Anti-myeloma Effects Of Artesunate And Its Mechanism

Multiple myeloma (MM) is a malignant disease of plasma cells which characterised by over-secretion of paraprotein, anaemia,lytic bone lesions,and kidney dysfunction. In recent years,although the median survival time of patients with multiple myeloma has improved from seven months to five years with the treatment of new chemotherapy drugs such as a protein enzyme inhibitor,thalidomide,as well as high-dose chemotherapy with peripheral blood stem cells (PBSC) support,MM is still an incurable hematological malignancy due to the eventual emergence of drug resistance. Faced with these major limitations,we have an urgent need for new drugs.In recent years,most researches try to explore the traditional chinese medicinal(TCM) method to treat muitiple myeloma.Dozens of traditional chinese medicinal materials have been found to have good effect on muitiple myeloma. With the development of novel treatment options for malaria with a very good tolerability and very few side effects,the nature compound artemisinin(ARS) and its derivatives have been widely applied to clinical use because of its potent activity against drug-resistant strains of plamodium fciparum and plasmodium vivax. Alone with the progress of research, it was found that artemisinin compounds also have many other important pharmacological effect,such as anti-virus,anti-Schistosoma japonicum, immune regulation,anti-arrhythmia and anti-tumor. In particular, its anti-tumor effect has drawn many researchers'attention. Using a panel of 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute (USA), artesunate was most active against leukemia and colon cancercell lines(mean50% inhibition concentration (IC50) values:1 and 2μM, respectively).Non-small cell lung cancer celllines showed the highest mean IC50 value (26μM) indicating the lowest sensitivity to artesunate in this test panel. Intermediate IC50 values were obtained for melanomas,breast,ovarian, prostate,CNS,and renal cancer cell lines.Artemisinins and its derivatives revealed profound cytotoxic activity against tumor cells In many other experiments.A salient feature of artemisinins and its derivatives is the lack of cross- resistance with conventional chemotherapy drugs.It is not only active against multidrug resistant Plasmodium strains,but also active against drug-resistant tumor cells.Another propitious characteristics is its good tolerability and the lack of significant adverse side effects.Artemisinins may be promising for the treatment of refractory multiple myeloma. In this study, mouse myeloma cells SP2/0 were to be treated as research objects,we tried to explore the anti-myeloma effects of artesunate and its mechanism through theestablishment of multidrug-resistant mouse myeloma cell lines and solid tumors.This paper was divided into the following three parts:Part one Artesunate induces the apoptosis and inhibits the proliferation of SP2/0 by blocking nuclear factor kappa B signal pathway. Objective To investigate in vitro the effects of artesunate at different concentrations on the proliferation inhibition and apoptosis induction in mouse myeloma cell line SP2/0 cells and try to explain its mechanism preliminarily.Method The growing inhibition of Artesunate on SP2/0 cells was studied by MTT method after the cells were treated with Artesunate at different concentrations for 24 to 72 hours;The ratio of apoptotic cells and the change of cells cycle was analyzed with flow cytometry and AnnexinⅤ/PI double staining after artesunate treatment for 24 and 48 hours;The morphology change of apoptosis cells was observed under the microscope with conventional Giemsa staining,DAPI fluorescence staining and under transmission electron microscope;DNA ladder of apoptotic cells was checked on agarose gel electrophoresis;The expression of nuclear factor kappa B p65 (NF-κBp65) protein in nucleus and the inhibitor of NF-κB (IκBα) in cytoplasm was measured by western blotting;ArraystarTM transcription factor kit was used to measure