GRP78 is a key regulator of ER homeostasis due to its critical roles in protein folding, ER calcium binding, and activating transmembrane ER stress sensors. As a multifunctional chaperone, GRP78 promotes tumor cell proliferation, survival, metastasis, and resis tance to a variety of therapies. Combination therapy suppressing GRP78 expression and activity may repre sent an approach KPT-330 price toward improvement of the effective ness of cancer therapy. Recently, we and others demonstrated that taxol could induce GRP78 overex pression, XBP 1 splicing, and eIF2a phosphorylation, hallmarks of the unfolded protein response. GRP78 knockdown potentiates taxol induced JNK phos phorylation and protects breast cancer cells against paclitaxel induced apoptosis. In this regard, GRP78 may be a novel target to overcome taxol resistance.

Based on these earlier findings, we initiated this Inhibitors,Modulators,Libraries study to investigate whether EGCG is able to enhance the anti tumor effects of taxol in vivo. Materials and methods Reagents Paclitaxel were purchased from Sigma Aldrich, Inc. Ninety nine percent pure EGCG was purchased from MUST Biotech. The phosphorylated JNK antibody and JNK inhi bitor SP600125 were provided by Cell Signaling. GRP78 and b actin antibodies were pur chased from Santa Cruz Biotechnology. Cell culture Breast cancer cell lines were originally obtained from the American Tissue Culture Collection. Tumor cells were grown in tissue culture flasks Inhibitors,Modulators,Libraries at 37 C in a humidified atmosphere of 5% CO2 and were maintained as monolayer cultures in Dul beccos minimal essential medium supplemented with 5% fetal bovine serum, 100 U mL penicillin and 100 ug mL streptomycin.

4 1, 3 benzene disulfonate assay Cells Inhibitors,Modulators,Libraries were plated in 96 well plates at 5,000 cells per well. The next day, cells were treated with or without taxol and 20 uM EGCG in four replicates. After 24 or 48 h, cell viability was assessed by incubating cells with WST 1 reagent for 2 Inhibitors,Modulators,Libraries h and measuring the absorbance at 450 nm, and at 630 nm as reference, with a microplate reader. Hoechst 33342 staining Replicate cultures of 1 �� 106 cells per well were plated in 24 well plate. The cells were treated with or without 20 uM EGCG, 1 uM taxol and 10 uM SP600125. After a change of fresh medium 24 h later, the cells were incubated with 5 uL of Hoechst 33342 solution per well at 37 C for 10 minutes, followed by observation under a fluorescence microscope.

Strong fluorescence can be observed in the nuclei of apoptotic cells, while weak fluorescence was observed in non apoptotic cells. Quan tification of apoptotic cells was performed by counting cells in four random fields in each well. Western blot For cell cultures, cells were Inhibitors,Modulators,Libraries treated with 1 uM taxol, 20 uM EGCG, or combination for 24 h. The cells were washed selleck chem MG132 twice with phosphate buffered saline and har vested with cold lysis buffer containing protease inhibi tors or phosphatase inhibitors.