| Background and Objective:Given the rising demand for efficient Chinese herbal remedies in treating refractory diseases,efficacy and safety are two industrial bottlenecks for the efforts of modernization traditional Chinese Medicine（TCM）.Pancreatic cancer（PC）is one of the most malignant tumors.The onset of PC is insidious,and most patients are already at an advanced stage at the time of diagnosis,often accompanied by distant metastases.Gemcitabine（GEM）is widely used as the gold standard and basic regimen for the treatment of unresectable as well as postoperative adjuvant chemotherapy for resectable PC However,the objective remission rate of gemcitabine alone is less than 10%and is an important cause of metastatic progression and treatment failure in pancreatic cancer.Therefore,intensive research and discovery of safe and effective chemopotentiation agents to increase the therapeutic index of gemcitabine signifies an urgent need for PC patients.To maintain genomic stability,normal cells initiate a DNA damage response（DDR）to rapidly repair damaged DNA.Radiotherapy and chemotherapy,to some extent,kill cancer cells via the induction of DNA damage.To evade cell death,tumors become more reliant on DDR and develop resistance to chemotherapy or radiotherapy.Thus,targeting DDR signaling pathways has become an attractive strategy for overcoming resistance to cancer therapy.DDR is a complex signal transduction network from sensing DNA lesions,transducing the DNA damage signal downstream to elicit a series of biological responses,including cell cycle checkpoint activation and DNA damage repair.Checkpoint kinase 1（Chkl）,a serine/threonine protein kinase,is a major effector of DDR.Chk1 is an attractive target for DDR:（1）it participates in almost all the cell cycle checkpoints surveillance;（2）it functions in stabilizing replication forks.When cells encounter replication stress or DNA damage,Chkl facilitates late repair and maintains genomic instability by stalled replication forks;（3）it promotes the loading of key molecules of homologous recombination repair such as BRCA1/2 and RAD51 to the damaged site,thereby promoting DNA damage repair.Therefore,inhibition of Chkl can disrupt replication fork structure,inhibit HR repair,and ultimately lead to severe DNA double-strand break damage in tumor cells,thereby enhancing gemcitabine sensitivity.PC has been well-recognized and documented by Chinese medicine practice,which believes that dampness and heat toxicity and the interplay between the two factors predominantly contribute to the development of PC.Particularly,climbing vine herbs have significant activity for dispelling wind and dampness,removing blood stasis and clearing heat and toxicity.Celastrus orbiculatus Thunb.a woody vine of the family Celastraceae is widely distributed and used as a folk medicine in China.Our lab has conducted a systematic study on the chemical basis and pharmacological efficacy of Celastrus orbiculatus Thunb.against gastrointestinal tumors for more than 10 years,and has confirmed that the extract of Celastrus orbiculatus has significant anti-tumor activity against gastric,liver cancer,etc.We further obtained the active fraction,Total Terpenoids of C.orbiculatus（TTC）,from the ethyl acetate extact of C.orbiculatus through activity tracking,chemical isolation and structural identification.This thesis proposes to advance the knowledge of TTC against pancreatic cancer,and systematically answer whether TTC can enhance the chemotherapeutic sensitivity of pancreatic cancer to gemcitabine by inhibiting the key DDR signaling pathway.Therefore,this paper is aimed to clarify that whether TTC can enhance the antitumor activity of gemcitabine in PC models in vitro and in vivo,and to quantitatively evaluate the pharmacological synergistic effects between TTC and gemcitabine;to investigate the molecular mechanism by which TTC enhances the chemotherapeutic sensitivity of gemcitabine to PC cells by targeting Chkl/RAD51-mediated DNA damage repair.The paper provides an experimental basis for the development of naturally occurring DDR inhibitor used as a sensitizer of gemcitabine.Chapters:This thesis consists of three chapters:In the first chapter,the chemical composition of TTC was characterized by high performance liquid chromatography,nuclear magnetic resonance and mass spectrometry.Two pancreatic cancer cell lines AsPC-1 and BxPC-3 were selected to study the in vitro antitumor activity of TTC,purified diterpenes and triterpenes using MTT cell