Studies On The Anticancer Effect And Mechanism Of 2β, 3β, 23-trihydroxyurs-12-en-28-oic Acid(TUA) Isolated From Actinidia Chinensis Radix On Basis Of NF-κB Signaling Pathway

Tumor is one of the most widespread and feared largely diseases in the world today,because it is known to be difficult to cure.In three major cancer therapies, chemotherapy is an important contributor to curative treatment of tumor, but because of the high price,few side effect and single-target,the application of chemotherapy drugs is limited. Traditional Chinese medicines(TCM) exert their anticancer effects through a multi-component,multi-link and multi-target way and TCM can improve the anti-tumor effect, reduce drug adverse reactions and the emergence of drug multi--resistantance.Therefore, How to explore effective anti-tumor components from Chinese herbal medicine and elucidate its mechanism of action has become an important part of the priority research for TCM. Heat-clearing and detoxicating drug,tonification drug, medicine for activating blood circulation and eliminating stasis,damp-clearing drug,these Chinese medicines are often used to treat tumor and the first kind TCM with cold property is made up the largest percentage of all. Tengligen(Actinidia chinensis Radix) is regarded by Chinese people as the common TCM for heat-clearing and detoxification and it has been used to treat unknown toxin(tumor) in the private sectors. Some ursolic triterpenoids were isolated from Tengligen on the basis of reseach on its chemical composition systematically, meanwhile the primary anti-tumor activity of four monomers A,B,C,D with relative high content(mg scale) was detected in vitro by MTT. The results showed that the compound D(2b,3β, 23-trihydroxyurs-12-en-28-oic acid,TUA)had the significant anti-tumor effect on NCI-H460 cells and HeLa cells(IC50 values:12.15±1.15μg·mL-1, 18.46±2.09 μg·mL-1). Unfortunately,there was few reports about the research on antitumor activities of TUA in recent years.[Objective]To study the anti-proliferation effect of TUA on lung cancer cell lines NCI-H460 and cervical cancer cell line HeLa based on the previous research,,then further explore its antitumor mechanism at the molecular level in order to illuminate its substance foundation for the anticancer effect of Tengligen and provide a theoretical basis for further research and development of this TCM.[Methods]Part One –Study on the Effects of TUA on lung cancer cell lines NCI-H460 and NF-κB signaling pathway1.Antitumor effect of TUA in vitroIn vitro, lung cancer cells(NCI-H460) were cultured in RPMI1640 medium. Afetr treatment with TUA of different concentrations, Cytotoxicity Detection KitPLUS was used todetect the LDH release rate and determine the cell activity; Immune colorimetry was used to test the cell proliferation; The cell viability was examined using CCK-8; Cell apoptosis was determined using Annexin V-FITC/PI apoptosis kit; Cell cycle analysis was performed on NCI-H460 cells incubated with various concentrations of TUA by Flow Cytometry(FCM);Real time PCR technology was used to detect the expression of bax,bcl-2,caspase3,p65,survivin genes involving in apoptosis; Western Blot was used to analyse the expression level of p65,IkBa, p-IkBa,bax,bcl-2,caspase3,survivin proteins involved in the NF-κB signaling pathways; Electrophoretic mobility shift assay(EMSA) was used to assess NF-kB- DNA binding activation; The expression of NF-kB(p65) and IkBa was assayed by immunocytochemical staining.2. Anti-tumor efficacy experiments of TUA using lung cancer xenograft models in nude mice in vivoNCI-H460 cells were implanted into nude mice and the transplantation tumor block from nude mice of more than 2 generations was inoculated to the right armpits of BALB/c mice with dissecting needle to establish lung cancer xenograft model.When the volume of transplanted was about 50mm3, the mice were randomly divided into 6 groups:(1)model group;(2)10mg/kg cisplatin group;(3) 10mg/kg PDTC group;(4) TUA high dose group(30 mg/kg);(5)TUA middle dose group(12 mg/kg);(6) TUA low dose group(6 mg/kg). Administration approach was intratumoral injection. The effects of each group on the weight of transplanted tumor animal, the volume and weight of tumor were continuously observed for 14 days.Tumor volume growth curve was drawned and tumor inhibitory rate and index were calculated;HE staining was used to observe nude mice tumor tissue pathological changes; The effects of TUA on NF-κB signaling pathway related proteins were detected by immunohistochemistry and Western blot.Part Two-Study on apoptosis of human cervical cancer HeLa cell induced by TUA and its primary mechanismIn vitro, human cervical cancer cells(HeLa) were cultured in E-MEM medium. Afetr treatment with TUA of different concentrations, Cytotoxicity Detection KitPLUS was used to detect the LDH release rate and determine the cell activity; Immune colorimetry was used to test the cell proliferation; The cell viability was examined using CCK-8; The change of morphoiogy of apoptosis HeLa cells was observed by DAPI fluorescence staining; Apo- ONE?Homogeneous protease-3/7 kit was used to measure the activity of Caspase-3/7 and the binding activity of nuclear extracts of cervical cancer HeLa cells NF-κB p65 with DNA was determined to investigate the activation level of NF-κB.