lethality-bioassay-using-artemia-salina-l

Lethality Bioassay Using <em>Artemia salina</em> L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

Universidade Lusófona de Humanidades e Tecnologias

Faculdade de Farmácia da Universidade de Lisboa

Medicinal plants are a good source of novel therapeutic drugs, due to the phytochemicals present. Artemia, commonly known as brine shrimp, is a tiny halophilic invertebrate belonging to class Crustacean, which plays an important role in saline aquatic and marine eco-systems. Besides its usage in aquaculture, it is also highly valued for its application in toxicity detection and it is used in areas such as Ecology, Physiology, Ecotoxicology, Aquaculture and Genetics. Furthermore, Artemia based lethality assay (brine shrimp lethality assay, BSLA) is rapid, convenient and low cost. Presently, brine shrimp lethality assays are enormously employed in research and applied toxicology. It has been used in the study of natural products as a preliminary toxicity assay to screen a large number of extracts and compounds for drug discovery in medicinal plants. The aim of this review paper is to collect, organize, select and discuss the existing knowledge about the different uses of Artemia salina as a bench-top bioassay for the discovery and purification of bioactive natural products.

Artemia species: An Important Tool to Screen General Toxicity Samples.

The development of multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy. Several abietane diterpenes with antitumoral activities have been isolated from  spp. such as 6,7-dehydroroyleanone (DHR, ) and 7α-acetoxy-6β-hydroxyroyleanone (AHR, ). Several royleanone derivatives were prepared through hemisynthesis from natural compounds  and  to achieve a small library of products with enhanced anti-P-glycoprotein activity. Nonetheless, some derivatives tend to be unstable. Therefore, to reason such lack of stability, the electron density based local reactivity descriptors condensed Fukui functions and dual descriptor were calculated for several derivatives of DHR. Additionally, molecular docking and molecular dynamics studies were performed on several other derivatives to clarify the molecular mechanisms by which they may exert their inhibitory effect in P-gp activity. The analysis on local reactivity descriptors was important to understand possible degradation pathways and to guide further synthetic approaches toward new royleanone derivatives. A molecular docking study suggested that the presence of aromatic moieties increases the binding affinity of royleanone derivatives toward P-gp. It further suggests that one royleanone benzoylated derivative may act as a noncompetitive efflux modulator when bound to the M-site. The future generation of novel royleanone derivatives will involve (i) a selective modification of position C-12 with chemical moieties smaller than unsubstituted benzoyl rings and (ii) the modification of the substitution pattern of the benzoyloxy moiety at position C-6.

Molecular Docking Studies of Royleanone Diterpenoids from  spp. as P-Glycoprotein Inhibitors.

Plants have been used for centuries to treat several illnesses. The  genus has a vast variety of species that has allowed the isolation of cytotoxic compounds with notable activities. The abietane diterpenes 6,7-dehydroroyleanone (DeRoy, ), 7α-acetoxy-6β-hydroxyroyleanone (Roy, ), and Parvifloron D (ParvD, ) were obtained from  spp. and showed promising biological activities, such as cytotoxicity. The inhibitory effects of the different natural abietanes (-) were compared in MFC7, SkBr3, and SUM159 cell lines, as well as SUM159 grown in cancer stem cell-inducing conditions. Based on the royleanones' bioactivity, the derivatives RoyBz (), RoyBzCl (), RoyPr (), and DihydroxyRoy (), previously obtained from , were selected for further studies. Protein kinases C (PKCs) are involved in several carcinogenic processes. Thus, PKCs are potential targets for cancer therapy. To date, the portfolio of available PKC modulators remains very limited due to the difficulty of designing isozyme-selective PKC modulators. As such, molecular docking was used to evaluate royleanones - as predicted isozyme-selective PKC binders. Subtle changes in the binding site of each PKC isoform change the predicted interaction profiles of the ligands. Subtle changes in royleanone substitution patterns, such as a double substitution only with non-substituted phenyls, or hydroxybenzoate at position four that flips the binding mode of ParvD (), can increase the predicted interactions in certain PKC subtypes.

Activity to Breast Cancer Cell Lines of Different Malignancy and Predicted Interaction with Protein Kinase C Isoforms of Royleanones.

 spp. is widely known for its medicinal properties and bioactive metabolites.The cytotoxic and genotoxic properties of the four known abietane diterpenoids: 7α-Acetoxy-6β-hydroxyroyleanone (Roy), 6,7-dehydroroyleanone (Deroy), 7β,6β-dihydroxyroyleanone6 (Diroy),and Parvifloron D (Parv), isolated from  (Roy, DeRoy, and Diroy) and (Parv) were evaluated. The tested compounds showed cytotoxic effects against the human leukemiacell line CCRF-CEM and the lung adenocarcinoma cell line A549. All tested compounds inducedapoptosis by altering the level of pro- and anti-apoptotic genes. The results show that from the testedditerpenoids, Roy and Parv demonstrated the strongest activity in both human cancer cell lines,changing the permeability mitochondrial membrane potential and reactive oxygen species (ROS)levels, and possibly inducing mtDNA or nDNA damage. In conclusion, the abietane diterpenoidstested may be used in the future as potential natural chemotherapeutic agents.

Diterpenoids from  spp. as Potential Chemotherapeutic Agents via Apoptosis.

