Anticancer Mechanism Of Riccardin D And Its Derivative And The Discovery Of Rhamnose Receptor

Natural plants have been an important part of natural medicines, but also a major source of treating and preventing human disease. The incidence of cancer are increasing every year. An increase number of reports of clinical resistance to anticancer agents highlight the need for searching for new anticancer drugs which may have a new target and good effect. So far the research on anticancer drugs has been supported by many countries. Millions of plants, animals, microorganisms, and marine organisms are an important source of new drug discovery.Bryophytes belong to the most primitive higher plant, which grow in the dark and damp environment. Considered being the first plant type to successfully make the transition from water to land, bryophytes are divided into three classes, liverworts (Hepaticae), mosses (Musci), and hornworts (Anthocerotae). Various types of lipophilic terpenoids and aromatic compounds, which show significant biological activities, are isolated from the bryophytes in the past decades. Bisbibenzyls, a class of characteristic compontents derived from liverworts, are attracting more and more attention because of their wide range of biological significance including antitumor, anti-bacterial and antifungal. Riccardin D (RD), a liverwort derived natural occurring macrocyclic bisbibenzyl, has been found to exert anticancer activity in multiple cancer cell types through induction of apoptosis. However, its underlying mechanisms remain undefined. In addition, whether RD induce other forms of cell death such as autophagy has not been investigated. In this paper, we studied the manner and mechanism of cell death RD induced in human osteosarcoma cells U2OS (p53wild) and Saos-2(p53null). The MTT assay revealed that RD induced concentration-and time-dependent cell death in U2OS and Saos-2cells, and minor cytotoxicity in human normal osteoblasts. Using flow cytometry, we found RD caused U2OS cells and Saos-2cells arrest in G1phase, associated with reduction of cyclin D and cyclin E and induction of p53and p21WAf1in U2OS cells. RD-mediated cell cycle arrest was accompanied with promotion of apoptosis, as indicated by the changes in the nuclear morphology and expressions of apoptosis-related proteins. Further studies revealed that antiproliferation of RD was not affected in the presence of p53inhibitor, but reversed partially by a pan-inhibitor of caspases, suggesting that p53was not required in RD-mediated apoptosis, and caspase-independent mechanisms involved in RD-mediated cell death. Except for apoptosis, RD induced autophagy as evidenced by accumulation of LC3B-II, formation of AVOs, punctate dots and increased autophagic flux. Pharmacological blockade of activation of autophagy markedly attenuated RD-mediated cell death. Moreover, RD-induced cell death was significantly restored in combination of autophagy and caspase inhibitors in osteosarcoma cells. Together, our study revealed RD-induced caspase-dependent apoptosis and autophagy in cancer cells.The research on anticancer activity of RD has made preliminary progress. However, the attractive but moderate biological effects of RD motivate us to synthesize RD derivatives, improve their bioactivities, and discover more potent anticancer agents. Among various derivatives, the aminomethylated derivative of RD, riccardin D-N (RD-N), is a potential anticancer candidate. In order to investigate the pharmacology and molecular biological mechanisms of RD-N, we tested the cell growth inhibition by MTT method. Our results indicated that this compound exhibited strong cytotoxic properties in prostate cancer cells, however, normal cells appeared to be more resistant than the cancer cells. Therefore, we used prostate cancer cells for further study. Apoptosis was evaluated by annexin V/PI double staining and the cleavage of PARP, and the results revealed RD-N induced apoptosis in prostate cancer cells. Cell membrane integrity was assessed by detecting the leakage of cytoplasmic content, the release of lactate dehydrogenase (LDH), and the uptake of propidium iodide (PI). The ability of RD-N to make PI permeate into cancer cells and LDH release suggests a killing mechanism that involves plasma membrane perturbation. In addition, ATP decreased after RD-N was administered at different concentrations. ATP depletion can change the mode of cell death from apoptotic to necrotic morphology, which suggests that RD-N induced prostate cancer cell necrosis. To study the subcellular distribution of RD-N, we used sucrose density gradient centrifugation