مقالات

Treatment with EGCG induced apoptosis by targeting cFLIP/Ku70 complex in gastric cancer cells

1400/7/3 15:34
مقدمه

Cancer stem cells (CSCs) are the main cause for the initiation, spread, and development of resistance to treatments in tumors. Chemotherapy and radiotherapy, two common therapies for gastric cancer (GC), have several side effects. Specific cancer molecular pathways, particularly in GC, can be targeted for effective treatments. In cancer cells, the apoptosis process is disturbed. Ku70 is an acetylation-capable non-histone protein that is cumulated in the cancer cell's cytoplasm and binds to antiapoptotic proteins like cFLIP. This binding stabilizes cFLIP and prevents apoptosis. The goal of this study was to use a natural histone deacetylase inhibitor called EGCG, the main component of green tea, to induce apoptosis in cancer cells by targeting cFLIP/Ku70 complex in GC stem cells.

روش کار

First, we used AnnexinV-FITC to determine the proper dose (IC50) of EGCG that induced apoptosis. Cell cycle analysis was performed using flow cytometry technique to determine the phase of the cells after treatments and prove the induction of apoptosis in them. Two indirect immunofluorescence techniques, as well as isolation of nucleus and cell cytoplasm with sub-cellular fractionation technique and subsequently Western blotting technique, were used to analyze protein expression profile and the location of the cFLIP/ku70 complex before and after treatment. Finally, the expression of CyclinD1, P21, P53, and c-MYC genes that are implicated in the apoptosis and cell cycle pathways was examined using the qRT-PCR method.

نتایج

A concentration of 100μm EGCG was chosen as the proper concentration in these cells based on previous studies. G2/M arrest can be caused by 100μm EGCG treatment. Furthermore, EGCG reduced the expression of c-MYC and CyclinD1 genes, which are involved in the regulation of the cell cycle in the G2 / M phase, and induced the expression of apoptotic genes like P53 and P21. In the EGCG-treated group, the level of cFLIP protein in the cell’s cytoplasm was reduced, and Ku70 protein was considerably translocated from the cytoplasm to the nucleus. Western blot analysis proved our hypothesis about protein expression level before and after treatment with 100mM EGCG. Finally, for a more detailed study of this phenomenon, by separating the core proteins from the cytoplasm using the subcellular fractionation technique and consequently western blot technique, we confirmed the reduction of cFLIP expression and the displacement of the Ku70 protein from the cytoplasm to the nucleus.

نتیجه‌گیری

EGCG, which is a natural histone deacetylase inhibitor, induces dissociation of cFLIP/Ku70 complex. The existence of the cFLIP/Ku70 complex in cancer cell’s cytoplasm is one of the main reasons for apoptosis suppression. As a result, the application of EGCG could be a practical strategy for targeted molecular therapies for dissociation of this complex and induce apoptosis effectively.