Antitumor Mechanisms of Curcumae Rhizoma Based on Network Pharmacology.

Curcumae Rhizoma, a traditional Chinese medication, is commonly used in both traditional treatment and modern clinical care. Its anticancer effects have attracted a great deal of attention, but the mechanisms of action remain obscure. In this study, we screened for the active compounds of Curcumae Rhizoma using a drug-likeness approach. Candidate protein targets with functions related to cancer were predicted by reverse docking and then checked by manual search of the PubMed database. Potential target genes were uploaded to the GeneMANIA server and DAVID 6.8 database for analysis. Finally, compound-target, target-pathway, and compound-target-pathway networks were constructed using Cytoscape 3.3. The results revealed that the anticancer activity of Curcumae Rhizoma potentially involves 13 active compounds, 33 potential targets, and 31 signaling pathways, thus constituting a "multiple compounds, multiple targets, and multiple pathways" network corresponding to the concept of systematic actions in TCM. These findings provide an overview of the anticancer action of Curcumae Rhizoma from a network perspective, as well as setting an example for future studies of other materials used in TCM.

Identification of neprilysin as a potential target of arteannuin using computational drug repositioning

ABSTRACT The discovery of arteannuin (qinghaosu) in the 20th Century was a major advance for medicine. Besides functioning as a malaria therapy, arteannuin is a pharmacological agent in a range of other diseases, but its mechanism of action remains obscure. In this study, the reverse docking server PharmMapper was used to identify potential targets of arteannuin. The results were checked using the chemical-protein interactome servers DRAR-CPI and DDI-CPI, and verified by AutoDock Vina. The results showed that neprilysin (also known as CD10), a common acute lymphoblastic leukaemia antigen, was the top disease-related target of arteannuin. The chemical-protein interactome and docking results agreed with those of PharmMapper, further implicating neprilysin as a potential target. Although experimental verification is required, this study provides guidance for future pharmacological investigations into novel clinical applications for arteannuin.

Activity of fusion prophenoloxidase-GFP and its potential applications for innate immunity study.

Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+) was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.

Existence of prophenoloxidase in wing discs: a source of plasma prophenoloxidase in the silkworm, Bombyx mori.

In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

Specific amino acids affecting Drosophila melanogaster prophenoloxidase activity in vitro.

Insect prophenoloxidase (PPO) is a key enzyme that induces melanization around invading pathogens and at wounds to prevent further infection. Drosophila melanogaster has three PPO genes which have different biochemical properties following over-expression in S2 cells. As shown by automatic melanization of S2 cells, recombinant PPO3 (rPPO3) became activated upon Cu(2+) addition (Cu(2+)-aided cells melanization without ethanol activation and substrate addition: +Cu(2+); -DOPA, -Ethanol). The exact reasons for this phenomenon are still unknown. In this study, using site-directed mutagenesis and over-expression methods, we found that the place holder, two independent amino acids (equal to Manduca sexta amino acid residues: F218 and S393 in MsPPO1, F224 and E395 in MsPPO2) in the active site pocket and a missing fragment (similar to (565)RPGDPGT(571) in MsPPO1 and (571)QGSDPRR(577) in MsPPO2) at the C-terminus of PPO3, affect rPPO3-S2 cells Cu(2+)-aided auto-melanization. Some mutations nearly rescued rPPO3 Cu(2+)-aided auto-activation, which suggests that the auto-activation of wild type rPPO3 was not due to cleavage by serine proteases. We also found that the corresponding amino acids in the active site pocket have similar effect on PPO1 as on PPO3. PPO1 staining activity (Cu(2+) added or not during PPO transfection; cells melanized after ethanol activation and substrate addition: ±Cu(2+); +DOPA, +Ethanol) has a positive relationship with the active site pocket size as does rPPO3. The fragment of rPPO1 corresponding to the one missing from the C-terminus of PPO3 has no influence on rPPO1 staining activity after it is deleted. However, the staining activities of rPPO2 mutants decreased after deletion of those corresponding amino acid sequences. When the corresponding fragments from PPO1 or PPO2 were inserted into PPO3, the mutant rPPO3 had no influence on staining activity, but had a significantly lowered Cu(2+)-aided auto-activation. Thus, we found that some amino acids are important for rPPO3 Cu(2+)-aided auto-activation as well as PPO staining activity in vitro.

Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli.

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu(2+), either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu(2+) associated proenzymes. Cu(2+) associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu(2+) showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu(2+) than rPPO2 did. Surprisingly, however, high concentration of Cu(2+) (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2