Cui-Rong Zhao

Riccardin D, a novel macrocyclic bisbibenzyl, induces apoptosis of human leukemia cells by targeting DNA topoisomerase II

SummaryWe studied the effect of riccardin D, a macrocyclic bisbibenzyl, which was isolated from the Chinese liverwort plant, on human leukemia cells and the underlying molecular mechanism. Riccardin D had a significant antiproliferative effect on human leukemia cell lines HL-60, K562 and its multidrug resistant (MDR) counterpart K562/A02 cells, but showed no effect on the topoisomerase-II-deficient HL-60/MX2 cells, as measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pBR322 DNA relaxation assay revealed that riccardin D selectively inhibited the activity of topoisomerase II (topo II). The suppression of topo II activity by riccardin D was stronger than that of etoposide, a known topo II inhibitor. After treatment with riccardin D, nuclear extracts of leukemia K562 and K562/A02 cells left the majority of pBR322 DNA in a supercoiled form. Further examination showed that riccardin D effectively induced HL-60, K562 and K562/A02 apoptosis as evidenced by externalization of phosphatidylserine and formation of DNA ladder fragments. The activation of cytochrome c, caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) was also enhanced, as estimated by Western blot analysis. By contrast, riccardin D was unable to induce apoptosis in the topoisomerase-II-deficient HL-60/MX2 cells, indicating that the induction of apoptosis by riccardin D was due to the inhibition of topo II activity. In addition, riccardin D was able to significantly decrease P-glycoprotein (P-gp) expression in K562/A02 cells. Taken together, our data demonstrate that riccardin D is a novel DNA topo II inhibitor which can induce apoptosis of human leukemia cells and that it has therapeutic potential for both regular and MDR strains of leukemia cells.

Des-γ-carboxyl prothrombin induces matrix metalloproteinase activity in hepatocellular carcinoma cells by involving the ERK1/2 MAPK signalling pathway

Des-γ-carboxy prothrombin (DCP), an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells, has been shown to be associated with the biological malignant potential of HCC. The aim of this study was to evaluate the effect of DCP on HCC cell growth and metastasis, and to explore the underlying molecular mechanisms. DCP significantly stimulated HCC cell growth, as measured by cell counting kit-8 assay. Transwell chamber assay showed that DCP increased HCC cell migration through reconstituted extracellular matrix (Matrigel). Gelatin zymography assay and Western blot analysis demonstrated that DCP increased the secretion and expression of matrix metalloproteinase (MMP)-2 and MMP-9 in the supernatant of cultured HCC cells and on tumour cell membranes. DCP was found to bind to the cell surface receptor Met, resulting in Met phosphorylation and subsequent activation of the epidermal growth factor receptor (EGFR). Western blot analysis demonstrated that DCP stimulated a sequential kinase phosphorylation cascade including ERK1/2, MEK1/2 and c-Raf, indicating activation of the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK1/2 MAPK) signalling pathway. Furthermore, blocking ERK1/2 MAPK activation with ERK1/2 inhibitor PD98059 essentially abolished the DCP-induced MMP-2 and MMP-9 activity, confirming the signalling pathway of DCP stimulation. Taken together, these results suggested that DCP stimulates HCC growth and promotes HCC metastasis by increasing the activity of MMP-2 and MMP-9 through activation of the ERK1/2 MAPK signalling pathway.

