Studies On The Mechanism Of NIP For Inhibiting Autophagy And Inducing The Apoptosis Of HepG2/ADM Cells

Autophagy is a self-protection mechanism for cells when they are under the metabolizing pressure such as starvation, energy deficiency etc. Autophagy has the functions of degrading the non-essential substances in cells and maintaining the cell homeostasis. The previous research has shown that the autophagy that happened within cancer cells would reduce the sensitivity of cells to chemotherapeutic drugs. However, by means of inhibiting autophagy, the expression level of p62protein will increase; as the over-expressed p62combine with Keapl, the Nrf2will enter into the nucleus and activate the downstream genes to maintain the cell homeostasis, which will affect the treatment badly. Therefore, the inhibition of both the autophagy process and the expression of Protein Nrf2might be a new way to improve the sensitivity of drugs. In the present study, a new type of chemical compound NIP7-(4-nitrophenyl)-5,8a-diphenyl-1,2,3,7,8,8α-hexahydroimidazo-[1,2-a]pyridine was used to investigate its relationship with the autophagy of cancer cells; and by jointly using NIP and adriamycin, an investigation on the mechanism of drug resistance of liver cancer cells was conducted in order to illustrate and sustain this new strategy for tumor treatment.Through the MTT examination, it was found that NIP has a selectively anti-proliferative effect on eight types of tumor cells, and is particularly sensitive to liver cancer cell HepG2for which the IC50value is12.5μM. The HepG2cells were treated with non-nutrient medium MEM/EBSS to initiate autophagy, and then treated with NIP; comparing with the control group, it was found that, the number of autophogosome stained with specific dye MDC among the sample has decreased. Further detection with the flow cytometry found that the flux of autophagy has dropped; and observed from the transmission electron microscope it was also found that the number of autophogosome has decreased. The observation by using the confocal laser scanning microscope has revealed that the expression of the tagged molecule LC3has decreased while for p62, it has increased. The results of western blot test have shown that the protein expression of LC3and Beclinl has declined; and the examination by RT-PCR test has revealed that the transcriptional level of p62mRNA has increased. As HepG2is induced by maintaining with2μM adriamycin, a HepG2/ADM drug-resistent cell model is constructed. In comparison with the control group by means of flow cytometry, it was found that the HepG2/ADM cells that are jointly treated by NIP and adriamycin become more sensitive to adriamycin, which induces the apoptosis. Through the examination of western blot test, it was demonstrated that the expression level for p62has increased; while for proteins such as LC3, Beclinl, and Nrf2, the expression level has descended; the cleavage of apoptosis molecule PARP has increased.In conclusion, the present study demonstrated that NIP has the following effects as to inhibit the autophagy within cells, to inhance the sensitivity of HepG2/ADM cells to adriamycin, and to induce the apoptosis of this drug-resistent cells.