leupeptins and Endometrial-Neoplasms

We examined how clomiphene citrate (CC) reduces estrogen receptor-α (ERα) in a human endometrial cancer cell line. Ishikawa human endometrial cancer cells were treated with ERα ligands such as 17β-estradiol (E2), CC, and the pure antiestrogen, ICI 182,780 (ICI). Thereafter, the expression levels of ERα protein and mRNA were analyzed by western blot and real-time quantitative PCR, respectively, and those of ubiquitinated ERα were analyzed by immunoprecipitation of ERα followed by immunoblotting with an anti-ubiquitin antibody. The expression levels of ERα protein after treatment with E2, CC, and ICI were significantly decreased compared to pre-treatment levels without a corresponding decrease in ERα mRNA. These ligands significantly increased the levels of ubiquitinated ERα compared to vehicle treatment. Co-treatment with the proteasome inhibitor, MG132, abrogated the decrease in ERα levels caused by treatment with the ligands only. We demonstrated, for the first time, a CC-induced decrease in ERα mediated by the ubiquitin-proteasome pathway in human endometrial cancer cells.

Topics: Cell Line, Tumor; Clomiphene; Down-Regulation; Endometrial Neoplasms; Estrogen Receptor alpha; Female; Green Fluorescent Proteins; Humans; Leupeptins; Ligands; Proteasome Endopeptidase Complex; Protein Transport; Proteolysis; RNA, Messenger; Signal Transduction; Ubiquitin; Ubiquitination

To study the anti-cancer activities of Z-Leu-Leu-Leu-cho (MG132) against human endometrial carcinoma HEC-1B and Ishikawa cells and the potential of MG132 in human endometrial cancer therapy.. HEC-1B and Ishikawa cells were treated with MG132. Cell proliferation was assessed by MTT while cell apoptosis rate and cell-cycle distribution were assessed by flow cytometry.. The proliferation of HEC-1B and Ishikawa cells was inhibited by MG132 and cell proliferation was significantly inhibited with the raised concentration of MG132 (P<0.01), Ishikawa cells were more sensitive than HEC-1B cells to MG132. MG132 could induce the apoptosis of HEC-1B and Ishikawa cells (P = 0.000). Cell cycle analysis indicated that the percentage of HEC-1B cells was increased in G2 phase (P<0.05) while Ishikawa cells' was increased in G1 and G2 phase (P<0.05).. Proteasome inhibitor MG132 could inhibit the proliferation, promote cell apoptosis, and block the cell cycle of HEC-1B and Ishikawa cells. MG132 may be a potential treatment for endometrial cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Endometrial Neoplasms; Female; Humans; Leupeptins; Proteasome Inhibitors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising antineoplastic agent because of its ability to selectively kill tumoral cells. However, some cancer cells are resistant to TRAIL-induced apoptosis. We have previously demonstrated that in endometrial carcinoma cells such resistance is caused by elevated FLICE-inhibitory protein (FLIP) levels. The present study focuses on the mechanisms by which FLIP could be modulated to sensitize endometrial carcinoma cells to TRAIL-induced apoptosis. We find that inhibition of casein kinase (CK2) sensitizes endometrial carcinoma cells to TRAIL- and Fas-induced apoptosis. CK2 inhibition correlates with a reduction of FLIP protein, suggesting that CK2 regulates resistance to TRAIL and Fas by controlling FLIP levels. FLIP downregulation correlates with a reduction of mRNA and is prevented by addition of the MG-132, suggesting that CK2 inhibition results in a proteasome-mediated degradation of FLIP. Consistently, forced expression of FLIP restores resistance to TRAIL and Fas. Moreover, knockdown of either FADD or caspase-8 abrogates apoptosis triggered by inhibition of CK2, indicating that CK2 sensitization requires formation of functional DISC. Finally, because of the possible role of both TRAIL and CK2 in cancer therapy, we demonstrate that CK2 inhibition sensitizes primary endometrial carcinoma explants to TRAIL apoptosis. In conclusion, we demonstrate that CK2 regulates endometrial carcinoma cell sensitivity to TRAIL and Fas by regulating FLIP levels.

Topics: Antineoplastic Agents; Apoptosis; Casein Kinase II; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line; Drug Resistance, Neoplasm; Endometrial Neoplasms; fas Receptor; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Neoplasm Proteins; Proteasome Endopeptidase Complex; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

We have recently demonstrated that proteasome inhibitors can be effective in inducing apoptotic cell death in endometrial carcinoma cell lines and primary culture explants. Increasing evidence suggests that reactive oxygen species are responsible for proteasome inhibitor-induced cell killing. Antioxidants can thus block apoptosis (cell death) triggered by proteasome inhibition. Here, we have evaluated the effects of different antioxidants (edaravone and tiron) on endometrial carcinoma cells treated with aldehyde proteasome inhibitors (MG-132 or ALLN), the boronic acid-based proteasome inhibitor (bortezomib) and the epoxyketone, epoxomicin. We show that tiron specifically inhibited the cytotoxic effects of bortezomib, whereas edaravone inhibited cell death caused by aldehyde-based proteasome inhibitors. We have, however, found that edaravone completely inhibited accumulation of ubiquitin and proteasome activity decrease caused by MG-132 or ALLN, but not by bortezomib. Conversely, tiron inhibited the ubiquitin accumulation and proteasome activity decrease caused by bortezomib. These results suggest that edaravone and tiron rescue cells of proteasome inhibitors from cell death, by inhibiting blockade of proteasome caused by MG-132 and ALLN or bortezomib, respectively. We also tested other antioxidants, and we found that vitamin C inhibited bortezomib-induced cell death. Similar to tiron, vitamin C inhibited cell death by blocking the ability of bortezomib to inhibit the proteasome. Until now, all the antioxidants that blocked proteasome inhibitor-induced cell death also blocked the proteasome inhibitor mechanism of action.

Topics: Antioxidants; Antipyrine; Apoptosis; Ascorbic Acid; Blotting, Western; Boronic Acids; Bortezomib; Butylated Hydroxyanisole; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Coumarins; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Edaravone; Endometrial Neoplasms; Ergothioneine; Female; Humans; Leupeptins; Oligopeptides; Proteasome Inhibitors; Pyrazines; Ubiquitin; Vitamin E; Vitamins