activities in a mouse model. This effect is likely to result from the stimulation of cell proliferation through the activation of PI3K/AKT pathway, the activation of which prevents cells from undergoing apoptosis. Disclosures: No relevant conflicts of interest to declare.

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