Dihydroartemisinin Downregulates Vascular Endothelial Growth Factor Expression And Induces Apoptosis In Chronic Myeloid Leukemia K562 Cells

Angiogenesis, the development of new blood vessels by sprouting from pre-existing vasculature, plays a fundamental role in the neoplastic process and is essential for local progression and metastatic spread of solid tumors. Among many kinds of activators of angiogenesis, vascular endothelial growth factor (VEGF) is considered as one of the most potent angiogenic factors. The expression of VEGF has been suggested to be related to some fundamental features of solid tumors, such as growth rate, microvessel density, and the development of tumor metastasis. Recently, there are increasing evidences indicating that VEGF also plays an important role in the development and progression of chronic myeloid leukemia (CML). In addition, the VEGF concentrations in the bone marrows were found to correlate inversely with the length of survival in patients having chronic phase CML. Therefore, VEGF has been expected as an emerging target for CML therapy and some corresponding strategies have been raised including decreasing the production of VEGF, blocking the binding of VEGF to its receptors, and inhibiting VEGF receptor tyrosine kinases. In present study, we wanted to investigate the effect of artemisinin analogs on VEGF production in CML.Artemisinin, a sesquiterpene lactone isolated from the plant Artemisia annua L., and its derivatives are presently used in various countries as an antimalarial drug with little toxicity tohuman and have a potent effect on chloroquine-resistant malarial parasites. Dihydroartemisinin (DHA) is the main active metabolite of artemisinin derivatives and is more water-soluble and effective anti-malaria than artemisinin. In our previous report, we have shown that DHA inhibits the VEGF expression in solid tumor xenograft and exhibits the potent antiangiogenic effect in solid tumors. However, to our knowledge, the inhibitory effect of artemisinin analogs on VEGF expression in leukemic cells has not yet been reported. In present study, we analysed the anti-proliferation and inducing apoptosis effect of DHA on K.562 cells, and assessed the inhibitory effect on expression of VEGF in K562 cells. Besides, the conditioned medium (CM) of K562 cells pretreated with DHA was assessed for its stimulating effect on proliferation and migration of endothelial cells and angiogenesis on chicken chorioallantoic membrane (CAM) model.RESULTS1. The anti-proliferation effect of DHA in K562 cellsIn the trypan blue exclusion experiment, it showed that the proliferation of K.562 cell could be effectively inhibited after incubated with 10 u mol/1 of DHA for 36h, and the number of viable cells was decreased to 71.9% compared to that of control group. This number was continued to decrease to 48.2% and 44.8% after incubated for 48h and 72h, respectively. The presence of DHA at concentrations of more than 2 u mol/1 for 48h inhibited the growth of K562 cells in a concentration dependent manner as shown in the MTT assay. DHA at 2, 5, 10, 20 and 40 n mol/1 inhibited the growth of cells by 3.9%, 18.9%, 48.1%, 67.5% and 82.1%, respectively. The IC50 value of DHA for growth inhibition of K562 cells was 13.08 u mol/1, and 95% confidence interval was 10.02-17.07 ji mol/1.2. Morphological changes of apoptosis in K562 cellsThe Hoechst 33342, a sensitive fluorochrome to DNA, was used to assess changes in nuclear morphology following DHA treatment. The nuclei in normal cells were normal and exhibited diffused staining of the chromatin. However, after exposure to 5 M mol/1 DHA for 48 h, K562 cells underwent typical morphologic changes of apoptosis such as chromatin condensation ,margination and shrunken nucleus. While the plasma membrane remained well defined. Therefore, these morphological changes suggested the occurrence of apoptosis in K562 cells after treated with DHA.3. Percentage of cells undergoing apoptosisMoreover, the morphological alterations of DNA in DHA-treated K562 cell were supported by flow cytometry analysis. The dose-related increase in DHA-induced K562 cell apoptosis was initially analyzed using flow cytometry. After cells were treated with 2 u mol/1 DHA for 48h, there was no obvious increase of apoptotic cells by Pl-flowcytometry assay. The percentage of apoptotic cells was increased to 6.9% and 15.8% when treated with 5 and 10 V- mol/1 DHA for 48h, respectively. (PO.001)4. Qualitative analyse of VEGF expression by immunohistochemistory assayBy