Experimental Study On Anti-tumor Effects And Action Mechanism Of Juglone

Malignant tumor is one of diseases which jeopardizes the human health and is the first killer to mankind in the new century.At present,more sophisticated treatments of malignant tumor includes surgery treatment,radiation therapy, pharmacotherapy and immunotherapy.And the cancer chemotherapy has made considerable progress,but the treatment of malignant solid tumor,one of the most serious diseases endangering human health and life,accounting for more than 90%of malignant tumors,failed to achieve satisfaction and the vast majority of anticancer drugs in inhibiting or killing tumor cells may induce destruction of normal tissues, inevitable damage of organs or other toxicity to the patients.Therefore,the discovery and study of new effective anti-cancer drugs is the major issue and a long-term task for the biomedical research.Germplasm of medicinal plants in our country is rich in resources,which containing the active ingredient of Chinese herbal anti-cancer has about 200 kinds,and confirmed by transplanted animal tumor cell lines in vivo and in vitro, more than 4000 kinds of plant composition have antitumor activity.The chemical structure and the mechanisms of drugs from plant sources has the diversity.At the same time,a wealth of information and experience have been accumulated in a long-term study on chinese medicine treatment for tumor,which contribute to the discovery and development of plant sources of anti-tumor drugs.Therefore,to obtain drugs from natural ingredients and to make it the lead compounds are one of the important ways for research and development of anti-cancer drugs. Juglone(Nucin),is known as 5 - hydroxy -1,4 - naphthoquinone,5 - hydroxy -1,4 - naphthalene dione.Being the main toxic substances of juglans,it is separated from the root bark,branch bark,green peel of Juglans mandshurica and belongs to the hydroxy-naphthoquinone compounds.Juglans were used as a non-governmental anti-cancer walnut recipes in China for a long history.Juglone,as the main component of Juglans mandshurica,its anti-cancer activity has been reported.In recent years, some studies have showed juglone have various biological activities,such as anti-fungal,anti-Chlamydia or HIV virus,anti-cancer,lowering blood glucose and pesticides,and has been paid more and more attention.Therefore,further developing anti-tumor activity of juglone and understanding its mechanism has a very important practical significance.ObjectiveTo screen sensitive cell lines and learn the anti-cancer mechanism of junlone.To evaluate the inhibitory effect and toxicity of juglone and investigate the inhibitory effect of juglone on the tumor metastasis.To study the plasma protein binding rate of juglone.Methods1.Rates of growth inhibition of juglone on different cell lines were obstained by MTT method.2.BEL-7402 cells were treated with juglone at different concentration in vitro,probed with Flow-3/AM and the intracellular calcium concentrations were detected.Flow cytometry was used to observe cell apoptosis and mitochondrial membrane potential changes.The fluorescence depolarization method was used to measure values of fluorescence anisotropy,fluorescence polarization as well as microviscosity in BEL-7402 cells and microviscosity in liposome continuously for 30 min.3.BEL-7402 cell were incubated with 12.5μmol/Ljuglone for 24 hours,and fixed in 2.5%glutaraldehyde for HE,coomassie brilliant blue staining,scanning electron microscope and transmission electron microscopy observation.4.The effect of a series of concentration of juglone on the growth of EAHY.926 were measured by MTT.Serum-free culture of the rat aorta was conducted,with 100μmol/L,50μmol/L,25μmol/L and 12.5μmol/L dosages of juglone added,to observe its effect on crawling of the vascular endothelial cells and formation of capillary structure.7-day-old chick embryoes were selected for experimental angiogenesis model to explore the impact of juglone on neovascularization.5.Human hepatic carcinoma BEL-7402 cells were injected into nude mice subcutaneously.The resultant tumor were taken and prepared into small tissue blocks,which were implanted subcutaneously into nude mice to establish mouse models bearing human hepatoma.Different doses of juglone were administatored by intraperitoneal and local injection.The general condition of the tumor-bearing mice and the growth of the tumors were observed for pharmacodynamic study of quinone.6.B16-F10 cells were injected into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model,and the effect of juglone on lung metastasis was observed.7.The ultrafiltration was employed to determine the plasma protein binding rate of juglone.The plasma concentrations of juglone were measured by HPLC.Results1.Sensitivities of different cell lines to juglone were different:IC₅₀ of human hepatocellular carcinoma