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C4. Expression of immune regulatory genes and involvement of nitric oxide after BCG treatment of urinary bladder cancer
Available online at www.sciencedirect.com
Nitric Oxide 17 (2007) S20–S29 www.elsevier.com/locate/yniox
Poster abstracts C1. Nitric oxide donors for treating IBD and related diseases P. Assaf, J. Tarsis Renopharm Ltd., POB 2252, Nazareth, Israel Nitric oxide is a ubiquitous molecule involved in a variety of biological processes. In recent years, nitric oxide (NO), a gas previously considered to be potentially toxic chemical, has been established as a diffusible universal messenger that mediates cell cell communication throughout the body. The specific action of NO depends on its enzymatic sources namely neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) and all three isoforms have been localized in the gastrointestinal tract. Constitutive synthesis of NO by nNOS or eNOS isoforms is involved in the maintaining of the gastrointestinal mucosal integrity through modulation of gastric mucosal blood flow, epithelial secretion and barrier function. However, NO has also been associated with the initiation and maintenance of inflammation in human inflammatory bowel disease (IBD). Recent studies on animal models of experimental IBD have shown that constitutive and inducible NO production seems to be beneficial during acute colitis, but sustained up-regulation of NO is detrimental. This fact is also supported by studies on mice genetically deficient in various NOS isoforms. However, the mechanism by which NO proceeds from being an indispensable homeostatic regulator to a harmful destructor remains unknown. Recently, results from our laboratory show that our lead compound, a nitric oxide donor, when compared to 5-ASA, is more effective for the treatment of ulcerative colitis-IBD. doi:10.1016/j.niox.2007.09.046
C2. iNOS down-regulation induced by flavopiridol, hyperforin and polyphenols in CLL cells is a caspase-dependent mechanism C. Billard, C. Quiney, A. Ensaf, J.P. Kolb Centre de Recherche des Cordeliers, Equipe 18, UMR 872 INSERM, Universite´s Pierre et Marie Curie et Paris Descartes, 15 Rue de l ’Ecole de Me´decine, 75006 Paris, France Chronic lymphocytic leukemia (CLL) is characterized by a remarkable apoptosis deficiency of the leukemic cells rather than lymphoproliferation deregulation. We previously reported that iNOS is constitutively expressed in CLL cells and that the NO produced has anti-apoptotic properties in these cells possibly through S-nitrosylation of the active site of caspases. Flavopiridol and various plant-derived compounds such as polyphenols or hyperforin are able to induce the apoptosis of CLL cells ex vivo. We have shown that these compounds share the property to inhibit iNOS expression, suggesting that this effect could be a molecular switch contributing to apoptosis induction. However, flavopiridol-induced iNOS downregulation is totally prevented by z-VAD-fmk, a general caspase inhibitor, demonstrating that this effect is caspase-dependent. No cleavage fragment of iNOS was yet detected upon treatment of CLL cells with flavopiridol or incubation of recombinant iNOS with caspase-3, favoring the idea that
doi:10.1016/j.niox.2007.09.045
iNOS is not a caspase substrate. Our data indicate that the inhibition of the NO pathway is not a primary event in apoptosis triggering, but is rather one of the effector mechanisms of the apoptotic machinery triggered by flavopiridol and other plant-derived molecules in CLL cells. doi:10.1016/j.niox.2007.09.047
C3. Mechanisms of human colon cancer cells sensitization to nitric oxideinduced apoptosis M. Cortier, J.F. Jeannin, A. Bettaieb UMR866, INSERM, Ecole Pratique des Hautes Etudes, Universite´ de Bourgogne, Faculte´ de me´decine et de pharmacie, 7 Bd Jeanne d’Arc, Dijon, France We have previously reported that the nitric oxide donor glyceryl trinitrate (GTN) induced death of human colon cancer cells when used at high concentrations (500 lM). However, low doses of GTN failed to induce cell death. Then we explored whether GTN could induce colon cancer cells death at lower concentrations when combined with inhibitors of various proteins including MAPK. Among these inhibitors, the H89 {N-[2-(pBromo cinnamylamino) ethyl] -5- isoquinoline sulfonamide 2HCL}, a well known inhibitor of protein kinases including PKA, sensitized the human SW480 colon cancer cells to apoptosis