| At present,there are many anti-cancer drugs in clinic,and the drugs for treating breast cancer are mainly paclitaxel and cyclophosphamide.In clinical treatment,the application of chemotherapeutic drugs has achieved good results,but the chemotherapeutic drugs are also a double-edged sword,because they have large side effects on patients,such as damage to the human immune system,digestive tract,and blood production.System,immune system,skin and mucosal reactions,nervous system,liver damage,heart reaction,pulmonary toxicity,renal dysfunction,and other reactions.Complications sometimes occur.Common infections,bleeding,perforation,uric acid crystals Wait.How to reduce or bring a series of side effects to patients while maintaining or even improving the efficacy of patients has been plagued by cutting-edge medical workers.As a tumor gene therapy,oncolytic virus therapy has become one of the important means of treating cancer.This experimental group constructed a dual cancer-specific anti-tumor recombinant adenovirus in a previous study and named it Ad-apoptin-hTERTp-E1a(Ad-VT).That is,a tumor-specific promoter(hTERTp,human telomerase reverse transcriptase)and a specific tumor suppressor gene(apoptin)are inserted into the original adenovirus,and a tumor-specific promoter(hTERTp,human telomerase reverse transcriptase))Start the E1A gene(The genes necessary for virus replication allow the adenovirus vector to replicate in cells that stably express E without the helper virus’s participation.Therefore,it belongs to the helper virus-free adeno vector.It only replicates in cancer cells and induces cancer apoptosis.)、cytomegalovirus(CMV)promoter activates the apoptin gene,so Ad-VT is a bispecific oncolytic adenovirus that has both tumor-specific killing and tumor-specific replication capabilities.The method of imaging firefly luciferase labeled cancer cells in vivo can be used to construct cell lines and visual animal models that stably express luciferase.Compared with other methods,this method is stable in biological characteristics,non-toxic,does not interfere with the anti-cancer effect of drugs,does not affect the treatment effect by affecting tumor growth,and overcomes high-intensity background noise during testing.Will be affected by non-specific fluorescence,its detection sensitivity,its wavelength advantage makes the emitted light easier to pass through the tissue,strong fluorescent signal,can show a stronger response in a short time,and relatively long half-life,metabolism Slow,fluorescein is completely eliminated after 3hours,the specificity is higher,and the risk of false positive expression is reduced.At present,the prevalence of female breast cancer patients ranks first in female cancer,and the comprehensive clinical treatment has caused great physical and mental harm to female patients.Various effective treatments with small side effects are constantly emerging,which will bring dawn to cancer treatment.Therefore,the purpose of this study was to explore the visualization of breast cancer using recombinant adenovirus Ad-apoptin-hTERTp-E1a(Ad-VT)and its synergistic effect with paclitaxel(PTX)on the inhibition of breast cancer proliferation and the effect of attenuated and synergistic effects.Strive to bring new benefits to breast cancer patients,and new treatment methods are also expected to become the hope of unsolved clinical problems.Method:1.MDA-MB-231 cells were transfected with pGL4.51 plasmid to construct tumor cells expressing stable luciferase(mda-mb-231-luc)and visualize the combination of Ad-VT and paclitaxel(PTX)for breast cancer2.Use Calcusyn software to analyze the synergy between Ad-VT and paclitaxel(PTX)and determine the final drug concentration.3.Inhibition of breast cancer cells by Ad-VT and paclitaxel(PTX)was examined by crystal violet staining assay and WST-1 assay.4.The effects of viral and chemotherapeutic drugs on the apoptosis level of breast cancer cells were examined by Hoechst,Annexin V,JC-1 staining and ROS assay.5.The effects of viral and chemotherapeutic drugs on the apoptosis level of breast cancer cells were examined by detection of caspase activity and detection of caspase3,PARP and cytochrome c protein levels.6.The inhibition of migration and invasion of MCF-7 and MDA-MB-231 cells by combination of ad-vt and paclitaxel was detected using Scratch,Transwell migration and invasion assay.7.The inhibitory effect of Ad-VT and paclitaxel on MCF-7 and MDA-MB-231cell metastasis was analyzed by detecting MMP2,MMP9,E-cadherin,Vimentin,and Snail protein levels.8.The tumor-bearing nude mouse model and metastasis model were established to confirm the tumor inhibition effect of the combination of Ad-VT and paclitaxel in vivo.Result:1.Cross-combination of different toxic values??Ad-VT with different concentrations of chemotherapeutic drugs,the inhibition rate data obtained in the WST-1 experiment is introduced into the calcusyn software,and it can be concluded that the CI index of some range data is less than 1.2.In the crystal violet staining results,we found that Ad-VT can cause significant cytotoxicity in MCF-7 and MDA-MB-231 cells,but is substantially non-toxic to MCF-10A cells.Paclitaxel has a certain cytotoxic effect on MCF-10A cells.When Ad-VT and paclitaxel are used in combination,it can increase the killing effect on breast cancer cells and reduce the toxicity to normal cells.In the WST-1 experimental