Semi-synthetic Derivative Of Artemisinin Research Articles

Sfrps Family Invovled in Growth Inhibition of K562 Cells Induced by Artesunate

Abstract 5015 Introduction:Studies have shown that abnormal activation of wnt signaling pathway is closely related to tumor genesis. Cell surface coil protein (frizzled, fzd) is a specific receptor for wnt pathway to activate its downstream signaling through β-catenin to stimulate the growth of a range of tumor cells. Secreted frizzled related proteins (sfrps) are the main antagonists of wnt pathway. sfrps can inhibit the function of wnt pathway via competing with fzd receptor. Recent studies have found that sfrps family memberes expressed at very low levels in a variety of tumor cells,which was closely related to the methylation of sfrps gene promoters. DNA methyltransferase (dnmts) are the key enzymes involved in DNA methylation, which can promote the methylation of tumor suppressor genes’ promoters and inhibit transcription of these genes, and induce tumor genesis. Artesunate, a semi-synthetic derivative of artemisinin, is very effective in antimalaria. Recent studies have shown that artesunate has anti-tumor functions. However, the effect of artesunate on the expression of sfrps- and dnmts- mRNAs and β-catenin protein are not clear. To evaluate the growth inhibition effect of Artesunate (at final concentrations of 0μg/ml, 4μg/ml, 10μg/ml, 20μg/ml and 40μg/ml) on K562 cells and to investigate its potential mechanism by detecting the expression levels of sfrps- and dnmts- mRNAs and β-catenin protein in K562 cells treated with artesunate. Methods:Cell growth inhibition rate and cell cycle distribution of K562 cells induced by artesunate treatment were measured by MTT assay and flow cytometry, respectively. The mRNA expression levels of sfrp1, sfrp2, sfrp4 and dnmt1, dnmt3a, dnmt3b in K562 cells which had been treated with or without artesunate for 48 hours were evaluated with reverse transcription-polymerase chain reaction (RT-PCR). Protein expression level of β-catenin in K562 cells were detected by Western blot. Results:Artesunate treatment significantly induced growth inhibition of K562 cells in a concentration-dependent manner after the cells were treated with artesunate for 48 hours(p < 0.05). The inhibition rate of 4,10,20 and 40(μg/ml)artesunate exposure were 54.29%, 58.03%, 69.33% and 77.98% respectively.Flow cytometry analysis showed that K562 cells were arrested at G0/Gl and G2/M phase in concentration-dependent manner after 48 hours exposure of artesunate (p < 0.05).After treated with artesunate at the final concentrations of 0, 4, 10, 20 and 40μg/ml, the relative expression levels of sfrp1, sfrp2 and sfrp4 mRNA in K562 cells increased, while the expression levels of dnmtl, dnmt3a, dnmt3b mRNA decreased significantly compared with the control group.Results from Western blot showed that β-catenin protein levels decreased in a concentration-dependent manner, when compared with that of the control group (P<0.05). Conclusion: Results from the present study indicated that artesunate could inhibite the mRNA expression of dnmts family and minimize the methylation of sfrps gene promoter. Therefore, sfrps could inhibit the function of wnt pathway through competing with fzd receptors. Meanwhile, artesunate could decrease the expression of β-catenin protein in K562 cells, and could further inhibit the function of wnt pathway. Therefore, data from the present experiment provides a new theoretical basis for clinical application of artesunate in leukemia treatment. Disclosures:Ying:Nature science foundation of Hebei Province: Research Funding. Nan:Nature science foundation of Hebei Province: Research Funding. Jun:Nature science foundation of Hebei Province: Research Funding. Hai:Nature science foundation of Hebei Province: Research Funding. Kai:Nature science foundation of Hebei Province: Research Funding. Xu:Nature science foundation of Hebei Province: Research Funding. Pan:Nature science foundation of Hebei Province: Research Funding.

Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin

Artemisia annua L. (sweet wormwood, qinhao) has traditionally been used in Chinese medicine. The isolation of artemisinin from Artemisia annua and its worldwide accepted application in malaria therapy is one of the showcase success stories of phytomedicine during the past decades. Artemisinin-type compounds are also active towards other protozoal or viral diseases as well as cancer cells in vitro and in vivo. Nowadays, Artemisia annua tea is used as a self-reliant treatment in developing countries. The unsupervised use of Artemisia annua tea has been criticized to foster the development of artemisinin resistance in malaria and cancer due to insufficient artemisinin amounts in the plant as compared to standardized tablets with isolated artemisinin or semisynthetic artemisinin derivatives. However, artemisinin is not the only bioactive compound in Artemisia annua. In the present investigation, we analyzed different Artemisia annua extracts. Dichloromethane extracts were more cytotoxic (range of IC₅₀: 1.8-14.4 μg/ml) than methanol extracts towards Trypanosoma b. brucei (TC221 cells). The range of IC₅₀ values for HeLa cancer cells was 54.1-275.5 μg/ml for dichloromethane extracts and 276.3-1540.8 μg/ml for methanol extracts. Cancer and trypanosomal cells did not reveal cross-resistance among other compounds of Artemisia annua, namely the artemisinin-related artemisitene and arteanuine B as well as the unrelated compounds, scopoletin and 1,8-cineole. This indicates that cells resistant to one compound retained sensitivity to another one. These results were also supported by microarray-based mRNA expression profiling showing that molecular determinants of sensitivity and resistance were different between artemisinin and the other phytochemicals investigated.

Anti-malarial drug artesunate attenuates experimental allergic asthma via inhibition of the phosphoinositide 3-kinase/Akt pathway.

BackgroundPhosphoinositide 3-kinase (PI3K)/Akt pathway is linked to the development of asthma. Anti-malarial drug artesunate is a semi-synthetic derivative of artemisinin, the principal active component of a medicinal plant Artemisia annua, and has been shown to inhibit PI3K/Akt activity. We hypothesized that artesunate may attenuate allergic asthma via inhibition of the PI3K/Akt signaling pathway.Methodology/Principal FindingsFemale BALB/c mice sensitized and challenged with ovalbumin (OVA) developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Artesunate dose-dependently inhibited OVA-induced increases in total and eosinophil counts, IL-4, IL-5, IL-13 and eotaxin levels in bronchoalveolar lavage fluid. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, IL-17, IL-33 and Muc5ac in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, artesunate blocked epidermal growth factor-induced phosphorylation of Akt and its downstream substrates tuberin, p70S6 kinase and 4E-binding protein 1, and transactivation of NF-κB. Similarly, artesunate blocked the phosphorylation of Akt and its downstream substrates in lung tissues from OVA-challenged mice. Anti-inflammatory effect of artesunate was further confirmed in a house dust mite mouse asthma model.Conclusion/SignificanceArtesunate ameliorates experimental allergic airway inflammation probably via negative regulation of PI3K/Akt pathway and the downstream NF-κB activity. These findings provide a novel therapeutic value for artesunate in the treatment of allergic asthma.

Antiparasitic Therapy

Parasitic diseases affect more than 2 billion people globally and cause substantial morbidity and mortality, particularly among the world's poorest people. This overview focuses on the treatment of the major protozoan and helminth infections in humans. Recent developments in antiparasitic therapy include the expansion of artemisinin-based therapies for malaria, new drugs for soil-transmitted helminths and intestinal protozoa, expansion of the indications for antiparasitic drug treatment in patients with Chagas disease, and the use of combination therapy for leishmaniasis and human African trypanosomiasis.

