Inhibition Effect Of Cytochrome C Mediated By Lentivirus On The Growth Of Breast Cancer Cells In Vitro And In Vivo

Breast cancer is a serious threat to human health.Aspongopus chinensis Dallas has been used as a traditional Chinese medicine and exhibits good clinical anti-tumor effects.In the present study,the anti-tumor protein named Cytochrome c(Cyt c)has been purified from A.chinensis for the first time,which could inhibit the proliferation and induce apoptosis of human cancer cell lines in vitro and in vivo,and it shows good prospects for application in anti-cancer.However,Cyt c is water-soluble and has a large molecular weight,which makes it difficult to penetrate the cell membrane.Then,how does exogenous Cyt c enter into the cancer cells more effectively? In this paper,we have developed a novel gene therapy with Cyt c protein targeting to breast cancer cells.Base on HIV-1 Gag characteristics of self-assembling and carrying other macromolecules into the Virus-like particles(VLP),Cyt c was cloned and carried by lentiviral vector.When virus packages and infects breast cancer cells,Cyt c can be packaged with Gag into VLP and expressed in breast cancer cells,thus achieving the purpose of treating breast cancer.In addition,this paper also studied the anti-tumor effect of Cyt c-coated VLP on Balb/c mice allograft,so as to provide experimental basis for the development of safe,efficient and proprietary anti-tumor drugs,as well as reference for the development and utilization of other traditional Chinese medicine.The main research results are as follows:1.Effect of Cyt c on the growth of breast cancer cells in vivo and in vitroThe purified protein was identified as Cyt c by SDS-PAGE electrophoresis,Western blot and MALDI-TOF-TOF-MS.MTT and morphological observation were used to detect the anti-proliferative effect of Cyt c on breast cancer cells.The results showed that low dose of Cyt c(5 ?g/m L)could promote the proliferation of MDA-MB-453 and HCC-1937 cells of breast cancer(P<0.05).Medium dose(10?g/m L)and high doses(20 ?g/m L))of Cyt c could significantly inhibit the proliferation of MDA-MB-453 and HCC-1937 cells in vitro(P<0.05).Moreover,Cyt c(40 mg/kg/2 d)could inhibit the growth of breast cancer cells in vivo.2.Construction of lentiviral expression plasmid with Cyt c and the package of VLPTotal RNA was extracted by TRIzol method and Cyt c gene was cloned.The open reading frame contained 324 bases,which codes for 108 amino acids.The lentiviral expression plasmid with Cyt c was amplified by using specificity primer with the enzyme site of Not ?,Bam H ?,the recombinant lentivirus expression plasmid PWSLV-Cyt c was obtained by digesting with double enzyme,ligating with T4 ligase,transformation,PCR identification,plasmid extraction,identification with double enzyme digestion,recombinant plasmid sequencing.VLP were produced in 293 T cells transfected with recombinant lentiviral expression plasmids PWSLV-Cyt c,lentiviral packaging plasmids ps PAX2 and PLTR-G.The expression of red fluorescent protein mcherry was observed under fluorescence microscopy.Western blot was used to detect the expression of viral capsid protein p24 in infected cells,indicating that VLP have been successfully packaged.3.Inhibitory effect of Cyt c-coated VLP on the growth of breast cancer cells in vitroMDA-MB-453 cells infected by Cyt c-coated VLP(mcherry-Cyt c-VLP)were detected by immunofluorescence,and the results showed that the expression of red fluorescent protein mcherry was observed,which indicated that mcherry-Cyt c-VLP had entered MDA-MB-453 cells,and high expression of Cyt c could only be observed in the MDA-MB-453 cells infected by mcherry-Cyt c-VLP.The anti-proliferative effect of mcherry-Cyt c-VLP on the growth of MDA-MB-453 cells was detected by CFDA-SE fluorescence labeling.The results showed that the number of cells labeled with green fluorescence in the mcherry-Cyt c-VLP infection group was decreased and the fluorescence was stronger,with a dose-dependent effect,which suggested that mcherry-Cyt c-VLP could inhibit the proliferation of breast cancer cells.Annexin?