induced by subtoxic concentrations of GTN (10 lM). This synergic effect seems to be specific to GTN, because H89 failed to potentiate cell cytotoxicity induced by other chemotherapeutic agents such as cisplatin and 5-fluorouracil. In addition, H89 did not sensitize GTN-induced cell death via PKA inhibition since the knockdown of PKA protein expression did not potentiate apoptosis. Furthermore, the combination of GTN with inhibitors of the other protein kinases also inhibited by H89, did not induce apoptosis. We also observed that GTN/H89-induced apoptosis required caspases activation and reactive oxygen species (ROS) production, since apoptosis was significantly inhibited after pre-incubation of cells with respectively the pan-caspases inhibitor Z-VAD or the ROS scavenger N-acetyl cysteine (NAC). In conclusion, a better understanding of the molecular mechanisms involved in GTN/H89-induced apoptosis in colon cancer cells may lead to the development of new cancer therapeutic strategies. doi:10.1016/j.niox.2007.09.048
C4. Expression of immune regulatory genes and involvement of nitric oxide after BCG treatment of urinary bladder cancer T. Thiel, N.P. Wiklund, P. De Verdier Department of Molecular Medicine and Surgery, Forskningscentrum M3:02, Urologlab, 171 76 Stockholm, Sweden To identify ‘‘immune genes’’ involved in the cytotoxic response seen in bladder tumours after treatment with bacillus Calmette-Guerin (BCG) and
Poster abstracts / Nitric Oxide 17 (2007) S20–S29 to study how expression of these is influenced by nitric oxide (NO). Background: High-risk bladder tumours are either surgically removed by cystectomy or treated with BCG. The exact mechanisms behind BCG treatment are not clear but it is known that the production of NO increases dramatically in the bladder after treatment. High levels of NO are cytotoxic while low levels increase cell proliferation. Approach: We used an array to identify mRNA expression of 96 different ‘‘immune genes’’ after BCG treatment. Mouse bladder cancer cells (MBT2) and macrophages (RAW264.7) were treated with BCG alone and with BCG and/or L-NAME (NO inhibitor). MBT2 cells were also treated with supernatant from untreated or BCG-treated (BCG sup) RAW cells. Results and conclusion: In total, 45 different genes showed up or down regulated mRNA expression in MBT2 cells. Cells treated with BCG sup showed different expression from cells treated with BCG only and also generally much higher levels of expression than when treated with BCG only. Cell death was more profound in cells treated with BCG sup than in cells treated with BCG only. Many genes were differently expressed in cells treated with BCG compared to cells treated with L-NAME and BCG. We conclude that factors provided by stimulated macrophages are necessary for regulation of many genes after BCG treatment and that some of these genes are differently expressed depending on the presence or absence of NO. doi:10.1016/j.niox.2007.09.049
C5. Production of NO, superoxide anion, H2O2 and MDA in vitro by B lymphocytes of B-chronic lymphocytic leukemia patients P. Djurdjevic, I. Zelen, P. Ristic, I. Jovanovic, V. Jakovljevic, D. Baskic, S. Popovic, N. Arsenijevic School Of Medicine, Sv. Markovica 69, Kragujevac, Serbia B-CLL is neoplastic disease characterized by the accumulation of monoclonal CD5+ morphologically mature, functionally immature B lymphocytes. Expansion of malignant cells clone appears to be due to an underlying defect in its ability to undergo apoptosis. The precise mechanisms underlying apoptosis are still remained unknown. It has been proposed that molecular defects such as oxidative stress, although by yet unknown mechanism, may induce the constitutive activation of several transmembrane signaling pathways that regulate the differentiation, cell cycle progression and apoptosis of B-cells. The goal of our investigation was to determine in vitro lymphocytes production of nitric oxide, superoxide anion, hydrogen peroxide and malondialdehyde in B-chronic lymphocytic leukemia patients as markers of oxidative stress. Eighteen untreated B-chronic lymphocytic leukemia patients in the A stage of disease classified by Binet, sixteen patients in the stages B and C of disease classified by Binet and twenty healthy volunteers as a control group were examined. Our results showed that in vitro lymphocytes production of superoxide anion, hydrogen peroxide and malondialdehyde was increased in B-chronic lymphocytic leukemia patients than in control group, while there were no statistical significant differences of in vitro nitric oxide production between tested groups. The result of the present study showed that oxidative stress and lipid peroxidation are accelerated in patients with B-CLL. doi:10.1016/j.niox.2007.09.050
