Study on Anti-colorectal Cancer Mechanism of Gallic Acid Based on JAK/STAT Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate the effects of gallic acid （GA） on human colon cancer HCT-116 and Caco-2 cell activities， intracellular Janus kinase （JAK）/signal transducer and activator of transcription factor （STAT） signaling pathway， and the expression of anti-apoptotic protein B-cell lymphoma-2 （Bcl-2） and pro-apoptotic protein B-cell lymphoma-2-associated X protein （Bax）， so as to explore its underlying molecular mechanism. MethodFollowing the classification of cells into GA group， blank group， and 5-fluorouracil （5-FU， 0.05 g·L-1） group， the HCT-116 and Caco-2 cells were treated with GA （0.02， 0.05， 0.1， 0.15， 0.2 g·L-1） for 12， 24， 48， and 72 h， respectively， and the cell proliferation inhibition rats were determined by cell counting kit-8 （CCK-8） assay to select the GA concentration that effectively inhibited proliferation. The colony formation ability was detected by crystal violet staining and the migration of cells by scratch test. The level of reactive oxygen species （ROS） was measured using a fluorescent probe （DCFH-DA）. The expression of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in cell supernatant were determined using the enzyme-linked immunosorbent assay （ELISA） kits. The expression levels of JAK2， phosphorylated （p）-JAK2， STAT3， p-STAT3， Bcl-2， and Bax were assayed by Western blot. ResultCCK-8 assay showed that after 12， 24， 48， and 72 h of treatment， GA （0.02， 0.05， 0.1， 0.15， 0.2 g·L-1） inhibited the proliferation of HCT-116 and Caco-2 cells in a dose- and time-dependent manner， and the inhibition rates were higher than those in the blank control group. Compared with the 5-FU group， GA （0.2 g·L-1） enhanced the inhibition of cell proliferation in a time-dependent manner. Compared with the blank control group， GA （0.1， 0.15， and 0.2 g·L-1） significantly decreased the number of cell colonies （P<0.01）， increased the inhibition rate of cell colony formation （P<0.01）， diminished the scratch healing rate （P<0.05， P<0.01）， elevated the fluorescence intensity of intracellular ROS （P<0.01）， and down-regulated the expression of IL-6 and TNF-α in the supernatant （P<0.01） in a dose-dependent manner. Compared with the 5-FU group， GA （0.2 g·L-1） decreased the scratch healing rate （P<0.01）， enhanced the fluorescence intensity of intracellular ROS （P<0.01）， and down-regulated the levels of IL-6 and TNF-α in cell supernatant （P<0.01）. According to Western blot analysis， compared with the blank control group， GA （0.1， 0.15， 0.2 g·L-1） obviously lowered the expression of p-JAK2， p-STAT3， Bcl-2， p-JAK2/JAK2， p-STAT3/STAT3， and Bcl-2/Bax （P<0.01） and raised Bax protein expression （P<0.05， P<0.01） in a dose-dependent manner. Compared with the 5-FU group， GA （0.2 g·L-1） down-regulated the expression of p-JAK2， p-STAT3， Bcl-2， p-JAK2/JAK2， p-STAT3/STAT3， and Bcl-2/Bax （P<0.05， P<0.01） and up-regulated the expression of Bax protein （P<0.05， P<0.01）. ConclusionGA significantly inhibits the proliferation of HCT-116 and Caco-2 cells， which may be related to the increased accumulation of intracellular ROS， down-regulation of inflammatory factors IL-6 and TNF-α， p-JAK2 and p-STAT3 protein expression in JAK/STAT signaling pathway， and Bcl-2， and up-regulation of Bax.