immunostaining, VEGF positive K562 cells could be distinguished by the brownstained cytoplasm. Most control K562 cells showed strong expression of VEGF, but the expression in experimentally treated K562 cells was weaker. No significant immunohistochemical reaction occurred in the negative control slips.5. DHA downregulates VEGF expression in K562 cellsIn order to quantitative analyse the effect of DHA on VEGF expression in K562 cells, we adopted westernblot method. We treated K562 cells in vitro with various concentrations of DHA, and found that increasing concentrations of DHA lead to a stepwise reduction in VEGF expression. Compared with vehicle control, the levels of VEGF in K562 cells were decreased by 24.0%, 37.6% and 92.7% after treated with 2, 5,10 u mol/1 of DHA for 48h, respectively (PO.05).6. Effect of DHA on VEGF mRNA expression in K562 cellsRT-PCR analysis showed that two forms of transcripts were detected, which encoded for VEGFni and VEGF|65. DHA could significantly suppress VEGF mRNA expression even at very low concentration of 2 u mol/1. The levels of VEGFi65 and VEGF,2i mRNA in K562 cells pretreated with 2 u mol/1 DHA were decreased by 27.1% and 11.7% respectively (PO.05) and were stepwise decreased in a concentration dependent manner.7. DHA decreases the VEGF secretion from K562 cellsELISA analysis was performed to determine the amount of secreted VEGF protein in conditioned media. K562 cells pretreated with DHA were grown in serum-free medium for 24h,and the secreted protein of VEGF in culture media were determined by ELISA. Compared to normal K562 cells, K562 cells pretreated with 2 u mol/1 DHA showed a 25.7% decrease of the secreted VEGF level (PO.01).8. Endothelial cell proliferation assayTo evaluate the effects of decreased level of VEGF on the proliferation of endothelial cells, CM of K562 cells were assayed for their potential on vascular endothelial cell line EA.hy926. Compared to the cells proliferation in CM from control K562 cells, the proliferation of EA.hy926 induced by the CM from K.562 cells pretreated with 2, 5, 10 u mol/1 DHA were reduced by 20.8%, 29.4% and 48.9%, respectively (P<0.05).9. Endothelial cell migration assayThe migration effect of endothelial cells in response to the CM from drug-pretreated K.562 cells was assayed with millicell culture plate inserts (Millipore). The results demonstrated that compared with the CM from control K562 cells, the migration effect of EA.hy926 induced by the CM from K562 cells pretreated with DHA was significantly reduced, and the number of migrated endothelial cell was obviously decreased.10. Results of chicken chorioallantoic membrane (CAM) assayMacroscopically, CM-loaded sponges displayed a stimulating angiogenic response, numerous microvessels converged towards the sponges in a radial pattern. The angiogenic activity was decreased in response to the CM from K562 cells pretreated with DHA in a dose-dependent manner. Compared with CM from normal K562 cells, the number of microvessels stimulated by CM from K562 cells pretreated with 2, 5, 10 u mol/1 DHA was decreased by 24.5%, 50.5% and 65.9%, respectively. Furthermore, there was no significant difference between the microvessel number of negative control group (RPMI-1640 medium alone) and that of CM group from 10 n mol/1 DHA pretreated K562 cells. (P>0.05) It implied that DHA could effectively suppress the stimulating angiogenic ability of CM by reacting with K.562 cells. Statistical analysisAll experiments were performed in triplicate unless otherwise noted, results were expressed as mean ± standard deviation. Dunnett's t test was used to compare the mean differences between samples using the statistical software SPSS version 10.0. Throughout the work, P values less than 0.05 were considered to be statistically significant.DISCUSSIONVEGF is a multifunctional cytokine that acts as both a potent inducer of vascular permeability and a specific endothelial cell mitogen. And it is commonly expressed in a wide variety of human tumor cells and has been associated with angiogenesis, growth, metastasis, and poor outcome in solid tumors. Recently, it was reported that, in addition to express VEGF, CML cells also expressed the VEGF-related receptors flk-1/KDR and Fit-1. Consequently, CML cells release VEGF, which can either bind to the receptors on their own surface and stimulate CML cell proliferation (autocrine loop) or bind to the receptors of endothelial cells and activate various functions of the cell including stimulation of growth, migration of