Molecular Mechanism Study On The Anti-tumor Activity Of Novel Pentacyclic Triterpenoid Gold Derivatives By Inhibiting TrxR Expression

Disturbance of tumor redox system is a significant feature of tumorigenesis.When tumor cells proliferate in large numbers,it will lead to the abnormal activation of intracellular signaling pathways and increase the reactive oxygen species(ROS)level continuously.With the increase of ROS level,the ability of tumor cells to produce antioxidant substances is enhanced to resist the cell death induced by ROS.Thioredoxin reductase(TrxR)is an important antioxidant enzyme that can regulate a variety of biological functions and has been proven to be closely related to the occurrence of human malignant tumors and other diseases.It has become one of important anti-tumor targets.ROS is important to maintain the homeostasis of the endoplasmic reticulum.During the folding process of the endoplasmic reticulum,oxidized proteins stimulate TrxR to transfer electrons to thioredoxin(Trx),which destroys oxidation by removing ROS.The latest researches show that the activation of endoplasmic retieulum stress(ERS)is highly correlated with the degree of tumor damage and pathological progress in the design of drugs targeting TrxR,which become a potential downstream mechanism of TrxR.Interestingly,the generation of ERS and ROS is an important part of generating tumor immunogenic cell death(ICD).The co-existence of ERS and excess ROS increases the characteristic discharges of different damage-associated molecular patterns(DAMPs),including the exposure of Calreticulin(CRT)and high mobility protein(HMGB1)on the cell surface and the release of ATP,these signals are key to the immunogenicity of tumor cells in the ICD process.The release of DAMPs enhances the immunogenicity of tumor cells,improves the antigen presentation ability of dendritic cells(DCs),and triggers a series of T cell-dependent immune responses.After T cells specifically bind to tumor cells,it destroys its cell membrane,directly kills tumor cells,or releases lymphokines to expand and enhance the immune effect.Studies have shown that pentacyclic triterpenoids of traditional Chinese medicine have very broad application prospects in anti-tumor research.However,due to the various degrees of defects in the clinical efficacy of these compounds,they can only be used as adjuvant therapy.Preliminary studies of our lab have shown that gold compounds have clear TrxR inhibitory activity and show obvious advantages in anti-tumor.Therefore,in this study,the traditional Chinese medicine pentacyclic triterpenoids are effectively coordinated with gold to play the dual function of the pentacyclic triterpenoids and gold ions,so as to achieve the powerful anti-tumor compounds.Taking into account the combination of the active ingredients of traditional Chinese medicine and the mechanism of action of metals,the design and synthesis of novel moleculars possessing dual functions have become the focus of innovative drug research and development,especially in the design of immune anti-cancer drugs.Mainly by reversing the tumor immune escape,directly promoting the function of immune cells,thereby enhancing the anti-cancer effects.We propose that effective coordination of traditional Chinese medicine pentacyclic triterpenoids-gold compounds can obtain a new class of compounds that can effectively inhibit TrxR activity,which directly leads to the increase of ROS level during the process of redox regulation.Further,these compounds could induce the ICD effects of tumor cells by activating ERS signal,which finally achieve the purpose of strong anti-tumor ability.In this thesis,we synthesis a series of pentacyclic triterpenoids(Oleanolic acid(OA),Glycyrrhetinic acid(GA),Betulinic acid(BA),Ursolic acid(UA))-alkynyl gold(I)compounds and tested their anti-tumor activities in different tumor cells(breast cancer cell MCF-7,human colon cancer cell HT-29,liver cancer cell HepG2,ovarian cancer cell A2780).We finally select the dominant compound(OA-Au)for the following step to explore the mechanism and evaluate its activity in vitro.The results account for that OA-Au has better proliferation inhibitory activity in human ovarian cancer cell A2780 compared with the raw material drug OA.The compound OA-Au significantly inhibits the enzymatic activity of TrxR.Electron microscopy results show that OA-Au can significantly promote the expansion of the endoplasmic reticulum lumen,swelling of the endoplasmic reticulum,and a large number of translucent vacuoles formed in the ERS state.It showed that OA-Au significantly promoted the occurrence of ERS in A2780 cells.Next,we use ROS scavenger NAC and ERS inhibitor Salubrinal for the reverse verification.Western blot and immunofluorescence analysis consistently showed that inhibition of ROS expression could significantly inhibit the overexpression of ERS markers CHOP and Calnexin.Similarly,Salubrinal,an ERS inhibitor,can also counteract the level of apoptosis of tumor cells and the inhibitory activity of TrxR.The results of in vivo experiments are consistent with those in vitro.The gold compound of OA significantly inhibits the growth of transplanted tumors in nude mice.There is a higher TrxR expression in tumor model tissues,and the administration group significantly decreases.The results of immunohistochemistry and immunofluorescence analysis also consistently showed that the expression of ERS and apoptosis markers in tumor tissues decreased,and the expression of these factors was significantly increased after administration.Therefore,we infer that OA-Au may further activate ERS expression by inhibiting TrxR activity,thereby promoting tumor cell apoptosis.In addition,we also synthesized a series of pentacyclic triterpene nitrogen heterocyclic carbene gold(?)compounds.In the same way,the anti-tumor activity and TrxR inhibitory effects of the compounds were evaluated.The dominant compound gold(?)glycyrrhetinic acid(GA-Au)was screened for the following mechanism study.Since the co-existence of ROS and ERS is a prerequisite for ICD,we further tested ERS-related indicators and the expression of Ca2+,confirming that the endoplasmic reticulum has a significant stress regulation with the OA-Au treatment.And the morphological changes of the endoplasmic reticulum were verified by ultramicroscopic observation of electron microscope.Next,we tested the effects of the compounds on the tumor immune system and the expression of DAMPs-related signals.Western blot and laser confocal microscopy observations showed that GA-Au significantly promoted the exposure of calreticulin(CRT)on the cell surface.Following,the expression of HMGB1 in the supernatant was detected by ELISA and western blot assays.Extracellular transport,and detection of the release of ATP,these signals are the key to the immunogenicity of tumor cells in the ICD process.Finally,isolate and prepare dendritic cells(DCs)derived from C57BL/6 mouse bone marrow,and co-culture them with tumor cells to verify the maturation of DCs,and detect the cytokines IL-6,TNF-?,and IL-12 in the supernatant.Because IL-6 is an important cytokine for B cell stimulation in humoral immunity,TNF-? is a typical marker for cellular immunity and can directly kill cancer cells,and IL-12 can stimulate the proliferation of activated T cells and induce the cytotoxic activity of the CTL and NK cells.The results show that the compound significantly promotes the maturation of DCs,activates the corresponding cytokines and the proliferation of T cells,and then leads to the immune response of tumor cells.This also further explains the mechanism of the compound's potent anti-tumor effect.In summary,this study has confirmed that pentacyclic triterpene gold(?)compounds can significantly improve the anti-tumor activity of OA,GA,UA,and BA.Further studies on the mechanism of the dominant compound have shown that it inhibits TrxR activity and increases ROS level.It accumulates ROS in a large amount,and induces the activation of ERS-related signal pathways,and finally induces the occurrence of ICD in tumor cells.This thesis confirms the anti-tumor effect of traditional Chinese medicine pentacyclic triterpenoids from the overall level,cell level and molecular level,and investigates its in-depth mechanism,opening up a new idea for screening reasonable and effective anti-tumor drug candidates.