Extracting The Specific Inhibitor Of NF-κB From Abrotani Herba And Its Effecttion On Multicellular Resistance To Chemotherapeutic Drug Of Colon Cancer

Background and ObjectiveAnticancer drug resistance is the underlying cause of treatment failure in a significant number of patients with cancer. The transcription factor NF-kB is reportedly activated by anti-cancer chemotherapeutic compounds in many cancer cell lines and NF-kB activation is one mechanism by which tumors become resistant to apoptosis. In addition to this constitutive activity, the accumulating evidences has been clearly demonstrated that many anti-cancer agents can activate NF-κB in cancer cells. Either constitutive or treatment-induced activity, NF-κB mainly both acts as an inhibitor of apoptosis. Indeed, inhibition of NF-κB by genetic or chemical inhibitors can induces the apoptosis of various tumor cells and/ or restores the apoptotic response after treatment with ionizing radiations or chemotherapeutic agents, thus reversing NF-κB -linked radiotherapy or chemotherapy resistance in many models. Therefore, a precise knowledge of the signalling pathways controlling NF-κB as well as of the NF-κB target genes is essential in order to define precisely the field of application of anti- NF-κB drugs for the treatment of human cancers.Activation of NF-κB in cancer cells has been shown to attenuate apoptosis induced by chemotherapy or radiotherapy. The results of our previous studies suggest that NF-κB is an important molecular target for enhancing chemosensitivity. Consequently, the development of novel agents blocking the NF-κB activity has become a major goal for numerous laboratories and companies.Preclinical studies are being conducted regarding the inhibition of NF-κB activation in several laboratories.Tumor spheroids represent a excellent model to study drug resistance and in particular the so-called multicellular-mediated resistance(MCR) to anticancer agents.Classically,this type of resistance has been demonstrated in EMT6 tumors in mice in which resistance was inherently induced, whereas it was completely lost when the cancer cells were isolated and grown in monolayers; multicellular resistance, however, could be fully recapitulated when cells were cultured as multicellular spheroids.Chinese medicine is our unique advantage, there are many traditional Chinese drugs or treatments being treated to tumor.It has been found that many traditional Chinese drugs can augmented sensitivity to chemotherapeutic drugs and decrease cancer cells resistance to chemotherapy. Many of studies found that abrotani herba, triptolide, astragalus and other traditional Chinese drugs can inhibit the activation of NF-κB, artemisinin, rhein, baicalin, and other active ingredients of traditional Chinese drugs also can inhibit NF-κB activation. Therefore, it will provide a new way to find effective inhibitor of NF-κB if we could extract the inhibitor of NF-κB from the traditional Chinese drugs. Because of the complex composition of traditional Chinese drug, it is great significance to chose a effective method to obtain the inhibitor of NF-κB extracting from traditional Chinese drugs. So in this study,we used of biosensor technology, extracted the inhibitor of NF-κB from abrotani herba and identificated its structure .Secondly, studed the effection of the inhibitor on NF-κB activity , sensitivity to 5-FU,apoptosis and expression of apoptostic proteins in HT-29 spheroids.Finally,we studied effetion of the inhibitor in chemotherapy in xenograft experiment.Methods1. The specific inhibitor of NF-κB from abrotani herba was extracted by biosensor technology, organic solvent extraction, silica gel column chromatography, thin layer chromatography and other methods;2. The physico-chemical property of the specific inhibitor was tested .Then infrared Spectrometer(IR), high-pressure liquid chromatograph(HPLC), nuclear magnetic resonance spectroscopy(NMR), mass spectrometry(MS) were tested, and the structure was identificated;3. In vitro,effection of the inhibitor on NF-κB activity and the impact of chemotherapy sensitivity were studied in three-dimensiona colon cancer HT-29 cells;4. The cell cycle