| Background and Objective:The chemotherapy regimen based on gemcitabine is a first-line clinical option for many solid tumors.However,single usage of gemcitabine is not sensitive,with an overall objective response rate of less than 10%,and the issue of clinical drug resistance is particularly prominent.Research has found that gemcitabine resistance may be related to the DNA damage response and DNA repair mediated by the activated checkpoint kinase Chkl.Traditional Chinese medicine has broad application prospects in enhancing tumor chemotherapy sensitivity and reversing tumor resistance.In recent years,we have payed much attention on Celastrus Orbiculatus Thunb.,a traditional Chinese medicine from the family Celastraceae and genus Celastrus,and has found that it has good drug development prospects in the field of anti-tumor therapy.Previous studies have shown that the total terpenoid extracts of C.orbiculatus can enhance the sensitivity of pancreatic cancer to gemcitabine in vivo and in vitro,but its specific material basis and mechanism of action are still unclear.This study aims to further isolate and purify the active components of C.orbiculatus,focus on the Chkl mediated DNA damage response signaling pathway,and explore the pharmacological material basis and molecular mechanism of C.orbiculatus enhancing the sensitivity to gemcitabine chemotherapy,providing new scientific basis for the clinical application and development of C.orbiculatus in anti-tumor therapy.Contents and Methods:This paper is divided into four chapters:In the first chapter,extraction and separation of the main anti-tumor active triterpenoid component betulinic acid from C.orbiculatu was conducted.Extract from C.orbiculatus were prepared using conventional high-temperature heat reflux extraction(HRE)method and low-temperature ultrasound assisted extraction(UAE)method.In non-small cell lung cancer H1299 and pancreatic cancer BxPC-3 cell lines,MTT cell viability test was used to compare the anti-tumor activity of the two extracts in vitro;Using high-performance liquid chromatography,nuclear magnetic resonance,and mass spectrometry to separate and identify the main peak component of the extract,triterpenoid betulinic acid was identified.MTT assay,clone colony formation assay,and EdU proliferation assay were used to further evaluate the anti-proliferative activity of betulinic acid.In the second chapter,the anti-tumor activity of betulinic acid on both sensitive and resistant cell lines to gemcitabine.The Combenefit quantitative pharmacology software and Chou-Talalay combination index were used to analyze the in vitro synergistic anti-tumor effect between betulinic acid and gemcitabine.The colony formation assay and Annexin V/PI apoptosis assay were used to investigate the in vitro tumor growth inhibition and apoptosis induction effects of the combination treatment.A gemcitabine-resistant cell line with overexpression of cytidine deaminase was established,and the resistance level and drug metabolism level of this cell line to gemcitabine were measured.The MTT assay and colony formation assay were used to study the anti-tumor activity of betulinic acid on the gemcitabine-resistant cell line.In the third chapter,we investigated the DNA damage effect and molecular mechanism of the combination of gemcitabine and betulinic acid on tumor cells.Alkaline comet assay,chromosome spreading assay,and immunofluorescence assay were used to study the effects of gemcitabine and/or betulinic acid on DNA double-strand breaks,chromosome structure,and the formation of DNA damage marker yH2AX foci.Western blotting and co-immunoprecipitation experiments were used to investigate the effect of betulinic acid on the Chk1 signaling pathway,degradation pathway,and degradation mechanism.In the last chapter,in vivo validation of the anti-tumor effect and molecular mechanism of the combination of gemcitabine and betulinic acid was performed.Lentiviral mCherry was used to construct the red fluorescent BxPC-3 cells,and nude mice subcutaneous xenograft model was established.Live imaging technology was used to study the anti-tumor effect of the combination of gemcitabine and betulinic acid.Western blotting was used to detect the expression level of Chk1 in the xenografts,and the correlation between Chkl level and the tumor weight was analyzed.Results:In the first chapter,betulinic acid is the major anti-tumor triterpenoid component of C.orbiculatus and is thermally unstable.It was found that the extraction temperature is a key factor affecting the yield and anti-tumor activity of C.orbiculatus,and