Anti-tumor Activity Of The Macrocyclic Bisbibenzyl Compound Riccardin D

BackgroudCarcinogenesis is a multistep process which is involved in cell proliferation and apoptosis, cell cycle regulation, angiogenesis, cell differentiation, cell invasion and metastasis. The agents that could targetone or more of these processes should be ideal anti-cancer agents.Angiogenesis is a physiological process of micro vascular generation and growth, and plays an important role in the growth and spread of cancer. Angiogenesis involves an interplay between various regulatory proteins, proangiogenic stimuli, endothelial cell activation, as well as proliferation and migration. Currently, more than a dozen different molecules have been identified as angiogenesis-stimulating factors, including EGF receptor, VEGF, VEGF receptor and MMPs. The blockade of stimulation of angiogenic molecules could effectively decrease angiogenesis in tumor tissues. Therefore, anti-angiogenictherapy is one of the fundamental ways of cancer treatment. Telomerase is an RNA-protein complex which utilizes its RNA as a template for the addition of TTAGGG repeats to the 3’ends of chromosomes, thereby compensating for losses due to the end-replication problem. Telomerase activity has been detected in 85-95% of all human cancers and cancer cell lines, whereas adjacent normal tissue and mortal cells in culture are generally telomerase negative. Reactivation or upregulation of telomerase is found in the majority of human cancers and appears to be responsible for the limitless replicative potential of malignant cells, a hall mark of cancer.Riccardin D, a macrocyclic bisbibenzyl compound isolated from Chinese liverwort Dumortiera hirsute, has been demonstrated that possesses anti-cancer activity on many tumor cell lines. Riccardin D can significantly reduce the amount of polyps and adenomas in APCmin+ mice, indicating its activity in chemoprevention. Based on the previous study, and for the further development of Riccardin D as the anti-cancer candidate agent, we devised this project to study the anti-cancer activites and mechnisms of Riccardin D, mainly from the following aspects:cell angiogenesis and telomerase activity.1. Effect of Riccardin D on the tumor angiogenesisMethods:To investigate the effect of Riccardin D on angiogenesis, HUVEC cells were introduced in this study. MTT assay was used to test the anti-proliferation effect of Riccardin D (2.5,5,10,20,30μM) on the proliferation of HUVEC. The scratch wound experiment was used to test the effect of Riccardin D on the motility and migration of HUVEC. Capillary tube formation assay was used to test the effect of Riccardin D on the tube formation of HUVEC on a 3D matrigel. Western blot and RT-qPCR was used to examine the protein levels and mRNA levels of angiogenic factors including VEGF, P-VEGFR2 EGFR, and MMPs in HUVEC cells. The mRNA level of VEGF was also detected in Riccardin D-treated human lung cancer H460 cells. To evaluate the effect of Riccardin D on the tumor angiogenesis, a H460 xenograft mouse model was employed. Riccardin D was injected intravenously once every three days for 3 weeks, with Etoposide as the positive control drugs. The inhibition of cancer growth was defined as a ratio to the tumor weight of animals injected with nanosuspension without Riccardin D. The specimens were removed for immunohistochemical staining and antiangiogenesis evaluation.Results:MTT assay showed that Riccardin D weakly prevent HUVEC cell proliferation. In the concentration range of 2.5-30μM of Riccardin D, the rates of inhibition varied from 2.2% to a maximum of 28.5%. Scratch-wound assay showed the distances of cell migration were significantly reduced in the presence of Riccardin D in a time-and dose-dependent manner. In the range of 1.25-20μM of Riccardin D, the percentage of inhibition was increased from 3.8 to a maximum inhibition of 58.1%, for a 12 h exposure, from 5.0% to a maximum inhibition of 95.0% for a 24 h exposure. The capillary tube formation of HUVEC cells on 3-D Matrigel assay showed that the branch points of capillary tube were reduced after incubation with Riccardin D and the activity of capillary tube formation of HUVEC cells was significantly inhibited. At the concentrations of 5,10 and 20 μM of Riccardin D, the branch points of capillary tube were 78.7,59.0 and 29.5% of vehicle control, respectively. Western blot and RT-PCR showed that the expressions of angiogenic molecules, including EGF receptor, VEGF, phospho-VEGF receptor 2 and MMP-2 in HUVEC cells were significantly reduced after 48 h of Riccardin D exposure. Treatment with Riccardin D resulted in a significant decrease of MMP-2 and remarkable in pro MMP-2 more than in active MMP-2. The decrease of VEGF was also detected in Riccardin D-treated human lung cancer H460 cells. H460 xenografts mice model showed that 20 mg/kg of Riccardin D delayed the growth of H460 xenografts by 44.5%. Riccardin D treatment was generally well tolerated by mice with no obvious loss of body weight. Etoposide (20 mg/kg) inhibited the growth of H460 xenografts by 59.7%. However, a significantly