BEL-7402 cell line,Human lung cancer A549 cell line,Human breast cancer MCF-7 cell line,human nasopharyngeal carcinoma CNE2 cell line,human cervical carcinoma Hela cell line,human hepatocellular carcinoma HepG2 cell line were 14.664μol/L,24.414μmol/L,20.788μmol/L,25.317μmol/L, 27.907μmol/L,10.938μmol/L,respectively.2.The results showed that juglone at different dosages could inhibit the growth of human liver cancer BEL-7402,and induce cell apoptosis,IC₅₀ was 13.78μmol/L.Juglone induced a significant decrease of fluorescence anisotropy, fluorescence polarization as well as microviscosity in BEL-7402 cells (P＜0.05),particularly in the first 1 min.After 1 min,the values of anisotropy were remained lower levels.There was no change of intracellular Ca²⁺ concentration of BEL-7402 cells with juglone for 30min.The results suggested that juglone may induce apoptosis through Capase.3.It was found that changes developed in the cytomorphology and cytoskeleton of these cells.According to the scanning electron microscope,we found that the volume of cell bodies were decreased,microvilli on cell surface crimped and aberrated. intercellular junction was ruptured and the intercellular space was enlarged.4.We found that juglone of 200μmol/L,100μmol/L and 50μmol/L could effectively inhibit the proliferation of EAHY.926 in a dose-dependent manner(P＜0.05),with an IC₅₀ of 122.848μmol/L according to the results of MTT.While juglone of 25μmol/L,12.5μmol/L and 6.25μmol/L could promote growth of EAHY.926(P＜0.05 ). juglone of 100μmol/L,50μmol/L and 25μmol/L could completely inhibit the formation of new vessels,and juglone of 12.5μmol/L could reduce the number of endothelial cells and slow its crawling speed(P＜0.01).Juglone of 200μmol/L, 100μmol/L,50μmol/L and 25μmol/L have vascular stimulation,hemolyzation, agglutination as a dose-dependent manner and juglone could inhibit angiogenesis.5.The inhibitory effect of juglone of 600μg/kg,300μg/kg and 150μg/kg on the growth of human hepatocellular carcinoma Bel-7402 in nude mice was studied.The results revealed that NK cell activity decreased in nude mice with juglone of 600μg/kg, 300μg/kg.Compared with the positive control group(5-Fu),NK cell activity of nude mice with juglone of 600ug/kg group was no significant difference(P＞0.05).Juglone of600μg/kg,300μg/kg and 150μg/kg had no significant impact on tumor growth.Juglone of 600μg/kg,300μg/kg and 150μg/kg in PH=7.4 and PH=4.0 respectively were given by topical injection to human hepatocellular carcinoma BEL-7402 cells transplanted subcutaneously in nude models,we found 600μg/kg, 300μg/kg concentration juglone in different PH could effectly inhibit the growth of tumor and compared to the positive control group(5-Fu),there was no significant difference between all groups(P＞0.05),and the anti-tumor effect of the same concentration between different PH were no significant difference(P＞0.05).Juglone of 4.5mg/kg,3mg/kg and 1.5mg/kg were given by intraperitoneal injection to human liver cancer BEL-7402 cells transplanted subcutaneously in nude model,the results showed that the juglone could effectly inhibit the tumor growth, the tumor inhibitory rate of juglone of 4.5mg/kg,3.0 mg/kg,1.5 mg/kg were 78.24%,66.57%and 48.94%respectively.4.5mg/kg and 3mg/kg juglone had the same anti-tumer effect when compared with the positive control group(P＞0.05,P＞0.05). But there were significantly reduced subcutaneous fat,weight loss,and death cases in 4.5 mg/kg juglone group.6.The number of metastases in the animals that were given juglone of 4.5mg/kg,3mg/kg,1.5mg/kg and 7.5mg/kg was significantly reduced as compared with the vehicle control(P＜0.05),and the inhibition rates were 27.30%,55.35%,31.52%,25.34%,respectively.Compared with the positive control group,the inhibitory rate of 3mg/kg juglone was no significant difference(P＞0.05). The inhibitory rate of 4.5mg/kg juglone are low probably because the juglone could decrease immune function of nude mice.7.The plasma protein binding rate of juglone with SD rat plasma at the concentration of 100μg/ml was more than 90%.Conclusion1.Juglone could effectively inhibit proliferation of human origin cancer and sensitivities of different cell lines to juglone were different.2.Juglone could significantly increase the membrane fluidity of BEL-7402 cells,and promote the drug to enter cells,enhance the toxicity of drugs on cells.3.Juglone could effectively change the submicroscopic structure of human hepatocellular carcinoma BEL-7402 cells.4.Juglone had the role of anti-angiogenesis.5.Juglone could inhibit the growth of human liver cancer BEL-7402 cells in nude mice transplanted tumor,but there is a certain side effects,and the scope of drug safety were smaller.6.Juglone could inhibit melanoma metastasis of B16F10.7.The binding rate of juglone with SD rat plasma protein was so high that it would have anti-cancer effect in high doses.