Cancer is among the leading causes of death worldwide. One of the most challenging obstacles in cancer treatment is multidrug resistance (MDR). Overexpression of P-glycoprotein (P-gp) is associated with MDR. The growing incidence of cancer and the development of MDR drive the search for novel and more effective anticancer drugs to overcome the MDR problem. Royleanones are natural bioactive compounds frequently found in  spp. The cytotoxic diterpene 6,7-dehydroroyleanone () is the main component of the  (Pers.) Benth. essential oil, while 7α-acetoxy-6β-hydroxyroyleanone () can be isolated from acetonic extracts of  Gürke. The reactivity of the natural royleanones  and  was explored to obtain a small library of new P-gp inhibitors. Four new derivatives (6,7-dehydro-12---butyl-carbonate-royleanone (), 6,7-dehydro-12--methylroyleanone (), 6,7-dehydro-12--benzoylroyleanone (), and 7α-acetoxy-6β-hydroxy-12--benzoylroyleanone () were obtained as pure with overall modest to excellent yields (21-97%). P-gp inhibition potential of the derivatives - was evaluated in human non-small cell lung carcinoma NCI-H460 and its MDR counterpart NCI-H460/R with the P-gp overexpression, through MTT assay. Previously prepared diterpene 7α-acetoxy-6β-benzoyloxy-12--(4-chloro)benzoylroyleanone (), has also been tested. The P-gp inhibiting effects of compounds - were also assessed through a Rhodamine 123 accumulation assay. Derivatives  and  have significant P-gp inhibitory potential. Regarding stability and P-gp inhibition potential, results suggest that the formation of benzoyl esters is a more convenient approach for future derivatives with enhanced effect on the cell viability decrease. Compound  presented higher anti-P-gp potential than the natural diterpenes ,  and , with comparable inhibitory potential to Dexverapamil. Moreover, derivative  showed the ability to sensitize the resistant NCI-H460/R cells to doxorubicin.

Royleanone Derivatives From  spp. as a Novel Class of P-Glycoprotein Inhibitors.

Activation of CXCR2 by chemokines such as CXCL1 and CXCL2 increases aggressiveness of breast cancer, inducing chemoresistance, hence CXCR2 antagonists are in clinical trials. We previously reported that inhibition of CXCR2 increases MIP-2 (CXCL2), which may inhibit anti-tumoral effects of CXCR2 antagonists. This seems to be due to inhibition of protein kinase C (PKC) by CXCR2 antagonist since specific inhibitor of PKC also enhances MIP-2 secretion. We here examined whether CXCR2 inhibitor also increases KC (CXCL1) secretion, ligand for CXCR2 involved in metastasis and PKC activators can prevent increases in chemokine secretion. We used SB 225002, which is a specific CXCR2 antagonist. The effects of PKC activators that have documented anti-tumoral effects and activates multiple isozymes of PKC such as Ingenol-3-angelate (I3A) and bryostatin-1 were examined here. In addition, FR236924, PKCε selective and 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), PKCδ selective activators were also tested. The effects of activators were determined using brain metastatic (4TBM) and heart metastatic (4THM) subset of 4T1 breast carcinoma cells because these aggressive carcinoma cells with cancer stem cell features secrete high levels of KC and MIP-2. Inhibition of CXCR-2 activity increased KC (CXCL1) secretion. PKC activators prevented SB225002-induced increases in KC and MIP-2 secretion. Different activators/modulators induce differential changes in basal and SB225002-induced chemokine secretion as well as cell proliferation and the activators that act on PKCδ and/or PKCε such as bryostatin 1, FR236924 and Roy-Bz are the most effective. These activators alone also decrease cell proliferation or chemokine secretion or both. Given the role of KC and MIP-2 in drug resistance including chemotherapeutics, activators of PKCε and PKCδ may prevent emerging of resistance to CXCR2 inhibitors as well as other chemotherapeutics.

Rebound increases in chemokines by CXCR2 antagonist in breast cancer can be prevented by PKCδ and PKCε activators.

According to the present knowledge, this is the first report on establishing transformed root cultures of  after -mediated transformation. The preliminary phytochemical analysis showed differences in the content of phenols and flavonoids in transformed and nontransformed roots. The dominant compounds in the analyzed extracts were (+)-catechin (5464 and 6808 µg/g DW), -coumaric acid (2549 and 4907 µg/g DW), -coumaric acid (1508 and 2048 µg/g DW) and rosmarinic acid (1844 and 2643 µg/g DW) for nontransformed (LNNR) and transformed (LNTR4) roots, respectively. Initial biological studies carried out on LNNR, and LNTR4 extracts showed a cytotoxic effect on the A549 lung, HCC1937 breast and leukemia NALM-6 cell lines, antioxidants, as well as repair and protection against DNA damage induced by HO in HUVEC cells. Due to the stronger effect of the LNTR4 root extract, which can be a relatively efficient and cheap source of bioactive secondary metabolites, further biological analyses are needed to discover in detail their potentially valuable biological properties.

Preliminary Phytochemical Analysis and Evaluation of the Biological Activity of  (L.) R. Br Transformed Roots Extracts Obtained through -Mediated Transformation.

Vera M. S. Isca

CBIOS - Lusófona University's Center for Research in Biosciences & Health Technologies

Universidade Lusófona de Humanidades e Tecnologias

Lethality Bioassay Using Artemia salina L.