Investigation of mitochondria-dependent apoptosis pathway and lipid peroxidation level induced by biosynthesized silver nanoparticles: caspase-3 activation, BAK1/BCLx regulation and malondialdehyde production

Nowadays, silver nanoparticles (AgNPs) have attracted the attention of many researchers due to their special physical, chemical, and biological properties. There is strong evidence that biogenic AgNPs can act as potent anticancer agents through the production of reactive oxygen species (ROS) and initiate the mitochondrial pathway of apoptosis. That is why we decided to use Nepeta bracteata Benth flower extract for the first time to bio-synthesize AgNPs and study their cytotoxic and apoptotic effects on SK-BR-3 cells. AgNPs were biosynthesized at 70 degrees C after mixing silver nitrate and flower extract with a specific ratio and concentration, then were characterized using various analytical techniques, such as FESEM, FTIR, EDS, and zeta potential. Studies have shown that AgNPs have an irregular and circular shape, with about 99% by weight of silver, carbon, and oxygen. On the other hand, the appropriate size (below 57 nm) and surface charge (- 11.52 mV) make them stable in biological fluids. The better cytotoxic effect of AgNPs compared to flower extract on SK-BR-3 cells was investigated using the MTT method. The positive effect of AgNPs on inhibiting the growth of SK-BR-3 breast cancer cells was again confirmed by the sulforhodamine B staining method, so that AgNPs were able to decrease the density of cancer cells in a concentration-dependent manner. In addition, the flow cytometry test proved that biosynthesized AgNPs using Nepeta bracteata Benth flower extract can induce apoptosis in SK-BR-3 cancer cells. Real-time PCR then proved that the ratio of Bak1/Bclx, as well as caspase-3 expression, was increased due to active ROS-producing biomolecules present in the plant extract, and therefore, AgNPs can activate the mitochondria-dependent apoptosis pathway in breast cancer cells. Finally, their negligible oxidative stress on erythrocytes was confirmed by the lipid peroxidation method and showed that biosynthesized AgNPs can be used for breast cancer treatment without showing adverse effects on erythrocytes.