| The mammalian tachykinins is a family of evolutionary conserved peptides that share a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 including substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and the novel members including hemokinin-1 discovered in 2000. Most of their bioactivities are mediated through three receptors of G protein-coupled receptor family named as neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3). The most well known member of tachykinins is SP. The hemokinin-1 family including r/mHK-1, hHK-1 and hHK-1(4-11), we call them Hemokinins (HKs). HKs show preferential binding affinity for NK1 receptor just like SP. However, In contrast to SP which is largely expressed in neuronal tissues, HKs show much broader distribution in peripheral tissues. SP has been shown to induce angiogenesis through NK1 receptor. However, as naturally occurring peripheral SP-like agonists to NK1, the effects of HKs on endothelial cells and angiogenesis have not been studied. In the present study, for the first time, we demonstrated that r/mHK-1, hHK-1 and hHK(4-11) could stimulate angiogenesis.HUVECs are commonly used endothelial cell model for the investigation of angiogenesis. The mRNAs for NK1 and NK2 receptors have been detected in HUVECs. In order to mimic physiological conditions, HUVECs used in our experiments were freshly isolated from healthy mother. We demonstrated that HKs dose-dependently stimulate HUVECs proliferation, migration, adhesion and tube formation. In addition, HKs at 10 nM increased the G2/M and S cell fraction and reduced the G0/G1 cell fraction, suggesting that HKs drive DNA synthesis and mitosis in HUVECs. Based on these we further used CAM model to confirm the in vivo pro-angiogenic effects of HKs. We found that HKs increased capillary bed area and the number of order 3 blood vessels but had little effects on the numbers of order 1 and order 2 blood vessels, indicating that the pro-angiogenic effects of HKs may be due to the stimulation of the outgrowth of smaller branched vessels rather than the continued growth of the present large blood vessels.The pro-angiogenic effects of HKs in vivo and in vitro were notably inhibited by the non-peptide NK1 receptor antagonist L-732,138 but not by NK2 receptor antagonist MEN 10376, which suggested that at least in the vascular endothelium system HKs primary act through NK1 receptor. It is interesting to observe the bell-shaped dose-response curves for HKs in all of the experiments, with a maximum effect at 10 nM or 100 nM and lower response at 1 ?M and 0.01 nM. We further showed that HKs promoted ERK1/2 phosphorylation, eNOS activation and VEGF expression via NKl receptor in HUVECs, which might contribute to the angiogenesis induced by HKs.It is well known that one of the most important features of the chiral drugs is the well-timed chiral switch and only one enantiomer has a desired beneficial effect while others may cause undesired or serious side effects. Currently cancer remains a serious threat to human health. The main approaches for cancer therapy include surgery, radiotherapy, and chemotherapy. Cancer is known to exhibit six biological capabilities acquired during cancer development, including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Recently, agents that target these specific hallmarks of cancer have been shown to improve response rates of cancer to standard chemotherapeutic regimens.Thiazole heterocycle is ubiquitous amongst numerous natural molecules, in particular in the compounds isolated from the marine environment. The thiazole derivatives have shown promising antibacterial, anti-viral, immunosuppressive and anti-tumor activities. To develop more effective chiral anticancer analogues, in this study we synthesized a series of new chiral 2-(ethylthio)-thiazolone analogues. Based on the development of Computer Aided Drug Design (CADD) technology, we first investigated the structure-activity relationships of chiral 2-(ethylthio)-thiazolone analogues and their bioactivities through the IC50 values and computer analysis. We demonstrated that the nitro functionality is an important candidate for the bioactivity of chiral 2-(ethylthio)-thiazolone analogues, and the modification of substituents R could further improve their activities in the presence of the nitro group. In the following experiments, we provide several lines of evidence to reveal the mechanisms of the anti-cancer effects of compound Is, the most effective compound we synthesized. Compound Is exhibited strong cytotoxicity on five different cancer cell lines in a time and dose dependent manner. Since compound Is is a potent inducer of apoptosis in HeLa cell, we characterized the underlying mechanism. The results showed that apoptosis induced by Is is associated with (1) morphological change, (2) loss of the mitochondrial potential (??m), (3) activation of caspase-3/9, and (4) upregulation of intrinsic pro-apoptotic factor Bax and downregulation of intrinsic anti-apoptotic factor Bcl-2. Furthermore, we found that Is did not markedly affect the expression of the death receptors FAS and caspase-8 (date not shown). These results suggest that Is activates mitochondria apoptotic pathway but not extrinsic apoptotic pathway to induce cancer cell apoptois. Notably, in the present study, compound Is inhibited pro-angiogenic functions of HUVECs in a dose depend manner by inhibiting the growth, migration, adhesion and tube formation of human endothelial cells. The maximal dose we used was 5 ?M, which appeared to be a tolerant dose for HUVECs. These effects may be direct rather than cytotoxity. To confirm the inhibitory effects of Is on cancer cell growth we performed in vivo mice xenograft experiments and the results demonstrated that Is inhibited the growth of xenografted s-180 tumor. Therefore, both in vitro and in vivo experiments suggest the strong anti-tumor effects of Is.Progargyl alcohol is an important structure in the drugs and nature products. It is widely known for its cytotoxicity, antibacterial, antitumor, anti-HIV and inhibitor of enzymes. Based on the methodology discovered by our group, we synthetized a series of y-hydroxy propiolic acid ester analogues. We first investigated the structure-activity relationships of 7-hydroxy propiolic acid ester analogues and their bioactivities through the IC50 values and computer analysis. We demonstrated that the aryl group is an important candidate for the bioactivity of y-hydroxy propiolic acid ester analogues, and the substitute of halogen at 2'and 5'position could further improve their activities in the presence of the aryl group. In that case, we synthetized more optimized compounds and two of them exhibited great anti-cancer ability. Cell cycle analysis shows that LS-11 and LS-45 caused an increase in the percentage of HeLa cells in the S phase in 24h because of diminished DNA synthesis. However, they induce the apoptosis in 46h. It is generally agreed that S phase arrest was a common hallmark of the death induced by DNA-Top I inhibitors. LS-11 and LS-45 presents significant inhibitory effects on Top I and influences on Topi protein expression at the cellular level. We next evaluated the possibility that the pro-apoptotic activity of LS-11 and LS-45 in HeLa cells was attributable to a higher level of induced DNA damage and/or altered DNA repair. Next, we characterized the underlying mechanism. The results showed that apoptosis induced by LS-11 and LS-45 is associated with morphological change, loss of the mitochondrial potential (??m), activation of caspase-3/9, and upregulation of intrinsic pro-apoptotic factor Bax and downregulation of intrinsic anti-apoptotic factor Bcl-2. These results suggest that LS-11 and LS-45 activates mitochondria apoptotic pathway to induce cancer cell apoptois. To confirm the inhibitory effects of them on cancer cell growth we performed in vivo mice xenograft experiments and the results demonstrated that LS-11 and LS-45 inhibited the growth of xenografted s-180 tumor. Therefore, both in vitro and in vivo experiments suggest the strong anti-tumor effects of LS-11 and LS-45. |
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