[Results]1.Antitumor effect of TUA in vitroCompared with the control group,the results indicated that the LDH release rate in NCI-H460 cells increased after treatment of TUA with different concentration((10, 20,30μg/mL),the results of CCK-8 also proved that the cell viability decreased continuously with the increase of concentration and exposure time. It was found that the effect of cytotoxicity of TUA were both concentration-dependent and time- dependent. Results of the BrdU proliferation assay also showed the NCI-H460 cell proliferation was significantly increased in a dose- and time-dependent manner. FCM results indicated that the rate of apoptosis increased with the increase of TUA concentration and the NCI-H460 cells were arrested in G0/G1 phase.The results of RT-PCR analysis suggested the expression of apoptosis related gene p65,Survivin,Bcl-2 mRNA down-regulated and meanwhile Bax, Caspase-3 mRNA up-regulated. Both 30 μg/mL TUA and 10 μg/mL PDTC(positive control drug) had the similar effects. The Western blot results indicated that TUA decreased p-IkBa, p65,Survivin,Bcl-2 expression level while increasing the expression level of IkBa, Bax,Caspase-3.In addition, the effect of TUA on the NF-κB-related protein expression were significantly increased or decreased in a dose-dependent manner. EMSA results indicated compared with the control group, TUA significantly reduced the expression of NF-κB activity and TUA significantly decreased NF-κB DNA-binding activity in a dose-dependent manner. ICC results also indicated that TUA decreased p65 expression level while increased the expression level of IkBa.2. Anti-tumor efficacy experiments of TUA using lung cancer xenograft models in nude mice in vivoIn vivo experiments showed that the transplanted tumors in nude mice became small compared with the models. With the increase of TUA dose, the tumor tissue became more and more small, especially in TUA high dose(30 mg/kg), it had the similar size with the NF-κB inhibitor PDTC(10 mg/kg) group. HE dyeing observation results confirmed the degree of tumor necrosis and fission in TUA treated tumor tissues obviously decreased.Immunohistochemical results showed that the TUA treatment group compared with model group, p65 expression in tumor tissues was reduced, and expression of IκBa increased;Western blot results also showed that the NF-κB related p65 protein expression levels decreased, at the same time IκBa protein expression level increased; the apoptosis related proteins Survivin protein expression was depressed, Caspase-3 protein expression was promoted.3. Study on apoptosis of human cervical cancer HeLa cell induced by TUA and its primary mechanismAfter HeLa cells were treated by TUA, the leakage of LDH increased in a dosage and time dependent manner. In the process of determination of BrdU and CCK-8, the inhibitory effect of TUA to HeLa cell was stronger than standard drugs, and showed obviously inhibitory effect on the HeLa cell proliferation with the increasing of concentration and the acting time.By using the DAPI fluorescence dyes, the HeLa cell cytoplasm concentrating, nucleolus dispersion and chromatin aglomeration were observed. TUA enhanced the activity of Caspase-3/7 in a dose-dependent manner. TUA exerted remarkable inhibitory effect on the activation of subunit p65 of NF-κB, and this inhibitory effect was found dose-independent to some extent.[Conclusions]1. TUA has a significant inhibiting effect on tumour cells, especially on NCI-H460 and HeLa cells, and TUA can inhibit the proliferation of NCI-H460 and HeLa cells in a time-and dose-dependent manner.2. TUA can cause cell-cycle arrest and induce apoptosis of NCI-H460 cells by regulating apoptosis-related protein expression, which is mainly on NF-kB and the related signal transduction protein. It is proved that TUA has an anti-tumor effect on lung carcinoma cells by down-regulating the expression of NF-κB in vitro and in vivo.3. TUA can significantly induce the apoptosis of HeLa cells in a concentration-dependent manner, which is probably related to the activation of Caspase-3/7 and inhibition of NF-κB.It’s suggested that the apoptosis-inducing effect of TUA on HeLa cells is related to down-regulation of NF-κB, which established a rock-solid foundation of further studying on apoptosis-inducing effects of TUA.4. TUA could be used as a NF-κB inhibitor, which deserve further in-depth investigation.