Study On Antihepatoma Activity And Mechanism Of Xanthone Natural Product Isojacareubin

1、BackgroundClarify the changes of the molecules from cell signaling pathway during the progress of tumor development and progression, which can help us find effective ways to prevent and treat cancer. For the recent two or three decades hepatoma (mostly is hepatocellular carcinoma, HCC) has become the world’s fifth-most common cancer and the third leading cause of cancer-related mortality worldwide. The Chinese incidence of hepatoma has accounted for over 55% of the the world’s incidence. Currently, hepatoma has become the second cancer-related mortality in China, accounting for 19.33% of the total tumor.For many years in spite of biomedical and clinical medical workers trying to conquer hepatoma, but little progress has made. At present, surgery is an effective means, but most of the patients diagnosed with hepatoma have been found to the advanced period (most of them have been metastasis), which are not suitable for surgery. Systemic is commonly used as the only treatment options. Discovery the new targets for the hepatoma chemotherapy, not only can reveal the roles of new signaling pathways during the progress of hepatoma occurrence and development, and provide effective targets for clinical chemotherapy.The development of anticancer drugs is one of the direction of drug research. The natural product is a chemical compound or substance found in nature that usually has a biological or pharmacological activity for use in pharmaceutical drug design and discovery. After long-term clinical applications, most of the natural products are effective and safe to use. At present, almost 50% clinical drugs are derived from natural products and their derivatives. Polyoxygenated-xanthones have broad pharmacological activities, such as anti-bacterial, anti-inflammatory, anti-tumor, anti-oxidative, antihypertensive. Therefore, looking for the natural active products which are highly diversity in structure and function and rich in natural resources, has become a hot research.Protein kinase C (PKC), a family of serine/threonine kinases, based on their second messenger requirements, can be divided into three groups:conventional PKC (cPKCs a, βⅠ,βⅡ,γ), novel PKC (nPKCs δ,ε,η,θ,μ), and atypical PKC (aPKCs ζ,λ/ι). To date, these PKC isoforms are believed to be involved in a number of cellular processes. Many reports have shown that PKC overexpression is correlated with progression and prognosis of malignant diseases including HCC. Preventing PKC translocation from the cytoplasm to membranes inhibits the early response of phosphorylation of specific substrates and late response of PKC function, which may offer a unique therapeutic approach to HCC.Currently PKC inhibitors that are used in clinical have strong toxicity. Therefore, it is urgent to find compounds with high specificity and selectivity. The search for effective antitumor compounds from natural products often considered as preferable research direction of anti-tumor drug. A large number of studies have found that xanthones have significant antitumor efficacy. Accordingly, in our study polyoxygenated-xanthone based PKC inhibitor are applied in vitro and in vivo, through their inhibitory effect on hepatoma cells and human colon HCC xenografts, the changes associated with apoptosis, migration and invasion are examined, and the selectivity of PKC is clarified, which will provide a scientific basis for the design of clinical drugs for hepatoma treatment and lead compounds.2、Methods(1) Through cytotoxic test experiments, screening the compounds that have potential antitumor activity in HCC cell lines. Evaluating the in vitro migration, invasion and pro-apoptosis activity of the compounds on cellular-level.(2) Screening of human HCC cell lines in vitro culture. The human HCC cell lines need to possess the following biological characteristics:HCC cell proliferation is fast; HCC cell has significant in vitro migration, invasion activity; the growth of HCC cell is rapid in nude mice.(3) HepG2 cells were harvested and subcutaneously injected into BALB/c nude mice right flank. Body weights and tumor volumes were measured in nude mice before each drug injection. H&E staining and histological analysis were performed on the hepatoma xenografts to reveal tumor tissue necrosis and apoptotic protein positive stainning.(4) Through enzyme activity analysis, searching for the small molecule compounds that have PKC inhibitory activity.