Dissertation > Medicine, health > Pharmacy > Pharmacology

The Discovery and Biological Activity Study of ER Stress Modulator

Author FanLiXia
Tutor LiJia
School East China Normal University
Course Biomedical
Keywords UPR IRE1-XBP1 high-throughput screening 2DG BBR AMPK GRP78
Type Master's thesis
Year 2011
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The unfolded protein response(UPR) is a eukaryotic cellular adaptive mechanism that functions to cope with the endoplasmic reticulum stress(ER Stress). Accumulating evidence demonstrates that UPR is crucial for many diseases including tumor and diabetes. Now regulating UPR is a hot spot for anti-tumor and anti-diabetic drug discovery. IRE1-XBP1, one of the three pathways that activate the UPR, plays important roles in many biological processes such as cell growth, angiogenesis, adipogenesis, lipid metabolism and glucose metabolism and many disease such as cancer and diabetes. This thesis is attempted to find new compounds which involved in the regulation of IRE1-XBP1 pathway by establishment of high-throughput screening model and to study their biological activities and mechanisms of action.Firstly, the pCAX-F-XBP1△DBD-luciferase plasmid was constructed and transfected into human embryonic kidney cells HEK293. The stable cell lines expressing XBP1△DBD-luciferase were selected with G418 and evaluated with endoplasmic reticulum stress inducer tunicamycin(TM) for the sensitivity of ER stress response. Then clone named 6 was used for further study. Z’factor of up to 0.62 was obtained by a series of model optimization including the cell density, the final concentration of DMSO and TM as well as the incubation time, which means the cell-based high-throughput screening model targeting to IRE1-XBP1 signaling pathway was established. Then, a chemical molecule compound library containing 56,000 compounds were screened. And nine compounds were validated as the inhibitor of the XBP1 splicing with RT-PCR method. Among them, IC50 of three compounds against stable Hek293 cell line was below 1μg/ml. Then the inhibition of potent hits on cell growth was evaluated under both low-sugar condition induced by 2-Deoxyglucose(2DG) and normal culture. Compounds named 2 and 17 significantly inhibited tumor cell growth in low-sugar condition but had low toxic at normal culture. At last, compound named 2 displayed significant anti-tumor effects on human A549 lung cancer xenograft model with the T/C(%)value of 39.18. Berberine(BBR), a traditional Chinese medicine with a variety of biological activity, was found as the inhibitor of XBP1 splicing in the cellular model targeting to IRE1-XBP1 signaling pathway. And BBR in combination with 2DG enhanced growth Inhibition in many tumor cell lines, but not in the normal cell line WI-38. However, BBR in combination with TM had no enhanced effect on the inhibition of tumor cell growth. Although the activation of AMPK was observed when BBR was applied in combination with 2DG, the cell growth inhibitory effect failed to change significantly when endogenous AMPK were interfered with non-active AMPKα1 overexpressing, indicating that the significant inhibition of tumor cell growth by 2DG together with BBR was not depended on AMPK activation.Then the impact of 2DG combined with BBR on UPR was examined with. Western blot, luciferase reporter and RT-PCR methods, BBR alone has no effect on the UPR, and 2DG alone could activate the UPR. It is interesting that BBR could inhibited the expression of the key UPR downstream genes GRP78 and CHOP activated by 2DG in a dose depend manner, indicating that UPR was disrupted when 2DG was combined with BBR. Furthermore, all the three UPR signaling pathway including IRE1-XBP1, ATF6, and PERK-eIF2-ATF4 activated by 2DG were inhibited by BBR. However, BBR did not inhibit the UPR signaling pathway induced by TM. BBR selectively and dose-dependently suppressed 2DG-induced UPR and cell proliferation when A549 cells were treated with 2DG or tunicamycin in the presence or absence of BBR, suggesting that UPR inhibition is one of the possible reasons that UPR may be one of the reasons.Over-expression of GRP78 can relieve endoplasmic reticulum stress and promote cell survival. Then, GRP78 was over-expressed to investigate its contribution to the joint use of 2DG and BBR. The results showed that over-expression of GRP78 could partially reverse cell growth inhibitory effects of 2DG combined with BBR, indicating the important role of GRP78 in the enhanced anti-tumor activity of 2DG and BBR.In summary, we have established a high-throughput screening model targeting IRE1-XBP1 pathway at the cellular level and found a number of small molecule inhibitors, which had novel structures and displayed good biological activity, laying a tight basis for further drug discovery of targeting IRE1-XBP1 We also found that the joint application of traditional Chinese medicine berberine and 2DG could enhance the anti-tumor activity, and such enhancement was AMPK independent, but associated with the severely damage of UPR. Our work provides a new idea on the development of anticancer drug based on 2DG and BBR.

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