Macrocyclic Bisbibenzyl Compound Research Articles

Determination of the in vitro metabolic stability and metabolites of the anticancer derivative riccardin D-N in human and mouse hepatic S9 fractions using HPLC-Q-LIT-MS

Riccardin D-N (RD-N) is an aminomethylated derivative of the macrocyclic bisbibenzyl compound riccardin D (RD), which has shown stronger activity against cancer cells than RD. However, there has been no research on the metabolism of RD-N. The present study aimed to characterize the in vitro metabolism and metabolic stability of RD-N after incubation with mouse and human hepatic S9 fractions using high performance liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry (HPLC-Q-LIT-MS). Multiple ion monitoring (MIM) and multiple reaction monitoring (MRM)-information dependent acquisition-enhanced product ion (MIM/MRM-IDA-EPI) scans were used to identify the metabolites formed. MRM scans were also used to quantify the changes in the amount of RD-N and to semi-quantify the main metabolites. Twenty-eight metabolic products were detected and 25 structures were predicted. Hydroxylation, dehydrogenation, glucuronidation, and methylation were proposed to be the principle metabolic pathways in the in vitro incubation with human and mouse hepatic S9 fractions. There were differences in the number and abundance of RD-N metabolites between the human and mouse hepatic S9 fractions. RD-N was shown to have good metabolic stability. After 2 h of incubation, 44% of the original RD-N remained in the human hepatic S9 fraction compared with 22% in the mouse. The major metabolites of RD-N, M4, M8, M20 and M21, were monitored semi-quantitatively using the typical transitions. Finally, HPLC-Q-LIT-MS was used for the identification and quantitation of the metabolites of R D-N, which is a simple and efficient method to rapidly screen potential drug candidates.

Evaluation of anti-melanoma and tyrosinase inhibitory properties of marchantin A, a natural macrocyclic bisbibenzyl isolated from Marchantia species

Macrocyclic bisbibenzyl compound, marchantin A, naturally occurring in liverworts belonging to genus Marchantia, was isolated and assessed for their cytotoxicity against two different lines of human skin cells - immortalized human keratinocytes HaCaT and human malignant melanoma cells A375. The purpose of the studies was also to examine the anti-hyperpigmentation effect of marchantin A, especially used in concentrations assessed as safe to normal human keratinocytes while simultaneously toxic for malignant melanoma cells.The data showed that marchantin A was cytotoxic for A375 melanoma cells (IC50 = 11.97, 7.45 and 9.80 μg/mL), nevertheless less cytotoxic for normal keratinocytes. Assay conducted for mushroom tyrosinase activity showed also that this compound did not affect the dopachrome formation during enzymatic reaction in concentrations previously assessed as safe for normal human cell. To our knowledge this is the first report showing anti-melanoma activity of marchantin A and assessing its activity against tyrosinase. Obtained results indicate potential application of marchantin A as a potential active ingredient for pharmaceutical and dermatological products.

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

BackgroundRiccardin 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. MethodsExperiments 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. ResultsRiccardin D-26 significantly inhibited cancer growth in both KB and KB/VCR cells. Riccardin D-26's 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. ConclusionsRiccardin D-26 possessed an anti-proliferation activity against both sensitive KB and MDR KB/VCR cancer cells. General significanceRiccardin D-26 could be a promising agent for treatment of cancers with sensitive or drug resistant cells.

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 p21Waf1/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 APC(Min/+) mice by Riccardin D, a natural product derived from liverwort plant Dumortiera hirsuta.

BackgroundMutation 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.ResultsRiccardin 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.ConclusionsOur 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.

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.

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.

Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G 2/M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21 Waf1/Cip1. In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B 1, a cyclin required for progression through the G 2/M phase. Taken together, DHA induces G 2/M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.

Bisbibenzyl derivatives sensitize vincristine-resistant KB/VCR cells to chemotherapeutic agents by retarding P-gp activity

P-glycoprotein (P-gp) is known to mediate multidrug resistance (MDR) by acting as an efflux pump to actively transport chemotherapeutic agents out of carcinoma cells. Inhibition of P-gp function may represent one of the strategies to reverse MDR. We have previously reported that marchantin C (MC), a macrocyclic bisbibenzyl compound from liverworts, exerts anti-tumor activity as an antimitotic agent. This study was designed to evaluate the possible modulatory effect of MC and its three synthetic derivatives (MC1, MC2 and MC3) on P-gp in VCR-resistant KB/VCR cells. Results of the cytotoxicity assay revealed that MC was the most potent inhibitor of cell proliferation in both KB and KB/VCR cells among these four compounds, while the three MC-derived chemicals had little anti-proliferative activity under the same condition. However, in P-gp-expressing MDR cells, analysis of potency of these compounds in enhancing cytotoxicity of VCR led to the identification of MC2 as a more effective chemical on reversal of resistance. Further study showed that MC2 was able to reduce efflux of rhodamine-123, and in turn, increase the accumulation of rhodamine-123 and adriamycin in KB/VCR cells, indicating that MC2 re-sensitized cells to VCR by inhibition of the P-gp transport activity. In addition, the combination of MC2 and VCR at a concentration that does not inhibit cell growth resulted in an induction of apoptosis in KB/VCR cells. These results suggest that MC2, as a novel and effective inhibitor of P-gp, may find potential application as an adjunctive agent with conventional chemotherapeutic drugs to reverse MDR in P-gp overexpressing cancer cells.

Reversal effect of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in adriamycin-resistant K562/A02 cells

Plagiochin E is a new macrocyclic bisbibenzyl compound isolated from Marchantia polymorpha. In the previous studies, we reported that when combined with fluconazole, plagiochin E had synergetic effects against the resistant strain of Candida albicans. Herein, we examined the reversal effect of plagiochin E on multidrug resistance in adriamycin-induced resistant K562/A02 cells and the parental K562 cells. Its cytotoxicity and reversal effects on multidrug resistance were assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assay. Apoptosis percentage of cells was obtained from Annexin V/fluorescein isothiocyanate (FITC) and propridium iodide (PI) double-staining. The effects of plagiochin E on P-glycoprotein activity were evaluated by measuring rhodamine 123 (Rh123)-associated mean fluorescence intensity and P-glycoprotein expression on the basis of the flow cytometric technology, respectively. The results showed that plagiochin E ranging from 2 to 12 μg/ml had little cytotoxicity against K562/A02 cells. When combined with adriamycin, it significantly promoted the sensitivity of K562/A02 cells toward adriamycin through increasing intracellular accumulation of adriamycin in a dose-dependent manner. Further study demonstrated that the inhibitory effect of plagiochin E on P-glycoprotein activity was the major cause of increased stagnation of adriamycin inside K562/A02 cells, indicating that plagiochin E, as a new class of mutidrug resistance inhibitor, may effectively reverse the multidrug resistance in K562/A02 cells via inhibiting expression and drug-transport function of P-glycoprotein.