The redox antimalarial dihydroartemisinin targets human metastatic melanoma cells but not primary melanocytes with induction of NOXA-dependent apoptosis

Recent research suggests that altered redox control of melanoma cell survival, proliferation, and invasiveness represents a chemical vulnerability that can be targeted by pharmacological modulation of cellular oxidative stress. The endoperoxide artemisinin and semisynthetic artemisinin-derivatives including dihydroartemisinin (DHA) constitute a major class of antimalarials that kill plasmodium parasites through induction of iron-dependent oxidative stress. Here, we demonstrate that DHA may serve as a redox chemotherapeutic that selectively induces melanoma cell apoptosis without compromising viability of primary human melanocytes. Cultured human metastatic melanoma cells (A375, G361, LOX) were sensitive to DHA-induced apoptosis with upregulation of cellular oxidative stress, phosphatidylserine externalization, and activational cleavage of procaspase 3. Expression array analysis revealed DHA-induced upregulation of oxidative and genotoxic stress response genes (GADD45A, GADD153, CDKN1A, PMAIP1, HMOX1, EGR1) in A375 cells. DHA exposure caused early upregulation of the BH3-only protein NOXA, a proapototic member of the Bcl2 family encoded by PMAIP1, and genetic antagonism (siRNA targeting PMAIP1) rescued melanoma cells from apoptosis indicating a causative role of NOXA-upregulation in DHA-induced melanoma cell death. Comet analysis revealed early DHA-induction of genotoxic stress accompanied by p53 activational phosphorylation (Ser 15). In primary human epidermal melanocytes, viability was not compromised by DHA, and oxidative stress, comet tail moment, and PMAIP1 (NOXA) expression remained unaltered. Taken together, these data demonstrate that metastatic melanoma cells display a specific vulnerability to DHA-induced NOXA-dependent apoptosis and suggest feasibility of future anti-melanoma intervention using artemisinin-derived clinical redox antimalarials.

Abstract 4217: The anti-malarial compound artesunate induces reactive oxygen species-dependent killing of breast cancer cells

Abstract Introduction: Artesunate is a semi-synthetic derivative of artemisinin, a natural compound from the herb Artemisia annua L. Artemisinin has been used in traditional Chinese medicine while artesunate has recently been used as an anti-malarial drug. Artesunate is also cytotoxic to human cancer cells. Since the use of artesunate as an anti-malarial agent is associated with few adverse effects, artesunate may represent a less toxic alternative to conventional chemotherapy. This study investigates the cytotoxic effect of artesunate on breast cancer cell lines and the mechanism(s) underlying its activity. Methods and Results: Artesunate exhibited a time- and dose-dependent cytotoxic effect on all breast carcinoma cell lines examined (MCF-7, T47D, SK-BR-3, MDA-MB-231, MDA-MB-468). Further examination of MDA-MB-468 cells using Oregon Green-488 and Annexin-V-FLUOS/propidium iodide staining indicated that artesunate had an anti-proliferative effect that was associated with apoptosis induction. Cell cycle arrest occurred in the G2 phase, and was evident as early as 24h after artesunate treatment. Artesunate-induced apoptosis was low in normal fibroblasts at concentrations cytotoxic to breast cancer cells, although fibroblast proliferation was inhibited. DiOC6 staining revealed dose-dependent destabilization of the mitochondrial membrane in artesunate-treated MDA-MB-468 cells. Artesunate-treated MDA-MB-468 cells also displayed cytosolic cytochrome c and PARP cleavage, as demonstrated by Western blotting, which was consistent with apoptosis induction. Pretreatment with reduced glutathione rescued MDA-MB-468 cells from artesunate-induced apoptosis, indicating that reactive oxygen species (ROS) mediate artesunate-induced cytotoxicity. In addition, MDA-MB-468 cells that were pretreated with holotransferrin showed increased killing by artesunate, suggesting that iron promotes artesunate-mediated apoptosis. Induction of the endoplasmic reticulum stress pathway in artesunate-treated cells was demonstrated by Western blotting, suggesting involvement of this apoptosis-inducing pathway in artesunate-mediated cytotoxicity. Conclusions: These data show that artesunate causes reactive oxygen species-dependent induction of apoptosis in breast cancer cells and therefore warrants further investigation as a possible treatment for breast cancer. Supported by NSERC and the Canadian Breast Cancer Foundation-Atlantic Region Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4217. doi:10.1158/1538-7445.AM2011-4217

Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria.