results,we also found that Ad-VT has significant inhibitory effect on both breast cancer cells,the inhibition rate can reach about 40%,and has no toxic effect on normal breast epithelial cells,while paclitaxel has significant cytotoxicity on normal breast epithelial cells(P<0.01).When Ad-VT and paclitaxel were used in combination,the killing effect on normal cells was significantly lower than that on cancer cells(P<0.05),and the inhibitory effect of breast cancer cells after combination therapy was also significantly higher than that of Ad-VT or paclitaxel alone(P<0.05),the inhibition rate was higher than 65%when combined.3.In the Hoechst staining results,after the combination of Ad-VT and paclitaxel,the proportion of apoptosis was significantly higher than that of the Ad-VT group and the paclitaxel group;In the Annexin V flow results,after the combination of Ad-VT and paclitaxel,the apoptosis level of the two types of breast cancer cells was significantly increased,reaching about 75%(p<0.05).It was found by JC-1 staining that the combination of Ad-VT and paclitaxel found that the mitochondrial membrane potential changes of the two breast cancer cells were more significant.In the red-green ratio results,we also found that the mitochondrial membrane potential of the Ad-VT group was significantly lower than that of the control group and the paclitaxel-treated group(p<0.01),and this phenomenon was more sinificant after the combination.ROS test results showed that Ad-VT increased the level of reactive oxygen species in breast cancer cells,and the effect was more significant when combined with paclitaxel.4.Caspase activity detection and apoptosis-related protein detection found that Ad-VT can significantly increase the level of apoptotic proteins,and this phenomenon is even more pronounced when combined with paclitaxel.5.In the transwell migrate and scratch assay,it was found that Ad-VT,paclitaxel and combination therapy can inhibit migration ability of MCF-7 and MDA-MB-231 cells.The effect of Ad-VT was significantly higher than that of paclitaxel,and the effect after combination therapy was significantly higher than that of Ad-VT group and paclitaxel group(p<0.01).Similar results were also observed in the Transwell invasion assay.6.MMP2,MMP9,E-cadherin,Vimentin,and Snail protein levels were detected,and Ad-VT and Ad-VT combined with paclitaxel can reduce the levels of MMP2,MMP9,Vimentin,and Snail proteins and increase E-cadherin protein levels.7.The change in the bioluminescence intensity of the tumor was observed using a living body imaging system,and was continuously observed for 6 weeks.at 4-6weeks,the average bioluminescence intensity of the 1×10~9 PFU/100μl Ad-VT+20mg/kg paclitaxel treatment group and 1×10~9 PFU/100μl Ad-VT+10 mg/kg paclitaxel treatment group was significantly lower than that of control group(P<0.05).The inhibitory effect of Ad-VT combined with paclitaxel was significantly higher than that of the virus or drug alone.After the combination,the survival of the mice was significantly prolonged,the average survival times of 1×10~9 PFU/100μl Ad-VT+20 mg/kg paclitaxel treatment group and 1×10~9 PFU/100μl Ad-VT+10mg/kg paclitaxel treatment group were about 40.2 d and 39.6,respectively.And at 42days,the survival rate of the two treated mice were 80%and 70%,respectively.8.Observing the metastasis model using the in-vivo imaging system,it was found that in addition to the 1×10~9 PFU/100μl Ad-VT+20 mg/kg paclitaxel treatment group and 1×10~9 PFU/100μl Ad-VT+10 mg/kg paclitaxel treatment group,the phenomenon of tumor metastasis can be clearly seen in the lungs of the other treatment groups and the control group.Conclusion:1.Successfully constructed MDA-MB-231-LUC,a human breast cancer cell expressing luciferase,to visualize the efficacy of the combination of bispecific recombinant oncolytic adenovirus Ad-VT and paclitaxel in nude mice with breast cancer.2.Use Calcusyn software to analyze the synergistic effect of the combined application of Ad-VT and paclitaxel(PTX),and determine the final drug concentration.At the same time,it has less toxic and side effects on normal breast epithelial cells.3.The combined application of Ad-VT and paclitaxel can significantly increase the inhibition of breast cancer cell migration and invasion,and induced the apoptosis of MCF-7 and MDA-MB-231 cells through the mitochondrial pathway.4.The combined application of Ad-VT and paclitaxel can inhibit tumor growth and metastasis in vivo without obvious toxic and side effects in vivo.Application of luciferase-labeled human breast cancer cell MDA-MB-231-LUC has no effect on the anti-cancer.In summary,the recombinant adenovirus Ad-VT has tumor-specific replication and specific killing properties,and can inhibit the growth of breast cancer cells and promote their apoptosis.At the same time,the combined use with paclitaxel has a synergistic effect and plays a role in reducing toxicity and increasing efficiency.And the killing of triple negative breast cancer cells is basically consistent with the inhibitory effect of hormone-dependent breast cancer cells.This will increase the efficacy of breast cancer patients while reducing toxic and side effects.It may also bring dawn to patients with triple negative breast cancer with poor prognosis and provide new clues for the comprehensive treatment of breast cancer. |
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