C6. Effect of different concentrations of L-arginine on proliferation of human cervical carcinoma cell line (Hela) D. Dong Jin, T. Yaping, F. Zhengyu Department of Clinical Biochemistry, Fuxing Road 28#, PLA General Hospital, Beijing, China Nitric oxide (NO) is a molecule that plays crucial roles in many diseases, such as vascular disease, tumor, etc. L-arginine (L-arg) is one of important donors in providing NO in vivo, the dose-dependent of which may affect the
S21
NO synthesis and regulating metabolism and cytotoxicity in some extent. The increase of L-arg may elevate iNOS activation and leads to NO synthesis. As is known that in tumor cells growth and aggression, NO also has important effects which is released by tumor cells and help to itself invading to normal tissues. Our purpose is to investigate the proliferation of Hela cells affected by the different concentrations of L-arg that acts as the donor of NO. The MTT assay had been used to detect the proliferation of Hela cells and Griess assay was used to evaluate the cellular metabolites of NO derivatives, which indirectly showed the synthesis of NO. The result showed that L-arg increased NO2- concentration in culture medium, 1–30 mmol/L L-arginine inhibited the activity of Hela cells, under 50–100 mmol/L enhance the activity of the cells, but the growth style was changed from adhere to the wall to the flouting, and the number of cells was limited. L-arginine has the bi-direct effects on tumor cell proliferation, which increases exogenous NO and decreases tumor cells growth. doi:10.1016/j.niox.2007.09.051
C7. Modulation of NO biosynthesis in A-375 melanoma cells by plasma membrane cholesterol S. Durocher, B. Mutus, D. Shum, C. Hamm Department of Chemistry and Biochemistry University of Windsor, Windsor Regional Hospital, Windsor Regional Cancer Centre, 401 Sunset Ave, Windsor, Canada Nitric oxide (NO) can be both beneficial and detrimental to cancer cells. Therefore, understanding intrinsic mechanisms for augmenting and reducing levels of NO may be of interest from a therapeutic point of view. This in vitro study examines the plasma membrane (PM) cholesteroldependent effects on nitric oxide synthase isozymes in A-375 melanoma cells and normal fibroblasts. Specifically, here we show that melanoma cells produced 2-fold higher NO than fibroblasts. With both cell types NO levels were attenuated (3-fold) by the removal of PM-cholesterol with methyl-b-cyclodextrin (MbCD) and potentiated with cholesterolloaded-MbCD. We also demonstrated that PM-cholesterol could also be increased with endoplasmic reticulum (ER) stressors like thapsigargin in normal and melanoma cells and that the elevation of PM-chol was related to increased iNOS expression and NO biosynthesis. The melanoma cells became more susceptible to apoptosis as a result of ER stress. This was in agreement with previous studies. The role of NO in ER stress induced apoptosis was demonstrated by the fact that melanoma cells treated with either the iNOS inhibitor RW1400 or the chemical chaperone (reduces ER stress) 4-phenylbutyric (PBA) were less susceptible to apoptosis. Acknowledgments: Supported by a Windsor Regional Cancer Centre (WRCC) Local Investigator Award and an NSERC Discovery Grant to B.M. doi:10.1016/j.niox.2007.09.052
C8. Artemisinin derivatives induce generation of nitric oxide and reactive oxygen species in cell lines of hematopoietic origin V.B. Konkimalla, M. Blunder, E. Thedinga, B. Korn, R. Bauer, T. Efferth German Cancer Research Center, Pharmaceutical Biology (C015), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany Artemisinin is the active principle of the Chinese herb Artemisia annua L. In addition to its anti-malarial activity, artemisinin and its derivatives exert profound anti-cancer and antiviral activities [Efferth et al., J. Mol. Med. 2002; 80:233–42; Efferth et al., Mol. Pharmacol. 2003; 64:382–94]. The endoperoxide moiety in the chemical structure of artemisinin is thought to be responsible for the bioactivity. We analyzed the cytotoxicity and generation of nitric oxide (NO) and reactive oxygen species (ROS) in a panel of 10 artemisinin derivatives.