Ozonide OZ439 is a synthetic peroxide antimalarial drug candidate designed to provide a single-dose oral cure in humans. OZ439 has successfully completed Phase I clinical trials, where it was shown to be safe at doses up to 1,600 mg and is currently undergoing Phase IIa trials in malaria patients. Herein, we describe the discovery of OZ439 and the exceptional antimalarial and pharmacokinetic properties that led to its selection as a clinical drug development candidate. In vitro, OZ439 is fast-acting against all asexual erythrocytic Plasmodium falciparum stages with IC(50) values comparable to those for the clinically used artemisinin derivatives. Unlike all other synthetic peroxides and semisynthetic artemisinin derivatives, OZ439 completely cures Plasmodium berghei-infected mice with a single oral dose of 20 mg/kg and exhibits prophylactic activity superior to that of the benchmark chemoprophylactic agent, mefloquine. Compared with other peroxide-containing antimalarial agents, such as the artemisinin derivatives and the first-generation ozonide OZ277, OZ439 exhibits a substantial increase in the pharmacokinetic half-life and blood concentration versus time profile in three preclinical species. The outstanding efficacy and prolonged blood concentrations of OZ439 are the result of a design strategy that stabilizes the intrinsically unstable pharmacophoric peroxide bond, thereby reducing clearance yet maintaining the necessary Fe(II)-reactivity to elicit parasite death.

Artesunate mitigates proliferation of tumor cells by alkylating heme-harboring nitric oxide synthase

Artesunate (ART), a semi-synthetic derivative of antimalarial artemisinin, kills cancer cells with uncertain mechanisms. Here, we report for the first time that ART may exert the anti-tumor activity by conjugating the prosthetic heme of hemoproteins in a hepatoma cell line, HepG2, which was evident by monitoring the shift of absorbance from heme (A 415) to the ART-heme adduct (A 476). Accordingly, a transient elevation of A 415 was observed with a synchronous burst of nitric oxide (NO) and a high rate of survival following incubation of HepG2 with 50 μM ART. In contrast, ART at above 100 μM led to an abrogation of NO generation and a decline of the survival rate in HepG2. These data implied that heme-containing nitric oxide synthase (NOS) may represent a major cellular target of ART in killing tumor cells.

General Pharmacology of Artesunate, a Commonly used Antimalarial Drug: Effects on Central Nervous, Cardiovascular, and Respiratory System

Artesunate, a semi-synthetic derivative of artemisinin, is used primarily as a treatment for malaria. Its effects on the central nervous system, general behavior, and cardiovascular, respiratory, and other organ systems were studied using mice, rats, guinea pigs, and dogs. Artesunate was administered orally to mice at doses of 125, 250, and 500 mg/kg and to rats and guinea pigs at 100, 200, and 400 mg/kg. In dogs, test drugs were administered orally in gelatin capsules at doses of 50, 100, and 150 mg/kg. Artesunate induced insignificant changes in general pharmacological studies, including general behavior, motor coordination, body temperature, analgesia, convulsion modulation, blood pressure, heart rate (HR) , and electrocardiogram (ECG) in dogs in vivo; respiration in guinea pigs; and gut motility or direct effects on isolated guinea pig ileum, contractile responses, and renal function. On the other hand, artesunate decreased the HR and coronary flow rate (CFR) in the rat in vitro; however, the extent of the changes was small and they were not confirmed in in vivo studies in the dog. Artesunate increased hexobarbital-induced sleeping time in a dose-related manner. Artesunate induced dose-related decreases in the volume of gastric secretions and the total acidity of gastric contents, and induced increases in pH at a dose of 400 mg/kg. However, all of these changes were observed at doses much greater than clinical therapeutic doses (2.4 mg/kg in humans, when used as an anti-malarial) . Thus, it can be concluded that artesunate is safe at clinical therapeutic doses.

