Rapid decrease of intracellular pH associated with inhibition of Na+/H+ exchanger precedes apoptotic events in the MNK45 and MNK74 gastric cancer cell lines treated with 2-aminophenoxazine-3-one.

The effects of Phx-3 on changes in intracellular pH (pHi) in the MKN45 and MKN74 human gastric cancer cell lines were evaluated in order to determine the mechanism for the proapoptotic effects of 2-aminophenoxazine-3-one (Phx-3) on these cells. Phx-3 (100 µM) reduced pHi in MKN45 from 7.45 to 5.8, and in MKN74 from 7.5 to 6.2 within 1 min of engagement with these cells. Such a decrease of pHi was closely correlated with the dose of this phenoxazine and continued for 4 h. The activity of Na+/H+ exchanger isoform l (NHE1), which is involved in H+ extrusion from the cells, was dose-dependently suppressed by Phx-3 in these cells, and was greatly suppressed in the presence of 100 µM Phx-3. This result indicates that the decrease of pHi in MKN45 and MKN74 cells is closely associated with the inhibition of NHE1 in these cells. The morphology of these cells at 24 h after treatment with Phx-3 indicated shrinkage of the cells and condensation of the nuclear chromatin structure, which are characteristic of the apoptotic events in these gastric cancer cells. Cytotoxicity of Phx-3 against MKN45 and MKN74 cells was extensive because almost all MKN45 cells lost viability at 24 h in the presence of 20 µM Phx-3, and nearly 50% of the MKN74 cells lost viability in the presence of 50 µM Phx-3. These results suggest that rapid and extensive decrease of pHi in human gastric cancer MKN45 and MKN74 cells caused by Phx-3 might disturb intracellular homeostasis, leading to apoptotic and cytotoxic events in these cells. Phx-3 is a good candidate for therapeutics of gastric cancer that is intractable to conventional chemopreventive therapies.

Combined treatment with bortezomib plus bafilomycin A1 enhances the cytocidal effect and induces endoplasmic reticulum stress in U266 myeloma cells: crosstalk among proteasome, autophagy-lysosome and ER stress.

Bortezomib (BZ), a first line 26S proteasome inhibitor, induces a potent cytocidal effect with caspase-3 activation in multiple myeloma (MM) cell lines. Since IκBα is a substrate of the proteasome, the initial rationale for using BZ in MM has been to inhibit NF-κB. However, BZ rather activated NF-κB activity in U266 cells. BZ induces autophagy as well as endoplasmic reticulum (ER) stress in various cell lines tested. Inhibition of initial autophagosome formation by treatment with either 3-methyladenine or siRNA for LC3B in U266 cells and knockdown of the atg5 gene in a murine embryonic fibroblastic cell line all resulted in attenuation of BZ-induced cell death. In contrast, combined treatment with BZ and bafilomycin A1 (BAF), which is a specific inhibitor of vacuolar-ATPase and is used as an autophagy inhibitor at the late stage, resulted in synergistic cytotoxicity, compared with that by either BZ or BAF alone. BAF treatment also induced ER stress, but the kinetics of inductions of ER stress-related genes [e.g. CHOP (GADD153) and GRP78] completely differed between BZ- and BAF-treatments: BZ induced these ER stress markers within 8 h, whereas treatment with BAF required more than 48 h in U266 cells. In order to synchronize ER stress, we pre-treated U266 cells with BAF for 48 h, followed with BZ for 48 h. The sequential treatment with BAF and BZ induced a further enhanced cytotoxicity, compared with the simultaneous combination of BAF and BZ. These data suggest crosstalk among the ubiquitin-proteasome system, the autophagy-lysosome system, and ER stress. Controlling these interactions and kinetics appears to have important implications for optimizing clinical cancer treatment including MM-therapy.