betulinic acid was identified as the main triterpenoid component.Compared to heat reflux extraction,low-temperature ultrasound-assisted extraction could increase the extraction rate of betulinic acid by 3.6-folds,and the total extract obtained by ultrasound extraction showed 1.6 and 2.1-fold higher anti-tumor activity against BxPC-3 and H1299 cells compared to the total extract obtained by heat reflux.MTT,colony formation,and EdU proliferation assays showed that betulinic acid inhibited the proliferation of BxPC-3 and H1299 cells in a dose-dependent manner in vitro.In the second chapter,betulinic acid enhanced the anti-tumor sensitivity of gemcitabine in vitro and reverses gemcitabine resistance.Quantitative pharmacology experiments of drug combination activity showed that betulinic acid has a synergistic anti-tumor effect with gemcitabine.Clonogenic assays showed that low concentrations of betulinic acid further enhanced the clonogenic inhibition ability of gemcitabine.Annexin V/PI cell apoptosis experiments showed that low concentrations of betulinic acid could further enhance the apoptosis ability of gemcitabine.H1299 cell line overexpressing cytidine deaminase was successfully constructed,with a resistance index of 11.4.At the same time,overexpression of cytidine deaminase(CDA)can promote the deamination and inactivation metabolism of gemcitabine.MTT and clonogenic assay results showed that overexpression of cytidine deaminase had no effect on the anti-tumor activity of betulinic acid,indicating that betulinic acid has the same anti-tumor sensitivity to gemcitabine-resistant cell lines.In the third chapter,betulinic acid promoted gemcitabine-induced DNA damage and Chkl ubiquitination degradation.Alkaline comet assay results showed that betulinic acid further extended the comet tail lengths of BxPC-3 and H1299 tumor cells induced by gemcitabine.Chromosomal metaphase spreading experiments indicated that betulinic acid further promoted gemcitabine-induced chromatid breakage and chromosomal structural abnormalities.Immunofluorescence experiments demonstrated that betulinic acid further promoted the formation of DNA damage marker nuclear yH2AX foci induced by gemcitabine.These results suggest that betulinic acid enhanced gemcitabine-induced DNA double-strand break damage.Immunoblotting experiment showed that betulinic acid dose-dependently inhibited the phosphorylation activation of Chkl induced by gemcitabine and suppressed Chkl protein expression.Using proteasome inhibitor MG132,protein hydrolysis enzyme inhibitor leupeptin,and late-stage autophagy inhibitor Bafilomycin A1 to study the Chkl degradation pathway,it was found that only MG 132 could reverse the betulinic acid-mediated Chkl degradation.Immunoprecipitation experiment showed that betulinic acid could reverse the ubiquitination of Chkl induced by MG132.In the last chapter,betulinic acid in combination with gemcitabine inhibits the growth of pancreatic cancer xenograft tumors and reduced Chk1 expression in vivo.After drug administration for 5 weeks,gemcitabine(20 mg/kg,i.p,biw)or betulinic acid(40 mg/kg,i.g,qd)alone inhibited the relative growth of BxPC-3 xenograft tumors by 38.8%and 34.8%,respectively,while combination of two drugs significantly increased the relative growth inhibition rate to 78.9%.Therefore,compared with the gemcitabine group,the combination group significantly reduced the volume of the tumor grafts.There was no significant change in the body weight of mice in each treatment group compared to the model group.Protein immunoblotting experiments of the tumor grafts showed that betulinic acid alone or in combination with gemcitabine could inhibit the expression level of Chkl in the tumor grafts.Pearson correlation analysis of the expression level of Chkl and tumor weight in each group showed a significant positive correlation between the expression level of Chk1 and tumor mass(r=0.5919,p=0.0023).Conclusions:In summary,betulinic acid is an important substance basis for the anti-tumor activity of C.orbiculatus.Betulinic acid can enhance the anti-tumor effect of gemcitabine both in vivo and in vitro by promoting the degradation of Chkl,inhibiting the DNA damage response mediated by Chk1,and ultimately inducing DNA double-strand breaks to promote tumor cell death.These findings provide new experimental evidence for further understanding the chemotherapy enhancing effect,substance basis and molecular mechanism of C.orbiculatus,and provide a new scientific basis for its clinical application and development. |
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