Abstract:

Objective To discuss the induction effect of wedelolactone （WEL） on cuproptosis in the human pancreatic cancer cells （PANC-1）， and to clarify its molecular mechanism. Methods The PANC-1 cells were treated with different concentrations （0-300 μmol·L^-1） of WEL for 12， 24， and 48 h， respectively， cell counting kit-8 （CCK-8） method was used to detect the survival rates of the cells after treated with different concentrations of WEL to determine the drug concentration and action time for subsequent experiments. The human pancreatic cancer PANC-1 cells were divided into control group （0 μmol·L^-1 WEL）， 8.75 μmol·L^-1 WEL group， 17.50 μmol·L^-1 WEL group， and 35.00 μmol·L^-1 WEL group. Colony formation assay was used to detect the colony formation rates of the PANC-1 cells in various groups； 5-ethynyl-2'-deoxyuridine （EdU） staining was used to detect the EdU positive cell rates of the cells in various groups； lactate dehydrogenase （LDH） kit was used to detect the LDH release in supernatant of the cells in various groups. After treating PANC-1 cells with caspase inhibitor （Z-VAD-FMK）， cuproptosis inhibitor tetrathiomolybdate （TTM）， ferroptosis inhibitor ferrostatin-1 （Fer-1）， and necroptosis inhibitor necrostatin-1 （Nec-1） combined with 35.00 μmol·L^-1 WEL for 48 h， CCK-8 method was used to detect the cell survival rates of the PANC-1 cells after treated with different inhibitors， and to screen the death mode induced by WEL； cell copper （Cu2?） colorimetric assay kit was used to detect the intracellular Cu2? levels of the cells in various groups； transmission electron microscope was used to observe the mitochondrial ultrastructure of the PANC-1 cells in various groups； mitochondrial membrane potential assay kit （JC-1） was used to detect the mitochondrial membrane potential of the cells in various groups； immunofluorescence staining was used to detect the expression and mitochondrial co-localization of dihydrolipoamide S-acetyltransferase （DLAT） in the cells in various groups； Western blotting method was used to detect the expression levels of ferredoxin 1 （FDX1）， lipoic acid synthetase （LIAS）， DLAT， and dihydrolipoamide S-succinyltransferase （DLST） proteins in the cells in various groups. Results The CCK-8 assay results showed that compared with control group， the survival rates of the PANC-1 cells after treated with different concentrations of WEL for 12， 24， and 48 h were significantly decreased （P<0.05）， with the most significant inhibitory effect at 48 h； therefore， 0， 8.75， 17.50， and 35.00 μmol·L^-1 WEL were selected to treat the PANC-1 cells. The colony formation assay results showed that compared with control group， the colony formation rates of the PANC-1 cells in 8.75， 17.50， and 35.00 μmol·L^-1 WEL groups were decreased （P<0.01）. The EdU assay results showed that compared with control group， the EdU positive cell rates of the PANC-1 cells in 8.75， 17.50， and 35.00 μmol·L^-1 WEL groups were decreased （P<0.01）. The LDH assay results showed that compared with control group， the LDH release in supernatant of PANC-1 cells in 8.75， 17.50， and 35.00 μmol·L^-1 WEL groups was increased （P<0.01）. The cell Cu2? colorimetric assay kit results showed that compared with control group， the Cu2? levels in the PANC-1 cells in 8.75， 17.50， and 35.00 μmol·L?1 WEL groups were increased （P<0.01）. The inhibitor intervention assay results showed that compared with control group， the survival rate of the PANC-1 cells in 35.00 μmol·L?1 group was decreased（P<0.01）； compared with 35.00 μmol·L?1 WEL group， the survival rates of the PANC-1 cells in WEL+Z-VAD-FMK group and WEL+TTM group were increased （P<0.01）. The transmission electron microscope results showed that the mitochondria in the PANC-1 cells in 35.00 μmol·L^-1 WEL group exhibited membrane rupture， reduced number of cristae， and sparse arrangement. The JC-1 staining results showed that compared with control group， the mitochondrial membrane potential in the PANC-1 cells in 17.50 and 35.00 μmol·L^-1 WEL groups was significantly decreased （P<0.01）. The immunofluorescence staining results showed that compared with control group， the DLAT fluorescence intensity in the PANC-1 cells in 17.50 and 35.00 μmol·L^-1 WEL groups was significantly increased （P<0.01） and co-localized with mitochondria. The Western blotting method results showed that compared with control group， the expression level of FDX1 protein in the PANC-1 cells in 8.75 μmol·L?1 WEL group was increased （P<0.01）， the expression levels of DLAT， DLST， and FDX1 proteins in the PANC-1 cells in 17.50 and 35.00 μmol·L?1 WEL groups were significantly increased （P<0.01）， while the expression level of LIAS protein was significantly decreased （P<0.01）. Conclusion WEL can induce cuproptosis in the PANC-1 cells， and its mechanism may be related to increasing the Cu2? level and up-regulating the expression levels of key cuproptosis proteins DLAT， DLST， and FDX1 in the PANC-1 cells.

Key words: Wedelolactone, Pancreatic neoplasm, Cuproptosis, Mitochondria, Dihydrolipoamide S-acetyltransferase