Reversal of tamoxifen resistance by artemisinin in ER+breast cancer:bioinformatics analysis and experimental validation

Breast cancer is the leading cause of cancer-related deaths in women worldwide,with Hormone Receptor(HR)+being the predominant subtype.Tamoxifen(TAM)serves as the primary treatment for HR+breast cancer.However,drug resistance often leads to recurrence,underscoring the need to develop new therapies to enhance patient quality of life and reduce recurrence rates.Artemisinin(ART)has demonstrated efficacy in inhibiting the growth of drug-resistant cells,positioning art as a viable option for counteracting endocrine resistance.This study explored the interaction between artemisinin and tamoxifen through a combined approach of bioinformatics analysis and experimental validation.Five characterized genes(ar,cdkn1a,erbb2,esr1,hsp90aa1)and seven drug-disease crossover genes(cyp2e1,rorc,mapk10,glp1r,egfr,pgr,mgll)were identified using WGCNA crossover analysis.Subsequent functional enrichment analyses were conducted.Our findings confirm a significant correlation between key cluster gene expression and immune cell infiltration in tamoxifen-resistant and-sensitized patients.scRNA-seq analysis revealed high expression of key cluster genes in epithelial cells,suggesting artemisinin’s specific impact on tumor cells in estrogen receptor(ER)-positive BC tissues.Molecular target docking and in vitro experiments with artemisinin on LCC9 cells demonstrated a reversal effect in reducing migratory and drug resistance of drug-resistant cells by modulating relevant drug resistance genes.These results indicate that artemisinin could potentially reverse tamoxifen resistance in ER-positive breast cancer.

Designing Artemisinins with Antimalarial Potential, Combining Molecular Electrostatic Potential, Ligand-Heme Interaction and Multivariate Models

Artemisinins tested against W-2 strains of malaria falciparum are investigated with molecular electrostatic potential (MEP), in an attempt to identify key features of the compounds that are necessary for their activities, as well as to investigate likely interactions with the receptor in a biological process and to use that information to propose new molecules. In order to discover the best geometry involving the ligand-receptor complexes (heme) studied and help in the proposition of the new derivatives, molecular simulations of interactions between the most negative charged region around the peroxide and heme locates (the ones around the Fe2+ ion) were carried out. In addition, PCA (principal components analysis), HCA (hierarchical cluster analysis), SDA (stepwise discriminant analysis), and KNN (K-nearest neighbor) multivariate models were employed to investigate which descriptors are responsible for the classification between the higher and lower antimalarial activity of the compounds, and also this information was used to propose new potentially active molecules. The information accumulated in studies of MEP, molecular docking, and multivariate analysis supported the proposal of new structures with potential antimalarial activities. The multivariate models constructed were applied to the new structures and indicated numbers 19 and 20 as the most prominent for syntheses and biological assays.

Antitumor Research on Artemisinin and Its Bioactive Derivatives

Cancer is the leading cause of human death which seriously threatens human life.The antimalarial drug artemisinin and its derivatives have been discovered with considerable anticancer properties.Simultaneously,a variety of target-selective artemisinin-related compounds with high efficiency have been discovered.Many researches indicated that artemisinin-related compounds have cytotoxic effects against a variety of cancer cells through pleiotropic effects,including inhibiting the proliferation of tumor cells,promoting apoptosis,inducing cell cycle arrest,disrupting cancer invasion and metastasis,preventing angiogenesis,mediating the tumor-related signaling pathways,and regulating tumor microenvironment.More importantly,artemisinins demonstrated minor side effects to normal cells and manifested the ability to overcome multidrug-resistance which is widely observed in cancer patients.Therefore,we concentrated on the new advances and development of artemisinin and its derivatives as potential antitumor agents in recent 5 years.It is our hope that this review could be helpful for further exploration of novel artemisinin-related antitumor agents.

Protecting future antimalarials from the trap of resistance:Lessons from artemisinin-based combination therapy (ACT) failures

Having faced increased clinical treatment failures with dihydroartemisinin-piperaquine(DHA-PPQ),Cambodia swapped the first line artemisinin-based combination therapy(ACT)from DHA-PPQ to artesunate-mefloquine given that parasites resistant to piperaquine are susceptible to mefloquine.However,triple mutants have now emerged,suggesting that drug rotations may not be adequate to keep resistance at bay.There is,therefore,an urgent need for alternative treatment strategies to tackle resistance and prevent its spread.A proper understanding of all contributors to artemisinin resistance may help us identify novel strategies to keep artemisinins effective until new drugs become available for their replacement.This review highlights the role of the key players in artemisinin resistance,the current strategies to deal with it and suggests ways of protecting future antimalarial drugs from bowing to resistance as their predecessors did.

