Mechanism Analysis Of Doxorubicin Resistance In Human Breast Cancercell MCF7

At present, one of the key reasons for the failure of the treatment of tumors is the tumor’sown multi-drug resistance. Although research shows that a variety of possible mechanisms, but itsdefinite mechanism is still unknown. In this thesis, the cancer treatment commonly used drugdoxorubicin as experimental subjects, mainly by analyzing the distribution of doxorubicin in thewild-type cells (MCF7/W), and drug-resistant cells (MCF7/ADM), metabolic changes,transcription factors differentially expressed and drug resistance marker protein analysis, vitaminE analogues of γ-tocotrienol on cell resistance reversal effect of experimental means to explorethe mechanism of multidrug resistance.This subject first use the autofluorescence of doxorubicin, through qualitative analysis byfluorescence microscope and quantitative analysis of subcellular localization. Found that theuptake of doxorubicin in the wild-type cells MCF7/W is much more than doxorubicin resistantcell MCF7/ADM and mainly in the nuclei; uptake in MCF7/ADM is less and is mainly present inthe cytoplasm. TOF-MS analysis of the metabolism of doxorubicin in the cells found that thedifference in peak doxorubicin in the drug resistant cells with wild-type cells, suggesting thatdoxorubicin may undergo a certain modification in the resistant cells.As a fat-soluble small molecule drug, doxorubicin can freely enter the nucleus of cells, but inthe resistant cells did not meet it, so we speculated that may be in the cell, the cytosolic proteinbinding to interception, or modification to obstruct the nuclear pore complex, so that doxorubicinis difficult enter into the nucleus; may be on the cell membrane P-glycoprotein (P-gp), orintracellular vesicals, the role of P-gp or vesicles will let the intracellular drugs out of cells, so thatthe intracellular doxorubicin content decreased.With these questions, the access to this topic were to extract the nucleoprotein of MCF7/W,and MCF7/ADM cell for transcription factor membrane analysis, select the currently studied inthe P-gp protein upstream transcription factor YB-1and autophagy formation marker protein LC-3upstream transcription factor TFEB. Weatern blot analysis showed that, compared with wild-typecells, P-gp and LC-3expression in the resistant cells was significantly enhanced.On the other hand, γ-tocotrienol resistance reversal effect is affected by the intracellularlocalization of P-gp and small G proteins, membrane localization of P-gp dependent on thehydrophobic isoprene-based (FPP GGPP)which is covalently linked to the small G protein (Rab,Rho). Use a specific CaaX in the end of fluorescent fragments, with the enzyme reaction, the FPP,GGPP is combined to the fluorescent end, and then by HPLC detection cell of the FPP, GGPP.Compared with MCF7/W cells, the content of cell FPP, GGPP in MCF7/ADM cells is low. And the the FPP,&GGPP content decreased in the cells with the treatment time.The promptγ-tocotrienol content of the FPP&GGPP is, thus affecting the degree of isoprenylation of small Gprotein, thereby affecting the positioning of P-gp reversal resistant cells.