distribution,apoptosis and expression of apoptostic protein Bcl-2, FAS, and FAS-L on three-dimensiona HT-29 cells were detected by administration the NF-κB inhibitor extracting from abrotani herba with flow cytometry;5. In vivo, useing of HT-29 cells transplantation tumor subcutaneously in nude mice model, the effect of the inhibitor combination with chemotherapy in tumor growth was studied;6. With EMSA and immunohistochemical method, activity of NF-κB and expression of apoptostic protein Bcl-2, FAS, and FAS-L on tumors in xenograft experiments combination inhibitor with 5-FU were detected.Results1. The specific inhibitor of NF-κB from abrotani herba was successfully extracted ,and the physico-chemical property of the specific inhibitor was that slightly soluble in water, soluble in ethanol, soluble in chloroform, soluble in ethyl acetate, in the melting point 202-203℃.2. IR, NMR and MS of the inhibitor of NF-κB extracting from abrotani herba were tested, the structure of the inhibitor was identified,and it named AH-I temporary.Results followed: IRvKBrmax cm-1: 3334.7,1701.1,1566.1,1290.3. 1H-NMR(CDCl3)δppm:3.959(3H,s,OCH3),6.207(1H,s,OH),6.276(1H,d,J=9.2,H-3),6.852(1H,s,H-8),6.923(1H,s,H-5),7.608(1H,d,J=10,H-4). 13C-NMR(CDCl3)δppm:161.383(C-2),113.433(C-3),143.236(C-4),107.542(C-5),149.733(C-6),150.301(C-7),103.213(C-8),144.029(C-9),111.509(C-10),56.434(-OCH3).3. Administration AH-I, NF-κB activity in three-dimensiona HT-29 cells was inhibited, and combination AH-I with 5 - FU, the activity of NF-κB inhibition was stronger than 5– FU alone.4.Compared with the control, cells majority in the G1 phase and cell cycle was not changed significantly in three-dimensiona HT-29 cells treated with AH-I alone. While AH-I and 5 - FU combination, cell cycle obviously were changed in three-dimensiona HT-29 cells, the number of cells in G1 phase was decreased significantly ,and the number of cells in S phase was increased, both with the manner of dose-independent.5. AH-I and 5 - FU combination, apoptosis in three-dimensiona HT-29 cells was significantly increased compared with chemotherapy alone, and as the AH-I dose increasing, rates of apoptosis in three-dimensiona HT-29 cells were more obvious increased , with a dose-dependent effect.6. Combination AH-I plus 5 - FU, expression of anti-apoptosis protein Bcl-2 was decreased compared with chemotherapy alone group. But expression of pro-apoptotic protein FAS and FAS-L were increased significantly than chemotherapy alone group and both in a dose-dependent manner.7. Administration of AH-I on three-dimensiona HT-29 cells, the sensitivity to 5 - FU was enhanced, and with the AH-I dose increasing, the sensitivity to 5 - FU of three-dimensiona HT-29 cells was more obvious , and also in a dose-dependent manner8. In nude mice, AH-I and 5 - FU combination, volumes of tumors were decreased than 5-FU alone,weights of tumours were in the same changes with volumes ,growth of tumour was inhibited.9. In nude mice , NF-κB activity in tumour was inhibited in AH-I alone group.Combination AH-I and 5 - FU , NF-κB activity in tumour was inhibited more obviously than chemotherapy alone, also with a dose-dependent effect.10. In nude mice , combination AH-I and 5 - FU ,expression of the anti-apoptotic protein Bcl-2 in tumour was decreased ,but expression of apoptotic protein FAS and FAS-L in tumour were increased , and all of them were in dose-dependent effect.Conclusion1. The specific inhibitor of NF-κB from abrotani herba was successfully extracted ,then the inhibitor was identified and was named of AH-I temporary.2. NF-κB ativity was inhibited and sensitivity to 5 - Fu was enhanced in HT-29 spheroids by AH-I. 3. AH-I combination with 5–FU , cell cycle distribution was effected and expression of apoptostic proteins Bcl-2, FAS and FAS-L were effected aslo, apoptosis was induced in HT-29 spheroids in vitro.4. AH-I combination with 5–FU , growth of tumours was inhibited and effect of chemotherapy was enhanced in xenograft experiment.5. AH-I combination with 5–FU , cell cycle distribution was effected and expression of apoptostic proteins Bcl-2, FAS and FAS-L were effected aslo, apoptosis was induced in xenograft experiment in vivo.