Artemisinin, the Magic Drug Discovered from Traditional Chinese Medicine

Artemisinin and its derivatives represent the most important and influential class of drugs in the fight against malaria. Since the discovery of artemisinin in the early 1970s, the global community has made great strides in characterizing and understanding this remarkable phytochemical and its unique chemical and pharmacological properties. Today, even as artemisinin continues to serve as the foundation for antimalarial therapy, numerous challenges have surfaced in the continued application and development of this family of drugs. These challenges include the emergence of delayed treatment responses to artemisinins in malaria and efforts to apply artemisinins for non-malarial indications. Here, we provide an overview of the story of artemisinin in terms of its past, present, and future. In particular, we comment on the current understanding of the mechanism of action (MOA) of artemisinins, and emphasize the importance of relating mechanistic studies to therapeutic outcomes, both in malarial and non-malarial contexts.

Potential applications of artemisinins in ocular diseases

Artemisinin, also named qinghaosu, is a family of sesquiterpene trioxane lactone originally derived from the sweet wormwood plant(Artemisia annua), which is a traditional Chinese herb that has been universally used as anti-malarial agents for many years. Evidence has accumulated during the past few years which demonstrated the protective effects of artemisinin and its derivatives(artemisinins) in several other diseases beyond malaria, including cancers, autoimmune disorders, inflammatory diseases, viral and other parasiterelated infections. Recently, this long-considered antimalarial agent has been proved to possess anti-oxidant, anti-inflammatory, anti-apoptotic and anti-excitotoxic properties, which make it a potential treatment option for the ocular environment. In this review, we first described the overview of artemisinins, highlighting the activity of artemisinins to other diseases beyond malaria and the mechanisms of these actions. We then emphasized the main points of published results of using artemisinins in targeting ocular disorders, including uveitis, retinoblastoma, retinal neurodegenerative diseases and ocular neovascularization. To conclude, we believe that artemisinins could also be used as a promising therapeutic drug for ocular diseases, especially retinal vascular diseases in the near future.

Artemisinin resistance or tolerance in human malaria patients

Malaria is a major cause of morbidity and mortality in the developing world.This situation is mainly due to emergence of resistance to most antimalarial drugs currently available. Artemisinin-based combination treatments are now first-line drugs for Plasmodium falciparum (P.falciparum) malaria.Artemisinin(qinghaosu) and its derivatives are the most rapid acting and efficacious antimalarial drugs.This review highlights most recent investigations into the emergence of artemisinin resistance in falciparum malaria patients on the Thai-Cambodian border,a historical epicenter for multidrug resistance spread spanning over 50 years.The study presents the first evidence that highlights the parasites reduced susceptibility to artemisinin treatment by prolonged parasite-clearance times,raising considerable concern on resistance development.Although the exact mechanism of action remains unresolved,development of resistance was proposed based from both in vitro experiments and human patients.Lines of evidence suggested that the parasites in the patients are in dormant forms,presumably tolerate to the drug pressure.The World Health Organization has launched for prevention and/or containment of the artemisinin-resistant malaria parasites.Taken together,the emergence of artemisinin resistance to the most potent antidote for falciparum malaria,poses a serious threat to global malaria control and prompts renewed efforts for urgent development of new antimalarial weapons.

More than anti-malarial agents: therapeutic potential of artemisinins in neurodegeneration

Artemisinin,also called qinghaosu,is originally derived from the sweet wormwood plant(Artemisia annua),which is used in traditional Chinese medicine.Artemisinin and its derivatives(artemisinins)have been widely used for many years as anti-malarial agents,with few adverse side effects.Interestingly,evidence has recently shown that artemisinins might have a therapeutic value for several other diseases beyond malaria,including cancers,inflammatory diseases,and autoimmune disorders.Neurodegeneration is a challenging age-associated neurological disorder characterized by deterioration of neuronal structures as well as functions,whereas neuroinflammation has been considered to be an underlying factor in the development of various neurodegenerative disorders,including Alzheimer’s disease.Recently discovered properties of artemisinins suggested that they might be used to treat neurodegenerative disorders by decreasing oxidation,inflammation,and amyloid beta protein(Aβ).In this review,we will introduce artemisinins and highlight the possible mechanisms of their neuroprotective activities,suggesting that artemisinins might have therapeutic potential in neurodegenerative disorders.