Inhibition of angiogenesis involves in anticancer activity of riccardin D, a macrocyclic bisbibenzyl, in human lung carcinoma

Riccardin D is a novel macrocyclic bisbibenzyl compound extracted from Chinese liverwort plant Dumortiera hirsuta. Our previous studies showed that riccardin D is a DNA topo II inhibitor and has therapeutic potential for treatment of cancers. In this combined in vitro and in vivo study, we examined the inhibitory effects of riccardin D on tumor angiogenesis and the subsequent effect of anticancer activity was evaluated. Incubation with riccardin D weakly inhibited the proliferation of human umbilical vascular endothelial cells (HUVEC) as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The scratch wound experiment showed that riccardin D effectively decreased the motility and migration of HUVEC cells. Riccardin D inhibited the formation of capillary tube as demonstrated by decrease of branch points formed by HUVEC cells on 3-D Matrigel. We examined the levels of angiogenic factors including vascular endothelial growth factor (VEGF), VEGF receptor 2, epidermal growth factor receptor (EGF receptor), and matrix metalloproteinase (MMPs) in HUVEC cells. The expressions of VEGF, phospho-VEGF receptor 2, EGF receptor and MMP-2 were significantly reduced by riccardin D as estimated by Western blot assay and real-time quantitative PCR analysis. The decrease of VEGF was also detected in riccardin D-treated human lung cancer H460 cells. The anticancer activity of riccardin D was then evaluated in a mouse model in which riccardin D delayed the growth of H460 xenografts without obvious toxicity to animals after three weeks injection. To evaluate the role of antiangiogenesis of riccardin D in mice, CD34 immunohistochemical staining was employed to analyze the mean vascular density in H460 xenograft tissues. The number of blood vessels was significantly decreased after riccardin D treatment. These results suggest that riccardin D display the inhibitory effect on growth of human lung carcinoma cells and that the inhibition of angiogenesis may involve in anticancer activity of riccardin D.

Inhibitory effect of riccardin D on growth of human non-small cell lung cancer: in vitro and in vivo studies.

Riccardin D is a macrocyclic bisbibenzyl compound extracted from liverwort plant Dumortiera hirsuta. Our previous study showed that riccardin D induced apoptosis of human leukemia cells by targeting DNA topoisomerase II (topo II). Riccardin D has been considered as a novel DNA topo II inhibitor and potential chemotherapeutic agent for treatment of cancers. In this study, we evaluated the inhibitory effects of riccardin D on growth of human non-small cell lung cancer (NSCLC) both in vitro and in vivo. Riccardin D effectively inhibited the proliferation of NSCLC cells as estimated by the MTT assay. Further examination showed that the ability of invasion and migration of NSCLC cells was suppressed on exposure to riccardin D as estimated by the assays of scratch and transwell chamber. The anticancer activity of riccardin D was verified in mice bearing human NSCLC H460 xenografts. Riccardin D injection produced a 44.5% inhibition of cancer growth without apparent signs of toxicity to animals. Further, riccardin D induced apoptosis of NSCLC cells as evidenced by the increases of cells with externalization of phosphatidylserine and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive in H460 xenografts. The analysis of apoptotic proteins showed that riccardin D activated the caspases cascade signaling pathway as demonstrated by the increases of cleaved caspase-3 and cleaved PARP in NSCLC cells in vitro and in H460 xenografts in mice. The pBR322 DNA relaxation assay indicated that riccardin D inhibited the activity of DNA topo II in H460 and A549 cells, suggesting the mechanism of riccardin D in induction of NSCLC apoptosis. In addition, we studied the activity and expression of matrix metalloproteinases (MMPs) in NSCLC cells. The activities of MMP-2 and MMP-9 in supernatants of NSCLC cells were suppressed on exposure to riccardin D as estimated by gelatin zymography assay. The inhibitory effects of riccardin D on expressions of MMP-2 and MMP-9 were verified in H460 xenografts in mice and the decreases of vascular endothelial growth factor (VEGF) and Erk1/2 might associate with the inhibition of MMPs and NSCLC growth. Together, our results suggest that riccardin D has a high inhibitory effect on human NSCLC growth through induction of apoptosis.

Synthesis of indazole based diarylurea derivatives and their antiproliferative activity against tumor cell lines.