for isolation of lysosomes and mitochondrial and quantitation of compounds by HPLC. The amounts of RD-N that accumulated in the lysosomes and mitochondrial were determined. RD-N was found to preferentially accumulate within lysosomes in PC3cells. RD-N accumulation in the lysosome caused the volumes of lysosomes significantly increased and lysosomal membrane permeabilization (LMP). LMP was detected by Lysotracker Red and AO staining. The damaged lysosomes released cathepsins into the cytoplasm. The inhibitor of cathepsin B but not cathepsin D and L can significantly reverse RD-N-induced cell death. In addition, RD-N caused cell cycle arrest at the G2/M phase by reducing the expression of tubulin and actin. In vivo tumor xenograft model indicated treatment of RD-N significantly reduced size and weight of the tumor compared with vehicles. H&E and TUNEL staining indicated that RD-N induced apoptosis in the tumor tissues of nude mice. Therefore, RD-N induced cell death by lysosomal rupture in vitro and suppressed tumor growth in vivo.It has been reported that RD-N trigger lysosomal membrane permeabilization followed by cathepsin B-dependent programmed cell death in prostate cancer cell lines. However, not much work has been done in investigating cathepsin B binding partners in the cell death pathways. Inhibition of cathepsin B activity with cathepsin B inhibitors or depletion of cathepsin B protein using small interfering RNA abolishes cathepsin B-mediated DNA damage signaling pathway and chromatinolysis. While, cathepsin B overexpression sensitizes prostate cancer cells to RD-N killing and chromatinolysis. Furthermore, we found that after RD-N treatment cathepsin B moved from the lysosomes to the nucleus with the expression and activity of cathepsin B increased. During RD-N-induced apoptosis, DNA damage occurred, which was evidenced by the activation of ATM-Chk2and ATR-Chk1pathways, the phosphorylation of H2AX and BRCA1. To identify potential nuclear components downstream of CTSB, we performed immunofluorescence staining, protein docking, co-IP and knockdown of endogenous CTSB with siRNA, and found that cathepsin B translocated into the nucleus, degraded BRCA1, interfered with DNA repair and promoted DNA damage.About the anticancer activity of steroidal alkaloid glycoside isolated from Chinese herb Solanum Solanum nigrum L including solamargine, solasonine, solasodine and its two derivatives, we found rhamnose moiety played an important role in causing cell death and solamargine containing two rhamnoses showed strongest anti-cancer activity. Futher test revealed that solamargine also showed different cytotoxicity to different cancer cell lines. PC3and KB cells were more sensitive to solamargine than HT-29and MCF-7. To investigate the role of rhamose moiety, we designed and synthesized biotinylated-chacotriose,-rhamnose and-glucose as molecular probes that were bound to quantum dots (QDs) with bioconjugated streptavidin to visualize the expression of RBL receptor in situ on cancer cell membranes and found the fluorecence in rhamnose-QDs and chacotriose-QDs was obviously stronger than that in glucose-QDs. As we know that the rhamose has high affinity with rhamose lectin, we proved that RBL receptor mediate the anti-cancer activity of glycosides. Through the combination of biotinylated carbohydrates and streptavidin quantum dots, a method for in situ observation of RBL receptor on cell surface was established. According to this method, we detected the expression of RBL receptor and found different cancer cells had different RBL receptor levels and more RBL receptors on cancer cell surface mediated more effective cancer cell killings. These findings may provide a strategy to help design the chemotherapy agents by introducing rhamnose into the structure to increase the cell uptake or cancer targeting effects and also to prognosis on the chemotherapy sensitivity to different cancer cells.In this manuscript, we reported RD induced apoptosis and autophagic cell death for the first time. In order to improve the effects of RD, we synthesized new derivative RD-N, and proved RD-N was a lysosomotropic drug inducing cancer cell apoptosis and necrosis via LMP. After LMP, cathepsin B released from the lysosomes to the nucleus, degraded BRCA1and promoted DNA damage, which was a new function of cathepsin B. We also studied RBL receptor on cancer cell membranes and found different cancer cells had different RBL receptor expression. Cancer cells with more RBL receptors on cell surface were more sensitive to solamargine, which provided the foundation for cancer diagnosis and drug sensitivity research.