Differential Effect of Artemisinin Against Cancer Cell Lines

The present study aims at defining the differential cytotoxicity effect of artemisinin toward P815(murin mastocytoma)and BSR(kidney adenocarcinoma of hamster)cell lines.Cytotoxicity was measured by the growth inhibition using MTT assay.These in vitro cytotoxicity studies were complemented by the determination of apoptotic DNA fragmentation and Annexin V-streptavidin-FITC assay.Furthermore,we examined the in vitro synergism between artemisinin and the chemotherapeutic drug,vincristin.The in vivo study was investigated using the DBA2/P815(H2d)mouse model.While artemisinin acted on both tumor cell lines,P815 was much more sensitive to this drug than BSR cells,as revealed by the respective IC50 values(12 lM for P815 and 52 lM for BSR cells).On another hand,and interestingly,apoptosis was induced in P815 but not induced in BSR.These data,reveal an interesting differential cytotoxic effect,suggesting the existence of different molecular interactions between artemisinin and the studied cell lines.In vivo,our results clearly showed that the oral administration of artemisinin inhibited solid tumor development.Our study demonstrates that artemisinin caused differential cytotoxic effects depending not only on the concentration and time of exposure but also on the target cells.

Holotransferrin Enhances Selective Anticancer Activity of Artemisinin against Human Hepatocellular Carcinoma Cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

Trichome-Specific Expression of Amorpha-4,11-Diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in <i>Artemisia annua</i>L., as Reported by a Promoter-GUS Fusion

Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.

Antimalarial qinghaosu/artemisinin: The therapy worthy of a Nobel Prize

Malaria is a major cause of human morbidity and mortality in the tropical endemic countries worldwide. This is largely due to the emergence and spread of resistance to most antimalarial drugs currently available. Based on the World Health Organization recommendation, artemisinin-based combination therapies are now used as first-line treatment for Plasmodium falciparum malaria. Artemisinin or qinghaosu(Chinese name) and its derivatives are highly potent, rapidly acting antimalarial drugs. Artemisinin was discovered in 1971 by a Chinese medical scientist Youyou Tu, who was awarded the Nobel Prize in 2015 on her discovering the antimalarial properties of qinghaosu from the traditional Chinese qinghao plant. Nevertheless, artemisinin resistance in falciparum malaria patients has first emerged on the Thai-Cambodian border in 2009, which is now prevalent across mainland Southeast Asia from Vietnam to Myanmar. Here, we reviewed malaria disease severity, history of artemisinin discovery, chemical structure, mechanism of drug action, artemisinin-based combination therapies, emergence and spread of drug resistance, including the recent findings on mechanism of resistance in the falciparum malaria parasite. This poses a serious threat to global malaria control and prompts renewed efforts for the urgent development of new antimalarial drugs.

Effects of arteannuin B,arteannuic acid and scopoletin on pharmacokinetics of artemisinin in mice

Objective:To explore the effects of arteannuin B,arteannuic acid and scopoletin on the pharmacokinetics of artemisinin in mice.Methods:Artemisinin and a combination of artemisinin,arteannuin B,arteannuic acid and scopoletin were administered together to mice via oral administration.Blood samples were collected at different time intervals and pretreated by liquid-liquid extraction.The contents of four compounds in mouse plasma were determined by a validated HPLC-MS/MS method.Results:Compared to single artemisinin group,the C_(max) values from the combination group rose from 947 ng/mL to 1 254 ng/mL.AUC_((0-t))(2 371 h·ng/mL) was significantly higher than that from single artemisinin group(747 h·ng/mL).The peak time lag and the CL values reduced at a proportion of 66%.Conclusions:Arteannuin B,arteannuic acid and scopoletin can markedly affect the pharmacokinetics of artemisinin.

Pharmacological and analytical aspects of artemisinin for malaria: Advances and challenges

Malaria remains a major tropical health burden owing to the development of resistance and decreased sensitivity to the frequently used conventional antimalarial drugs. The drug like artemisinin possesses potent antimalarial activities, but has some limitations. Therefore, new strategies are to be implemented for optimal utilization of artemisinin to improve its therapeutic effectiveness and to overcome its limitations. The present review focuses on present scenario of malaria and pharmacological as well as analytical aspects of artemisinin. Data from 2000 to 2018 were collected from NCBI for understanding the various analytical techniques used for estimation of artemisinin. This review will reveal the facts about artemisinin which can be utilized to develop novel drug delivery system either in a combination or as alone for the wellbeing of the patients suffering from malaria.

