Citrus polymethoxyflavone tangeretin (5,6,7,8,4'-pentamethoxyflavone, TAN) displays multiple biological activities, but previous reports showed that TAN failed to induce MCF-7 human breast cancer cells apoptosis. Herein, we prepared 5-acetyl-6,7,8,4'-tetramethylnortangeretin (5-ATAN), and evaluated its cytotoxicity on MCF-7 cells. 5-ATAN revealed stronger cytotoxicity than that of parent TAN in the growth inhibition of MCF-7 cells. 5-ATAN induced apoptosis via both caspase-independent and -dependent pathways, in which 5-ATAN induced the translocation of apoptosis inducing factor and phosphorylation of H2AX as well as poly (ADP-ribose) polymerase cleavage, caspase-3 activation. However, 5-ATAN did not affect extrinsic markers caspase-8, BID, and FADD. Further, 5-ATAN induced the loss of mitochondrial membrane potential (??m) by regulating the Bax/Bcl-2 ratio. Loss of ??m led to the mitochondrial release of cytochrome c which triggered activation of caspase-9. In conclusion, these data indicate that 5-ATAN plays pro-apoptotic cytotoxic roles in MCF-7 cells through both caspase-dependent intrinsic apoptosis and caspase-independent apoptosis pathways.
Human platelet factor 4 (hPF4) was evaluated as a clinical alternative to protamine for heparin neutralization, a protector against radiation injury and an anti-neoplastic. To achieve high-level expression of hPF4, expression vectors pET-28a(+)-nf PF4 (n=4, 5, 6) containing n tandem repeats of PF4 were constructed and transformed into the Escherichia coli BL21(DE3) strain. A higher expression level, about 45% of the total proteins (TP), was obtained for E. coli BL21(DE3)/pET28a(+)-nf PF4 (n=4, 5, 6). The purified His-PF4 protein was further identified by cleavage with enterokinase and MS, and its heparin-neutralizing activity was determined by colony formation assay. This study represents a novel approach to large-scale production of PF4 in E. coli, one that might be applied to large-scale production of PF4 protein for possible clinical application. It also provides theoretical points for the expression and purification of other small-molecule peptides.
CX3CR1 is an important chemokine receptor and regulates the chemotactic migration of pancreatic ductal adenocarcinoma (PDAC) cells. Up to now, its regulatory mechanism remains largely undefined. Here, we report that hypoxia upregulates the expression of CX3CR1 in pancreatic cancer cells. When hypoxia-inducible factor (HIF)-1? expression was knocked down in vitro and in vivo, the expression of CX3CR1 was significantly decreased. Chromatin immunoprecipitation assay demonstrated that HIF-1? bound to the hypoxia-response element (HRE; 5-A/GCGTG-3) of CX3CR1 promoter under normoxia, and this binding was significantly enhanced under hypoxia. Overexpression of HIF-1? significantly upregulated the expression of luciferase reporter gene under the control of the CX3CR1 promoter in pancreatic cancer cells. Importantly, we demonstrated that HIF-1? may regulate cancer cell migration through CX3CR1. The HIF-1?/CX3CR1 pathway might represent a valuable therapeutic target to prevent invasion and distant metastasis in PDAC.
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