Panoply™ Human PDK1 Over-expressing Stable Cell Line

Metabolic disorder is a major characteristic of cancer cells, and controlling the genes involved in metabolic transformation is an effective strategy for cancer treatment. Andrographolide (AG) is a diterpene lactone widely considered a natural anticancer drug due to its ability to inhibit tumor growth. Here, researchers explored the mechanism by which AG exerts its anticancer effects by inhibiting pyruvate dehydrogenase kinase 1 (PDK1) expression and mediating mitochondrial pathways in lung cancer cells. After treating cells with AG, the expression levels of PDK1 mRNA and protein were detected by quantitative reverse transcription PCR and Western blotting, respectively. The results showed that AG significantly inhibited the viability of human lung cancer cells and suppressed aerobic glycolysis by reducing lactate production. AG decreased PDK1 protein and mRNA levels in a dose-dependent manner. AG-induced cell death was detected using flow cytometry and fluorescence microscopy. AG-induced apoptosis was associated with poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and mitochondrial damage, which in turn was associated with increased reactive oxygen species levels and decreased mitochondrial membrane potential. Overexpression of PDK1 in lung cancer cells partially inhibited AG-induced cell death. Therefore, the anticancer effect of AG on human lung cancer cells may be related to the negative regulation of PDK1 expression.

To determine whether AG-induced suppression of PDK1 expression is crucial for cancer cell death, researchers constructed a PDK1-overexpressing (PDK1-OE) H292 cell line (Figure 1A, B). In the MTT assay, AG-induced cytotoxicity was significantly lower in PDK1-overexpressing H292 cells compared to empty vector (EV) cells (Figure 1C). At the highest concentration of AG (75 µM), only 7.68% of empty vector cells survived, while the survival rate of PDK1-overexpressing cells was 31.7%. Figure 1D shows the morphological changes of PDK1-overexpressing H292 cells and EV cells after AG treatment. AG-induced cytotoxicity was weaker in PDK1-overexpressing H292 cells compared to control (EV) cells. The histogram also showed a slight decrease in the number of AG-induced dead cells in PDK1-overexpressing H292 cells compared to control cells (Figure 1E). The expression of apoptotic marker proteins (including cleaved PARP and cleaved caspase 3) was suppressed in PDK1-overexpressing H292 cells (Figure 1F). These results indicate that PDK1 expression is partially involved in the cytotoxic effect of AG. By inhibiting PDK1 expression with AG, the metabolic shift from glycolysis to oxidative phosphorylation leads to a significant increase in mitochondrial ROS production, which is another key factor in AG-induced cancer cell death (Figure 1G).

Figure 1. AG-induced cancer cell death is alleviated in PDK1-overexpressing H292 cells. (Yang E S, et al., 2023)