New series of indazole based diarylureas were synthesized and their anticancer activity against cancer cells H460, A549, OS-RC-2, HT-29, Lovo, HepG2, Bel-7402, SGC-7901 and MDA-MB-231 were examined. These derivatives of diarylureas, except azaindazole based diarylureas 5f, 5l and 5m, showed superior or similar activity against most of these selected cancer cell lines to the reference compound sorafenib. The effect of substituents on the indazole ring was also investigated. Derivatives with trifluoromenthy or halogen substituent on the indazole ring showed higher activity against the selected cancer cell lines than sorafenib. The acute toxicity assay showed that compounds 5a, 5b and 5i possessed lower toxicity than sorafenib. Compound 5i with 4-(trifluoromenthy)-1H-indazole and 4-(trifluoromenthy) benzene moieties exhibited the most potent anticancer activity.

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, inhibits human hepatocellular carcinoma growth through induction of apoptosis in p53-dependent way

Riccardin D-26 is a synthesized macrocyclic bisbibenzyl compound. We investigated the effect of Riccardin D-26 on human hepatocellular carcinomas. Riccardin D-26 possessed stronger activity against SMMC-7721 cells than human normal liver cells. Riccardin D-26 injection effectively delayed the growth of SMMC-7721 xenografts in mice without significant toxicity. This effect of Riccardin D-26 was associated with the status of p53 and its targets, bax and p21(Waf1)(/)(Cip1). Riccardin D-26 activated p53 expression and induced cancer cells to apoptosis through the p53-mediated transcription-dependent and -independent pathway. Overall, Riccardin D-26 may inhibit hepatocellular carcinoma growth through induction of apoptosis in p53-dependent pathway.

Inhibition of Intestinal Adenoma Formation in APCMin/+ Mice by Riccardin D, a Natural Product Derived from Liverwort Plant Dumortiera hirsuta

Background Mutation of tumor suppressor gene, adenomatous polyposis coli (APC), is the primary molecular event in the development of most intestinal carcinomas. Animal model with APC gene mutation is an effective tool for study of preventive approaches against intestinal carcinomas. We aimed to evaluate the effect of Riccardin D, a macrocyclic bisbibenzyl compound, as a chemopreventive agent against intestinal adenoma formation in APCMin/+ mice. Methods APCMin/+ mice were given Riccardin D by p.o. gavage for 7 weeks. Mice were sacrificed, and the number, size and histopathology of intestinal polyps were examined under a microscope. We performed immunohistochemical staining, western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in intestinal polyps to investigate the mechanism of chemopreventive effect of Riccardin D. Results Riccardin D treatment resulted in a significant inhibition of intestinal adenoma formation, showing a reduction of polyp number by 41.7%, 31.1% and 44.4%, respectively, in proximal, middle and distal portions of small intestine. The activity of Riccardin D against polyp formation was more profound in colon, wherein Riccardin D decreased polyp number by 79.3%. Size distribution analysis revealed a significant reduction in large-size polyps (2–3 mm) by 40.0%, 42.5% and 33.3%, respectively, in proximal, middle and distal portions of small intestine, and 77.8% in colon. Histopathological analysis of the intestinal polyps revealed mostly hyperplastic morphology without obvious dysplasia in Riccardin D-treated mice. Molecular analyses of the polyps suggested that the inhibitory effect of Riccardin D on intestinal adenoma formation was associated with its abilities of reduction in cell proliferation, induction of apoptosis, antiangiogenesis, inhibition of the Wnt signaling pathway and suppression of inflammatory mediators in polyps. Conclusions Our results suggested that Riccardin D exerts its chemopreventive effect against intestinal adenoma formation through multiple mechanisms including anti-proliferative, apoptotic, anti-angiogenic and anti-inflammatory activity.

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, inhibits human oral squamous carcinoma cells KB and KB/VCR: In vitro and in vivo studies.

