Anticancer Effects Of Nispex, An Extract Of Scurrula Parasitica L. Parasitized On Nernium Indicum Mill. And Its Components

Therioma is one of the most serious diseases that troubled human beings health greatly. The treatment of cancer with chemotherapeutic agents and radiation has two major problems: time-dependent development of tumor resistance to therapy (chemoresistance and radioresistance) and nonspecific toxicity toward normal cells. So, the search for new agents that effectively kill cancer cells, but that have minimal or no side effects on normal cells is always the aim of anticancer drug researchers. About 70% of the therapeutic drugs in use today are derived from plants. Paclitaxel, vincristine, camptothecin, etoposide and harringtonine are typical anticancer drugs that generally used in clinical therapy derived from plants. Except these individual component drugs, traditional herbal medicinal products have been commonly accepted and applied in European Union countries such as Germany, France, Switzerland and Italy et al. The herbalists have access to hundreds of years of observational data on the anticancer activity of many herbs. Laboratory studies are confirming the knowledge that is already documented in traditional texts.The induction, promotion and progression of cancer is a multistepprocess that involves biochemical interactions from the level of the genes, through cell-signaling pathways, intercellular communication mechanisms, supply of nutrients, channels for metastases, and a host immune response. An integrative approach for managing a patient with cancer should target the multiple biochemical and physiological pathways that support tumor development while minimizing normal tissue toxicity. Traditional herbal medicinal products always contain a range of complex organic chemicals that may have synergistic activity. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combination. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they may act as biological response modifiers potentially enhancing the efficacy of the so-called conventional therapies and protecting from therapy-associated toxicities.Research of the last few years has shown that many plant products exhibit chemopreventive effect on carcinogenesis. Many plant-derived polyphenols have been studied intently for their potential chemopreventive properties and are pharmacologically safe. Recent research has suggested that these plant polyphenols might be used to sensitize tumor cells to chemotherapeutic agents and radiation therapy by inhibiting pathways that lead to treatment resistance. These agents have also been found to be protective from therapy-associated toxicities.Nispex is an extract mainly composed by polyphenols of Scurrula parasitica L., a Chinese medicinal herb, which parasitized on Nernium indicum Mill. . It inhibits human cancer cells proliferation and induces human cancer cells apoptosis, but not murine cancer cells.In this doctoral dissertation, we described the preparation method of Nispex, analyzed its components preliminarily, and studied its anticancer effects in vitro and in vivo, its mechanisms of anticancer. The anticancer effects of extracts of Scurrula parasitica L. parasitized on other host plant had compared here, also.PartⅠ: in this part, the preparation method of Nispex was described. Leaves of Scurrula parasitica L. was extracted by 80% ethanol. And the extracts were purified by polyamides column chromatography further, the eluates of 30%, 50%, 70% and 90% ethanol were mixed being called Nispex (means extracts of Scurrula parasitica L. on Nernium indicum Mill.). Colour reactions indicted that Nispex consisted mainly of flavonoids, cardiac glycosides and alkaloids. Nispex contained 72.37% flavonoids, determined by colorimetry with rutin as the reference substance. There were about 30 components in Nispex by LC-MS analysis, including rutin, hyperoside, quercetin, quercitroside, kaempferol, genistin, liquidamboside, epicatechingallate, avicularin, liquiritin, daidzein4, 7-diglucoside, adynerin and oleandrin derivative, odoroside H, uzarigenin, et al.PartⅡ: In this part, the LD50 value of Nispex to mice was detected by 126.81 mg/kg. Pathological examination showed that the major toxicity presented in heart, cardiac muscle cells