ABSTRACT
Objective:To explore the mechanism of lysosomal membrane permeabilization(LMP)inuranyl acetate-induced death of human kidney proximal tubular epithelial HK-2 cells.Methods:HK-2 cells were exposed to uranyl acetate at concentrations of 100, 300 and 600 μmol/L for 24 h, then in tracellular reactive oxygen species (ROS)and mitochondrial superoxide were measured by DCFH-DA and MitoSOX probe, respectively. HK-2 cells were divided into four groups: blank control group, NAC or CA-074 Me group, uranyl acetate exposure group and uranyl acetate exposure plus NAC or CA-074 Me group. Two-color immune of luorescence staining was used to detect the co-localization of galectin-1 and lysosomal associated membrane protein-1 (LAMP-1) to measure the extent of LMP, and to detect the non- co-localization of cathepsin B and LAMP-1 to reflect the release of cathepsin B in lysosomes. Calcein-AM/PI double staining method was used to detect cell death. One-color immune of luorescence staining of cleaved-caspase-3 expression was used to detect apoptosis. Results:Intracellular ROS and mitochondrial superoxide levels were significantly increased in HK-2 cells after exposure with 100, 300 and 600 μmol/L uranyl acetate for 24 h, about 1.1-2.5 times or 4.0-28 times, respectively( tROS=17.98, 11.84, 11.75, P< 0.05; tmitochondrial superoxide=6.14, 16.02, 13.06, P< 0.05), and they also increased with uranyl acetate concentrations ( tROS=10.10, 10.37, 5.59, P< 0.05; tmitochondrial superoxide=21.50, 15.16, 5.93, P< 0.05). The percentage of co-localization of galectin-1 and LAMP-1 and the percentage of non- co-localization of cathepsin B and LAMP-1 were markedly increased in HK-2 cells after exposure with 600 μmol/L uranyl acetate for 24 h, 5.4-6.7 times or 1.5-2.1 times, respectively ( tGalectin-1=15.85, 12.70, P< 0.05; tCathepsin B=5.95, 6.69, P< 0.05), but these increases were inhibited by NAC ( tGalectin-1=4.74, P<0.05; tCathepsin B=4.51, P< 0.05). Moreover, the cell death rate and the cleaved-caspase-3 expression level were also significantly increased in HK-2 cells after exposure with 600 μmol/L uranyl acetate for 24 h, about 28-47 times or 2.4-6.0 times, respectively( tPI=30.40, 10.34, P<0.05; tCleaved-caspase-3=18.49, 9.52, P<0.05), and these increases were obviously diminished by CA-074 Me ( tPI= 6.76, P<0.05; tCleaved-caspase-3=13.47, P<0.05). Conclusions:Exposure to uranyl acetate induces a burst of intracellular ROSthat leads to LMP and consequently causes leakage of cathepsin B from lysosomes to cytoplasm, in turn triggering the lysosomal-dependent cell death and mitochondrial-regulated apoptosis of HK-2 cells.
ABSTRACT
Objective:To investigate the radiosensitizing effect of Ta 4C 3-PVP nanosheets on the tumor of 4T1 murine triple-negative breast cancer cells planted in mice. Methods:4T1 tumor-bearing mice model was established by subcutaneous injection of 4T1 cells into the right flank of the female BALB/c mice. The mice were divided into four groups uniformly according to their tumor size: blank control group, Ta 4C 3-PVP group, ionizing radiation (IR) group and Ta 4C 3-PVP plus IR group. A single dose of 8 Gy X-ray local irradiation was given to xenograft tumor at 24 h after tail intravenous injection of Ta 4C 3-PVP (20 mg/kg). The xenograft tumor volume and weight, the pathological changes of tumor tissue, the expression of tumor proliferative marker Ki-67 protein, and the formation of γ-H2AX foci [a DNA double-strand breaks (DSBs) molecular marker] were detected. Tumor growth curve was established, and enhancement factor (EF) and tumor inhibition rate were calculated. Results:Compared with the blank control group, tumor growth was significantly inhibited ( t=5.41, 9.59, P < 0.05) and tumor weight was markedly decreased ( t=2.67, 4.40, P < 0.05) in both IR group and Ta 4C 3-PVP plus IR group at day 16 after IR. The EF in Ta 4C 3-PVP plus IR group was 1.57, and tumor inhibition rate in Ta 4C 3-PVP plus IR group were about 64%, which was much higher than that of IR group alone(42%). Immunohistochemistry and immunofluorescence histochemistry assays showed that the expression of Ki-67 protein was obviously decreased and the amount of γ-H2AX foci was significantly increased in both IR group and Ta 4C 3-PVP plus IR group in comparison with the blank control group ( t=5.73, 8.02, 2.97, 9.86, P < 0.05). Moreover, the inhibition of Ki-67 protein expression and the increase of γ-H2AX foci were much higher in Ta 4C 3-PVP plus IR group than that in IR group ( t=4.75, 4.42, P < 0.05). Conclusions:Ta 4C 3-PVP nanosheets could enhance radiosensitivity of xenograft tumor in 4T1 tumor-bearing mice through increasing the IR-induced DNA DSBs.