-FITC/PI assay was used to detect the pro-apoptosis effect of mcherry-Cyt c-VLP on breast cancer cells.The results showed that the early apoptosis rate of MDA-MB-453 cells treated with mcherry-Cyt c-VLP after 48 h was 20.27±0.33%,which was significantly different from that of the control group(1.23±0.34%)(P<0.0001).The rate of late apoptosis was 1.50±0.06%,which was significantly different from that of the control group(0%)(P<0.001),and the results showed that mcherry-Cyt c-VLP could induce apoptosis of MDA-MB-453 cells.The ultrastructure change of MDA-MB-453 cells treated with mcherry-Cyt c-VLP was detected by Transmission Electron Microscopy(TEM),the size of cell body and nucleus was shrunk,and the cell membrane was relatively complete,but the mitochondrial structure was incomplete,which were the ultrastructural characteristics of cell apoptosis.The expression of Fibroblast Growth Factor Receptor(FGFR)is higher in 14.5%of breast cancer,according to this characteristic,FGFR was chosen as the target for breast cancer therapy,lentivirus expression plasmid p WSLV-Cyt c-P2A-FGF1 has been constructed,which was identified with double enzyme digestion and recombinant plasmid sequence.Annexin ?-FITC/PI assay was used to detect the pro-apoptotic effect of Cyt c-P2A-FGF1-VLP on breast cancer cells,and the results showed that the early apoptosis rate of MDA-MB-453 cells treated with Cyt c-P2A-FGF1-VLP was 61.67%,which was significantly different from that of the blank control group(1.23±0.34%)and negative control group(20.27±0.33%)(P<0.0001).The early apoptosis rate of HCC-1937 cells treated with Cyt c-P2A-FGF1-VLP after was 30.07%,which was significantly different from that of the control group(1.37±0.09%)(P<0.001),but significantly lower than that of the mcherry-Cyt c-VLP group(32.97±0.99%)(P<0.01),indicating that Cyt c-P2A-FGF1-VLP had a strong pro-apoptotic effect on MDA-MB-453 and HCC-1937 cells,it is more effective in inducing apoptosis of MDA-MB-453 cells with FGF receptor overexpression.4.The inhibitory effect of Cyt c-coated VLP on the growth of transplanted breast cancer4T1 cells of mouse breast cancer were cultured in vitro,and Balb/c mouse model of breast cancer were constructed by injecting 4T1 cells into the fat pads of female Balb/c mice.mcherry-Cyt c-VLP were suspended in PBS and injected in situ into Balb/c mice.The mice were weighed every other day,the long and short diameters were measured with vernier calipers.2 weeks later,the volume and weight of tumor were measured.The influence of mcherry-Cyt c-VLP on the tumor,liver,spleen,lung and kidney of the mice were detected by HE staining,and the results showed that mcherry-Cyt c-VLP can significantly inhibit the growth of tumors,the number of tissues sinus capillaries were much less in the mice treated with low,middle and high doses of Cyt c,Moreover,the larger the dose,the fewer capillaries there were.In the negative control group and the empty carrier control group,the tumor cells and its nucleus were larger in size,clearer in cell contour,more uniform in chromatin distribution,and the mitochondrial ridge was complete.However,medium dose(5mg/kg),high dose(10 mg/kg)of Cyt c and low dose(10 ?g/kg),medium dose(50?g/kg),high dose(100 ?g/kg)of mcherry-Cyt c-VLP group showed significant reduction in the volume of tumor cell,nucleus shrinkage and nucleus chromatin aggregation.In the mcherry-Cyt c-VLP group,low dose and high dose of mcherry-Cyt c-VLP group possess smaller volume of cell body and nucleus,and the structure of intracellular organelles was incomplete,suggesting that mcherry-Cyt c-VLP may induce the occurrence of apoptosis in breast cancer.The results of the microscopy of liver tissue showed that a large number of lymphocytes were observed near the interlobular vein,interlobular bile duct and central vein of hepatic lobule in the portal region of liver tissues of mice treated with low,medium and high dose of Cyt c.However,there was no abnormal number of lymphocytes in the liver of mice treated with mcherry-Cyt c-VLP,which may be related to the decreased immune response caused by VLP.No abnormality was found in HE staining sections of spleen,lung and kidney.In summary,the results of this study showed that medium and high dose of Cyt c could inhibit the growth of breast cancer cells,while low dose of Cyt c could promote the growth of breast cancer cells in vitro.Lentivirus mediated the delivery of Cyt c into breast cancer cells,which could inhibit the growth and induce the apoptosis of breast cancer cells in vitro and in vivo,and its anti-cancer effect has been improved.