of the sudden death mice caused by Nispex becoming swollen and disturbance. The anticancer effect of Nispex in vitro was studied by MTT assay and colony forming assay with human acute myeloid leukemia cell line HL-60. Nispex inhibited HL-60 cells proliferation significantly in time and dose dependent manner, the IC50 was 0.54μg/ml when treated 48 hours. Colony forming assay indicted that proliferating-cell population were more sensitive to Nispex. Xenograft of HL-60 was established by subcutaneous implantation of cultured HL-60 cells in BALB/c nude mice. Tumor growth inhibition in these mice was used to evaluate the anticancer activity of drugs in vivo. Mice used in these experiments were randomly divided into six groups: control group, ADR 15 mg/kg group, 30 mg/kg/day Nospex-ip group, 10 mg/kg/day Nospex-ip group, 10 mg/kg/day Nospex-it group, 10 mg/kg/day Nospex-ip combined with ADR 15 mg/kg group. ADR were injected through caudal vein once only at the first day; Nospex treatment, 14 days. Tumor growth inhibition was evaluated by relative tumor volume (RTV) and tumor weight (TW). HL-60 grafts of 30 mg/kg/day Nospex-ip were inhibited by 60.6% according to RTV, 31.2% according to TW(P<0.05); grafts of 10 mg/kg/day Nospex-it were inhibited by 59.4% according to RTV, 45.3% according to TW(P<0.05); and grafts of ADR wre inhibited by 65.2% (RTV), 60.4% (TW), grafts of 10 mg/kg/day Nospex-ip combined with ADR were inhibited by 94.4% according to RTV, 92.9% according to TW(P<0.01), the coefficient of drug interaction (CDI) was 0.24<0.7, meaning the synergistic effect was very significant. Enhancement of Adriamycin by Nispex was explored further by 10 mg/kg/day Nospex-ip with ADR 15mg/kg, ADR 10 mg/kg and ADR 5 mg/kg. The inhibition of tumor growth by ADR was enhanced significantly combined by Nispex 10 mg/kg/day. CDI was 0.43 when combined with ADR 15 mg/kg, 0.80 when combined with ADR 10mg/kg. Apoptotic HL-60 cells induced by Nispex were detected by staining with AO/EB, flow cytometry analysis, TUNEL assay and DNA fragmentation analysis. The cell cycles were mainly arrested at G0/G1 after treated by Nispex. Nispex was confirmed to be a natural inhibitor of NF-κB pathway by EMSA, Western blot and immunofluorescence assay. VEGF levels were reduced significantly in the supernatant of HL-60 cell cultures treated by Nispex with ELISA .PartⅢ: In this part, MTT assay and colony forming assay was used to detected the inhibition of cell proliferation by Nispex on 8 human cancer cells (human acute lymphoblastic leukemia cell line Molt4, human small cell lung cancer cell line NCI-H446, human myeloma cell line U266, human acute promyelocytic leukemia cell line NB4, human chronic myelocytic cell line K562, human nasopharyngeal carcinoma cell line CNE, human cervical carcinoma cell line Hela, human T cell leukemia cell line Jurkat ) , primary human chronic myelocytic cells, and 5 murine cancer cell lines. Nispex inhibited human cancer cells proliferation significantly in time and dose dependent manner. But Nispex had no cytotoxicity on murine cancer cells. Nispex also inhibited 293T cells (a human embryoic kidney cell line) proliferation, but not showing dose dependent manner, Nispex did not induce 293T cells apoptosis as high as 50.0μg/ml drug concentration.PartⅣ: In this part, MTT assay was used to compare the effects of inhibiting human cancer cells proliferation by extracts of Scurrula parasitica L. from four diffenent host, Nernium indicum Mill., Morus alba L., Opsmanthus fragrans Lours., and Sapindus mulorossi Gaertn. . Extract of Scurrula parasitica L. parasitized on Nernium indicum Mill. was the most effective, and extract of Scurrula parasitica L. parasitized on Morus alba L. taken the second place. Extract of Scurrula parasitica L. parasitized on Opsmanthus fragrans Lours. had no effectiveness even if as high as 50.0μg/ml drug concentration.Conclusion: Nispex, an extract of Scurrula parasitica L. parasitized on Nernium indicum Mill. , has effective anticancer activity in vitro and in vivo; it selectively kills human cancer cells, but not murine cancer cells. Nispex enhanced Adriamycin anticancer effects significantly. Nispex's anticancer effects may be partially ascribed to the inhibition of activation of NF-kB and suppression of VEGF secretion.