Dihydroartemisinin Induces Apoptosis by a Bak-Dependent Intrinsic Pathway

The sesquiterpene lactone dihydroartemisinin (DHA), a semisynthetic derivative of the herbal antimalaria drug artemisinin, is cytotoxic to human tumor cells. Treatment of Jurkat T-lymphoma cells with DHA induced a breakdown of the mitochondrial transmembrane potential, release of cytochrome c, activation of caspases, and DNA fragmentation indicative of apoptosis induction. Although the absence of FADD or caspase-8 did not alter apoptosis rates in Jurkat cells, overexpression of dominant-negative caspase-9 or of antiapoptotic Bcl-xL or Bcl-2 largely decreased the cytotoxicity of DHA, demonstrating a role of the intrinsic death pathway. The proapoptotic Bcl-2 effector protein Bak and the Bcl-2 homology domain 3-only protein NOXA turned out to be important mediators of DHA-induced apoptosis in Jurkat cells. DHA treatment triggered the expression of NOXA and the activation of Bak. Furthermore, DHA-induced apoptosis was completely abrogated by loss of Bak and largely reduced in cells with siRNA-mediated downregulation of Bak or NOXA. Proapoptotic signaling of DHA also involved the formation of reactive oxygen species and membrane oxidation. Pretreatment with the lipophilic radical scavenger vitamin E or the hydrophilic radical scavengers glutathione and N-acetylcysteine reduced DHA-induced membrane oxidation and apoptosis, respectively. Oxidative changes also occurred in cells with disruption of the mitochondrial death pathway, suggesting a role of reactive oxygen species and oxidative membrane changes in death signaling upstream of the mitochondria. Interestingly, DHA increased the cytotoxic action of ionizing radiation and of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand in Jurkat cells, suggesting a potential benefit of DHA in combined treatment strategies.

Artesunate: The Best Drug in the Treatment of Severe and Complicated Malaria

This review summarizes progress in treating severe and complicated malaria, which are global problems, claiming at least one million lives annually, and have been accompanied by advances in our understanding of the pathogenesis of severe malaria complications. New drugs such as intravenous artesunate (AS) and intramuscular artemether (AM) are improving outcomes and decreasing malaria deaths. Trials comparing AM to the traditional parenteral drug, quinine, have not demonstrated however convincing evidence of a mortality advantage for AM. The South East Asian Quinine Artesunate Malaria Trials (SEAQUAMAT), a multicenter, randomized, open-label study comparing AS with quinine showed that parenteral AS was shown to be associated with a 35% reduction in the risk of mortality compare to quinine, and is now the recommended treatment by the WHO for severe and complicated malaria in low-transmission areas and in the second and third trimesters of pregnancy, with almost all the benefit reported in those with high parasite counts. Artesunate is a semisynthetic derivative of artemisinin whose water solubility facilitates absorption and provides an advantage over other artemisinins because it can be formulated as oral, rectal, intramuscular, and intravenous preparations. Artesunate is rapidly hydrolyzed to dihydroartemisinin, which is the most active schizonticidal metabolite. Injectable AS results in a more rapid systemic availability of AS compared with intramuscular AM. This pharmacokinetic advantage may provide a clinical advantage in the treatments of severe and complicated malaria.

Rationale Design of Biotinylated Antimalarial Endoperoxide Carbon Centered Radical Prodrugs for Applications in Proteomics

The semisynthetic artemisinin derivatives such as artesunate and artemether, along with the fully synthetic endoperoxide antimalarials (e.g., OZ277, Nature 2004, 430, 900-904), are believed to mediate their antimalarial effects by iron-induced formation of carbon-centered radicals capable of alkylating heme and/or protein. Here, we describe the design and synthesis of a series of biotinylated endoperoxide probe molecules for use in proteomic studies. The target molecules include derivatives of the artemisinin and OZ families, and we demonstrate that these conjugates express nanomolar in vitro activity versus cultured strains of Plasmodium falciparum. We also describe the synthesis of chemically cleavable linked conjugates designed to enable mild elution of labeled proteins during target protein identification.

