The newly synthesized naftopidil analogue HUHS1015 reduced viability of MKN28 and MKN45 human
gastric cancer cells in a concentration (0.3-100 μM)-dependent manner, with the potential greater than that for naftopidil.
In the cell cycle analysis, HUHS1015 significantly increased the proportion at the subG1 phase of cell cycling in MKN28
cells. In the flow cytometry using propidium iodide (PI) and annexin V, HUHS1015 significantly increased the
populations of PI-positive/annexin V-negative and PI-positive/annexin V-positive MKN28 cells, corresponding to primary
necrosis and late apoptosis/secondary necrosis, respectively. HUHS1015-induced MKN28 cell death was attenuated by the
necroptosis inhibitor Nec-1. In the enzymatic caspase assay, caspase-3, -4, -8, and -9 were not sufficiently activated by HUHS1015.
HUHS1015 increased nuclear localization of apoptosis-inducing factor-homologous mitochondrion-associated inducer of death (AMID),
without affecting expression of the AMID mRNA and protein in MKN28 cells. HUHS1015 caused nuclear fragmentation and
condensation in MKN28 cells treated with HUHS1015. Taken together, these results of the present study indicate that HUHS1015
induces both necroptosis and caspase-independent apoptosis of MKN28 cells, possibly the latter effect being due to AMID accumulation
in the nucleus.