viability assay and EdU proliferation assay.The DNA double-strand break damage effect of TTC was investigated using comet assay and metaphase chromosome spread assay.Western blot assay was used to study the effect of TTC on DNA damage response and repair pathways including Chkl/RAD51.The objective of this chapter is to confirm that TTC exerts anti-tumor effects by inhibiting HR repair and triggering DNA double-strand breaks in pancreatic cancer.In the second chapter,we analyzed the synergistic effect of TTC on gemcitabine using Combenefit software.The in vitro chemosensitization activity of TTC combined with gemcitabine was investigated using clone formation assay,EdU proliferation assay and Annexin V/PI apoptosis assay.The effects of TTC and/or gemcitabine on DNA damage response and repair pathways were examined using western blot assay.BxPC-3 cells were stably transduced with mCherry using lentiviral particles was implanted into nude mice to establish orthotopic xenograft model,which was used to study the in vivo antitumor activity and safety of TTC in combination with gemcitabine.The objective western blot assay of this chapter is to clarify that TTC exerts its gemcitabine-sensitizing effect by reversing the Chkl-mediated DNA damage response and HR repair activated by gemcitabine and enhancing DNA damage in PC cells in vitro and in vivo.In the third chapter,the molecular mechanisms of TTC on Chkl,RAD51,and p53 protein stability was investigated using the protein synthesis inhibitor cycloheximide and the protein ubiquitin proteasome pathway inhibitor MG132,in combination with Western blotting assay.To investigate the effect of mutant p53 on Chkl/RAD51 signaling pathway and the effect of TTC,three hotspot mutant p53（R175H,R248W and R273H）were transiently transfected in p53-null AsPC-1 cells.The objective of this chapter is to confirm that TTC increases gemcitabine efficacy via targeting Chkl/RAD51 signaling molecules of DDR and mutant p53.Rresults:As a result,TTC,purified diterpenes and triterpenes exhibited certain cytotoxic effects on AsPC-1 and BxPC-3 cells.In comparison,TTC showed lower cytotoxicity in normal human gastric epithelial cells（GES-1）.Comet assay and metaphase chromosome spreading assay confirmed that TTC could induce DNA double-strand breaks and chromosomal structure abnormalities in pancreatic cancer cells.Besides,TTC attenuated the total reactive oxygen species and we ruled out the possibility of oxidative DNA damage.Western blot assay showed that TTC dose and time-dependently suppressed the expression levels of Chkl and RAD51,which which were essential DNA repair.Moreover,accumulation of yH2AX,a biomarker of DNA double-strand breaks,was observed in TTC-treated PC cells.Quantitative synergy analysis using Combenefit software analysis indicated that the most intense region of synergy attained across combined concentrations under sub-IC50 values.EdU assay revealed that TTC conferred sensitivity by further depleting cells at the G1/S boundary in the presence of gemcitabine.Gemcitabine plus TTC could stimulate an enhanced pro-apoptotic potential in PC cells.Western blot assay revealed that gemcitabine elicited the phosphorylation of Chkl and nuclear loading of RDA51,which could be partially or completely ablated by TTC.In an orthotopic mouse model of pancreatic cancer,TTC could further attenuate the growth of primary tumor by weighting pancreas tissues compared with gemcitabine treatment reversed gemcitabine-mediated hepatotoxicity.Mechanism study showed that TTC triggered proteasomal degradation of Chkl and RAD51.Furthermore,TTC also targeted mutant p53 and inhibited mutant p53-mediated activation of Chk/RAD51 signaling pathway.Conclusion:Our data suggest that TTC displays single-agent anti-tumor activity and increases gemcitabine efcacy via suppression of Chkl/RAD51-mediated DNA damage response and repair.Mechanistically,TTC promotes proteasomal degradation of Chkl and RAD51,culminating in DSBs with apoptotic cell death.Besides,TTC is found to destabilize the mutant forms of p53 in PC cells,in which RAD51 transcription is activated.In summary,our study could provide a plausible candidate of novel combination options for augmentation of the therapeutic index of gemcitabine.In sum,we believe that TTC holds encouraging preclinical data to test the combination of gemcitabine and Chkl-RAD51 pathway inhibitors in metastatic and recurrent PC patients with mutant p53... |
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