Nitric Oxide 17 (2007) S20–S29 www.elsevier.com/locate/yniox
Poster abstracts C1. Nitric oxide donors for treating IBD and related diseases P. Assaf, J. Tarsis Renopharm Ltd., POB 2252, Nazareth, Israel Nitric oxide is a ubiquitous molecule involved in a variety of biological processes. In recent years, nitric oxide (NO), a gas previously considered to be potentially toxic chemical, has been established as a diffusible universal messenger that mediates cell cell communication throughout the body. The specific action of NO depends on its enzymatic sources namely neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) and all three isoforms have been localized in the gastrointestinal tract. Constitutive synthesis of NO by nNOS or eNOS isoforms is involved in the maintaining of the gastrointestinal mucosal integrity through modulation of gastric mucosal blood flow, epithelial secretion and barrier function. However, NO has also been associated with the initiation and maintenance of inflammation in human inflammatory bowel disease (IBD). Recent studies on animal models of experimental IBD have shown that constitutive and inducible NO production seems to be beneficial during acute colitis, but sustained up-regulation of NO is detrimental. This fact is also supported by studies on mice genetically deficient in various NOS isoforms. However, the mechanism by which NO proceeds from being an indispensable homeostatic regulator to a harmful destructor remains unknown. Recently, results from our laboratory show that our lead compound, a nitric oxide donor, when compared to 5-ASA, is more effective for the treatment of ulcerative colitis-IBD. doi:10.1016/j.niox.2007.09.046
C2. iNOS down-regulation induced by flavopiridol, hyperforin and polyphenols in CLL cells is a caspase-dependent mechanism C. Billard, C. Quiney, A. Ensaf, J.P. Kolb Centre de Recherche des Cordeliers, Equipe 18, UMR 872 INSERM, Universite´s Pierre et Marie Curie et Paris Descartes, 15 Rue de l ’Ecole de Me´decine, 75006 Paris, France Chronic lymphocytic leukemia (CLL) is characterized by a remarkable apoptosis deficiency of the leukemic cells rather than lymphoproliferation deregulation. We previously reported that iNOS is constitutively expressed in CLL cells and that the NO produced has anti-apoptotic properties in these cells possibly through S-nitrosylation of the active site of caspases. Flavopiridol and various plant-derived compounds such as polyphenols or hyperforin are able to induce the apoptosis of CLL cells ex vivo. We have shown that these compounds share the property to inhibit iNOS expression, suggesting that this effect could be a molecular switch contributing to apoptosis induction. However, flavopiridol-induced iNOS downregulation is totally prevented by z-VAD-fmk, a general caspase inhibitor, demonstrating that this effect is caspase-dependent. No cleavage fragment of iNOS was yet detected upon treatment of CLL cells with flavopiridol or incubation of recombinant iNOS with caspase-3, favoring the idea that
doi:10.1016/j.niox.2007.09.045
iNOS is not a caspase substrate. Our data indicate that the inhibition of the NO pathway is not a primary event in apoptosis triggering, but is rather one of the effector mechanisms of the apoptotic machinery triggered by flavopiridol and other plant-derived molecules in CLL cells. doi:10.1016/j.niox.2007.09.047
C3. Mechanisms of human colon cancer cells sensitization to nitric oxideinduced apoptosis M. Cortier, J.F. Jeannin, A. Bettaieb UMR866, INSERM, Ecole Pratique des Hautes Etudes, Universite´ de Bourgogne, Faculte´ de me´decine et de pharmacie, 7 Bd Jeanne d’Arc, Dijon, France We have previously reported that the nitric oxide donor glyceryl trinitrate (GTN) induced death of human colon cancer cells when used at high concentrations (500 lM). However, low doses of GTN failed to induce cell death. Then we explored whether GTN could induce colon cancer cells death at lower concentrations when combined with inhibitors of various proteins including MAPK. Among these inhibitors, the H89 {N-[2-(pBromo cinnamylamino) ethyl] -5- isoquinoline sulfonamide 2HCL}, a well known inhibitor of protein kinases including PKA, sensitized the human SW480 colon cancer cells to apoptosis induced by subtoxic concentrations of GTN (10 lM). This synergic effect seems to be specific to GTN, because H89 failed to potentiate cell cytotoxicity