(5) Because each kind of PKC subtype can arise unique biological effect through its downstream particular molecules of signaling pathways. For the further research, targeting different PKC subtypes, the small molecule compounds have PKC inhibitory activity and selectivity. The binding property of small molecule compound with PKC isoforms is also examined. At the same time detection of small molecule compounds on HCC cells apoptosis, migration and invasion.3、Results(1) A preliminary screening polyoxygenated-xanthones against HepG2 and QGY-7703 cells was carried out at 50μM. After the second screening, we found ISJ and its intermediate compound 7 (7,9-dihydroxy-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]xanthen-6-one) possessed antiproliferative activities against HCC and exerted no apparent toxicity on normal cells. To further explore the potential mechanisms of antiproliferative effects induced by compounds 7 and ISJ, Annexin V-FITC/PI double staining was performed to quantify cell apoptosis by flow cytometry. The results demonstrated that compound 7-and ISJ-induced apoptosis of HepG2 at least partially contributed to its antiproliferative effect.(2) After carring out wound healing and matrigel invasion assays, and injecting HCC cells into BALB/c nude mice, we found HepG2 cells possess the satisfied properties, such as rapid proliferation, significant in vitro migration, invasion activity, and the growth is rapid in nude mice.(3) To evaluate the antitumor activities of ISJ in vivo, HepG2 cells were harvested and a total of 5×106 cells were subcutaneously injected into the right flank. Once the volume of tumor reached 75-100 mm3, ISJ was injected every other day for two weeks. ISJ treatment at a dose of 5 mg/kg significantly inhibited the growth of tumor over time and reduced tumor weight compared with control. There is no significant difference of body weights between ISJ and control group. H&E staining analysis revealed significant tissue death in ISJ-treated tumors, in contrast to the tumors from mice treated with vehicle controls. Moreover, immunohistochemistry (IHC) of the tumors revealed upregulation in c-PARP, cleaved caspase (c-Cas) 3 and c-Cas9 apoptotic proteins levels in the tumors treated with ISJ. Conversely, the vehicle control tumors showed much weaker staining. To further validate the results in vivo, an immunoblotting assay was used to detect antibodies specific to c-PARP, c-Cas3 and c-Cas9 from tumor tissue samples. Compared with vehicle controls, these proteins levels were increased, especially the ISJ could remarkably induce apoptosis.(4) The activity of PKC in HepG2 cells was assessed 3 h after the addition of compounds 7 and ISJ. Compared to the basal control (in the absence of any compound), the PKC activity, as measured by the phosphorylation state of a PKC-specific target peptide, was inhibited by compounds 7 and ISJ, especially ISJ at the concentration of 5 μM.(5) To further explore whether compounds 7 and ISJ were potential candidates of selective PKC expression inhibitors against hepatoma, the expressions of PKC isoforms in HepG2 cells were characterized by Western blot. In order to identify whether ISJ and compound 7 can target PKC in cells, we performed pull-down experiments using ISJ-and compound 7-coupled sepharose beads with cell lysates of HepG2. Targeting different PKC isotypes, ISJ possessed various action mechanism. ISJ selectively inhibited expression of aPKC (PKC ζ) in cytosol and translocation of cytosolic aPKC (PKCζ) to membrane, meanwhile the direct interactions between ISJ and cPKC (PKC a) or nPKC (PKCδ, PKCε and PKCμ) were observed.(6) Different acting mechanisms of ISJ lead to the inhibition of the early response of major MAPKs phosphorylation and the late response of HCC cell invasion and proliferation.4、Conclusion In summary, we had synthesized and biologically evaluated a novel class of polyoxygenated-xanthone based PKC inhibitors. Especially the natural product ISJ, demonstrated more efficacious antiproliferative activity towards HCC cells, together with pro-apoptotic effects on HepG2 and impairment of HepG2 cell migration and invasion. Moreover, we found ISJ exhibited better PKC inhibition potency. Therefore, ISJ was identified as a preferential inhibitor of PKC and further mechanistic investigations suggested that targeting various PKC isotypes, ISJ possessed differential action mechanism. ISJ selectively inhibited expression of aPKC (PKCζ) in cytosol and translocation of cytosolic aPKC (PKC Q to membrane, meanwhile the direct interactions between ISJ and cPKC (PKC a) or nPKC (PKC 8, PKC s and PKCμ) were observed, which resulted in the inhibition of the early response of major MAPKs phosphorylation and the late response of HCC cell invasion and proliferation. Furthermore, in hepatoma-xenograft model, pretreatment of ISJ possessed potent antihepatoma activity in vivo. Taken together, our current findings indicated that the lead polyoxygenated-xanthone based PKC inhibitor may be developed as a novel and promising antitumor drug to target HCC with overexpression of PKC.