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.

Biotransformation of Artemisinin by Hairy Root Cultures of Rheum palmatum L.

The biotransformation of artemisinin by hairy root cultures ofRheum palmatum L. was investigated for the first time. The main product, deoxyartemisinin, was isolated and characterized on the basis of its spectral data.

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr.) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae , the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.

Elicitation on Artemisinin Biosynthesis in Artemisia annua Hairy Roots by the Oligosaccharide Extract from the Endophytic Colletotrichum sp. B501

The oligosaccharide elicitor from the mycelial wall of an endophytic Colletotrichum sp. B501 promoted the production of artemisinin in Artemisia annua L. hairy root culture. When hairy roots of 22-day-old cultures (later growth phase) were exposed to the elicitor (20 mg/L) for 4 d, the maximum content of artemisinin reached 1.15 mg/g, a 64.29% increment over the control. The electron X-ray microanalysis disclosed the rapid accumulation of Ca 2+ in the elicited cortical cells of hairy root. The electronic microscope observation revealed the high electron density area in vacuole of elicited cells. During the first day of elicitation the peroxidase activity of hairy roots was improved sharply. Some cellular morphological changes including cell shrinkage, condensation of cytoplasm and nuclear fragmentation, coincident with the appearance of DNA ladders, were observed after the third day of elicitation. It was suggested that the oligosaccharide elicitor triggered the programmed cell death, which may provide the substance or chemical signal for artemisinin biosynthesis.

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.

The Search for Antimalarial Drugs and the Discovery of Artemisinin

With its novel chemical structure, artemisinin is an antimalarial component isolated from the traditional Chinese medicine qinghao(Artemisia annua L.). Nowadays, artemisinin and its derivatives are used compatibly with new synthesized chemical antimalarial compounds to create artemisinin-based combination therapies(ACTs). These have become the first choice in treating malaria p.f. all over the world, providing an effective solution for the global challenge of curing drug-resistant malaria. Among the five ACTs recommended by the WHO, two were initiated in China and are used as the first-line treatment of falciparum malaria in all malaria endemic areas. As the use of artemisinin-based compound drugs have made such significant contributions to rolling back malaria, regarded as one of the great achievements globally in public health of the early twenty-first century, Tu Youyou, one of the most important researchers in the discovery of artemisinin, was made the first Nobel Prize laureate in Physiology or Medicine from the Chinese mainland. Artemisinin was discovered in a special social and cultural context through a combination of the exploration of traditional Chinese medical literature with the modern research approach of pharmaceutical sciences. This(Project 523) is a typical case of goal-oriented research leading to scientific advance, and the result of scientific research driven by the national needs.

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.

Research Progresses on Extraction and Detection Techniques of Artemisinin

[Objective] The aim was to make a summary on research progresses on extraction and detection techniques of artemisinin. [Method] In the research, the concerning progress was made based on physicochemical property, source, extraction methods and detection technology of artemisinin. [Result] Artemisinin is colorless acicular crystal and easier to be dissolved in organic solvent. The extraction methods of artemisinin include organic solvent extraction, ultrasonic extraction, microwave extraction and supercritical fluid extraction; the detection technologies include ultraviolet spectrophotometry, high performance liquid chromatography and HPLC-evaporative light-scattering. [Conclusion] The research laid foundation for further exploration and global market.

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.

Induction of apoptosis by artemisinin relieving the severity of inflammation in caerulein-induced acute pancreatitis

AIM： To observe the apoptosis and oncosis of pancreatic acinar cells and secondary inflammatory reaction in pancreatic tissue from rats with acute pancreatitis （AP）, and the influences of artemisinin on them.METHODS： AP was induced by 4 intraperitoneal iojections of caerulein at 1 h intervals. To induce apoptosis, solution of artemisinin （50 mg/kg） was given intraperitoneally 1, 12, 24 and 36 h after the last caerulein injection. Histological examination of impairment of pancreatic tissue and detection of serum amylase were performed to evaluate the severity of acute pancreatitis. Apoptosis and oncosis were detected with acridine orange （AO） and ethylene dibromide （EB） staining. Caspase-3 and myeloperoxidase （MPO） activity were measured by colorimetric assay. Nuclear factor-kappa B （NF-KB） activation was detected by flow cytometry. Macrophage inflammatory protein-lα（MIP-1α） protein was measured by Western blot. Interleukin- 1β（IL-1β） mRNA was detected by RT-PCR.RESULTS： Addition of artemisinin increased the number of apoptotic cells （11.7%±1.4% vs 6.3%± 0.7%, P 〈 0.05）, while reduced the number of oncotic cells （13.0% ±2.4% vs 17.5%±2.2%, P 〈 0.05）. The activity of caspase-3 speeded up （1.52±0.21 vs 1.03±0.08, P 〈 0.05）, the pancreas pathological impairment was relieved （3.0±0.5 vs 4.0± 0.5, P 〈 0.05） and the level of serum amylase decreased （5642±721 U/dL vs 7821±653 U/dL, P 〈 0.05）. The activation of NF-1α （29%±4.1% vs 42%±5.8%）, MIP-1α protein （3.7±0.5 vs 5.8±0.7）,MPO （0.52±0.06 U/g vs 0.68±0.09 U/g）, IL-1β mRNA （1.7 ±0.3 vs 2.4 ±0.4） in the apoptosis inducing group was obviously decreased （P 〈 0.05）.CONCLUSION： Inducing apoptosis can relieve pathological impairment and inflammatory reaction in AP rats.

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.

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.

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.

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.

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.

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.