Hydrophobic flavonoids from Scutellaria baicalensis induce colorectal cancer cell apoptosis through a mitochondrial-mediated pathway.

Scutellaria baicalensis extract (SbE) has been shown to exert chemopreventive effects on several types of cancer. Baicalin, a hydrophilic flavonoid found in SbE, may have opposing effects that decrease the antitumor potential of SbE against colorectal cancer. In this study, after removing baicalin, we prepared an aglycone-rich fraction (ARF) of SbE and evaluated its anti-proliferative activity and mechanisms of action. The flavonoids found in ARF, baicalin fraction (BF) and SbE were determined by high-performance liquid chromatography (HPLC). The effects of ARF, BF, SbE and representative flavonoids on the proliferation of HCT-116 and HT-29 human colorectal cancer cells were determined by an MTS assay. The cell cycle, the expression of cyclins A and B1 and cell apoptosis were assayed using flow cytometry. Apoptosis-related gene expression was visualized by quantitative real-time polymerase chain reaction (PCR), and mitochondrial membrane potential was estimated following staining with JC-1. HPLC analysis showed that ARF contained two hydrophobic flavonoids, baicalein and wogonin, and that BF contained only baicalin. SbE had little anti-proliferative effect on the colorectal cancer cells; cancer cell growth was even observed at certain concentrations. ARF exerted potent anti-proliferative effects on the cancer cells. By contrast, BF increased cancer cell growth. ARF arrested cells in the S and G2/M phases, increased the expression of cyclins A and B1, and significantly induced cell apoptosis. Multiple genes in the mitochondrial pathway are involved in ARF-induced apoptosis, and subsequent cellular functional analysis validated the involvement of this pathway. These results suggest that removing baicalin from SbE produces an ARF that significantly inhibits the growth of colorectal cancer cells, and that the mitochondrial apoptotic pathway plays a role in hydrophobic flavonoid-induced apoptosis.

Paraptosis and NF-κB activation are associated with protopanaxadiol-induced cancer chemoprevention.

BACKGROUND: Protopanaxadiol (PPD) is a triterpenoid that can be prepared from steamed ginseng. PPD possesses anticancer potential via caspase-dependent apoptosis. Whether paraptosis, a type of the caspase-independent cell death, is also induced by PPD has not been evaluated. METHODS: Cell death, the cell cycle and intracellular reactive oxygen species (ROS) were analyzed by flow cytometry after staining with annexin V/PI, PI/RNase or H2DCFDA. We observed morphological changes by crystal violet staining assay. Mitochondrial swelling was measured by ultraviolet-visible spectrophotometry. The activation of NF-κB was measured by luciferase reporter assay. RESULTS: At comparable concentrations of 5-fluorouracil, PPD induced more cell death in human colorectal cancer cell lines HCT-116 and SW-480. We demonstrated that PPD induced paraptosis in these cancer cells. PPD treatment significantly increased the percentage of cancer cells with cytoplasmic vacuoles. After the cells were treated with PPD and cycloheximides, cytoplasmic vacuole generation was inhibited. The paraptotic induction effect of PPD was also supported by the results of the mitochondrial swelling assay. PPD induced ROS production in cancer cells, which activated the NF-κB pathway. Blockage of ROS by NAC or PS-1145 inhibited the activation of NF-κB signaling. CONCLUSIONS: PPD induces colorectal cancer cell death in part by induction of paraptosis. The anticancer activity of PPD may be enhanced by antioxidants such as green tea, which also inhibit the activation of NF-κB signaling.