Suppression of PMA-induced tumor cell invasion by dihydroartemisinin via inhibition of PKCα/Raf/MAPKs and NF-κB/AP-1-dependent mechanisms

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently been shown to possess antitumor activity in various cancer cells. However, the effects of DHA in preventing the invasion of cancer cells have not been studied. In the present study, we investigated the inhibitory effects of DHA on tumor invasion and migration and the possible mechanisms involved using human fibrosarcoma HT-1080 cells. DHA reduced PMA-induced activation of MMP-9 and MMP-2 and further inhibited cell invasion and migration. DHA suppressed PMA-enhanced expression of MMP-9 protein, mRNA, and transcriptional activity through suppressing NF-κB and AP-1 activation without changing the level of tissue inhibitor of metalloproteinase (TIMP)-1. DHA also reduced PMA-enhanced MMP-2 expression by suppressing membrane-type 1 MMP (MT1-MMP), but did not alter TIMP-2 levels. DHA-inhibited PMA-induced NF-κB and c-Jun nuclear translocation, which are upstream of PMA-induced MMP-9 expression and invasion. Furthermore, DHA strongly repressed the PMA-induced phosphorylation of Raf/ERK and JNK, which are dependent on the PKCα pathway. In conclusion, we demonstrated that the anti-invasive effects of DHA may occur through inhibition of PKCα/Raf/ERK and JNK phosphorylation and reduction of NF-κB and AP-1 activation, leading to down-regulation of MMP-9 expression. The data presented show that DHA is an effective anti-metastatic agent that functions by down-regulating MMP-9 gene expression.

Artemisinin-based combination therapies: a vital tool in efforts to eliminate malaria

Plasmodium falciparum resistance to chloroquine and sulphadoxine-pyrimethamine has led to the recent adoption of artemisinin-based combination therapies (ACTs) as the first line of treatment against malaria. ACTs comprise semisynthetic artemisinin derivatives paired with distinct chemical classes of longer acting drugs. These artemisinins are exceptionally potent against the pathogenic asexual blood stages of Plasmodium parasites and also act on the transmissible sexual stages. These combinations increase the rates of clinical and parasitological cures and decrease the selection pressure for the emergence of antimalarial resistance. This Review article discusses our current knowledge about the mode of action of ACTs, their pharmacological properties and the proposed mechanisms of drug resistance.

Dihydroartemisinin improves the efficiency of chemotherapeutics in lung carcinomas in vivo and inhibits murine Lewis lung carcinoma cell line growth in vitro

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has exhibited the strongest antimalarial activity among the derivatives of artemisinin. There is growing evidence that DHA has some impact against tumors. Our purpose was to evaluate in vitro antitumoral properties of DHA in the murine Lewis lung carcinoma (LLC) cell line. At the same time, we observed the therapeutic effect of DHA combined with cyclophosphamide (CTX) in the LLC and combined with cisplatin (CDDP) in the human non-small cell lung cancer A549 xenotransplanted carcinoma in vivo. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, apoptosis was measured by AO/EB double staining and flow cytometry. The expression of vascular endothelial growth factor (VEGF) receptor KDR/flk-1 was analyzed by western blotting and RT-PCR. In vivo activity of DHA combined with CTX or CDDP was assayed through tumor growth and metastasis. Dihydroartemisinin exhibited high anti-cancer activity in LLC cell line. DHA also induced apoptosis of LLC cells and influenced the expression of VEGF receptor KDR/flk-1. Furthermore, in both tumor xenografts, a greater degree of growth inhibition was achieved when DHA and chemotherapeutics were used in combination. The affection by DHA combined CTX on LLC tumor metastasis was significant. Dihydroartemisinin is a potent compound against LLC cell line in vitro. In vivo, the combination strategy of DHA and chemotherapeutics holds promise for the treatment of relatively large and rapidly growing lung cancers.