induced by other chemotherapeutic agents such as cisplatin and 5-fluorouracil. In addition, H89 did not sensitize GTN-induced cell death via PKA inhibition since the knockdown of PKA protein expression did not potentiate apoptosis. Furthermore, the combination of GTN with inhibitors of the other protein kinases also inhibited by H89, did not induce apoptosis. We also observed that GTN/H89-induced apoptosis required caspases activation and reactive oxygen species (ROS) production, since apoptosis was significantly inhibited after pre-incubation of cells with respectively the pan-caspases inhibitor Z-VAD or the ROS scavenger N-acetyl cysteine (NAC). In conclusion, a better understanding of the molecular mechanisms involved in GTN/H89-induced apoptosis in colon cancer cells may lead to the development of new cancer therapeutic strategies. doi:10.1016/j.niox.2007.09.048
C4. Expression of immune regulatory genes and involvement of nitric oxide after BCG treatment of urinary bladder cancer T. Thiel, N.P. Wiklund, P. De Verdier Department of Molecular Medicine and Surgery, Forskningscentrum M3:02, Urologlab, 171 76 Stockholm, Sweden To identify ‘‘immune genes’’ involved in the cytotoxic response seen in bladder tumours after treatment with bacillus Calmette-Guerin (BCG) and
Poster abstracts / Nitric Oxide 17 (2007) S20–S29 to study how expression of these is influenced by nitric oxide (NO). Background: High-risk bladder tumours are either surgically removed by cystectomy or treated with BCG. The exact mechanisms behind BCG treatment are not clear but it is known that the production of NO increases dramatically in the bladder after treatment. High levels of NO are cytotoxic while low levels increase cell proliferation. Approach: We used an array to identify mRNA expression of 96 different ‘‘immune genes’’ after BCG treatment. Mouse bladder cancer cells (MBT2) and macrophages (RAW264.7) were treated with BCG alone and with BCG and/or L-NAME (NO inhibitor). MBT2 cells were also treated with supernatant from untreated or BCG-treated (BCG sup) RAW cells. Results and conclusion: In total, 45 different genes showed up or down regulated mRNA expression in MBT2 cells. Cells treated with BCG sup showed different expression from cells treated with BCG only and also generally much higher levels of expression than when treated with BCG only. Cell death was more profound in cells treated with BCG sup than in cells treated with BCG only. Many genes were differently expressed in cells treated with BCG compared to cells treated with L-NAME and BCG. We conclude that factors provided by stimulated macrophages are necessary for regulation of many genes after BCG treatment and that some of these genes are differently expressed depending on the presence or absence of NO. doi:10.1016/j.niox.2007.09.049
C5. Production of NO, superoxide anion, H2O2 and MDA in vitro by B lymphocytes of B-chronic lymphocytic leukemia patients P. Djurdjevic, I. Zelen, P. Ristic, I. Jovanovic, V. Jakovljevic, D. Baskic, S. Popovic, N. Arsenijevic School Of Medicine, Sv. Markovica 69, Kragujevac, Serbia B-CLL is neoplastic disease characterized by the accumulation of monoclonal CD5+ morphologically mature, functionally immature B lymphocytes. Expansion of malignant cells clone appears to be due to an underlying defect in its ability to undergo apoptosis. The precise mechanisms underlying apoptosis are still remained unknown. It has been proposed that molecular defects such as oxidative stress, although by yet unknown mechanism, may induce the constitutive activation of several transmembrane signaling pathways that regulate the differentiation, cell cycle progression and apoptosis of B-cells. The goal of our investigation was to determine in vitro lymphocytes production of nitric oxide, superoxide anion, hydrogen peroxide and malondialdehyde in B-chronic lymphocytic leukemia patients as markers of oxidative stress. Eighteen untreated B-chronic lymphocytic leukemia patients in the A stage of disease classified by Binet, sixteen patients in the stages B and C of disease classified by Binet and twenty healthy volunteers as a control group were examined. Our results showed that in vitro lymphocytes production of superoxide anion, hydrogen peroxide and malondialdehyde was increased in B-chronic lymphocytic leukemia patients than in control group, while there were no statistical significant differences of in vitro nitric oxide production between tested groups. The result of the present study showed that oxidative stress and lipid peroxidation are accelerated in patients with B-CLL. doi:10.1016/j.niox.2007.09.050