NF-κB p65 transcription activity.Result 1 Artesunate inhibited the proliferation of SP2/0 cells in dose- and time -dependent manners.By artesunate-treatment for 24 hours,artesunate at the concentration of 2μg/ml inhibited the proliferation of SP2/0 cells and the inhibition rate was 32.97%.The inhibitory effect on cell proliferation enhanced with the increasing of artesunate concentration. On the other hand,the inhibitory effect of artesunate at different concentration enhanced,according to the treatment time extended.After 72 hours' treatment, the inhibition rate in SP2/0 cells treated with 40μg/ml artesunate was 91.86%.The growth inhibition effect of artesunate was in a time-dependence (P=0.0000) and dose-dependence (P=0.0023) manner,but its effect was weaker than adriamycin under the same conditions.2 There were no morphological sign of apoptosis in SP2/0 cells without artesunate treatment. The cells were plump, the cytoplasm was abundant and the cell nucleus was complete and homogeneous.Cells had no fluorescent labeling under the fluorescence microscope.Observed under the transmission electron microscope,the chromatin was delicate and homogeneous,the nucleolus was clear,the organelles complete and the microvilli on the cells were clear and visible. While after artesunate treatment,SP2/0 cells shrank and displayed the morphological sign of apoptosis:chromatin condensation, marginalization,vacuole or crescent shape,nuclear pyknosis and fragmentation, the appearance of apoptotic bodies.Under the transmission electron micros- cope,it was shown that the microvilli on the cells disappeared and structure of the organelles was incomplete.3 After treated with artesunate for 24 hours,180 to 200bp DNA bands extracted from the SP2/0 cells appeared on agarose gel electrophoresis.DNA ladder also appeared and they were multiple small bands. There were intervals between the fragments,which were the duplicated DNA fracture among the nucleosomes.The cells in control group did not show the phenomenon of DNA degradation.4 After treated with 2μg/ml artesunate for 24 hours,the apoptosis rate in SP2/0 cells increased statistically significant,compared with that in SP2/0 cells without artesunate treatment (P<0.01). The apoptosis rate fluctuated between 10% and 20%.The apoptosis rate fluctuated between 50% and 80% after treated for 48 hours. The apoptosis rate in SP2/0 cells treated with 40μg/ml artesunate was 78.7%(compared with that of 0μg/ml ,P<0.05). The apoptosis induction effect of artesunate was remarkable.5 After cultured with different concentrations of artesunate for 24 and hours, the number of SP2/0 cells in each cell cycle phase was detected by flow cytometry. The results showed that the cell number increased in G0/G1 phase, while decreased in G2/M and S phase statistically significant, when compared with that in SP2/0 without artesunate treatment.6 The result of western blotting showed that, the expression level of NF-κB p65 protein in SP2/0 cells decreased gradually with the extension of time of artesunate treatment.The change in each group was statistically significant after artesunate treatment. The gray value was 5294.23±240.25 in cells without artesunate treatment,2609.13±126.32 in cells treated with artesunate for 24 hours,558.58±111.25 for 48 hours,552.83±98.56 for 72 hours and 545.9±79.62 for 96 hours,respectively.The results also demonstrated an increase in IκBαprotein level in SP2/0 cells with the extension of time of artesunate treatment.The gray value was 6986.47±356.96 in cells without artesunate treatment,7207.58±256.24 in cells with artesunate treatment for 24 hours,7936.46±126.37 for 48 hours,8212.23±246.31 for 72 hours,and 9227.30±330.12 for 96 hours.7 The transcriptional activity of NF-κB p65 decreased significantly after ART of 10μg/ml treatment from 24 to 96 hours(P<0.01). The inhibition rate was 66.3%in cells treated with artesunate for 24 hours,68.4%for 48 hours,76.0%i for 72 hours and 89.6%for 96 hours,respectively.The effect was time-dependent the decreasing