Preclinical efficacy and toxicity studies of a highly specific chimeric anti-CD47 antibody.

Cluster of differentiation 47 (CD47) is a widely expressed self-protection transmembrane protein that functions as a critical negative regulator to induce macrophage-mediated phagocytosis. Overexpression of CD47 enables cancer cells to escape immune surveillance and destruction by phagocytes both in solid tumours and leukaemia. The usefulness of anti-CD47 antibody has been demonstrated in multiple immunotherapies associated with macrophages. However, antigen sinks and toxicity induced by inadvertent binding to normal cells restrict its clinical applications. Here, a novel anti-human CD47 antibody, 4D10, was generated, and its variable regions were grafted onto a human IgG4 scaffold. Compared with the anti-CD47 antibody Hu5F9, the resulting chimeric antibody (c4D10) has consistently demonstrated good tolerance in in vitro and in vivo toxicity studies. Additionally, c4D10 showed effective therapeutic potential through inducing the eradication of human cancer cells. Thus, c4D10 is a promising candidate therapeutic antibody with higher efficacy and reduced side effects compared to earlier antibodies, and its use may reduce the dose-limiting toxicity of CD47 antagonists for immunotherapy.

Chaetominine induces cell cycle arrest in human leukemia K562 and colon cancer SW1116 cells.

Chaetominine is a cytotoxic alkaloid that has been demonstrated to promote apoptotic cell death in human leukemia K562 cells. In the present study, chaetominine inhibited K562 (IC50 34 nM) and SW1116 (IC50 46 nM) cell growth. However, it remains unclear whether the inhibition of cell growth is associated with the cell cycle. To assess this potential relationship, the effect of chaetominine on the cell cycle of K562 and SW1116 cells was examined. Chaetominine treatment caused cell apoptosis and G1-phase arrest in SW1116 cells. Conversely, K562 cells underwent S-phase arrest according to flow cytometric analysis. The present study also aimed to elucidate the molecular mechanisms underpinning cell cycle regulation following the incubation of the associated cells with chaetominine. Western blot and reverse transcription-quantitative polymerase chain reaction analyses suggested that chaetominine treatment facilitated the expression of p53, p21, checkpoint kinase 2 (Chk2) and phosphorylated ataxia telangiectasia mutated (p-ATM) and caused a reduction in the mRNA levels of cyclin E and cyclin-dependent kinases (CDKs) 2 and 4. These results suggest that chaetominine may be involved in the regulation of p53/p21 and ATM and Rad3-related (ATM)/Chk2 signaling in SW1116 cells. Previous studies have demonstrated that these signaling pathways are responsible for G1-phase arrest. Results of the present study demonstrated that the expression of p-ATR and Chk1 were increased in K562 cells. Additionally, cdc25A levels were decreased, while protein and gene expression levels of cyclin A and CDK2 were repressed. These results elucidated the role of chaetominine in in the regulation of ATR/cdc25A/Chk1 expression in K562 cells. These proteins are thus important determinants in the initiation of S-phase arrest. These data support the hypothesis that chaetominine is a potential anti-cancer therapeutic agent that targets the cell cycle.

Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells.

Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line.

Assessment of the Cytotoxic and Apoptotic Eἀects of Chaetominine in a Human Leukemia Cell Line.

Chaetominine is a quinazoline alkaloid originating from the endophytic fungus Aspergillus fumigatus CY018. In this study, we showed evidence that chaetominine has cytotoxic and apoptotic effects on human leukemia K562 cells and investigated the pathway involved in chaetominine-induced apoptosis in detail. Chaetominine inhibited K562 cell growth, with an IC50 value of 35 nM, but showed little inhibitory effect on the growth of human peripheral blood mononuclear cells. The high apoptosis rates, morphological apoptotic features, and DNA fragmentation caused by chaetominine indicated that the cytotoxicity was partially caused by its pro-apoptotic effect. Under chaetominine treatment, the Bax/Bcl-2 ratio was upregulated (from 0.3 to 8), which was followed by a decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, and stimulation of Apaf-1. Furthermore, activation of caspase-9 and caspase-3, which are the main executers of the apoptotic process, was observed. These results demonstrated that chaetominine induced cell apoptosis via the mitochondrial pathway. Chaetominine inhibited K562 cell growth and induced apoptotic cell death through the intrinsic pathway, which suggests that chaetominine might be a promising therapeutic for leukemia.

Dimethyl Cardamonin Exhibits Anti-inflammatory Effects via Interfering with the PI3K-PDK1-PKCα Signaling Pathway.

Consumption of herbal tea [flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae)] is associated with health beneficial effects against multiple diseases including diabetes, asthma, and inflammatory bowel disease. Emerging evidences have reported that High mobility group box 1 (HMGB1) is considered as a key "late" proinflammatory factor by its unique secretion pattern in aforementioned diseases. Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone, DMC) is a major ingredient of C. operculatus flower buds. In this study, the anti-inflammatory effects of DMC and its underlying molecular mechanisms were investigated on lipopolysaccharide (LPS)-induced macrophages. DMC notably suppressed the mRNA expressions of TNF-α, IL-1ß, IL-6, and HMGB1, and also markedly decreased their productions in a time- and dose-dependent manner. Intriguingly, DMC could notably reduce LPS-stimulated HMGB1 secretion and its nucleo-cytoplasmic translocation. Furthermore, DMC dose-dependently inhibited the activation of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1), and protein kinase C alpha (PKCα). All these data demonstrated that DMC had anti-inflammatory effects through reducing both early (TNF-α, IL-1ß, and IL-6) and late (HMGB1) cytokines expressions via interfering with the PI3K-PDK1-PKCα signaling pathway.