C6. Effect of different concentrations of L-arginine on proliferation of human cervical carcinoma cell line (Hela) D. Dong Jin, T. Yaping, F. Zhengyu Department of Clinical Biochemistry, Fuxing Road 28#, PLA General Hospital, Beijing, China Nitric oxide (NO) is a molecule that plays crucial roles in many diseases, such as vascular disease, tumor, etc. L-arginine (L-arg) is one of important donors in providing NO in vivo, the dose-dependent of which may affect the
S21
NO synthesis and regulating metabolism and cytotoxicity in some extent. The increase of L-arg may elevate iNOS activation and leads to NO synthesis. As is known that in tumor cells growth and aggression, NO also has important effects which is released by tumor cells and help to itself invading to normal tissues. Our purpose is to investigate the proliferation of Hela cells affected by the different concentrations of L-arg that acts as the donor of NO. The MTT assay had been used to detect the proliferation of Hela cells and Griess assay was used to evaluate the cellular metabolites of NO derivatives, which indirectly showed the synthesis of NO. The result showed that L-arg increased NO2- concentration in culture medium, 1–30 mmol/L L-arginine inhibited the activity of Hela cells, under 50–100 mmol/L enhance the activity of the cells, but the growth style was changed from adhere to the wall to the flouting, and the number of cells was limited. L-arginine has the bi-direct effects on tumor cell proliferation, which increases exogenous NO and decreases tumor cells growth. doi:10.1016/j.niox.2007.09.051
C7. Modulation of NO biosynthesis in A-375 melanoma cells by plasma membrane cholesterol S. Durocher, B. Mutus, D. Shum, C. Hamm Department of Chemistry and Biochemistry University of Windsor, Windsor Regional Hospital, Windsor Regional Cancer Centre, 401 Sunset Ave, Windsor, Canada Nitric oxide (NO) can be both beneficial and detrimental to cancer cells. Therefore, understanding intrinsic mechanisms for augmenting and reducing levels of NO may be of interest from a therapeutic point of view. This in vitro study examines the plasma membrane (PM) cholesteroldependent effects on nitric oxide synthase isozymes in A-375 melanoma cells and normal fibroblasts. Specifically, here we show that melanoma cells produced 2-fold higher NO than fibroblasts. With both cell types NO levels were attenuated (3-fold) by the removal of PM-cholesterol with methyl-b-cyclodextrin (MbCD) and potentiated with cholesterolloaded-MbCD. We also demonstrated that PM-cholesterol could also be increased with endoplasmic reticulum (ER) stressors like thapsigargin in normal and melanoma cells and that the elevation of PM-chol was related to increased iNOS expression and NO biosynthesis. The melanoma cells became more susceptible to apoptosis as a result of ER stress. This was in agreement with previous studies. The role of NO in ER stress induced apoptosis was demonstrated by the fact that melanoma cells treated with either the iNOS inhibitor RW1400 or the chemical chaperone (reduces ER stress) 4-phenylbutyric (PBA) were less susceptible to apoptosis. Acknowledgments: Supported by a Windsor Regional Cancer Centre (WRCC) Local Investigator Award and an NSERC Discovery Grant to B.M. doi:10.1016/j.niox.2007.09.052
C8. Artemisinin derivatives induce generation of nitric oxide and reactive oxygen species in cell lines of hematopoietic origin V.B. Konkimalla, M. Blunder, E. Thedinga, B. Korn, R. Bauer, T. Efferth German Cancer Research Center, Pharmaceutical Biology (C015), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany Artemisinin is the active principle of the Chinese herb Artemisia annua L. In addition to its anti-malarial activity, artemisinin and its derivatives exert profound anti-cancer and antiviral activities [Efferth et al., J. Mol. Med. 2002; 80:233–42; Efferth et al., Mol. Pharmacol. 2003; 64:382–94]. The endoperoxide moiety in the chemical structure of artemisinin is thought to be responsible for the bioactivity. We analyzed the cytotoxicity and generation of nitric oxide (NO) and reactive oxygen species (ROS) in a panel of 10 artemisinin derivatives.