was most significant in SP2/0 cells treated with artesunate for 96 hours.Conclusion Artesunate could significantly inhibit proliferation and induce apoptosis of SP2/0 cells by blocking of NF-κB signal pathway.It is promising that artesunate will be useful in the treatment of patients with multiple myeloma.Part two Establishment of multidrug-resistant mouse myeloma cell line SP2/0/ADM and the mechanism of artesunate overcoming multidrug resistanceObjective To explore the mechanism of artesunate overcoming multi- drug resistance through establishment of multidrug-resistant mouse myeloma cell line SP2/0/ADMMethod A mltidrug-resistant mouse myeloma cell line SP2/0 /ADM was established from the parental cell line SP2/0 by exposing it to adriamycin with concentrations increased gradually over a period of 7 months;Its morpho- logical changes were observed by light microscopy;Population doubling time was calculated according to Patterson formula by cell counting;Flow cytometry cytometry (FCM) was performed to determine cell cycle and assess the expres- sion of P-glycoprotein (P-gp) by efflux the fluorescent dye rhodamine 123; multidrug resistance to multianticancer agents was evaluated by MTT assay, The same method was used to determine SP2/0/ADM cell proliferation of ART treatment for 72 hours and observe effect on reversal multidrug resistance of ART at 0.3μg/ml(IC20);After treated with 10μg/ml artesunate for 24 to 96 hours, real-time quantitative PCR was used to detect the expession of MDR1 mRNA and western blotting was used to detect the expression of P-gp protein.Result1 In the course of induction,SP2/0/ADM cells grew slowly.Most cells did not have significant morphological changes.A few irregular cells with increased cytoplasm granular could be found occasionally.In light microscope, as its parental cells, SP2/0/ADM cells were round and plump with round nuclei,nomal cytoplasm granular and delicate,homogeneous chromatin and clear border.All cells with homogeneous attributes were single-layer growth in culture flask. the doubling time of SP2/0/ADM (35.28±7.56 hours) was not statistically significant when compared with that of SP2/0 cell(33.84±5.68 hours)(P>0.05).The ratio of cells in G0/G1 phase was not statistically significant(P>0.05),but the ratio of S phase increased (P<0.05)while that of G2/M phase decreased(P<0.05) when compared with SP2/0 cell.2 SP2/0/ADM cells developed drug-resistance not only to adriamycin but also to other chemotherapeutics,such as mitoxantrone,etoposide,methotre- exate with the resistance indexes of 22.7 for adriamycin,4.2 for mitoxantrone, 2.9 for etoposide and 5.3 for methotrexate,respectively.3 The function of p-glycoprotein was assessed with flow cytometry by monitoring of rhodamine-123 uptake. The fluorescent intensity of SP2/0/ADM cells was weaker than that of SP2/0 cells due to the increased P-gp protein discharging most rhodamine-123.4 ART inhibited the proliferation of SP2/0 cells in dose-dependent manners(P<0.05). According to the result,0.3μg/ml ART was chosen to reverse the resistance of SP2/0/ADM cells for its IC20 value was 0.29μg/ml.The result showed the IC50 values of adriamycin were 17.80μg/ml and 4.27μg/ml in the absence and presence of 0.3μg/ml ART with the reversal index of 4.16(P<0.05).5 The result of PCR showed that the expression level of MDR1 mRNA in SP2/0/ADM cells decreased gradually with the extensionof time of artesunate treatment,The change in each group was statistically significant after artesunate treatment(P<0.05),2-△△Ct was 0.0887±0.0023 in cells treated with artesunate for 24 hours,0.0556±0.0036 for 48 hours,0.0338±0.0031 for 72 hours and 0.0186±0.0014 for 96 hours,respectively.6 The result of western blotting showed that the expression level of P- coprotein protein in SP2/0/ADM cells decreased gradually with the extension of time of artesunate treatment.The change in each group was statistically significant after artesunate treatment. the gray value was 6568.23±156.95 in cells without artesunate treatment(P<0.05),4459.13±297.12 