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

BackgroundDihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood.MethodsIn this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (ΔΨm) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis.ResultsOur results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of ΔΨm and the activation of caspase-3.ConclusionThese results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma.

Dihydroartemisinin inhibits growth of pancreatic cancer cells in vitro and in vivo

Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has recently shown antitumor activity in various cancer cells. Its effect on pancreatic cancer is, however, unknown and the mechanism is unclear. The study aims to investigate its antitumor activity and underlying mechanisms in human pancreatic cancer BxPC-3 and AsPC-1 cells in vitro and subcutaneous BxPC-3 xenograft tumors in mice. The MTT assay was used to evaluate cell viability, and flow cytometry and laser scanning confocal microscopy were used to detect apoptosis, for cultured cells. Pancreatic tumors were established by subcutaneous injection of BxPC-3 cells in nude BALB/c mice, and DHA was administered intraperitoneally to the mice. The size of tumors was monitored and they were harvested after the mice had been killed. Tumor sections were immunostained with an anti-Ki-67 Ab to assess the proliferation index, or stained with TUNEL to evaluate in-situ cell apoptosis. The gene expression in cells and tumors was evaluated by western blot analysis. In the cultured cells, DHA inhibited cell viability, downregulated the expression of proliferating cell nuclear antigen and cyclin D1, and upregulated p21(WAF1/CIP1); and induced apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9, in a dose-dependent manner. Similarly, in mice bearing BxPC-3 xenograft tumors, administration of DHA inhibited tumor growth in a dose-dependent manner, and modulated tumoral gene expression consistent with the in-vitro observations. This study indicates that DHA may be a potent and promising agent to combat pancreatic cancer.

A Novel Putative Mechanism of Anti-Myeloma Activity Targeted Against Heat Shock Protein 27 by Derivative of Artemisinin, Artesunate.

Recent introduction of molecular targeted drugs such as bortezomib and IMIDs in the clinical settings has achieved the improved treatment outcome of multiple myeloma (MM). However, MM is still an incurable disease and these drugs also possess serious adverse reactions, therefore, safe and more effective therapy should be established. Artesunate (ART) is a semi-synthetic derivative of artemisinin and is widely used for the treatment of malaria as a salvage therapy. Recent in vitro studies showed that ART also has an anti-tumor activity against several cancer cell lines. We thus investigated whether ART could possess anti-myeloma activity and demonstrated that apoptosis of myeloma cells is strongly induced by ART. Interestingly, we also found that this activity is mediated through heat shock protein (Hsp) 27-dependent pathway. Several MM cell lines (IM9, OPM2, RPMI8226, U266) were treated with various concentrations of ART for 48 hours, and MTT assays were performed to assess the anti-myeloma activity. IM9, OPM2, RPMI8226 cells showed the striking reduction of viability (up to 40% at 1μM and up to 90% at 10μM) in a dose- and time-dependent manner, whereas ART has less inhibitory effect on U266 cell line. ART induced G1 arrest of IM9 cells, and apoptosis was confirmed by decreased mitochondrial membrane potential, and flow cytometric analysis using AnnexinV/PI staining. Colony assay showed that ART has no growth inhibitory effect on normal CD34-positive bone marrow cells even at a concentration of 10μM. Immunoblot analyses demonstrated the activation of caspases-3 and -9, and the decreased expression of pro-apoptotic protein Bid. Interestingly, heat shock protein (Hsp) 27 was downregulated in myeloma cell lines (IM9, RPMI8226) which are sensitive to the ART treatment. In contrast, downregulation of Hsp 27 was not observed in U266 cells which are resistant to ART. Other anti-apoptotic Hsps (Hsp70, 90), as well as Bcl-2 family proteins (Bcl-2, Bax, Bad, Bcl-xL), Akt, MDM2, p53 and MAPKs (SAPK/JNK, p38, ERK1/2) were not affected by ART. Quantitative RT-PCR analysis showed that ART did not influence the mRNA expression of Hsp27, suggesting that ART could downregulate the Hsp27 protein at a posttranscriptional or posttranslational levels. Overexpression of Hsp27 cDNA by transfection method in ART-sensitive myeloma cell lines demonstrated that these cell lines acquired resistance to ART and apoptosis was not induced at a concentration which ART is effective to the parental cells. Our preliminary data using knockdown procedure of Hsp27 mRNA by RNAi-expressing lentivirus showed the growth suppression of myeloma cells, which apparently suggested that Hsp27 could confer a critical function in the proliferation of myeloma cells and that downregulation of Hsp27 could result in induction of apoptosis in MM. Hsp27 has an important role in anti-apoptotic effect against various stresses to the cells. Our data implies a novel mechanism that downregulation of Hsp27 by ART could induce misfolding of several client proteins indispensable for the cell growth, which results in the induction of apoptosis in MM cells. In conclusion, ART could become a candidate of safe and effective therapeutic drug for MM, and Hsp 27 might be a potential molecular target for the treatment of MM.