decrease of body weight was observed, indicating the toxicity to animals. The number and size of blood vessel profiles were demonstrated by CD34 antigen which is considered to be a marker of capillary endothelial cells. Immunohistochemistry staining for CD34 showed that the number of blood vessels was markedly decreased in the Riccardin D-treated H460 xenograft tissues.Conclusions:These results indicated that the inhibition of angiogenesis may involve in the anticancer activity of Riccardin D.2. Effect of Riccardin D on human telomerase activityMethods:To investigate the effects of Riccardin D on human telomerase activity, experiments were performed on human breast cancer MCF-7 cells and MDA-MB-231 cells. TRAPeze(?) XL Telomerase Detection Kit was used to detect the the effects of Riccardin D on human telomerase activity in MCF-7 cells and MDA-MB-231 cells. Western blot was used to detect the protein expression of hTERT and TRF2 after incubation with Riccardin D. RT-PCR was used to detect the mRNA expression of hTERT and TRF2 after incubation with Riccardin D. Then, we test whether the inhibition of telomerase activity by Riccardin D could induce the DNA damage response. γ-H2AX foci formation was tested by immunofluorescence assay and western blot for the detection of DNA damage response activation. Western blot was used to detect the expression of p-ATM and p-Chk2 in MCF-7 and MDA-MB-231 cells after treatment with Riccardin D. In vitro, MTT assay was used to test the anti-proliferation effect of Riccardin D on MCF-7 cells and MDA-MB-231 cells. In vivo, the anti-tumor effect of Riccardin D was examined by MCF-7 cells and MDA-MB-231-D3H2LN xenografts bearing in mice. The effect of Riccardin D on cell apoptosis was estimated with Annexin V-FITC staining by using flowcytometry, western blot for the melocules was involved in apoptosis, such as cleaved PARP, cleaved caspase-3, cleaved caspase-9, bax, bcl-2, and TUNEL assay. Flowcytometry assay and western blot were used for the detection of cell cycle arrest in Riccardin D treated-MCF-7 and MDA-MB-231 cells.Results:TRAPeze(?) XL Telomerase Detection assay showed that Riccardin D significantly inhibited the telomerase activity in a does-dependent manner in MCF-7 cells and MDA-MB-231 cells. The activities of telomerase, at concentrations of 5,10 and 20μM Riccardin D, were 82.8%,48.4% and 19.3% of vehicle, respectively, in MCF-7 cells; and 87.6%,57.3% and 35.4% of vehicle, respectively, for MDA-MB-231 cells. Western blot assay showed that low levels of hTERT protein expression were detected in both MCF-7 and MDA-MB-231 cells after Riccardin D treatement. At concentrations of 2.5,5,10 and 20μM Riccardin D, the levels of hTERT in MCF-7 cells were decreased by 18.2%,21.6%,34.0% and 57.1%, respectively; the levels of hTERT in MDA-MB-231 cells were decreased by 9.8%,10.5%,28.0% and 41.6%, respectively. RT-PCR assay showed that Riccardin D significant inhibited the hTERT mRNA levels in MCF-7 and MDA-MB-231 cells. Westen blot and RT-PCR showed that Riccardin D exhibited the action of downregulated TRF2 at both protein and mRNA levels. Immuno fluorescence assay and western blot showed that Riccardin D significantly increased the γ-B2AX foci formation. Riccardin D was found to increase the phosphorylation of ATM and Chk2 in both MCF-7 and MDA-MB-231 cells by western blot. MTT assay showed that Riccardin D significantly inhibited the growth of MCF-7 cells and MDA-MB-231 cells in a dose-and time-dependent manner. In vivo, at the dosages of 20 mg/kg of Riccardin D, the inhibition rates on the growth of MCF-7 and MDA-MB-231-luc-D3H2LN exnografts were 47.2% and 38.5%, respectively. Epotoside at 20 mg/kg inhibited the growth of MCF-7 and MDA-MB-231-luc-D3H2LN exnografts by 57.5% and 49.3%. However, mice treated with Epotoside showed a significant loss in body weight. Annexin V-FITC/PI staining showed that the apoptosis rates after treatment with Riccardin D for 48 h were 32.0% and 22.1%, respectively, in MCF-7 cells and MDA-MB-231 cells. Western blot showed that the molecules involved in apoptosis, such as cleaved PARP, cleaved caspase-3, cleaved caspase-9, bax/bcl-2 was increased after incubation with Riccardin D. TUNEL staining showed that the TUNEL stained cells increased in specimen from MCF-7 and MDA-MB-231-luc-D3H2LN xenografts treated with Riccardin D. Riccardin D was found to induce the accumulation of MCF-7 cells (p53-proficient) in G1 phase, and for MDA-MB-231 cells (p53-deficient), in G2/M phase. Western blot showed that in p53-proficient MCF-7 cells, Riccardin D induced the activation of p53 and p21, which was noticeably absent in p53-deficient MDA-MB-231 cells.Conclusions:Riccardin D could inhibit the telomerase activity, resulting telomere dysfunction. Telomerase inhibition and telomere dysfunction could induce the DNA damage response, which finally leads to cell apoptosis and cell cycle arrest.In a word, Riccardin D exerts the anti-cancer activities by various pathways, mainly manifested in restraining agiogenesis and inhibiting telomerase activity to accelerate apoptosis and inducing cell cycle arrest.