in cells treated with artesunate for 24 hours,4218.31±153.25 for 48 hours,3558.58±241.32 for 72 hours and 3024.83±157.35 for 96 hours, respectively.Conclusion SP2/0/ADM cell line was a stable multidrug resistance cell line;ART could reverse SP2/0/ADM cell drug-resistance by inhibiting the expression of MDR1/P-gp gene.Part three The experimental research of artesunate inhibiting angiogenesisObjective To study the effects of artesunate inhibiting angiogenesis in vitro and in vivo.Method The effect of ART on the migration of human microvascular endothelial cell (HMVEC) was investigated by establishing three-dimensional culture systems;The effect of ART inhibiting angiogenesis was observed in chick chorilallantoic membranes(CAM) model;The effect of artesunate inhibiting tumor growth was investigated by establishing SP2/0 solid tumor in BALB/C mice;The transplanted tumor was used to study anti-angiogenesis effect of ART by CD34 immunohistochemical,detect the level of VEGF mRNA by Real time quantitative PCR,measure the expession of VEGF protein by western blotting.Result1 ART inhibited the sprouts of HUVEC in a dose-dependent manner. After ART at different concentrations treatment for five days, the mean sprout length of HUVEC was 10.3±1.03μm in control,8.2±1.5μm in 10μg/ml ART group,5.3±1.8μm in 20μg/ml ARTgroup,2.68±0.95μm in 40μg/ml ART group, respectively.2 ART significantly suppressed blood vessels'formation in chickembryo choriallantoic membrane (CAM). In control group,blood vessels surrounding gelatin sponge grew well.It was tree-like and well-branched.But in ARTgroup, avascular area surrounding gelatin sponge could be found,its blood vessels were in amount than those of the control group.3 ART inhibited the growth of SP2/0 transplantable tumor(P<0.05), the inhibition rate was 17.83% in artesunate group of 50m g/kg.d-1,36.54% in artesunate group of 100mg/kg.d-1 and 47.95% in artesunate group of 200 mg/ kg.d-1 ,respectively.The inhibition effect of 200mg/kg.d-1 ART group was prominent,but it was weaker than that of cyclophosphamide group of 10 mg/kg. d-1. 4 The result of CD34 immunohistochemical showed that ART could inhibit angiogenesis of tumor tissue in a dose-dependent manner(P<0.05).The mean optical density(MOD) was 0.811±0.059 in control,0.602±0.049 in artesunate group of 50mg /kg.d-1,0.531±0.087 in artesunate group of 100mg /kg.d-1,0.529±0.026 in artesunate group of 50mg /kg.d-1 and 0.201±0.018 in cyclophosphamide group of 10mg /kg.d-1,respectively.5 The result of PCR showed that,the expression level of VGEF mRNA mRNA In transplantable tumor tissue decreased gradually with the increase of dose of artesunate and cyclophosphamide treatment. The change in each group was statistically significant(P<0.05),2-△△C t values was 0.1086±0.0012 in artesunate group of 50mg /kg.d-1, 0.0838±0.0021in artesunate group of 100mg /kg.d-1, 0.0456±0.0054 in artesunate group of 200mg /kg.d-1, 0.0288±0.0043 in cyclophosphamide group of 10mg /kg.d-1,respectively.6 The result of Western blotting showed that,the expression level of VEGF protein in transplantable tumor tissue decreased gradually with the increase of dose of artesunate and cyclophosphamide treatment. The change in each group was statistically significant(P<0.05).The gray value was 8924.47±250.35 in control,8126.35±345.32 in artesunate group of 50mg /kg.d-1, 4527.13±112.96 in artesunate group of 100mg /kg.d-1,4051.72±168.24 in artesunate group of 50mg /kg.d-1 and 2773.77±302.56 in cyclophosphamide group of 10mg /kg.d-1, respectively.Conclusion ART can inhibit growth of SP2/0 solid tumors in vivo,the mechanism is relataed with anti-agiogenesis by supressing the expression of VEGF gene; Artesunate may be an effective anti-myeloma drug. Summary This paper showed that artesunate maybeis an effective anti- myeloma drug,its mechanism was relataed with proliferation inhibition, apoptosis induction, reversal multidrug resistance and anti-agiogenesis.As anti-myeloma agents used in clinical, It will have widespread prospect.