The role of calcium, P38 MAPK in dihydroartemisinin-induced apoptosis of lung cancer PC-14 cells

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, is an effective novel antimalarial drug. Recent studies suggest that it also has anticancer effect. The present study was designed to investigate the effects of DHA on cultured human lung cancer cells (PC-14 cells) to better understand its apoptosis and apoptosis-related factors in vitro. The cell viability was measured by MTT assay. The apoptosis induction was examined by DNA ladder and flow cytometry. The intracellular-free calcium concentration in the lung cancer cells were evaluated by laser scanning confocal microscopy with Fura-3/AM as probe. The associated gene expression was examined by Western blot. After treatment with DHA, a decrease in the viability of PC-14 cells and apoptosis were observed. DHA-induced apoptosis were accompanied by an increase of Ca(2+) and activation of p38. Deleted levels of Ca(2+) by BAPTA-AM 20 microM or inhibition of p38 by its selective inhibitor SB202190 then led to decreased DHA-induced apoptosis. These results demonstrated that DHA can induce apoptosis of lung cancer cell line PC-14 cells and calcium and p38 play important roles in the apoptotic signalling pathways.

Artesunate inhibits angiogenesis and downregulates vascular endothelial growth factor expression in chronic myeloid leukemia K562 cells

Artesunate (ART), a semi-synthetic derivative of artemisinin extracted from the Chinese herb Artemisia annua, is a safe and effective antimalarial drug. In the present investigation, we analyzed the inhibitory effects of ART on angiogenesis and on VEGF production in chronic myeloid leukemia (CML) K562 cells in vitro and in vivo. In order to analyze the effect of ART on VEGF secretion in K562 cells, we examined the level of VEGF secreted in conditioned media (CM) by ELISA assay. The result showed that ART could decrease the VEGF level in CM of K562 cells, even at a lower concentration (2 μmol/l, P < 0.01). The inhibitory effect of in vitro angiogenesis was tested on aortic sprouting in fibrin gel. ART could effectively suppress the stimulating angiogenic ability of CM by pretreated with K562 cells for 48 h in a time-dependent manner (days 3–14). The antiangiogenic effect of ART was further evaluated in vivo in chicken chorioallantoic membrane (CAM) neovascularization model. The result indicated that the stimulating angiogenic activity was decreased in response to the K562 cells treated with ART or the CM from K562 cells pretreated with ART in a dose-dependent manner (3–12 μmol/l). Furthermore, we analyzed the level of VEGF expression by western blot and detected the form of VEGF mRNA by RT-PCR in K562 cells. The experiments showed that ART could inhibit the VEGF expression, correlated well with the level of VEGF secreted in CM. These findings suggest that ART might present potential antileukemia effect as a treatment for CML therapy, or as an adjunct to standard chemotherapeutic regimens.