endothelial cells and angiogenesis in the bone marrow (paracrine loop). VEGF has been recognized to play an important role in the pathogenesis of CML by autocrine and paracrine regulation, and several research groups have displayed the antileukemia effect by downregulating VEGF expression in CML cells.In present study, we assessed the effect of DHA on VEGF expression in human CML cells. K562 cell line was used for our investigation because this cell line expresses high levels of VEGF mRNA, secretes the VEGF protein and expresses the VEGF receptors. We first investigated the anti-proliferation effect of DHA in K562 cells in vitro. The results indicated that DHA could effectively inhibit the proliferation of K562 cells, and the inhibitory effect of DHA on the proliferation of K562 cells was in a dose and incubation time dependent manner. Artemisinin derivatives in range of 20-180 u mol/1 have been reported to inhibit cancer cell proliferation in vitro. Our study also showed that the IC50 value of DHA on K562 cells is 13.08 u mol/1. In order to analyse the effect of DHA on VEGF expression in K562 cells, we assessed the level of VEGF expression by immunohistochemistory and Westernblot methods;detected the form of VEGF mRNA by RT-PCR and examined the level of VEGF secreted in CM by ELISA assay. All these experiments suggested that DHA could inhibit the VEGF expression and secretion effectively in K.562 cells, even at a lower concentration^ u mol/1, PO.05).Artemisinin is a sesquiterpene lactone peroxide containing an endoperoxide moiety. Previous studies have reported that in addition to its antimalarial effect, artemisinin and derivatives also showed antitumor activity both in vitro and in vivo. The mechanism of its anti-tumor effect was focus on the endoperoxide bridge structure, which react with a ferrous iron atom to form free radical and lead to cellular destruction directly. In present study, we reported that DHA could effectively inhibit the proliferation and downregulate VEGF expression in K562 leukemic cells. Ruan, G.R. et al have reported that VEGF play an important role on the abnormal proliferation of CML cells through autocrine mechanism and suppression of the VEGF expression will inhibit the CML cell proliferation. This suggested that the inhibitory effect of DHA on VEGF expression might also contribute to its antiproliferation effect in CML cells. In addition to its antiproliferation effect on CML cells, DHA was also found to induce K562 cells apoptosis from our data. This was consistent with previous reports which indicated that the anti-tumor effect of DHA was ascribe to the rapid induction of apoptosis in cancer cells after treatment with DHA. It has been showed that elevated circulating VEGF level will confer VEGFR+ expressing tumor cells with great survival potential and resistance to apoptosis in an autocrine fashion. So, it further confirmed that the antiproliferation and inducing apoptosis effect of DHA in K562 cells maybe also partly related to the downregulation of VEGF expression.Besides stimulating CML cell proliferation through autocrine mechanism, VEGF could also bind to the receptors on the surface of vascular endothelial cells and stimulate the growth, migration of endothelial cells and the angiogenesis in the bone marrow through paracrine mechanism. So we assessed the CM of K562 cells pretreated with DHA for its stimulating effect on proliferation and migration of endothelial cells and stimulating angiogenic activity on CAM model. The result indicated that compared with CM from normal K562 cells, the proliferation and migration effects of human vascular endothelial cell were decreased in response to the CM from DHA pretreated-K562 cells. Besides, the ability of stimulating angiogenesis of CM from K562 cells pretreated with DHA was also decreased. Combinded with the results from ELISA assay, it implied that DHA could also reduce the CML angiogenesis in partly by downregulating the VEGF expression in CML cells.In summary, in addition to its antiproliferation and inducing apoptosis effects in K562 cells, we have demonstrated that DHA could also downregulate the expression of VEGF and reduce the angiogenic activity of CM from K562 cells. These data further confirmed the role of VEGF in CML through autocrine and paracrine mechanism. The results from our study together with its known low toxicity make it possible that DHA might present potential antileukemia effect as a treatment for CML therapy, or as an adjunct to standard chemotherapeutic regimens.