Translocation Of TG6-CytC Into Cytoplasm Induces Apoptosis

Apoptosis is a programmed death,a process in which cells die in a regulated manner.The process of tumor transformation,progression and metastasis involves alterations in the normal apoptotic pathway.Exploring the signals and mechanisms of apoptosis can help develop better cancer therapy.Cytochrome C(CytC)is an essential regulatory factor in the apoptotic process.When cells are stimulated by signals such as DNA damage,oxidative stress,or growth factor deficiency,CytC is released from mitochondria into the cell matrix and forms an apoptosome with APAF1 and pro Caspase-9.Activated Caspase-9 cleaves activated Caspase-3,leading to the onset of irreversible apoptosis.Activated Caspase-9 cleaves activated Caspase-3,leading to irreversible apoptosis.In order to diagnose and treat tumors,targeting CytC in cell stroma has developed into a hotspot.It has been shown that CytC can be transported into the cancer cell stroma to induce apoptosis in a variety of ways.For example,microinjection assay,supramolecular carrier assay and nanoparticle assay.The number of cells that can be manipulated by microinjection is limited,while in other assays CytC enters the cell by endocytosis and has to escape from the endosome to the cytoplasmic matrix before it can act,which significantly reduces the efficiency of CytC action.In previous experiments,our group found that the small molecule carrier TG6,which can be modified on CytC,can deliver CytC directly into the cancer cell stroma.This transport method can maintain the protein activity of CytC in a high dimension.The main objective of this project is to investigate whether the synthesized substance TG6-CytC can efficiently enter the cell matrix and induce apoptosis in cancer cells,and to further investigate the mechanism of TG6-CytC-induced apoptosis.In this study,the binding number of small TG6 molecules was increased by sulfhydrylation modification of CytC.Mass spectrometry peaks were used to demonstrate that TG6-CytC was fabricated exactly as expected.In addition,the similarity of TG6-CytC and CytC protein bands by SDS-PAGE and the concordance of TG6-CytC with the spectral peak at 400 nm of CytC using ultra-micro spectrophotometer.The above results confirm that TG6-CytC has been successfully fabricated.The results showed that TG6-CytC could effectively induce apoptosis in He La cells.Furthermore,TG6-CytC activity to induce apoptosis in tumor cells in vitro was detected by FACS.Among them,the number of early apoptosis,late apoptosis and dead cells produced by TG6-CytC action on He La were higher than those in CytC,CytC-SH and control cell groups.The results indicated that TG6-CytC could efficiently induce apoptosis in He La cells.In order to investigate the inhibitory effect of TG6-CytC on the proliferation rate of different cancer cells,lung cancer A549 and liver cancer Hep G2 cells were selected for MTT experiments.The results indicated that TG6-CytC had a significant inhibitory effect on cancer cells of different tissue origins.The above experiments showed that TG6-CytC could induce apoptosis efficiently.Based on this result,we further investigated the mechanism of TG6-CytC induced apoptosis in cancer cells.DEVD is a small molecule inhibitor of Caspase,which itself has no effect on cell proliferation.He La cells were examined using EC50(0.03 μg/m L)of TG6-CytC and CytC action together with DEVD,and the results showed that DEVD rescued TG6-CytC-induced apoptosis with a minimum effective concentration of 20 μM.To better observe the phenomenon of TG6-CytC inducing early apoptosis,the cells were treated with DEVD at a final concentration of 20 μM for 2 h,followed by the addition of0.3 μg/m L of TG6-CytC for 1 h in He La cells.The data of red fluorescence intensity over green fluorescence intensity by mitochondrial membrane potential assay(JC-1)were analyzed,and the smaller the ratio obtained,the higher the degree of apoptosis.The results showed that the ratio was extremely small in the TG6-CytC group and increased in the TG6-CytC+DEVD group.It can be deduced that the action of TG6-CytC on He La cells decreased the membrane potential of mitochondria,resulting in the release of CytC from mitochondria into the cell matrix.Meanwhile,the results of JC-1 verified that DEVD could effectively inhibit TG6-CytC induced apoptosis.Using experimental conditions consistent with those described above,it was observed using immunofluorescence(IF)that green fluorescence(CytC)was more diffuse in TG6-CytC group than in the other groups,and that CytC was present in both mitochondria and the cell matrix.Western Blot(WB)results showed that TG6-CytC treated cells obtained higher cytoplasmic CytC protein and cleaved Caspase-3 protein than the other groups,while the amount of CytC protein in mitochondria was almost absent.All the above experiments can verify that TG6-CytC can efficiently enter the cell matrix and effectively stimulate the release of CytC from mitochondria into the cell matrix,thus inducing apoptosis.Meanwhile,this study infers that TG6-CytC-induced apoptosis is mediated by Caspase-3.In summary,TG6-CytC synthesized and fabricated in this study can efficiently enter the stroma of cancer cells and induce apoptosis.Based on the experiments,it was tentatively concluded that TG6-CytC induced apoptosis was mediated by Caspase-3.The development and clinical application of TG6-CytC antitumor agent designed in this study is of great significance.