Efficient Somatic Embryogenesis and Organogenesis of Self-Pollination <i>Artemisia annua</i>Progeny and Artemisinin Formation in Regenerated Plants

To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants and artemisinin formation in regenerated plants. The first through sixth nodal leaves of seedlings are used as explants. On agar-solidified MS basal medium supplemented with TDZ (0.6 mg/l) and IBA (0.1 mg/l), all explants after inoculation of less than 3 weeks start to form embryogenic calli, which further produce globular, torpedo, heart and early cotyledon embryos. In all six positional leaves, explants from the sixth leaf show the rapidest responses to induction of embryogenic calli and somatic embryos. On this medium, somatic embryos continuously develop into adventitious buds, which can form adventitious roots on a rooting medium containing NAA (0.5 mg/l). Meanwhile, on agar-solidified MS basal medium supplemented with BAP (1 mg/l) and NAA (0.05 mg/l), approximately 100% of explants from leaves #3-6 form calli in less than 3 weeks of inoculation and adventitious buds via organogenesis in 3-4 weeks. In all six positional leaves, explants from the sixth leaf exhibit the rapidest response to induction of calli and adventitious buds. Nearly 100% adventitious buds can form adventitious roots on the rooting medium. Regenerated plants from both somatic embryogenesis and organogenesis complete self-pollination to produce seeds in 80-90 days of growth in growth chamber. LC-ESI-MS analysis demonstrates that regenerated plants biosynthesize artemisinin. These results show the highly efficient regeneration capacity of self-pollination A. annua plants that can form a new platform to enhance the understanding of artemisinin biosynthesis and metabolic engineering.

Overview of the improvement of the ring-stage survival assay - a novel phenotypic assay for the detection of artemisinin-resistant Plasmodium falciparum

Artemisinin resistance in Plasmodium falciparum threatens the remarkable efficacy of artemisininbased combination therapies worldwide. Thus, greater insight into the resistance mechanism using monitoring tools is essential. The ring-stage survival assay is used for phenotyping artemisinin-resistance or decreased artemisinin sensitivity. Here, we review the progress of this measurement assay and explore its limitations and potential applications.

Artemisinin protected human SY5Y and hippocampal neurons from H2O2-induced oxidative damage through activation of AMPK pathway

Oxidative stress is one of the main causes of neurodegenerative diseases such as Alzheimer disease(AD).Our previous studies have shown that artemisinin,a anti-malaria Chinese medicine,with neuroprotective effect,however,the antioxidative effect of artemisinin and its potential mechanism remain to be elucidated.In the present study,the protective effect and the underlying mechanism of artemisinin against injury of hydrogen peroxide(H_2O_2) in SH-SY5Y and hippocampal neurons were studied.Our results show that artemisinin protected SH-SY5Y and hippocampal neuronal cells from H_2O_2-induced cell death at clinically relevant concentrations in a concentration-dependent manner.Further studies showed that artemisinin significantly reduced cell death caused by H_2O_2 by restoring nuclear morphology,abnormal changes in intracellular ROS,activation of caspase 3,lactate dehydrogenase release and mitochondrial membrane potential.Hoechst staining and flow cytometry showed that artemisinin significantly reduced the apoptosis of SH-SY5Y cells exposed to H_2O_2.Western blotting analysis showed that artemisinin stimulated the phosphorylation and activation of AMP-activated protein kinase(AMPK) in SH-SY5Y cells in a time and concentration-dependent manner,whereas the application of AMPK inhibitor Compound C or decrease in expression of AMPKα with shRNA specific for AMPKα blocked the protective effect of artemisinin.Similar results were obtained in primary cultured hippocampal neurons.Taken together,these results indicate that artemisinin can protect neuronal cells from oxidative damage,at least in part through the activation of AMPK.Because artemisinin is relatively inexpensive and has few side effects,our findings support the role of artemisinin as a potential therapeutic agent for neurodegenerative diseases.