BACKGROUND Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, might possess anti-cancer properties. We aimed to evaluate the efficacy of Riccardin D-26 as a candidate compound for treatment of cancers with sensitive or drug resistant cells. METHODS Experiments were performed on human oral squamous carcinoma KB cells and vincristin-selected MDR KB/VCR cells. The inhibition of cell growth was evaluated by colorimetric and clonogenic assays. The apoptotic cells were determined by the Annexin V-FITC/PI staining assay. JC-1 fluorescence probe was used to examine the mitochondria membrane potential (MMP). Further experiments were performed in nude mice bearing KB or KB/VCR xenografts. Riccardin D-26 was administered by injection for 2weeks. The specimens of KB and KB/VCR xenografts were removed for TUNEL staining and Western blotting analysis. RESULTS Riccardin D-26 significantly inhibited cancer growth in both KB and KB/VCR cells. Riccardin D-26s activity in cancer cells was greater than that in human normal liver cells. In mice, Riccardin D-26 effectively prevented the growth of KB and KB/VCR xenografts without significant toxicity. Further studies suggested that Riccardin D-26 inhibited cancer growth by inducing apoptosis in the activation of mitochondria-mediated intrinsic apoptosis pathway. Riccardin D-26 also possessed this activity in regulation of mitogen-related protein kinases such as MAPK and PI3K/Akt, which is associated with its inhibitory effect on KB/VCR cells. CONCLUSIONS Riccardin D-26 possessed an anti-proliferation activity against both sensitive KB and MDR KB/VCR cancer cells. GENERAL SIGNIFICANCE Riccardin D-26 could be a promising agent for treatment of cancers with sensitive or drug resistant cells.

SL-01, an oral gemcitabine derivative, inhibited human cancer growth more potently than gemcitabine

SL-01, an oral gemcitabine derivative, was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl)pyrazine-2-carbonyl at the N4-position on the cytidine ring of gemcitabine. Our goal in this study was to evaluate the efficacy of SL-01 on the growth of human cancers with gemcitabine as control. Experiments were performed on human non-small cell lung cancer NCI-H460 and colon cancer HCT-116 both in vitro and in vivo. In vitro assays, SL-01 significantly inhibited the growth of cancer cells as determined by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Further studies indicated that SL-01 induced the cancer cells to apoptosis showing chromatin condensation and externalization of phosphatidylserine. In in vivo studies, we evaluated the efficacy of SL-01 in nude mice bearing human cancer xenografts. SL-01 effectively delayed the growth of NCI-H460 and HCT-116 without significant loss of body weight. Molecular analysis indicated that the high efficacy of SL-01 was associated with its ability to induce apoptosis as evidenced by increase of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining cells, activation of caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) in tumor tissues. SL-01 also increased Bax/Bcl-2 ratio in cancer cells. These biological activities of SL-01 were more potential than that of gemcitabine. Based on these in vitro and in vivo results, SL-01 is proposed as a potent oral anticancer agent that may supplant the use of gemcitabine in the clinic.

Synthesis and Biological Evaluation of Oral Prodrugs Based on the Structure of Gemcitabine

A series of oral prodrugs based on the structure of gemcitabine (2′,2′‐difluorodeoxycytidine) were synthesised by introducing an amide group at the N4‐position of the cytidine ring. A total of 16 compounds were obtained, and their chemical and biological characteristics were evaluated. The half‐maximal inhibitory concentrations (IC50s) for most of these compounds were higher than that of gemcitabine in vitro. Compounds 5d and 5m, the representative compounds, were examined in terms of their physiological stabilities and pharmacokinetics. Compound 5d showed good stability in PBS and simulated intestinal fluid, and an analysis of its pharmacokinetics in mice suggested that the introduction of an amide group to gemcitabine could greatly improve its bioavailability. Further evaluation of compound 5din vivo showed that this compound possesses higher activity than gemcitabine against the growth of HepG2 human hepatocellular carcinoma cells and HCT‐116 colon adenocarcinoma cells with less toxicity to animals. These results suggest that compound 5d could be further developed as a potential oral anticancer agent for clinical applications in which gemcitabine is currently used.