Inhibitory Effect Of Small Molecule Inhibitors On The Degradation Of Tumor Cells And The Regulation Of Endoplasmic Reticulum Stress In Tumor Stem Cells

Objective The purpose of this study was to characterise the inhibitory effect of Eerl. a small molecule targeting the key protein of the ERAD pathway, p97, alone or in combination with the traditional anticancer drugs on tumor cells.Methods The effect of Eerl singularly and combined with cisplatin (CDDP) or bortezomib (BTZ) on tumor cell proliferation and apoptosis was measured through MTS assay and flow cytometry. Western blot was used to detect the cleavge of PARP and GRP78 in SW480 and PANC-1 cells after treatments.Results Eerl significantly inhibited cell proliferation and induced apoptosis of SW480 and PANC-1 in a dose-dependent manner compared with the control group (P<0.01). MTS assay and flow cytometry assay show that the combination treatment remarkably enhanced the cytotoxic effects of CDDP and BTZ (P<0.01). Western blot results demonstrate the combination treatment further enhanced the PARP cleavage, which is a marker of cell apoptosis. Interestingly, the expression of BiP, a marker for the unfolded protein response, is elevated after treatment of BTZ.Conclusion Eerl combined with other chemotherapeutic drugs significantly inhibits the growth of SW480 and PANC-1, thus establishing p97 as a novel target in the treatment of colon and pancreatic cancer.Objective To enrich human breast cancer stem cells in MDA-MB-231 tumor cells using the method of mammosphere formation assay and investigate the endoplasmic reticulum stress (ERS) in the mammospheres. Moerover, to explore the influence of ERS on sternness and the potential mechanism between sternness and ERS.Methods MDA-MB-231 cells were cultured in super-low adherent plate with serum-free medium, mammosperes were harvested, dispersed and xanograft assay was performed in nude mices to estimate sphere cell tumorigenicity in vivo. Q-PCR, Western Blot were performed to detect the expression of sternness and ERS markers in both mRNA and protein levels; The ERS inducers were added into the mammosphere culture medium to detect their influence on sphere formation ability; crystal violet test was assessed to evaluate the effect of ERS inducer on clonogeic potential of MDA-MB-231 cell; the effect of Tunicamycin on MDA-MB-231 cell population with high ALDH enzymatic activity was analyzed by flow cytometry. Effects of ERS on the proteins related to sternness such as Sox2 were performed by Western blot.Results Mammospheres were usually formed at day 7 under the serum free medium. Mammospheres highly expressed sternness related makers such as Sox2、Nanog、Oct4 and possessed high tumorigenic ability. Compared with adherent cells, mammospheres show obviously ERS with significant expression of XBP-1 splicing, GRP78, chop, ATF4; endoplasmic reticulum stress significantly weaken the mammosphere formation and 2D clone forming ability and down-regulate the cell population with high ALDH enzymatic activity and the expression of sternness related makers such as Sox2.Conclusion The method of mammosphere formation assay can successfully enrich BCSCs; BCSCs have significant ERS compared with adherent cells; the sternness characters of BCSC could be reduced through high level of ERS.