Antimalarial activity of a novel series of artemisinin-derived 1, 2, 3-triazole dimers

Objective: To obtain suitable artimisinin-based drug candidates with high antimalarial activity.Methods: Three different reaction schemes were used to synthesize a total of 15 artemisininbased compounds.The first synthetic scheme involved the synthesis of diazido aliphatic and aromatic compounds from commercially available dihalides and azido derivatives of artemisinin.The second scheme consisted of the reaction of dibromoaliphatic compounds with sodium azide in dimethylformamide which yielded the desired compounds.Artemisinin-based compounds on treatment with sodium azide and bromotrimethylsilane in dichloromethane produced the most potent compound GB-2.Another potent compound GB-1 was synthesized from artemisinin by treatment with alcohols in the presence of Aberlyst-15 in anhydrous dichloromethane.The third scheme involved the Huisgen 1,3-dipolar cycloaddition between the synthesized aliphatic and aromatic diazides and two alkyne derivatives of artemisinin to obtain the desired artemisinin dimers with average yields.Results: The best in vitro antiplasmodial activity was shown by the compound GB-2 registering IC_(50) value 0.066 μg/mL against chloroquine-sensitive and 0.865 μg/mL against chloroquineresistant strains of Plasmodium falciparum.It suppressed 59.0% parasitaemia in vivo of rodent malaria parasite Plasmodium berghei in Swiss albino model at 50 μg/kg body weight dosage.Molecular docking interactions of Plasmodium falciparum ATP6(PfATP6) protein revealed strong bonding of GB-2 with Thr255 residue which is likely to be the reason for excellent antimalarial activity of this compound.Conclusion: Two compounds GB-1 and GB-2 exhibited excellent in vitro antiplasmodial activity and fair in vivo antimalarial activity.Of the two, GB-2 showed better activity which could be attributed to its strong bonding interactions with Thr255 as evidenced from the molecular docking study.Study helped in identifying artemisinin analogues possessing good antimalarial properties and further research in structural alterations of the selected molecules should be carried out which may result in obtaining potent drug candidates against the malarial parasite.

Fluorescence Determination of Artemisinin Using Hemoglobin as Catalyst and Pyronine B as Substrate

Fluorescence decrease ratio was applied to determine of arte misinin （qinghaosu, QHS） based on the catalytic effect of hemoglobin （Hb） using tetraethyldiaminoxanthenyl chloride （Pryonine B, PB） as monitor in this work, Experimental results show that the catalytic characteristics of Hb for QHS, being expressed as Michaelis-Menten parameters, Km, Vmax and Kcat are 2.8×10^-5 mol·L^-1, 4.2×10^-5 mol·L^-1·s^-1 and 280 s^-1 , respectively. Like hemin, enzymatic bioactivities of Hb is inhibited by both deactivated agents and high temperature whereas enhanced by ethanol. With the catalytic action of Hb, quantitative method of QHS can be established based on the fluorescence decrease of PB and linear relationship between the fluorescence decrease ratio F0/F and the concentration of QHS is in the range of （0.0-1.1）×10^-6 mol·L^-1 with detection limit （3σ） being 7. 2×10^-9 mol·L^-1, The concentration of QHS in the media of plasma or urine was detected using this method.

Toxic response of aquatic organisms to guide application of artemisinin sustained-release granule algaecide

In our previous study,we prepared the granules by embedding artemisinin into alginate-chitosan using microcapsule technology.These granules can release artemisinin sustainably and have a strong inhibitory effect on the growth of both single Microcystis aeruginosa and mixed algae.To safely and effectively use artemisinin sustained-release granules to control algal blooms,the ecotoxicity was studied by assessing their acute and chronic toxicity to Daphnia magna(D.magna)and Danio rerio(D.rerio),along with their antioxidant activities.The results showed that the 48-h median effective concentration(EC50)of pure artemisinin to D.magna was 24.54 mg/L and the 96-h median lethal concentration(LC50)of pure artemisinin to D.rerio was 68.08 mg/L.Both values were classified as intermediate toxicity according to the Organization for Economic Co-operation and Development(OECD).The optimal algae inhibitory concentration of artemisinin sustained-release granules(1 g/L)had low acute toxicity to both D.magna and D.rerio.The sustained-release granules had higher chronic toxicity to D.magna than to D.rerio.Partial indices of D.magna were inhibited by granules when the concentrations were larger than 0.1 g/L.Low granule concentration had an inductive effect on antioxidant enzyme activities in D.magna and D.rerio.With the increase of the exposure concentration and time,the enzyme activity presented a trend of first increasing and then decreasing,and the overall changes were significant.The change trend and range of enzyme activity indicated that the granules could cause serious oxidative stress to D.magna and D.rerio,and the changes were consistent with the results of toxicity experimentation.